WO2023155879A1

WO2023155879A1 - Methods for regulating immune cell mediated functions

Info

Publication number: WO2023155879A1
Application number: PCT/CN2023/076778
Authority: WO
Inventors: Li Yu; Haifeng JIAO; Cuifang ZHANG
Original assignee: Tsinghua University
Priority date: 2022-02-18
Filing date: 2023-02-17
Publication date: 2023-08-24

Abstract

Provided are methods and compositions for regulating an immune cell relating functions, by regulating the formation and/or function of a migrasome generated by another immune cell.

Description

METHODS FOR REGULATING IMMUNE CELL MEDIATED FUNCTIONS

BACKGROUND OF THE INVENTION

Secretion is a fundamental process of cells. Cells communicate with each other by secreting signaling ligands such as cytokines and neuron transmitter. It is known that secretion can occur on restricted portion of cell surface, however, highly localized secretion on specialized membrane structure have only been found in neuron cells, in which secretory vesicles are transported from cell body to axon terminal, where it fuses with membrane on axon terminal and releases neuron transmitters. The basic unit of secretion is secretory vesicles, once it fuses with plasma membrane, the cargos are released form the cells, however, whether there are specialized cellular structure which can package and release secretory cargos en masse is not known.

Migrasome is a newly discovered organelle in migrating cells. When cells migrate, they leave long membrane tether named retraction fibers on the trailing edge of the cells. Large vesicles named migrasome with a diameter of around 2μm grow on retraction fibers, inside the migrasome are numerous small intraluminal vesicles with unknown origin. Migrasomes have been observed in various biological settings and have been shown to play important physiological roles in vivo, such as during the embryogenesis process of zebrafish. However, the functions and regulation of immune cell derived migrasomes are less clear.

SUMMARY OF THE INVENTION

The present disclosure relates to the function, the characterization as well as the regulation of migrasomes generated by or derived from immune cells.

The present inventors found that in immune cells (such as monocytes and/or macrophages) , cytokines such as TNF-α and IL-6 are enriched in migrasomes. In vitro and in vivo, cytokine secretion is reduced when migrasome formation is compromised, which suggests that migrasomes are required for efficient secretion (such as secretion of signaling molecules, e.g., cytokines for immune cells) . Monocyte-derived, cytokine-enriched migrasomes were detected in blood upon pathogen stimulation or infection (e.g., as in LPS-treated mice) , which indicates that cytokines can be released en masse in migrasome-bound form. For immune cells, migrasomes mediate both localized secretion and packaged release of cytokines, and thus are portals for secretion in migrating cells.

In one aspect, the present disclosure provides a method for regulating the recruitment of a second immune cell by a first immune cell, comprising regulating the formation and/or function of a migrasome generated by the first immune cell.

In some embodiments, the method increases the recruitment of the second immune cell and comprises promoting the formation and/or function of the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in the first immune cell and/or in the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises overexpressing the tetraspanin protein, the functional fragment thereof, and/or the functional variant thereof in the first immune cell.

In some embodiments of the method, the tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the number of intraluminal vesicles in the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the transportation of intraluminal vesicles into the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a motor protein in the first immune cell.

In some embodiments of the method, the motor protein comprises a Myosin.

In some embodiments of the method, the motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing fusion of the membrane of the migrasome with an intraluminal vesicle therein.

In some embodiments of the method, increasing the fusion comprises increasing the amount and/or function of a SNARE complex in the migrasome.

In some embodiments of the method, increasing the fusion comprises increasing calcium in the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a chemokine in the first immune cell and/or in the migrasome.

In some embodiments of the method, the chemokine comprises CXCL12.

In some embodiments, the method decreases the recruitment of the second immune cell and comprises inhibiting the formation and/or function of the migrasome.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises inhibiting the expression and/or function of a tetraspanin in the first immune cell and/or in the migrasome. In some embodiments of the method, inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for the tetraspanin in the first immune cell. In some embodiments of the method, the tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises decreasing the number of intraluminal vesicles in the migrasome.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises inhibiting transportation of intraluminal vesicles into the migrasome.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises decreasing the amount and/or function of a motor protein in the first immune cell. In some embodiments of the method, the motor protein comprises a Myosin. In some embodiments of the method, the motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises inhibiting fusion of the membrane of the migrasome with an intraluminal vesicle therein.

In some embodiments of the method, inhibiting the fusion comprises decreasing the amount and/or function of a SNARE complex in the migrasome.

In some embodiments of the method, inhibiting the fusion comprises decreasing calcium in the migrasome.

In some embodiments of the method, decreasing calcium in the migrasome comprises administering a calcium chelator.

In some embodiments of the method, the calcium chelator comprises BAPTA-AM.

In some embodiments of the method, the intraluminal vesicle comprises one or more immune signaling molecules.

In some embodiments of the method, the immune signaling molecule comprises a cytokine and/or a flavonoid.

In some embodiments of the method, the cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.

In some embodiments of the method, the interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.

In some embodiments of the method, the interferon comprises IFN-γ.

In some embodiments of the method, the chemokine comprises CCL2 and/or CXCL12.

In some embodiments of the method, the Tumor Necrosis Factor comprises TNF-α.

In some embodiments of the method, the flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises decreasing the amount and/or function of a chemokine in the first immune cell and/or in the migrasome.

In some embodiments of the method, decreasing the amount and/or function of the chemokine comprises knocking out or knocking down the expression of a gene encoding for the chemokine in the first immune cell.

In some embodiments of the method, decreasing the amount and/or function of the chemokine comprises treating the migrasome with an agent capable of inhibiting the function of the chemokine.

In some embodiments of the method, the agent capable of inhibiting the function of the chemokine comprises a protease, a small molecule, and/or an antibody capable of inhibiting the activity of the chemokine.

In some embodiments of the method, the chemokine comprises CXCL12.

In some embodiments of the method, the first immune cell comprises a monocyte and/or a macrophage.

In some embodiments of the method, the first immune cell consists essentially of a monocyte and/or macrophage.

In some embodiments of the method, the second immune cell comprises a monocyte and/or a macrophage.

In some embodiments of the method, the second immune cell consists essentially of a monocyte and/or a macrophage.

In some embodiments of the method, the first immune cell is of the same type as the second immune cell.

In some embodiments of the method, the first immune cell is a monocyte and/or a macrophage, and the second immune cell is a monocyte and/or a macrophage.

In some embodiments of the method, the migrasome comprises and/or expresses a chemokine. In some embodiments of the method, the chemokine comprises CXCL12.

In some embodiments of the method, the second immune cell comprises and/or expresses a molecule capable of specifically recognizing the chemokine comprised and/or expressed by the migrasome.

In some embodiments of the method, the second immune cell comprises and/or expresses CXCR7 and/or CXCR4.

In another aspect, the present disclosure provides a method for regulating the migration of an immune cell towards a location, comprising regulating the amount and/or function of a migrasome present at or near the location.

In some embodiments, the method increases the migration of the immune cell and comprises increasing the amount and/or function of the migrasome.

In some embodiments of the method, increasing the amount of the migrasome comprises administering the migrasome to the location.

In some embodiments of the method, increasing the amount and/or function of the migrasome comprises promoting the formation and/or function of the migrasome generated by a local immune cell, the local immune cell is an immune cell at or near the location.

In some embodiments of the method, promoting the formation and/or function of the migrasome generated by the local immune cell comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in the local immune cell.

In some embodiments of the method, promoting the formation and/or function of the migrasome generated by the local immune cell comprises increasing the number of intraluminal vesicles in the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome generated by the local immune cell comprises increasing the transportation of intraluminal vesicles into the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome generated by the local immune cell comprises increasing the amount and/or function of a motor protein in the local immune cell.

In some embodiments of the method, the motor protein comprises a Myosin.

In some embodiments of the method, promoting the formation and/or function of the migrasome generated by the local immune cell comprises increasing fusion of the membrane of the migrasome with an intraluminal vesicle therein.

In some embodiments of the method, promoting the formation and/or function of the migrasome generated by the local immune cell comprises increasing the amount and/or function of a chemokine in the local immune cell and/or in the migrasome.

In some embodiments of the method, the chemokine comprises CXCL12.

In some embodiments, the method decreases the migration of the immune cell and comprises decreasing the amount and/or function of the migrasome.

In some embodiments of the method, decreasing the amount and/or function of the migrasome comprises inhibiting the formation and/or function of the migrasome generated by a local immune cell, the local immune cell is an immune cell at or near the location.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome generated by the local immune cell comprises inhibiting the expression and/or function of a tetraspanin in the local immune cell and/or in the migrasome. In some embodiments of the method, inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for the tetraspanin in the local immune cell. In some embodiments of the method, the tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome generated by the local immune cell comprises decreasing the number of intraluminal vesicles in the migrasome.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome generated by the local immune cell comprises inhibiting transportation of intraluminal vesicles into the migrasome.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome generated by the local immune cell comprises decreasing the amount and/or function of a motor protein in the local immune cell.

In some embodiments of the method, the motor protein comprises a Myosin.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome generated by the local immune cell comprises inhibiting fusion of the membrane of the migrasome with an intraluminal vesicle therein.

In some embodiments of the method, the calcium chelator comprises BAPTA-AM.

In some embodiments of the method, the interferon comprises IFN-γ.

In some embodiments of the method, the chemokine comprises CCL2 and/or CXCL12.

In some embodiments of the method, the Tumor Necrosis Factor comprises TNF-α.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome generated by the local immune cell comprises decreasing the amount and/or function of a chemokine in the local immune cell and/or in the migrasome.

In some embodiments of the method, decreasing the amount and/or function of the chemokine comprises knocking out or knocking down the expression of a gene encoding for the chemokine in the local immune cell.

In some embodiments of the method, the chemokine comprises CXCL12.

In some embodiments of the method, the local immune cell comprises a monocyte and/or a macrophage.

In some embodiments of the method, the local immune cell consists essentially of a monocyte and/or macrophage.

In some embodiments of the method, the immune cell that migrates towards the location comprises a monocyte and/or a macrophage.

In some embodiments of the method, the immune cell that migrates towards the location consists essentially of a monocyte and/or a macrophage.

In some embodiments of the method, the local immune cell is of the same type as the immune cell that migrates towards the location.

In some embodiments of the method, the local immune cell is a monocyte and/or a macrophage, and the immune cell that migrates towards the location is a monocyte and/or a macrophage.

In some embodiments of the method, the migrasome comprises and/or expresses a chemokine.

In some embodiments of the method, the chemokine comprises CXCL12.

In some embodiments of the method, the immune cell that migrates towards the location comprises and/or expresses a molecule capable of specifically recognizing the chemokine comprised and/or expressed by the migrasome.

In some embodiments of the method, the immune cell that migrates towards the location comprises and/or expresses CXCR7 and/or CXCR4.

In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process, comprising regulating the formation and/or function of a migrasome generated by an immune cell mediating the immune response.

In some embodiments of the method, the immune response and/or immune response mediated biological process comprises secretion (such as localized secretion, such as local packaged secretion) of a substance by the immune cell.

In some embodiments of the method, the immune response and/or immune response mediated biological process is regulated by the secretion of a substance by the immune cell.

In some embodiments of the method, the immune response and/or immune response mediated biological process comprises inflammatory response, and/or other diseases, conditions or disorders affected by the function of an immune cell.

In some embodiments, the method comprises promoting the formation and/or function of the migrasome.

In some embodiments, the method comprises inhibiting the formation and/or function of the migrasome.

In another aspect, the present disclosure provides a method for regulating the secretion of a substance by an immune cell, comprising regulating the formation and/or function of a migrasome generated by the immune cell.

In some embodiments of the method, the immune cell is a migrating immune cell.

In some embodiments of the method, the immune cell is a migrating immune cell in or from the blood.

In some embodiments of the method, the immune cell comprises a monocyte and/or a macrophage.

In some embodiments of the method, the substance comprises one or more immune signaling molecules.

In some embodiments of the method, the one or more immune signaling molecules comprise a cytokine and/or a flavonoid.

In some embodiments of the method, the interferon comprises IFN-γ.

In some embodiments of the method, the chemokine comprises CCL2 and/or CXCL12.

In some embodiments of the method, the Tumor Necrosis Factor comprises TNF-α.

In some embodiments, the method increases the secretion of the substance, and comprises promoting the formation and/or function of the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises promoting the generation of the migrasome by the immune cell.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in the immune cell and/or in the migrasome.

In some embodiments of the method, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a motor protein in the immune cell. In some embodiments of the method, the motor protein comprises a Myosin. In some embodiments of the method, the motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

In some embodiments, the method decreases the secretion of the substance, and comprises inhibiting the formation and/or function of the migrasome.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises inhibiting the expression and/or function of a tetraspanin in the immune cell and/or in the migrasome. In some embodiments of the method, inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for the tetraspanin in the immune cell. In some embodiments of the method, the tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.

In some embodiments of the method, inhibiting the formation and/or function of the migrasome comprises decreasing the amount and/or function of a motor protein in the immune cell.

In some embodiments of the method, the motor protein comprises a Myosin. In some embodiments of the method, the motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

In some embodiments of the method, the calcium chelator comprises BAPTA-AM.

In some embodiments of the method, the intraluminal vesicle comprises the substance to be secreted by the immune cell.

In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process in a subject in need thereof, comprising administering to the subject an effective amount of an immune cell derived migrasome.

In some embodiments of the method, the immune response and/or immune response mediated biological process is regulated by a secreted substance of the immune cell.

In some embodiments of the method, he immune response and/or immune response mediated biological process comprises inflammatory response and/or other diseases, conditions or disorders affected by the function of the immune cell.

In some embodiments of the method, the immune cell is a migrating immune cell.

In some embodiments of the method, the secreted substance of the immune cell comprises one or more immune signaling molecules.

In some embodiments of the method, the interferon comprises IFN-γ.

In some embodiments of the method, the chemokine comprises CCL2 and/or CXCL12.

In some embodiments of the method, the Tumor Necrosis Factor comprises TNF-α.

In some embodiments, the method further comprises administering to the subject an additional active molecule.

In some embodiments of the method, the additional active molecule comprises a caspase inhibitor.

In some embodiments of the method, the caspase inhibitor is a caspase 8 inhibitor.

In some embodiments of the method, the additional active molecule comprises z-VAD-FMK or a functional derivative thereof.

In another aspect, the present disclosure provides a method for monitoring an immune response and/or an immune response mediated biological process in a subject, comprising analyzing the presence, amount and/or function of a migrasome obtained from a biological sample of the subject.

In some embodiments of the method, the immune response and/or an immune response mediated biological process comprises inflammatory response and/or other diseases, conditions or disorders affected by the function of an immune cell.

In some embodiments of the method, the biological sample comprises a body fluid sample of the subject.

In some embodiments of the method, the biological sample comprises a blood sample of the subject.

In some embodiments of the method, an increase of the amount of the migrasome indicates an increase of the immune response.

In some embodiments of the method, analyzing the presence, amount and/or function of the migrasome comprises analyzing the presence and/or amount of a marker molecule of the migrasome.

In some embodiments of the method, analyzing the presence, amount and/or function of the migrasome comprises determining the presence and/or amount of Tspan4⁺, Integrin⁺, Pleckstrin Homology (PH) domain⁺, NDST1⁺, PIGK⁺, CPQ⁺, EOGT⁺, KUL01⁺, CD115⁺, and/or CCR2⁺ vesicles in the biological sample.

In some embodiments of the method, analyzing the presence, amount and/or function of the migrasome comprises staining the biological sample with wheatgerm agglutinin (WGA) .

In some embodiments of the method, the migrasome is KUL01⁺, CD115⁺, and/or CCR2⁺.

In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process in a subject, comprising:

i) monitoring the immune response and/or the immune response mediated biological process in the subject according to the present disclosure; and

ii) administering a regulating agent according to the result of step i) .

In some embodiments, the method is an in vitro or ex vivo method.

In some embodiments, the method is an in vivo method.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by a first immune cell, for use in recruiting a second immune cell to the first immune cell, according to the present disclosure.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome present at or near a location, for use in regulating the migration of an immune cell towards the location, according to the present disclosure.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by an immune cell mediating an immune response, for use in regulating the immune response and/or the immune response mediated biological process, according to the present disclosure.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by an immune cell, for use in regulating the secretion of a substance by the immune cell, according to the present disclosure.

In another aspect, the present disclosure provides an agent capable of detecting the presence, amount and/or function of a migrasome obtained from a biological sample of a subject, for use in monitoring an immune response and/or an immune response mediated biological process in the subject, according to the present disclosure.

In another aspect, the present disclosure provides an isolated migrasome derived from an immune cell. In some embodiments, the isolated migrasome is for use in regulating an immune response and/or an immune response mediated biological process.

In another aspect, the present disclosure provides an engineered immune cell with altered ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell, the engineered immune cell has been modified to alter its migrasome generation ability.

In some embodiments, the engineered cell has increased ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell.

In some embodiments, the engineered cell has decreased ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell.

In another aspect, the present disclosure provides an engineered immune cell with altered ability for regulating an immune response and/or an immune response mediated biological process comparing to a corresponding unmodified immune cell, the engineered immune cell has been modified to alter its migrasome generation ability.

In some embodiments, the engineered cell has increased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell.

In some embodiments, the engineered cell has decreased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell.

In another aspect, the present disclosure provides an engineered immune cell with altered secretion ability comparing to a corresponding unmodified immune cell, the engineered immune cell has been modified to alter its migrasome generation ability.

In some embodiments, the engineered cell has increased secretion ability comparing to a corresponding unmodified immune cell.

In some embodiments, the engineered cell has decreased secretion ability comparing to a corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to have increased ability for generating migrasomes.

In some embodiments, the engineered cell has been modified to increase the amount and/or function of a tetraspanin therein.

In some embodiments, the engineered cell has been modified to overexpress a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

In some embodiments of the engineered cell, the tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

In some embodiments, the engineered cell has been modified to have decreased ability for generating migrasomes.

In some embodiments, the engineered cell has been modified to decrease the amount and/or function of a tetraspanin therein.

In some embodiments of the engineered cell, the expression of a gene encoding for a tetraspanin has been knocked out or knocked down.

In some embodiments of the engineered cell, the tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.

In some embodiments, the engineered cell has been modified to generate a migrasome with increased number of intraluminal vesicles comparing to the corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to have increased ability to transport an intraluminal vesicle into the migrasome comparing to the corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to increase the amount and/or function of a motor protein therein.

In some embodiments, the engineered cell has been modified to generate a migrasome with decreased number of intraluminal vesicles comparing to the corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to have decreased ability to transport an intraluminal vesicle into the migrasome comparing to the corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to decrease the amount and/or function of a motor protein therein.

In some embodiments of the engineered cell, the motor protein comprises a Myosin.

In some embodiments of the engineered cell, the motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.

In some embodiments, the engineered cell has been modified to generate a migrasome with increased fusion ability of the migrasome membrane with an intraluminal vesicle therein comparing to the corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to increase the amount and/or function of a SNARE complex in a migrasome generated by the engineered immune cell.

In some embodiments, the engineered cell has been modified to increase calcium in a migrasome generated by the engineered immune cell.

In some embodiments, the engineered cell has been modified to generate a migrasome with decreased fusion ability of the migrasome membrane with an intraluminal vesicle therein comparing to the corresponding unmodified immune cell.

In some embodiments, the engineered cell has been modified to decrease the amount and/or function of a SNARE complex in a migrasome generated by the engineered immune cell.

In some embodiments, the engineered cell has been modified to decrease calcium in a migrasome generated by the engineered immune cell.

In some embodiments of the engineered cell, the intraluminal vesicle comprises one or more immune signaling molecules.

In some embodiments of the engineered cell, the immune signaling molecule comprises a cytokine and/or a flavonoid.

In some embodiments of the engineered cell, the cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.

In some embodiments of the engineered cell, the interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.

In some embodiments of the engineered cell, the interferon comprises IFN-γ.

In some embodiments of the engineered cell, the chemokine comprises CCL2 and/or CXCL12.

In some embodiments of the engineered cell, the Tumor Necrosis Factor comprises TNF-α.

In some embodiments of the engineered cell, the flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.

In some embodiments of the engineered cell, the immune cell comprises a monocyte and/or a macrophage.

In another aspect, the present disclosure provides use of the agent, the isolated migrasome and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the recruitment of a second immune cell to the first immune cell.

In another aspect, the present disclosure provides use of the agent, the isolated migrasome and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the migration of an immune cell towards the location.

In another aspect, the present disclosure provides use of the agent, the isolated migrasome and/or the engineered immune cell according to the present disclosure in the preparation of a regulator of an immune response and/or an immune response mediated biological process.

In another aspect, the present disclosure provides use of the agent, the isolated migrasome and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the secretion of a substance by the immune cell.

In another aspect, the present disclosure provides use of the agent according to the present disclosure in the preparation of an indicator for an immune response and/or an immune response mediated biological process in the subject.

In another aspect, the present disclosure provides a composition, comprising the agent, the isolated migrasome, and/or the engineered cell according to the present disclosure.

In some embodiments, the composition is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.

In another aspect, the present disclosure provides a kit, comprising the agent, the isolated migrasome, the engineered cell, and/or the composition according to the present disclsoure.

In another aspect, the present disclosure provides a method for characterizing a migrasome generated by a monocyte, comprising determining the presence and/or amount of C-C chemokine receptor type 2 (CCR2) .

In some embodiments, the method comprises determining the presence and/or amount of the CCR2 in the migrasome.

In some embodiments of the method, the migrasome is in or from a biological sample.

In some embodiments of the method, the biological sample comprises a body fluid sample.

In some embodiments of the method, the biological sample comprises a blood sample.

In some embodiments of the method, determining comprises using an agent capable of specifically identifying the CCR2.

In some embodiments of the method, the agent capable of specifically identifying the CCR2 comprises an anti-CCR2 antibody or an antigen binding fragment thereof.

In some embodiments of the method, the antigen binding fragment comprises Fab, F (ab) ₂, F (ab’) , F (ab’) ₂, scFv, affibody and/or VHH.

In some embodiments of the method, the agent capable of specifically identifying the CCR2 further comprises a detectable label.

In some embodiments of the method, the determining comprises detecting the presence and/or amount of a modified CCR2 containing a detectable label.

In some embodiments of the method, the detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

In some embodiments, the methods further comprises determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

In some embodiments, the method further comprises staining with wheatgerm agglutinin (WGA) .

In another aspect, the present disclosure provides a method for isolating and/or regulating a migrasome generated by a monocyte, comprising: i) characterizing the migrasome according to the method of the present disclosure; and ii) isolating the characterized migrasome, and/or administering a regulating agent to the characterized migrasome.

In some embodiments, the method is an in vitro or ex vivo method.

In some embodiments, the methods is an in vivo method.

In one aspect, the present disclosure provides an agent capable of determining the presence and/or amount of C-C chemokine receptor type 2 (CCR2) , for use in characterizing a migrasome generated by a monocyte.

In some embodiments, the agent is capable of specifically identifying the CCR2.

In some embodiments, the agent comprises an anti-CCR2 antibody or an antigen binding fragment thereof.

In some embodiments of the agent, the antigen binding fragment comprises Fab, F (ab) ₂, F (ab’) , F (ab’) ₂, scFv, affibody and/or VHH.

In some embodiments, the agent further comprises a detectable label.

In some embodiments, the agent comprises a modified CCR2 containing a detectable label.

In some embodiments of the agent, the detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

In one aspect, the present disclosure provides a composition, comprising the agent according to the present disclosure.

In some embodiments, the composition further comprises a second agent capable of determining the presence and/or amount of a migrasome.

In some embodiments of the composition, the second agent is capable of determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

In some embodiments of the composition, the second agent comprises WGA.

In some embodiments of the composition, the agent capable of determining the presence and/or amount of CCR2 is not mixed with the second agent.

In another aspect, the present disclosure provides a kit, comprising the agent and/or the composition according to the present disclosure.

In another aspect, the present disclosure provides use of the agent, the composition, and/or the kit according to the present disclosure, in the preparation of an indicator for a migrasome generated by a monocyte.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “FIG. ” , “Fig. ” and “FIG. ” herein) , of which:

FIG. 1A illustrates confocal image of WGAhigh and WGAlow cells in CAM from a 9-day chick embryo (CAM9d) . CAM was stained by WGA (wheat germ agglutinin) and observed by spinning disk microscopy. Scale bar, 5 μm. Enlarged images of migrasomes and retraction fibers emanating from WGAhigh CAM cells are shown in the lower panels.

FIG. 1B illustrates TEM image of CAM9d. Scale bar, 1 μm. Enlarged images of individual migrasomes are shown in the right panels.

FIG. 1C illustrates that migrasomes from FIG. 1B were quantified for their size and the number of intralumenal vesicles. 60 migrasomes were examined and quantified.

FIG. 1D-1E: FIG. 1D illustrates focused ion beam scanning electron microscope (FIB-SEM) analysis of CAM9d. Enlarged z-stack images of the same migrasome are shown in the lower panels to highlight the connection between the migrasome and the retraction fiber. Scale bar, 500 nm. FIG. 1E illustrates 3D reconstruction of a migrasome (arrowhead) . Scale bar, 1 μm.

FIG. 1F illustrates migrasome formation captured by spinning disk confocal microscopy. Scale bar, 5 μm.

FIG. 1G illustrates the distribution of WGA^high cells in CAM9d. Blood vessels were revealed by dextran staining. CAM was visualized by Dragonfly confocal z-stack imaging and presented as the maximum intensity projection. Scale bar, 15 μm.

FIG. 1H-1I: FIG. 1H illustrates trajectories of WGA^high cells in CAM9d were observed by time-lapse microscopy and analyzed by NIS-Elements Viewer. Scale bar, 10 μm. FIG. 1I illustrates enlargement of a WGA^high cell in E9d CAM from (H) . Scale bar, 5 μm.

FIG. 2A illustrates a diagram showing the procedure for isolation of WGA^high or WGA^low cells from CAM9d.

FIG. 2B illustrates isolated cells from WGA-stained CAM9d sorted by FACS according to WGA signal.

FIG. 2C illustrates WGA^high and WGA^low cells were observed by confocal microscopy. Scale bars, 10 μm.

FIG. 2D illustrates that cells from FIG. 2C were quantified for the number of migrasomes. Data are presented as means ± SEM; n = 40 cells per group pooled from three independent experiments. P values (*P < 0.0001) were calculated using a two-tailed, unpaired t-test.

FIG. 2E illustrates WGA^high cells were subjected to single-cell RNA-seq analysis. Data were analyzed by principal component analysis (PCA) .

FIG. 2F-2H: FIG. 2F illustrates heat map reporting scaled expression of discriminative marker gene sets for the two cell types identified in FIG. 2E. FIG. 2G illustrates cells isolated from CAM9d were stained by CD115 and sorted by FACS. FIG. 2H illustrates the CD115-positive cells were observed by Dragonfly spinning disk confocal microscopy. Scale bar, 5 μm.

FIG. 2I illustrates CAM9d was stained by WGA and anti-CD115 or KUL01 antibody. Immunofluorescence was visualized in CAMs by confocal z-stack imaging and presented as the maximum intensity projection. Scale bar, 15 μm.

FIG. 2J illustrates WGA^high cells were untreated or treated with GM-CSF for 72 h. Boiled yeast cells (strain BY4741) labelled with a red fluorescent tracer were added. Cells were observed by spinning disk confocal microscopy. White lines indicate the outlines of WGA^high cells. Arrows indicate the internalized yeast. Scale bar, 5 μm.

FIG. 2K illustrates quantification of the number of monocytes from (l) with engulfed yeast. Data are presented as mean ± SEM; n=22 from three independent experiments; *P < 0.0001.

FIG. 2L illustrates trajectories of cultured WGA^high and WGA^low cells in the same amount of time (12 h) . 20 cells were examined and quantified in each group.

FIG. 3A-3C: FIG. 3A illustrates migration of WGA^high and WGA^low cell was monitored by time-lapse confocal microscopy. Scale bar, 5 μm. FIG. 3B illustrates that cells from FIG. 3A were quantified for migration speed. Data are presented as means ± SEM; n = 21 cells per group pooled from three independent experiments. FIG. 3C illustrates that cells from FIG. 3A were quantified for size. Data are presented as means ± SEM; n = 21 cells per group pooled from three independent experiments. P values (*P < 0.0001) were calculated using a two-tailed, unpaired t-test.

FIG. 4A illustrates diagram of the migrasome isolation procedure.

FIG. 4B illustrates images of migrasomes purified from CAM9d. Left panel, confocal image of purified migrasomes stained by WGA, scale bar, 5 μm; right panel, TEM image of migrasomes isolated from CAM9d, scale bar, 500 nm.

FIG. 4C illustrates western blot analysis of isolated CAM9d migrasomes with the indicated antibodies.

FIG. 4D illustrates diagram of the procedure for TMT-labelling and quantitative mass spectrometry.

FIG. 4E illustrates volcano plot showing the mass spectrometry-based quantification of TMT-labelled proteins. The upper right dots represent a migrasome: cell abundance ≥ 2, P < 0.01; the upper left dots represent a migrasome: cell abundance < 0.5, P < 0.01. n = 6 biologically independent experiments. P values were calculated in Excel using a two-tailed, two-sample unequal variance t-test.

FIG. 4F illustrates that data from FIG. 4E were analyzed for the abundance of the indicated proteins.

FIG. 4G illustrates violin plots showing the mRNA levels of indicated genes from single-cell sequencing analysis of monocyte and epithelial cells.

FIG. 4H illustrates that cell bodies and migrasomes were analyzed by western blot using anti-CXCL12 and anti-VEGFA antibodies.

FIG. 4I illustrates monocytes isolated from CAM9d were stained with WGA and the indicated antibodies and visualized by confocal microscopy. Scale bar, 5 μm.

FIG. 4J illustrates CAM9d were stained with WGA and the indicated antibodies. CAM immunofluorescence was visualized by confocal z-stack imaging and presented as the maximum intensity projection. Scale bar, 10 μm.

FIG. 5A illustrates that migrasomes were delivered to CAM9d in low-melting-point agarose. After 48 h, CAMs were stained with WGA and visualized by spinning disk microscopy. Scale bar, 5 μm.The boxed areas are enlarged images.

FIG. 5B illustrates that CAMs from FIG. 5A were quantified for the number of WGA^high cells. Data are presented as mean ± SEM; n = 25 fields from three independent experiments; *P < 0.0001.

FIG. 5C-5E: FIG. 5C illustrates diagram of the transwell assay for recruitment of WGA^high or WGA^low cells. FIG. 5D illustrates that cells adhered to the underside of the transwell membrane were stained by DAPI and visualized by confocal microscopy. Scale bar, 30 μm. FIG. 5E illustrates statistical analysis of the migration indexes from FIG. 5D were calculated using one-way ANOVA. Data are presented as mean ± SEM; n = 35 fields from three independent experiments; N. S: no significance, *P < 0.01.

FIG. 6 illustrates a diagram showing the strategy for knocking out TSPAN4 and knocking in mCherry in chick embryos. The mCherry coding sequence was inserted into TSPAN4 at the position targeted by the sgRNA. Thus, an mCherry-positive signal indicates that native TSPAN4 gene expression was silenced simultaneously. s

FIG. 7A illustrates CAMs stained with WGA and visualized by confocal microscopy. Scale bar, 50 μm.

FIG. 7B illustrates CAMs from FIG. 7A were quantified for the number of WGA^high cells. Data are presented as mean ± SEM; n = 22 fields from three independent experiments, respectively; *P <0.01.

FIG. 8 illustrates violin plots showing the mRNA levels of TGFB3 from single-cell sequencing analysis of monocyte-like cells and epithelial cells.

FIG. 9 illustrates that CAM9d were stained with WGA and the indicated antibodies and visualized by confocal microscopy. Scale bar, 20 μm. Immunofluorescence in CAMs was visualized by confocal z-stack imaging and presented as the maximum intensity projection.

FIG. 10A illustrates that mouse monocytes were cultured in FN-precoated confocal dishes in the presence of 500 ng/mL LPS for 12 hr. Cells were then stained with CCR2 and WGA before visualization. Scale bar, 5 μm.

FIG. 10B illustrates representative TEM images of activated monocytes from FIG. 10A. Scale bar, 1 μm. Right panels, enlarged migrasomes containing intraluminal vesicles are shown. Scale bar, 200 nm.

FIG. 10C-10D illustrate immunostaining of endogenous TNF-α (10C) and IL-6 (10D) in activated monocytes as shown in FIG. 10A. Scale bar, 5 μm. The lower panels show enlarged migrasomes. Scale bar, 500 nm.

FIG. 10E illustrates western blot analysis of migrasomes purified from activated monocytes using the indicated antibodies. CPQ and Integrin α5 (Itg α5) were used as migrasome markers. Equal amounts of total protein from cell bodies (C) and migrasomes (M) were subjected to western blot analysis.

FIG. 10F illustrates confocal images of WT and Tspan9^-/- (T9 KO) monocytes plated on the indicated dishes. Scale bar, 5 μm. Quantification of the number of migrasomes per cell is shown as the mean ± SEM. n > 100 cells from three independent experiments were analyzed using the two-tailed unpaired t-test (right panel) . ***p < 0.001.

FIG. 10G illustrates TNF-α and IL-6 secretion in indicated monocytes. Cells were activated, primed with 500 ng/mL LPS, and seeded into control or FN-precoated dishes for 16 hr. Cell lysates and concentrated medium were collected and analyzed by western blot.

FIG. 10H illustrates that L929 cells were cultured in medium containing migrasomes isolated from activated monocytes in the presence of 10 μM zVAD for 18 hr. Cells undergoing necroptosis were detected by propidium iodide (PI) staining coupled with FACS analysis. The right panel shows statistical analysis of cell death. Error bar, mean ± SEM. Experiments were independently repeated three times. Two-tailed unpaired t tests were used for statistical analyses. **p < 0.01, ***p < 0.001.

FIG. 11A and FIG. 11B illustrate intravital imaging of mouse liver monocytes and monocyte derived migrasomes after LPS stimulation. Monocytes were detected with PE-labeled anti-mouse CCR2 antibody. FIG. 11A shows that time-lapse images were acquired at intervals of 12 s. Scale bar, 5 μm. FIG. 11B shows that WGA labels blood vessels, and arrowheads indicate free CCR2 positive migrasomes detached form retraction fibers. Time interval, 45 s. Scale bar, 5 μm.

FIG. 11C illustrates schematic illustration of monocyte derived migrasomes purification from mouse blood samples.

FIG. 11D illustrates representative scanning electron microscopy (SEM) images of migrasomes isolated from blood monocytes as shown in FIG. 11C. Scale bar, 500 nm.

FIG. 11E-11F illustrate immunofluorescence stained z-stack images of migrasomes purified from blood monocytes. Migrasomes were stained with CCR2, VAMP2, TNF-α (E) and IL-6 (F) . Z-stack images were acquired by confocal microscopy. Scale bar, 10 μm. Right panels, 3D reconstructions of enlarged migrasomes. Scale bar, 2 μm.

FIG. 11G illustrates that LPS (12 mg/kg) was injected into mice by intraperitoneal injection (i. p. ) . Cell bodies (C) and monocyte derived migrasomes (M) from mouse with or without LPS treatment were isolated from equal volume of blood, and then analysed by western blot using the indicated antibodies.

FIG. 11H illustrates western blot analysis, using the indicated antibodies, of migrasomes purified from blood monocytes. Lysates of cell bodies (C) and migrasomes (M) were normalized to equal total protein loading for western blot analysis.

FIG. 11I illustrates that equal numbers of WT and T9 KO monocytes were labelled with anti-CCR2 antibodies conjugated to different colored tags. The color-coded cells were combined for injection into WT mice and intravital imaging of mouse liver was performed. WGA labels blood vessels. Scale bar, 10 μm. The right panel shows statistical analysis of the number of migrasomes per cell. Shown are mean ± SEM of > 100 cells from three independent experiments. Two-tailed unpaired t-test was used to compare the datasets. ***p < 0.001.

FIG. 11J illustrates western blot analysis of monocyte derived migrasomes. Migrasomes were isolated from WT and T9 KO mouse blood in equal volume, and then subjected to western blot analysis.

FIG. 11K illustrates inflammatory cytokine protein profiles in WT and T9 KO mouse blood. After i.p. of LPS, inflammatory cytokines (TNF-α, IL-6, IFN-γ, MCP-1, IL-10 and IL-12p70) were measured in blood from WT and T9 KO mouse by cytometric bead array (CBA) . Data are presented as mean ± SEM. Experiments were independently repeated two times (n = 12-18 mice per group) . Two-tailed unpaired t-test was used for statistical analyses. *p < 0.05, **p < 0.01, NS, not significant.

FIG. 12A illustrates representative transmission electron microscopy (TEM) images of L929 cells. Scale bar, 10 μm. Middle panel, enlarged region of interesting (ROI) . Scale bar, 500 nm. Right panel, the relationship between the distance from migrasome to cell body with the diameter of migrasome and the number of intraluminal vesicles per migrasome was quantified. n=30 cells from three independent experiments.

FIG. 12B illustrates TEM images of high pressure freezing (HPF) samples of L929 cells. Left panel, retraction fiber. Right panel, migrasome. Scale bar, 500 nm.

FIG. 12C illustrates TEM images of L929 cells. Left panel, the entrance of retraction fibers. Right panel, detached migrasome. Scale bar, 500 nm.

FIG. 12D illustrates TEM images of L929 cells treated with 10 μM BAPTA-AM. Scale bar, 500 nm.Right panel, statistical analysis of the number of small vesicles per migrasome. Error bars, mean ± SEM. n > 100 migrasomes from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p < 0.001.

FIG. 12E illustrates L929-T4-mCherry cells were immunostained with antibody against Rab8a and then visualized. White dashed lines, the outline of the cell body. Scale bar, 20 μm. Right panels, enlarged ROI. Scale bar, 2 μm.

FIG. 12F illustrates structured illumination microscopy (SIM) images of L929 cells stably expressing GFP-Rab8a and T4-mCherry. Scale bar, 500 nm.

FIG. 12G illustrates representative TEM images of DAB staining pattern in L929 cells stably expressing APEX2-GFP-Rab8 and reacted with diaminobenzidine (DAB) . Scale bar, 100 nm.

FIG. 12H illustrates confocal images of GFP-Rab8a-and T4-mCherry-expressing L929 cells treated with 10 μM BAPTA-AM. Scale bar, 20 μm. The right panel shows statistical analysis of the number of GFP-Rab8a-puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p <0.001.

FIG. 13A illustrates confocal images of L929 cells stably expressing GFP-Myosin5a (Myo5a) and T4-mCherry. Scale bar, 20 μm. Lower panels, enlarged ROI. Scale bar, 2 μm.

FIG. 13B illustrates APEX2-based TEM images of L929 cells stably expressing APEX2-mCherry-Myo5a. Scale bar, 2 μm. The lower panels show higher-magnification images of vesicles from the cell body (C) , the base of a retraction fiber (B) and a migrasome (M) . Scale bar, 200 nm.

FIG. 13C illustrates time-lapse images of L929 cells stably expressing GFP-Myo5a and T4-mCherry. Time interval, 90 s. Scale bar, 5 μm.

FIG. 13D illustrates time-lapse Grazing Incidence-Structured Illumination Microscopy (GI-SIM) images of L929-GFP-Myo5a cells. Scale bar, 5 μm. Right panels, enlarged ROI. Time-lapse images were acquired at intervals of 30 s. Arrowheads indicate Myo5a transporting to the edge of the cell, Myo5a moving into migrasomes or Myo5a accumulating at the edge of cell and left on retraction fibers. Scale bar, 2 μm.

FIG. 13E illustrates TEM images of WT, Myosin5a-overexpression (Myo5a OE) and Myosin5a-knockout (Myo5a KO) L929 cells. Scale bar, 500 nm. The right panel shows the statistics of the number of small vesicles per migrasome. The error bars are mean ± SEM of > 100 migrasomes from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p <0.001.

FIG. 13F illustrates that stable expression of T4-mCherry or mCherry-Myo5a was established in L929-GFP-Rab8a cells. The cells were then subjected to confocal analysis. Scale bar, 20 μm. Right panel, statistical analysis of the number of GFP-Rab8a-puncta in migrasomes per cell. Data represent the mean ± SEM. n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p < 0.001.

FIG. 14A illustrates SIM images of L929 cells stably expressing GFP-VAMP2 and T4-mCherry. Scale bar, 200 nm.

FIG. 14B illustrates TEM images of L929 cells stably expressing APEX2-GFP-VAMP2 and reacted with DAB. Scale bar, 200 nm.

FIG. 14C illustrates immunostaining of endogenous VAMP2 in WT, Myo5a OE and Myo5a KO L929 cells. Scale bar, 20 μm. Right panel, statistical analysis of the number of VAMP2 puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p < 0.001.

FIG. 14D illustrates L929-T4-mCherry cells were immunostained with SNAP23 antibody and then visualized. Scale bar, 20 μm.

FIG. 14E illustrates western blot analysis of total plasma membrane proteins isolated from the cell bodies (C) or migrasomes (M) using the indicated antibodies.

FIG. 14F illustrates L929-T4-mCherry cells were infected with nonspecific (WT) or SNAP23-shRNA lentiviral constructs. Cells were then immunostained with VAMP2 antibody and subjected to confocal analysis. Scale bar, 20 μm. Right panel, statistical analysis of the number of VAMP2 puncta in migrasomes per cell. Error bars, mean ± SEM n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p < 0.001.

FIG. 14G illustrates L929 cells stably expressing GFP-VAMP2 were subjected to time-lapse imaging. Time-lapse images were acquired at intervals of 5s. Scale bar, 2 μm.

FIG. 14H illustrates confocal images of L929 cells stably expressing VAMP2-PHlourin and T4-mCherry. Scale bar, 20 μm. The right panel shows statistical analysis of the migrasome/cell fluorescence intensity ratio. Data represent the mean ± SEM n > 30 cells from three independent experiments. Two-tailed unpaired t tests were used for statistical analyses.

FIG. 15A illustrates confocal images of L929-T4-mCherry cells stably expressing the indicated forms of Myo5a. Full-length (FL) , motor domain (H) , tail domain (T) . Scale bar, 20 μm.

FIG. 16A illustrates confocal images of L929 cells stably expressing GFP-SNAP23 and T4-mCherry. Scale bar, 20 μm.

FIG. 16B illustrates that stable expression of T4-mCherry or mCherry-Myo5a was established in L929-GFP-VAMP7 cells. The cells were then subjected to confocal analysis. Scale bar, 20 μm. Right panel, statistical analysis of the number of GFP-VAMP7-puncta in migrasomes per cell. Data represent the mean ± SEM. n > 100 cells from three independent experiments. Two-tailed unpaired t-test was used for statistical analyses. ***p < 0.001.

FIG. 17A illustrates immunostaining of endogenous TACE in activated monocytes. Scale bar, 5μm. The lower panels show enlarged migrasomes. Scale bar, 500 nm.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used herein, the term “antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, the antibody can include an immunoglobulin composed of at one or more heavy (H) chains and/or one or more light (L) chains, and include any molecule including its antigen binding portion. The term “antibody" includes monoclonal antibodies, antibodies fragment or antibody derivatives, including but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-strand antibodies (e.g., scFv) , and antigen-binding fragments of antibodies (e.g., Fab, Fab’ , VHH and (Fab) 2 fragments) .

As used herein, the term “antigen-binding fragment” generally refers to one or more fragments of the antibody which serve to specifically bind to the antigen. The antigen binding function of the antibody may be implemented by the full-length fragment of the antibody. The antigen binding function of the antibody may also be implemented by the followings: a heavy chain comprising a fragment of Fv, ScFv, dsFv, VHH, Fab, Fab’ or F (ab’) 2, or a light chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab’ or F (ab’) 2. (1) Fab fragment, that is, a monovalent fragment comprising VL, VH, CL and CH domains; (2) F (ab’) 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (3) an Fd fragment comprising VH and CH domains; (4) an Fv fragment comprising VL and VH domains in one arm of an antibody; (5) a dAb fragment comprising a VH domain (Ward et al., (1989) Nature 341: 544-546) ; (6) isolated complementary determining region (CDR) ; and (7) a combination of two or more isolated CDRs which are optionally linked by a linker. Moreover, a monovalent single-strand molecule Fv (scFv) formed by pairing of VL and VH may further be included (see Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85: 5879-5883) .

As used herein, the term “CCR2” generally refers to C-C Motif Chemokine Receptor 2, which is a seven-transmembrane domain G-protein coupled chemotactic receptor. CCR2 is capable of binding to MCP-1, CCL8 (MCP-2) , CCL7 (MCP-3) and/or CCL13 (MCP-4) . CCR2 is also known as CMKBR2 and CKR2. Two alternatively-spliced forms of the CCR2, CCR2A and CCR2B, have been cloned which differ in their C-termini. The protein encoded by human CCR2 has the accession number of P41597 in UniProtKB/Swiss-Prot. The term also encompasses CCR2 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term CCR2 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term CCR2 encompasses the CCR2 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

As used herein, the term “cytokine” generally refers to the general class of biological molecules which effect/affect cells of the immune system. The definition is meant to include, but is not limited to, those biological molecules that act locally or may circulate in the blood, and which may serve to regulate or modulate an individual's immune response. Exemplary cytokines for use in practicing the inventions of the present disclosure include but are not limited to interferons, interleukins, tumor necrosis factors, erythropoietin (EPO) , MIP3a, monocyte chemotactic protein (MCP) -1, intracellular adhesion molecule (ICAM) , macrophage colony stimulating factor (M-CSF) , granulocyte colony stimulating factor (G-CSF) , granulocyte-macrophage colony stimulating factor (GM-CSF) and chemokines.

As used herein, the term "engineered" generally refers to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome, of a polypeptide, or of other components. The term "engineered" can refer to alterations, additions, and/or deletions of the genes, polypeptides or other components. The term "engineered cell" generally refers to a modified cell of human or non-human origin. For example, an engineered cell can refer to a cell with an added, deleted and/or altered gene, polypeptide or other components.

As used herein, the term “ex vivo method” generally refers to a method with substantially all steps performed outside of an organism (e.g., an animal or a human body) . For example, an ex vivo method may be performed in or on a tissue from an organism in an external environment with minimal alteration of natural conditions. Tissues may be removed in many ways, including in part, as whole organs, or as larger organ systems. For example, in an ex vivo method, the samples to be tested may have been extracted from the organism. For example, using living cells or tissue from the same organism may also be considered to be ex vivo. One widely performed ex vivo study is the chick chorioallantoic membrane (CAM) assay. In this assay, angiogenesis is promoted on the CAM membrane of a chicken embryo outside the organism (chicken) .

As used herein, the term “extracellular vesicle” or “EV” generally refers to a membrane-delimited (such as lipid-bilayer delimited) particle that is released from a cell or artificially generated. Unlike a cell, an extracellular vesicle generally cannot replicate. EVs range in diameter from near the size of the smallest physically possible unilamellar liposome (around 20-30 nanometers) to as large as 4000nm or more. EVs can be divided according to size and synthesis route into exosomes, microvesicles, apoptotic bodies, migrasomes and retractosomes. They may carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell. A wide variety of EV subtypes have been proposed, defined variously by size, biogenesis pathway, cargo, cellular source, and function.

As used herein, the term "functional fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.

As used herein, the term "functional variant" generally refers to a nucleic acid molecule, or a polypeptide having similar amino acid or nucleic acid sequences as the parent sequence and retain one or more properties of the parent sequence.

As used herein, the terms “immune cell mediated biological function” generally refers to an immune cell (e.g., T cell, B cell) mediated response, which could be influenced by modulation of the immune cell stimulation, activation and/or death. Exemplary immune cell mediated biological functions include T cell responses, e.g., cytokine production, and cellular cytotoxicity, antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. An immune cell mediated biological function also encompasses a non-immune response, such as tissue repair, wound healing, etc.

As used herein, the term “immune response” generally refers to the action of immune cells (e.g., lymphocytes, antigen presenting cells, phagocytes, granulocytes) and soluble macromolecules produced by such cells or the liver (including antibodies, cytokines, and complements) that results in the selective damage, destruction, or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells from the human body, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. The immune response can be protective, protective, preventive and/or therapeutic.

As used herein, the term “in vitro method” generally refers to a method performed with microorganisms, cells, or biological molecules outside their normal biological context. For example, an in vitro method may be performed in labware such as test tubes, flasks, Petri dishes, and microtiter plates. In vitro methods may be performed using components of an organism that have been isolated from their usual biological surroundings. For example, microorganisms or cells can be studied in culture media, and proteins can be examined in solutions.

As used herein, the term “in vivo method” generally refers to a method wherein the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism. For example, the in vivo method may be performed in a whole organism, rather than in isolated cells thereof.

As used herein, the term “intraluminal vesicle” generally refers to a membranous vesicle that is formed or present in a lumen, or inside the space of a luminal or tubular structure, such as an organelle or a larger vesicle. The term lumen is also used herein to describe the inside space of a cellular component or structure, such as the migrasome. For example, intraluminal vesicle may be generated from an organelle. For example, an intraluminal vesicle may refer to an intraluminal vesicle in a migrasome or an intraluminal vesicle that is destinated to be comprised in a migrasome.

As used herein, the term “knock down” generally refers to a measurable reduction in the expression of a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., siRNA, shRNA, microRNA, antisense RNA, or other RNA-mediated inhibition techniques, to knock down a target polynucleotide sequence.

As used herein, the term “knock out” generally includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence. For example, a knock-out can be achieved by altering a target polynucleotide sequence by inducing a deletion in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., CRISPR/Cas systems, ZFN, TALEN, TgAgo, to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

As used herein, the term “migrasome” generally refers to a membrane-bound cellular structure derived from or generated by a migrating cell. The term “migrasome” encompasses an organelle (also known as “pomegranate-like structure” or PLS) attached to a retraction fiber generated by a migrating cell. In some cases, the term “migrasome” also refers to a vesicle (e.g., an extracellular vesicle) already detached from the cell generating it. In the present disclosure, the term “migrasome” also refers to a vesicle (e.g., an artificial vesicle) with similar functions and/or compositions as such a vesicle or organelle derived from, and/or generated by migrating cells.

As used herein, the terms “migrating cell” and “circulating cell” are used interchangeably, and generally refer to a cell moving from one location to another location. In some cases, a migrating cell is a cell whose relative position, space, and/or contour has changed or is changing with time. A circulating cell comprises a cell circulating in the body fluid (e.g., blood or lymph) of an organism.

As used herein, the term “motor domain” generally refers to a mechanical element of a motor protein. For example, motor domain may hydrolyze GTP or ATP, and change the conformation of the microtubule-binding domains of the motor protein, which may result in the motion of the motor protein.

As used herein, the term “motor protein” generally refers to a molecular motor that can move along the cytoplasm of cells. For example, motor proteins may play roles in intracellular transportation and/or cell motility. For example, motor proteins may comprise actin motor and microtubule motor. For example, actin motors such as myosin may move along microfilaments through interaction with actin, and microtubule motors such as dynein and kinesin may move along microtubules through interaction with tubulin. For example, the term “motor protein” may encompass various isoforms of the motor protein, as well as naturally-occurring allelic and processed forms thereof.

As used herein, the term “myosin family member” generally refers to a member of the superfamily of Myosin motor protein. Myosins constitute a large superfamily of proteins that share a common domain which has been shown to interact with actin, hydrolyze ATP and produce movement in all cases examined to date. Myosins are typically constructed of three functional subdomains: (1) the motor domain which interacts with actin and binds ATP, (2) the neck domain which binds light chains or calmodulin, and (3) the tail domain which serves to anchor and position the motor domain so that it can interact with actin. The motor domains are relatively conserved. The tail domains are the most diverse domains and vary widely in length and in sequence. Functional motifs, such as SH3 domains, GAP domains, FERM domains, and pleckstrin homology (PH) domains are sometimes found in the tails of myosins. In addition, the tails of many myosins contain coiled-coil forming sequences which allow the molecules to dimerize and produce two-headed molecules. The number of myosin genes present in mammals is conservatively estimated at 25–30 from classes I (such as myosin1c) , II, III, V (such as myosin5a) , VI, VII, IX, X and XV.

As used herein, the term “pharmaceutically acceptable excipient” generally refers to any material, which is inert in the sense that it substantially does not have a therapeutic and/or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties.

As used herein, the term “recruitment” generally refers to a process by which a cell is selected for certain tasks or added into a certain cell population.

As used herein, the term “retraction fiber” or “RF” generally refers to actin-rich fibers exposed as the cell margin retracts. For example, the retraction fiber may include tubular strands left behind a cell during cell migration. During migration, RF may be pulled out at the trailing edge of cells, and migrasomes may form on the tips or branch points of the RF.

As used herein, the term “secretion” generally refers to the production and/or release of certain substances by an organ (e.g., a gland) , a cell, or an organelle. When a substance is secreted, it may depart from the organ, cell or organelle producing it.

As used herein, the term “signal peptide” generally refers to a peptide linked in frame to a polypeptide (e.g., to the amino terminus of a polypeptide) and directs the encoded polypeptide into a cell's secretory pathway.

As used herein, the term “signaling molecule” generally refers to a molecule that is responsible for transmitting information. For example, signaling molecule may comprise lipids, phospholipids, amino acids, monoamines, proteins, glycoproteins, and/or gases. For example, signaling molecule may interact with a target cell as a ligand to cell surface receptors, and/or by entering into the cell through its membrane or endocytosis for intracrine signaling.

As used hererin, the term “SNARE complex” generally refers to a protein complex formed by one or more components (e.g., subunits) that may mediate vesicle fusion. SNARE is also known as soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor. For example, SNARE may comprise vesicle-SNAREs or v-SNAREs, which are incorporated into the membranes of transport vesicles during budding, and target-SNAREs or t-SNAREs, which are associated with nerve terminal membranes. SNARE complex may be the components of the fusion machinery.

As used herein, the term “tetraspanin” generally refers to a membrane protein, which is also known as the transmembrane 4 superfamily (TM4SF) protein, and may have four transmembrane alpha-helices and two extracellular domains. For example, the term “tetraspanin” may encompass various isoforms of the tetraspanin, as well as the naturally-occurring allelic and processed forms thereof.

As used herein, the term “Tetraspanin 4 (TSPAN4) ” generally refers to a TSPAN4 gene and/or a protein that is encoded by the TSPAN4 gene. For example, the NCBI Entrez Gene for TSPAN4 may be 7106. For example, the UniProtKB/Swiss-Prot number for Tetraspanin 4 may be O14817. For example, the term “Tetraspanin 4” may encompass various isoforms of the Tetraspanin 4, the naturally-occurring allelic and processed forms thereof. The term also encompasses TSPAN4 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term TSPAN4 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term TSPAN4 encompasses the TSPAN4 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

As used herein, the term “Tetraspanin 9 (TSPAN9) ” generally refers to a TSPAN9 gene and/or a protein that is encoded by the TSPAN9 gene. For example, the NCBI Entrez Gene for TSPAN9 may be 10867. For example, the UniProtKB/Swiss-Prot number for Tetraspanin 9 may be O75954. For example, the term “Tetraspanin 9” may encompass the isoforms of the Tetraspanin 9, the naturally-occurring allelic and processed forms thereof. The term also encompasses TSPAN9 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term TSPAN9 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term TSPAN9 encompasses the TSPAN9 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

As used hererin, the term “t-SNARE” generally refers to a target-SNARE. For example, t-SNARE may be the part of the SNARE complex that is in the target membrane. For example, t-SNARE may compirse syntaxin, SNAP25, SNAP23, the isoforms thereof, as well as the naturally-occurring allelic and processed forms thereof.

As used herein, the term “v-SNARE” generally refers to a vesicle-SNARE. For example, v-SNARE may be the part of the SNARE complex that is in the vesicle. For example, v-SNARE may comprise VAMP2, VAMP3 and/or VAMP7, the isoforms of the VAMP2, VAMP3 and/or VAMP7, the naturally-occurring allelic and processed forms thereof.

Unless otherwise specified, “a” , “an” , “the” and “at least one” are used interchangeably and refer to one or more than one.

In the present disclosure, the term “comprise” also encompasses “is” , “has” and “consist of” . For example, “acomposition comprising X and Y” may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .

In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process in a subject, comprising: i) monitoring the immune response and/or the immune response mediated biological process in the subject according to the present disclosure; and ii) administering a regulating agent according to the result of step i) .

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by a first immune cell, for use in recruiting a second immune cell to the first immune cell.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome present at or near a location, for use in regulating the migration of an immune cell towards the location.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by an immune cell mediating an immune response, for use in regulating the immune response and/or the immune response mediated biological process.

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by an immune cell, for use in regulating the secretion of a substance by the immune cell.

In another aspect, the present disclosure provides an agent capable of detecting the presence, amount and/or function of a migrasome obtained from a biological sample of a subject, for use in monitoring an immune response and/or an immune response mediated biological process in the subject.

In another aspect, the present disclosure provides an isolated migrasome derived from an immune cell.

In another aspect, the present disclosure provides use of the agent according to the present disclosure, the isolated migrasome according to the present disclosure and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the recruitment of a second immune cell to the first immune cell.

In another aspect, the present disclosure provides use of the agent according to the present disclosure, the isolated migrasome according to the present disclosure and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the migration of an immune cell towards the location.

In another aspect, the present disclosure provides use of the agent according to the present disclosure, the isolated migrasome according to the present disclosure and/or the engineered immune cell according to the present disclosure in the preparation of a regulator of an immune response and/or an immune response mediated biological process.

In another aspect, the present disclosure provides use of the agent according to the present disclosure, the isolated migrasome according to the present disclosure and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the secretion of a substance by the immune cell.

In another aspect, the present disclosure provides use of the agent capable of detecting the presence, amount and/or function of the migrasome in the preparation of an indicator for an immune response and/or an immune response mediated biological process in the subject.

In another aspect, the present disclosure provides a kit, comprising the agent, the isolated migrasome, the engineered cell and/or the composition according to the present disclosure.

In another aspect, the present disclosure provides a method for isolating and/or regulating a migrasome generated by a monocyte, comprising: i) characterizing the migrasome according to a method of the present disclosure; and ii) isolating the characterized migrasome, and/or administering a regulating agent to said characterized migrasome.

In another aspect, the present disclosure provides an agent capable of determining the presence and/or amount of C-C chemokine receptor type 2 (CCR2) , for use in characterizing a migrasome generated by a monocyte.

In another aspect, the present disclosure provides a composition, comprising the agent capable of determining the presence and/or amount of CCR2 according to the present disclosure.

In another aspect, the present disclosure provides a kit, comprising the agent capable of determining the presence and/or amount of CCR2 according to the present disclosure, and/or the composition comprising such an agent.

In another aspect, the present disclosure provides use of the agent, the composition, and/or the kit of the present disclosure in the preparation of an indicator for a migrasome generated by a monocyte.

In the present disclosure, an immune cell mediated biological function may encompass any biological process involving the function or participation of an immune cell. The immune cell mediated biological function may comprise an immune response or a non-immune response (such as a response mainly involving other types of cells, but it could be triggered or promoted by an immune cell) .

For example, in the present disclosure, the immune cell mediated biological function may comprise the recruitment of a second immune cell by a first immune cell. The recruitment of the second immune cell may be increased by promoting the formation and/or function of the migrasome generated by the first immune cell. In some cases, the recruitment of the second immune cell is increased by administering an effective amount of migrasomes derived from the first immune cell. In some cases, the recruitment of the second immune cell is decreased by inhibiting the formation and/or function of the migrasome generated by the first immune cell.

For example, in the present disclosure, the immune cell mediated biological function may comprise the migration of an immune cell towards a location. The migration of the immune cell towards the location may be increased by increasing the amount and/or function of the migrasome present at or near the location. In some cases, the migration of the immune cell towards the location is increased by administering an effective amount of migrasomes at or near the location. In some cases, the migration of the immune cell towards the location is increased by promoting the formation and/or function of the migrasome generated by a local immune cell, the local immune cell is an immune cell at or near the location. In some cases, the migration of the immune cell towards the location is decreased or inhibited by decreasing the amount and/or function of the migrasome present at or near the location. In some cases, the migration of the immune cell towards the location is inhibited or decreased by inhibiting the formation and/or function of the migrasome generated by the local immune cell.

For example, in the present disclosure, the immune cell mediated biological function may comprise an immune response and/or an immune response mediated biological process. The immune response and/or immune response mediated biological process may comprise secretion of a substance by the immune cell. For example, the immune response and/or the immune response mediated biological process may be regulated by a secreted substance of the immune cell. For example, the immune response and/or immune response mediated biological process may comprise an inflammatory response, and/or other diseases, conditions or disorders affected by the function of an immune cell. The immune response and/or the immune response mediated biological process may be increased or promoted by increasing the amount and/or function of the migrasome generated by the immune cell mediating the immune response. In some cases, the immune response and/or the immune response mediated biological process is increase or promoted by administering an effective amount of the migrasome generated by the immune cell mediating the immune response. In some cases, the immune response and/or the immune response mediated biological process is decreased or inhibited by decreasing the amount and/or function of the migrasome generated by the immune cell mediating the immune response.

For example, in the present disclosure, the immune cell mediated biological function may comprise the secretion of a substance by an immune cell. The secretion of the substance may be increased or promoted by increasing or promoting the amount and/or function of the migrasome generated by the immune cell. In some cases, the secretion of the substance is increased or promoted by administering an effective amount of the migrasome generated by the immune cell. In some cases, the secretion of the substance is decreased or inhibited by decreasing the amount and/or function of the migrasome generated by the immune cell.

In the present disclosure, promoting the immune cell mediated biological function refers to causing a change in the process due to an increase of the amount and/or function of the migrasomes generated by the immune cell mediating such a process. In the present disclosure, inhibiting or decreasing the immune cell mediated biological function refers to causing a change in the process due to a decrease of the amount and/or function of the migrasomes generated by the immune cell mediating such a process.

According to any aspect of the present disclosure, the formation and/or function of a migrasome generated by an immune cell may be promoted or inhibited.

The formation and/or function of a migrasome may be regulated (i.e., promoted or inhibited, as appropriate) by any approach applicable. For example, the formation and/or function of a migrasome may be regulated by regulating migration of the cell generating the migrasome. For example, the formation and/or function of a migrasome may be regulated by regulating the formation of a retraction fiber of the cell generating the migrasome. For example, the formation and/or function of a migrasome may be regulated by regulating the amount and/or function of a tetraspanin protein (including its function fragment, and/or its functional variant) . For example, the formation and/or function of a migrasome may be regulated by regulating the amount of cholesterol in a cell generating the migrasome or in the migrasome.

In some cases, promoting the formation and/or function of the migrasomes comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in the immune cell generating the migrasome and/or in the migrasome. For example, this may be achieved by overexpressing the tetraspanin protein, the functional fragment thereof, and/or the functional variant thereof in the immune cell. For example, the tetraspanin may comprise TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

The overexpression may be achieved either by introducing an exogenous protein or an exogenous nucleic acid molecule encoding the protein, or by causing increased expression of the endogenous protein or the endogenous gene encoding for said protein. For example, such overexpression may be caused by a mutation in the regulatory region of a gene encoding for the protein. In some cases, the overexpression may be achieved by changing the function of one or more components of the transcriptional and/or translational machinery.

In some cases, promoting the formation and/or function of the migrasome comprises increasing the number of intraluminal vesicles in the migrasome. For example, the number of intraluminal vesicles that has been in the migrasome may be increased. For example, the number of intraluminal vesicles that would be transported into the migrasome may be increased. For example, increasing the number of intraluminal vesicles may comprise increasing the number of intraluminal vesicles in the cell generating the migrasome. For example, increasing the number of intraluminal vesicles may comprise increasing the transportation of the intraluminal vesicles into the migrasome. For example, the number of intraluminal vesicles in the migrasome may be analyzed by confocal imaging analysis and/or Transmission Electron Microscopy analysis. In some cases, increasing the number of intraluminal vesicles in the migrasome comprises increasing the amount and/or function of a motor protein in the immune cell generating the migrasomes.

In some cases, promoting the formation and/or function of the migrasome comprises increasing fusion of the membrane of the migrasome with an intraluminal vesicle therein. For example, the amount of the intraluminal vesicles, which is fusing or fused with the migrasomes, may be increased. For example, the amount of merged membrane, which is derived from the migrasome and the intraluminal vesicle therein, may be increased. For example, the amount of a marker, which is derived from the intraluminal vesicle, may be increased on the membrane of migrasome. For example, the amount of the marker may be analyzed by Western Blot, Immunofluorescence and/or quantitative mass spectrometry analysis. For example, fusion of the membrane of the migrasome with the intraluminal vesicle therein may be increased by increasing the amount and/or function of a SNARE complex in the migrasome. For example, fusion of the membrane of the migrasome with the intraluminal vesicle therein may be increased by increasing the amount and/or concentration of calcium in the migrasome. For example, the amount of calcium in the migrasome may be increased by at least 0.1%, at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 1000 times, or at least 10000 times. For example, the amount of calcium in the migrasome may be analyzed by a calcium assay kit.

In some cases, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a chemokine in the immune cell generating the migrasomes and/or in the migrasome. For example, the chemokine may comprise CCL2 (also known as CC chemokine, MCP-1, or monocyte chemotactic protein-1) and/or CXCL12.

In some cases, inhibiting the formation and/or function of the migrasome comprises inhibiting the expression and/or function of a tetraspanin in the immune cell generating the migrasome and/or in the migrasome. Inhibiting the expression and/or function of the tetraspanin may comprise knocking out or knocking down the expression of a gene encoding for the tetraspanin in the immune cell generating the migrasome. The tetraspanin may comprises tetraspanin 4 and/or tetraspanin 9.

In some cases, inhibiting the formation and/or function of the migrasome comprises decreasing the number of intraluminal vesicles in the migrasome. For example, the number of intraluminal vesicles that has been in the migrasome may be decreased. For example, the number of intraluminal vesicles that would be transported into the migrasome may be decreased. For example, decreasing the number of intraluminal vesicles may comprise decreasing the number of intraluminal vesicles in the cell generating the migrasome. For example, decreasing the number of intraluminal vesicles may comprise inhibiting the transportation of the intraluminal vesicles into the migrasome. For example, the number of intraluminal vesicles in the migrasome may be analyzed by confocal imaging analysis and/or Transmission Electron Microscopy analysis. In some cases, decreasing the number of intraluminal vesicles in the migrasome comprises decreasing the amount and/or function of a motor protein in the immune cell generating the migrasomes.

In some cases, inhibiting the formation and/or function of the migrasome comprises inhibiting fusion of the membrane of the migrasome with an intraluminal vesicle therein. For example, the amount of the intraluminal vesicles, which is fusing or fused with the migrasomes, may be decreased. For example, the amount of merged membrane, which is derived from the migrasome and the intraluminal vesicle therein, may be decreased. For example, the amount of a marker, which is derived from the intraluminal vesicle, may be decreased on the membrane of migrasome. For example, the amount of the marker may be analyzed by Western Blot, Immunofluorescence and/or quantitative mass spectrometry analysis. For example, fusion of the membrane of the migrasome with the intraluminal vesicle therein may be inhibited or decreased by decreasing the amount and/or function of a SNARE complex in the migrasome. For example, fusion of the membrane of the migrasome with the intraluminal vesicle therein may be inhibited or decreased by decreasing the amount and/or concentration of calcium in the migrasome.

For example, the amount and/or concentration of calcium in the migrasome may be decreased or reduced by administering an agent capable of regulating calcium in migrasome. For example, such an agent may comprise a calcium chelator. For example, the calcium chelator may comprise EGTA, EDTA, BAPTA, BAPTA-AM and/or derivatives thereof. For example, the amount of calcium in the migrasome may be reduced by at least 0.1%, at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 1000 times, or at least 10000 times. For example, the amount of calcium in migrasome may be analyzed by a calcium assay kit.

According to any aspect of the present disclosure, the intraluminal vesicle may comprise one or more immune signaling molecules (for example, the substance to secreted) . The signaling function of the intraluminal vesicle may be effected upon releasing of the immune signaling molecules. According to any aspect of the present disclosure, the immune cell mediated biological function may be regulated by one or more substances secreted or to be secreted by the immune cell, which may comprise one or more immune signaling molecules.

For example, the immune signaling molecule may comprise a signal peptide. For example, the immune signaling molecule may be involved in an immune process or an immune cell-mediated process. For example, the immune signaling molecule may comprise a cytokine and/or a flavonoid.

According to any aspect of the present disclosure, the cytokine may generally comprise any signaling molecule secreted by an immune cell. For example, the cytokine may comprise an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor. For example, the interleukin may comprise IL-6, IL-10, IL-12, and/or a functional subunit thereof. For example, the interferon may comprise IFN-γ. For example, the chemokine may comprise CCL2 and/or CXCL12. For example, the Tumor Necrosis Factor may comprise TNF-α. For example, the flavonoid may comprise 3’-O- (3-chloropivaloyl) quercetin.

In some cases, inhibiting the formation and/or function of the migrasome comprises decreasing the amount and/or function of a chemokine in the immune cell generating the migrasome and/or in the migrasome. For example, decreasing the amount and/or function of the chemokine may comprise knocking out or knocking down the expression of a gene encoding for the chemokine in the immune cell generating the migrasome. For example, decreasing the amount and/or function of the chemokine may comprise treating the migrasome with an agent capable of inhibiting the function of the chemokine. For example, the agent capable of inhibiting the function of the chemokine may comprise a protease, a small molecule, and/or an antibody capable of inhibiting the activity of the chemokine. For example, the chemokine may comprise CCL2 and/or CXCL12.

According to any aspect of the present disclosure, the motor protein may comprise any motor protein, a fragment thereof, or a domain thereof capable of binding to or transporting an intraluminal vesicle of the present disclosure. For example, the motor protein according to the present disclosure may comprise one or more actin based motor proteins. For example, the motor protein may comprise a Myosin or a member of the Myosin family. In some cases, the motor protein comprises Myosin1c, Myosin5a or comprises a motor domain thereof. In some cases, the Myosin5a comprises an amino acid sequence as set forth in SEQ ID NO: 10.

According to any aspect of the present disclosure, the SNARE complex is also known as SNAP Receptor (SNARE) . For example, formation of SNARE complex may mediate the fusion of the membrane of migrasome with the intraluminal vesicle. For example, the SNARE may comprise a vesicle-SNARE and/or a v-SNARE, which may be incorporated into the membrane of secretory vesicles during budding. For example, the v-SNARE may comprise SNAP23. For example, SNARE may comprise target-SNAREs or t-SNAREs. The SNARE complex may be components of the fusion machinery. For example, the t-SNARE may comprise VAMP2 and/or VAMP7. For example, during membrane fusion, v-SNARE and t-SNARE on separate membranes may combine to form a SNARE complex. Regulating the SNARE complex may comprise regulating the amount and/or function of member of SNARE complex family.

For example, increasing the amount and/or function of SNARE complex may comprise providing a member of the SNARE complex family, overexpressing a member of the SNARE complex family, and/or activating a member of the SNARE complex family. For example, increasing the amount and/or function of the SNARE complex may comprise introducing a member of the SNARE complex family and/or a gene encoding for a member of the SNARE complex family. For example, increasing the amount and/or function of the SNARE complex may comprise activating the interaction between the members of the SNARE complex family.

For example, decreasing the amount and/or function of the SNARE complex may comprise knocking out or knocking down the expression of a gene encoding for a member of the SNARE complex family, and/or treating the migrasome with an agent capable of inhibiting the function of a member of the SNARE complex family.

In the present disclosure knocking down a target (such as the tetraspanin 4, the tetraspanin 9, a Myosin or a SNARE complex member/component) refers to a process by which the expression of the target-encoding gene is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

The knocking down may be through a genetic modification or may be transient. If a DNA of an organism or cell is genetically modified, the resulting organism or cell may be referred to as a “knockdown organism” or a “knockdown cell” . If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result may be referred to as a “transient knockdown” .

In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of transcription (in the case of gene-binding) , the degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA) ) or RNase-H dependent antisense, or through the blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA (e.g. by morpholino oligos or other RNase-H independent antisense) .

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC) . The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.

In some cases, a shRNA is used for knocking down the target, for example. The shRNA may be introduced into the cell via a viral construct. In some cases, the viral construct is a lentiviral construct.

Knocking out the target (such as the tetraspanin 4, the tetraspanin 9, a Myosin or a SNARE complex member/component) refers to a genetic process in which the target-encoding gene is made inoperative ( “knocked out” ) . When the target-encoding gene is knocked out, it may comprise heterozygous knock out or homozygous knock out. In the heterozygous knock out, only one of two gene copies (alleles) is knocked out, in the homozygous knock out, both copies are knocked out.

Knockouts may be accomplished through a variety of techniques. In some cases, the knockouts may be naturally occurring mutations that are screened out or identified (e.g., by DNA sequencing or other methods) .

In some cases, the knockouts are generated by homologous recombination. For example, it may involve creating a nucleic acid (e.g., DNA) construct containing the desired mutation. The construct may also comprise a drug resistance marker in place of the desired knockout gene. The construct may further contain a minimum length (e.g., 2kb or above) of homology to the target sequence. The construct may be delivered to target cells (for example, through microinjection, electroporation or other methods, such as transfection, using a virus or a non-virus system) . This method then relies on the cell’s own repair mechanisms to recombine the nucleic acid construct into the existing DNA (e.g., the genome of the cell) . This may result in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all. The drug selection marker on the construct may be used to select for cells in which the recombination event has occurred. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection may be performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.

In some cases, the knockouts are generated using site-specific nucleases. Various methods may be used to precisely target a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell’s repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ) , which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene.

For example, a zinc-finger nuclease may be used to generate such knockouts. Zinc-finger nucleases comprise DNA binding domains that can precisely target a DNA sequence. Each zinc finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene.

As another example, Transcription activator-like effector nucleases (TALENs) may be used to generate such knockouts. TALENs contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region may comprise amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene.

As a further example, Clustered regularly interspaced short palindromic repeats (CRISPR) system may be used to generate such knockouts. The CRISPR/Cas9 method is a method for genome editing that contains a guide RNA complexed with a Cas9 protein. The guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing. The coupled Cas9 may cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene.

The knockout may also comprise a conditional gene knockout. A conditional gene knockout allows gene deletion in a tissue or cell when certain conditions are fulfilled, for example, in a tissue specific manner. It may be achieved by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.

According to any aspect of the present disclosure, the immune cell (e.g., the first immune cell, the second immune cell, the local immune cell; including the engineered immune cell of the present disclousre) may comprise a monocyte and/or a macrophage, including their progenitors and/or progenies (i.e., cells derived from the monocytes and/or the macrophage) . For example. the immune cell generating the migrasome (e.g., the first immune cell, or the local immune cell of the present disclosure) may consists essentially of a monocyte and/or macrophage. In some cases, the affected or targeted immune cell (e.g., the second immune cell of the present disclosure, or the immune cell to migrate) comprises a monocyte and/or a macrophage (including their progenitors and/or progenies) . In some cases, the affected or targeted immune cell (e.g., the second immune cell of the present disclosure, or the immune cell to be migrated) consists essentially of a monocyte and/or a macrophage. In some cases, the immune cell generating the migrasome is of the same type as the affected or targeted immune cell. For example, the immune cell generating the migrasome may comprise a monocyte and/or a macrophage, and the affected or targeted immune cell may also comprise a monocyte and/or a macrophage. In some cases, the migrasome generated by the immune cell comprises and/or expresses a chemokine (e.g., CXCL12) . In some cases, the affected or targeted immune cell comprises and/or expresses a molecule capable of specifically recognizing (e.g., specifically binding to) the chemokine comprised and/or expressed by the migrasome. In some cases, the molecule comprises CXCR7 and/or CXCR4.

According to any aspect of the present disclosure, the immune cell (including the engineered immune cell of the present disclousre) may be a migrating immune cell (including a circulating immune cell) . In some cases, the immune cell is a migrating immune cell in or from the blood.

The present disclosure also provides an engineered immune cell.

In some cases, the engineered immune cell has increased ability for recruiting a target immune cell (e.g., the second immune cell of the present disclosure) comparing to a corresponding unmodified immune cell. In some cases, the engineered immune cell has decreased ability for recruiting a target immune cell (e.g., the second immune cell of the present disclosure) comparing to a corresponding unmodified immune cell.

In some cases, the engineered immune cell has increased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell. In some cases, the engineered immune cell has decreased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell.

In some cases, the engineered cell has increased secretion ability comparing to a corresponding unmodified immune cell. In some cases, the engineered cell has decreased secretion ability comparing to a corresponding unmodified immune cell.

In some cases, the engineered cell has been modified to have increased ability for generating migrasomes. In some cases, the engineered cell has been modified to have decreased ability for generating migrasomes.

A cell may be modified by any approach applicable for the purpose of the present disclosure. For example, the modification may be a genetic modification. In some cases, the modification may comprise treating the cell with one or more agent causing the desired change or effect. The modification may be temporary, transient or may be stable or permanent. In some cases, the engineered cell may be a progeny of a parent cell that has been modified.

The present disclosure also provides use of the agent of the present disclosure in the preparation of the engineered immune cell of the present disclosure.

In one aspect, the present disclosure also provides a method for generating or making the engineered immune cell of the present disclosure, comprising modifying the migrasome generation ability of the immune cells, according to the description of the present disclosure.

The present disclosure also provides isolated migrasomes. In some cases, the present disclosure provides immune cell derived migrasomes, such as isolated migrasomes derived from the immune cells of the present disclosure. The migrasomes may be used for regulating an immune response and/or an immune response mediated biological process. In some cases, the migrasomes may be used from regulating the immune cell mediated biological function as described in the present disclosure.

According to any aspect of the present disclosure, the method may comprise administering to a subject in need thereof an effective amount of the migrasome (such as the immune-cell derived migrasome of the present disclosure, for example, the isolated migrasome of the present disclosure) .

The method may further comprise administering to the subject an additional active molecule. The additional active molecule may comprise a caspase inhibitor. For example, the caspase inhibitor may comprise a caspase 8 inhibitor. For example, the additional active molecule may comprise z-VAD-FMK or a functional derivative thereof.

In one aspect, the present disclosure provides a method for monitoring an immune response and/or an immune response mediated biological process in a subject. The method may comprise analyzing the presence, amount and/or function of a migrasome obtained from a biological sample of the subject. The subject may be a mammal, such as a human subject. The immune response and/or the immune response mediated biological process may comprise inflammatory response and/or other diseases, conditions or disorders affected by the function of an immune cell.

An increase of the amount of the migrasome may indicate an increase of the immune response. In some cases, an increase of the amount of the migrasome indicates progression of the immune response mediated biological process. decrease of the amount of the migrasome may indicate a decrease of the immune response. In some cases, a decrease of the amount of the migrasome indicates anesis or remission of the immune response mediated biological process.

Analyzing the presence, amount and/or function of the migrasome may comprise analyzing the presence and/or amount of a marker molecule of the migrasome. For example, analyzing the presence, amount and/or function of the migrasome may comprise determining the presence and/or amount of Tspan4⁺, Integrin⁺, Pleckstrin Homology (PH) domain⁺, NDST1⁺, PIGK⁺, CPQ⁺, EOGT⁺, KUL01⁺, CD115⁺, and/or CCR2⁺ vesicles in the biological sample. In some cases, analyzing the presence, amount and/or function of the migrasome comprises staining the biological sample with wheatgerm agglutinin (WGA) .

According to any aspect of the present disclosure, the migrasome (e.g., the immune cell derived migrasome , such as the monocyte derived migrasome of the present disclosure, such as the isolated migrasome) may be KUL01⁺, CD115⁺, and/or CCR2⁺. In some cases, the migrasome is KUL01⁺. In some cases, the migrasome is CD115⁺. In some cases, the migrasome is CCR2⁺. In some cases, the migrasome is KUL01⁺, CD115⁺, and CCR2⁺. In some cases, the migrasome is KUL01⁺ and CD115⁺. In some cases, the migrasome is KUL01⁺ and CCR2⁺. In some cases, the migrasome is CD115⁺ and CCR2⁺.

In the present disclosure, if a marker (for example, a target protein) can be detected (i.e., with a signal above the detection threshold) with any of the methods or approaches known to a skilled person, then, the detection result is considered to be positive for the marker. The sample (e.g., the biological sample, or the components in the biological sample, such as the cell or the migrasome) is considered to be positive for the marker, and can be referred to as the marker⁺ (e.g., KUL01, CD115, Tspan4, or CCR2 etc. ) .

In one aspect, the present disclosure also provides a method for regulating an immune response and/or an immune response mediated biological process in a subject, comprising: i) monitoring the immune response and/or the immune response mediated biological process in the subject according to the present disclosure; and ii) administering a regulating agent according to the result of step i) . For example, if an increased immune response is determined in step i) , an agent may be administered in step ii) to further boost or inhibit the immune response as needed.

In one aspect, the present disclosure also provides a method for characterizing (e.g., monitoring, detecting, tracing, revealing, etc. ) a migrasome generated by a monocyte, comprising determining the presence and/or amount of C-C chemokine receptor type 2 (CCR2) .

The isolation may be performed with a method known to a skilled person. Procedures for separation may include magnetic separation, using antibody-coated magnetic beads or dynabeads, affinity chromatography, affinity agents conjugated to a monoclonal antibody or used in conjunction with a monoclonal antibody, and “panning” with antibody attached to a solid matrix, e.g., plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc., as well as magnetic activated cell sorters. The antibodies (e.g., anti-CCR2 antibodies or the antigen binding fragments thereof) may be conjugated with markers, such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, such as FITC, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular target (e.g., a monocyte-derived migrasome) . Other techniques include, but are not limited to, dense particles for density centrifugation, an adsorption column, an adsorption membrane, and the like.

The regulating agent may be any agent suitable for the desired purpose, e.g., an agent capable of specifically regulating the function of a monocyte, a migrasome, and/or a monocyte-derived migrasome. The regulating agent may be a protein, a polypeptide, a small molecule compound, a nucleic acid, a cell, or any combination (such as a conjugate) thereof.

In another aspect, the present disclosure provides a composition, comprising agent capable of determining the presence and/or amount of the CCR2. The composition may further comprise a second agent capable of determining the presence and/or amount of a migrasome, as described in other parts of the present disclosure. For example, the second agent may be capable of determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT. In some cases, the second agent comprises WGA.

In another aspect, the present disclosure provides a kit. The kit may comprise the agent (e.g., the agent capable of determining the presence and/or amount of CCR2) and/or the composition according to the present disclosure.

In another aspect, the present disclosure provides use of the agent, the composition, and/or the kit of the present disclosure, in the preparation of an indicator for a migrasome generated by a monocyte.

According to the present disclosure, characterizing (e.g., monitoring, detecting, tracing, revealing, etc. ) a migrasome generated by a monocyte may comprise determining the presence and/or amount of the CCR2 in the migrasome. The migrasome may be in or from a biological sample (such as a body fluid sample, e.g., a blood sample) .

According to the present disclosure, determining the presence and/or amount of the CCR2 may comprise using an agent capable of specifically identifying the CCR2, such as contacting the biological sample to be analyzed with such an agent. The agent capable of specifically identifying the CCR2 may comprise an anti-CCR2 antibody or an antigen binding fragment thereof. The antigen binding fragment may comprise Fab, F (ab) 2, F (ab’) , F (ab’) 2, scFv, affibody and/or VHH. The agent capable of specifically identifying the CCR2 may further comprises a detectable label.

In some cases, determining the presence and/or amount of the CCR2 may comprise detecting the presence and/or amount of a modified CCR2 containing a detectable label. The detectable label may comprise a fluorescent label, a luminescent label, and/or a non-optically detectable label.

In some cases, characterizing (e.g., monitoring, detecting, tracing, revealing, etc. ) a migrasome generated by a monocyte may further comprise determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT. In some cases, characterizing (e.g., monitoring, detecting, tracing, revealing, etc. ) a migrasome generated by a monocyte may further comprise staining the biological sample with wheatgerm agglutinin (WGA) .

Thus, the method, agent, composition or use of the present disclosure may also involve detecting or analyzing an additional marker of the migrasome.

Migrasome formation or a change thereof (e.g., an increase in migrasome formation or a decrease in migrasome formation) may be monitored and/or determined by observation, e.g. using microscopy, such as scanning electron microscope (SEM) and/or transmission electron microscope (TEM) . For example, migrasomes may be identified as membrane-bound vesicular structures, either in the extracellular space or in the cell generating them. The migrasomes may be connected to or closely associated with retraction fibers. A migrasome may be oval shaped, with diameters from e.g. about 400 nm to about 3500 nm, the migrasomes may contain multiple smaller vesicles. For example, the structure of a migrasome may resemble opened pomegranates (e.g., also known as pomegranate-like structures, or PLS) .

In addition or alternatively, migrasome formation or a change thereof (e.g., an increase in migrasome formation or a decrease in migrasome formation) may be monitored and/or determined by detecting the expression and/or amount of a migrasome specific marker. Such detection may be at transcriptional level and/or at protein level. Such marker may include but not limited to Tetraspanin-4, integrin, pleckstrin homology (PH) domain, NDST1 (bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1) , PIGK (phosphatidylinositol glycan anchor biosynthesis, class K) , CPQ (carboxypeptidase Q) and/or EOGT (EGF domain-specific O-linked N-acetylglucosamine transferase) .

In some cases, migrasome formation or a change thereof (e.g., an increase in migrasome formation or a decrease in migrasome formation) may be monitored and/or determined by staining the cell or sample with a migrasome specific dye, for example, by using WGA (wheatgerm agglutinin, a sialic acid-and N-acetyl-D glucosamine-binding lectin) .

According to any aspect of the present disclosure, a detectable label may be attached to an analyte (such as an agent of the present disclosure) to render the reaction of the analyte detectable. For example, the detectable label may produce a signal that is detectable by visual and/or instrumental approaches. For example, the detectable label may comprise moieties that produce light, and/or moieties that produce fluorescence. For example, the detectable label may comprise a fluorescent label, a luminescent label, and/or a non-optically detectable label (for example, to be detected according to its specific mass, weight, shape and/or size) .

For example, the detectable label may be attached to a marker or agent, and the presence and/or amount of the signal produced by the detectable label may indicate the presence and/or amount of the marker or agent. For example, one or more analytes may be attached with one or more detectable labels. For example, each of one or more specific analytes may be attached with different detectable label. For example, different detectable label may have different color, or may have different types of labels, e.g., a fluorescent label, a luminescent label, and/or a non-optically detectable label.

According to any aspect of the present disclosure, the biological sample may be collected and/or analyzed. For example, the biological sample may comprise but not limited to biological fluids such as sputum, blood, serum, plasma, or urine. For example, the biological sample may comprise a blood sample. For example, the blood sample may comprise whole blood, plasma, and/or serum.

For example, the biological sample may be from a human and/or an animal. For example, the biological sample may be analyzed in vivo, e.g., without being removed from the human or animal, or the biological sample may be tested in vitro. For example, the biological sample may be analyzed after being processed, e.g., by isolating. For example, the biological sample may be freshly taken from a human or animal, or may be processed or stored.

For example, analyzing the biological sample may comprise assessing a change in migrasome level in the biological sample in comparison with a reference sample. For example, the amount and/or function of a migrasome in the biological sample may be lower than in the reference sample, which may indicate that the subject has decreases immune response. For example, the amount and/or function of a migrasome in the biological sample may be higher than in the reference sample, which may indicate that the subject has increased immune response (e.g., increase inflammation) . For example, the reference sample may be derived from the same subject, taken at a different time point or from other site of the body, and/or from another individual.

According to any aspect of the present disclosure, the method may be an in vivo method. In some cases, the method is a in vitro method. In some cases, the method is an ex vivo method.

In the present disclosure, an agent may be a small molecule compound, an antibody, a nucleic acid molecule, a polypeptide, or fragments thereof. In some cases, the agent may comprise one or more active components, present in a single molecule or as separate molecules.

The agent may be provided in a non-active form and be converted into an active form in vitro or in vivo before, during or after administration.

The agent may be a pharmaceutical agent or an agent for non-pharmaceutical use.

The agent may exert the desired functions directly or indirectly via the function of additional agents, compositions or cells.

The composition of the present disclosure may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient.

The composition may comprise an effective amount of the agent of the present disclosure. The effective amount may be an amount of the agent that when administered alone or in combination with another agent to a cell, tissue, or subject is effective to achieve the desired effect (e.g., regulating the immune cell mediated biological function) .

The compositions may further include pharmaceutically acceptable materials, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers. These carriers are involved in transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

The formulation and delivery methods will generally be adapted according to the site and the disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration The dosage of the agents of the disclosure will vary according to the extent and severity of the need for regulation, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

Agents as described herein can be incorporated into compositions suitable for administration. Such compositions typically comprise the agent and a pharmaceutically acceptable carrier. Supplementary active compounds can also be incorporated into the compositions. In yet other embodiments, the agents described herein are delivered locally. Localized delivery allows for the delivery of the agent non-systemically, for example, to the site of regulation in need.

In some cases, the composition may be detection or diagnosis composition for analyzing the biological sample and/or for monitoring the immune cell mediated biological function according to the description of the present disclosure.

The kit of the present disclosure may comprise the agent, the engineered cell, and/or the composition according to the present disclosure.

The agent, the engineered cell, and/or the composition may be comprised in suitable packaging, and written material that can include instructions for use, discussion of experimental studies (such as clinical studies) , listing of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, experimental results (such as clinical trial results) , and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent, the engineered cell and/or the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the users (such as health care provider or consumers) . Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.

The kit may further contain an additional agent. In some embodiments, the agent, engineered cell and/or the composition of the present invention and the additional agent may be provided as separate compositions in separate containers within the kit. In some embodiments, the agent, the engineered cell and/or the composition of the present disclosure and the additional agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to users (such as health providers) , including scientists, physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

The present disclosure also provides the following embodiments:

1. A method for characterizing a migrasome generated by a monocyte, comprising determining the presence and/or amount of C-C chemokine receptor type 2 (CCR2) .

2. The method of embodiment 1, comprising determining the presence and/or amount of the CCR2 in the migrasome.

3. The method of any one of embodiments 1-2, wherein said migrasome is in or from a biological sample.

4. The method of embodiment 3, wherein said biological sample comprises a body fluid sample.

5. The method of any one of embodiments 3-4, wherein said biological sample comprises a blood sample.

6. The method of any one of embodiments 1-5, wherein said determining comprises using an agent capable of specifically identifying said CCR2.

7. The method of embodiment 6, wherein said agent capable of specifically identifying said CCR2 comprises an anti-CCR2 antibody or an antigen binding fragment thereof.

8. The method of embodiment 7, wherein said antigen binding fragment comprises Fab, F (ab) ₂, F (ab’) , F (ab’) ₂, scFv, affibody and/or VHH.

9. The method of any one of embodiments 6-8, wherein said agent capable of specifically identifying said CCR2 further comprises a detectable label.

10. The method of any one of embodiments 1-9, wherein said determining comprises detecting the presence and/or amount of a modified CCR2 containing a detectable label.

11. The method of any one of embodiments 9-10, wherein said detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

12. The method of any one of embodiments 1-11, further comprising determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

13. The method of any one of embodiments 1-12, further comprising staining with wheatgerm agglutinin (WGA) .

14. A method for isolating and/or regulating a migrasome generated by a monocyte, comprising:

i) characterizing the migrasome according to the method of any one of embodiments 1-13; and

ii) isolating the characterized migrasome, and/or administering a regulating agent to said characterized migrasome.

15. The method of any one of embodiments 1-14, which is an in vitro or ex vivo method.

16. The method of any one of embodiments 1-14, which is an in vivo method.

17. An agent capable of determining the presence and/or amount of C-C chemokine receptor type 2 (CCR2) , for use in characterizing a migrasome generated by a monocyte.

18. The agent of embodiment 17, which is capable of specifically identifying said CCR2.

19. The agent of any one of embodiments 17-18, which comprises an anti-CCR2 antibody or an antigen binding fragment thereof.

20. The agent of embodiment 19, wherein said antigen binding fragment comprises Fab, F (ab) ₂, F (ab’) , F (ab’) ₂, scFv, affibody and/or VHH.

21. The agent of any one of embodiments 17-20, further comprising a detectable label.

22. The agent of embodiment 17, which comprises a modified CCR2 containing a detectable label.

23. The agent of any one of embodiments 21-22, wherein said detectable label comprises a fluorescent label, a luminescent label, and/or a non-optically detectable label.

24. A composition, comprising the agent according to any one of embodiments 17-23.

25. The composition of embodiment 24, further comprising a second agent capable of determining the presence and/or amount of a migrasome.

26. The composition of embodiment 25, wherein said second agent is capable of determining the presence and/or amount of Tspan4, Integrin, Pleckstrin Homology (PH) domain, NDST1, PIGK, CPQ, and/or EOGT.

27. The composition of any one of embodiments 25-26, wherein said second agent comprises WGA.

28. The composition of any one of embodiments 25-27, wherein said agent capable of determining the presence and/or amount of CCR2 is not mixed with said second agent.

29. A kit, comprising the agent according to any one of embodiments 17-23, and/or the composition according to any one of embodiments 24-28.

30. Use of the agent according to any one of embodiments 17-23, the composition according to any one of embodiments 24-28, and/or the kit according to embodiment 29, in the preparation of an indicator for a migrasome generated by a monocyte.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; r. t., room temperature; and the like.

Materials and Methods

Examples 1-6

Cultivation of SPF chick eggs

Fertilized SPF eggs (variety: White Leghorn; cleanliness: SPF) were bought from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd. The eggs were incubated in a hatching incubator at 37.5 ℃ with 60-70%humidity. Eggs were turned every 5 minutes.

Reagents

The antibody against VEGFA was generated by ABclonal Technology (Co. WG-04988, China) . The antibody against CXCL12 was from LSBio (Co. LS-B943-100, Seattle, USA) . The anti-Integrinα5 was from Cell Signaling Technology (4705S, Massachusetts, USA) . The antibody against GAPDH was from Proteintech (60060004-1-IG, Rosemont, USA) . The antibody against NDST was from Santa Cruz Biotechnology (sc-374529, Dallas, USA) . The antibody against CPQ was generated by Sigma (HPA023235, Shanghai, China) . The KUL01 antibody was from SouthernBiotech (8420-09, Birmingham, USA) . The antibodies against CD115 (CSF1R) , CD115-Alex488 and CD115- Alex647 were from Bio RAD (MCA5956GA, MCA5956GA488, MCA5956GA647, Hercules, USA) . The antibody against TSG101 was from Abcam (ab125011, Cambridge, USA) . The antibody against Calnexin was from Abcam (ab22595, Cambridge, USA) .

WGA (wheat-germ agglutinin) was from Life Technologies (W11261, Carlsbad, USA) . CellTracker^TM Red CMTPX was from Invitrogen Life Technologies (C34552, Carlsbad, USA) . GM-CSF (granulocyte-macrophage colony stimulating factor) was from PeproTech (315-03, Cranbury, USA) . Lipo-fectamine^TM3000 transfection reagent and P3000 reagent were from Invitrogen Life Technologies (L3000015, Carlsbad, USA) . Matrigel Basement Membrane Matrix was from Corning (356234, New York, USA) . Dextran was from Sigma-Aldrich (46945-100MG-F, USA) . PBS liposomes and Clodronate liposomes were from LIPOSOMA research (C-005, P-005, Amsterdam, The Netherlands) . Low melting agarose II was from AMRESCO (0815-25G, USA) .

Phosphate buffered saline was from Cytiva HyClone (SH30256.01, Marlborough, USA) . Endothelial Cell Medium was from ScienCell Research Laboratories (1001, Carlsbad, USA) . 0.25%Trypsin+0.02%EDTA solution was from Cienry (CR-25200, Hangzhou, China) . Penicillin&Streptomycin solution was from GENOM (GNM15140, Hangzhou, China) . GlutaMAX^TM I (100×) was from Gibco (35050-061, Carlsbad, USA) ; 4%Paraformaldehyde was from DINGGUO CHANGSHENG Biotechnology (ar-0211, Beijing, China) .

Collagenase Type II powder was from Gibco (17101-015, Carlsbad, USA) . Lysosome isolation kit was from Sigma-Aldrich (LYSISO1-1KT, Shanghai, China) .

2xRealStar green power mixture was from Gibco (A311-01, Carlsbad, USA) . TaKaRa MiniBEST Universal RNA Extraction kit was from TaKaRa (9767, Kusatsu, Japan) . Endofree plasmid Midi kit was from CWBIO (CW2105S, Taizhou, China) . TIANgel Midi Purification kit was from Tiangen (DP209-02, Beijing, China) .

Cell culture and treatment

HUVECs were grown in endothelial cell medium (ECM, ScienCell) and used between passages 3 and 5. HEK293A cells were cultured in DMEM (Gibco Life Technology) supplemented with 10%FBS. Cells were grown at 37 ℃ in a humidified incubator with 5%CO₂. WGA^high cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10%v/v fetal calf serum (Sigma-Aldrich, UK) , 2 mM L-glutamine, and 1%v/v (500 U/mL) penicillin/streptomycin. Cells were grown at 37℃ in a humidified incubator with 5%CO₂.

Migrasome purification

Migrasome purification was performed by iodixanol sucrose density-gradient centrifugation using an Opti-prep kit (LYSISO1, Sigma-Aldrich) . Chorioallantoic membranes were isolated from E9d chicken embryos (approximately 8 embryos for the rescue experiments and 30 embryos for quantitative mass spectrometry analysis) , then subjected to mechanical mincing. The chopped-up CAMs were then treated with collagenase II and trypsin. The samples were centrifuged at 1,000g for 5 min at 4 ℃ to remove the cell bodies, followed by 4,000g for 20 min at 4 ℃ to remove the cell fragments, and finally at 20,000g for 20 min at 4 ℃. The pellet containing the crude migrasome fraction was resuspended and lysed in extraction buffer (Sigma-Aldrich) and then fractionated at 150,000g for 4 h at 4 ℃ in a multistep Optiprep dilution gradient. The gradient was: 3, 5, 8, 12, 16, 19(sample) , 22.5 and 27%. Fractions were collected and added to 500 μl PBS. Centrifugation was then performed at 20,000g for 30 min at 4 ℃. The pellet was collected, washed once with PBS and centrifuged at 4 ℃, 2,000g for 10 min. The supernatant was collected and centrifuged at 4 ℃, 20,000g for 30 min to obtain migrasomes for TEM observation and injection into embryos.

Observation of migrasome formation in CAM in vivo

First, a square hole (about 1 cm) was cut in the eggshell. Second, WGA was diluted in 1×PBS (1: 500, 200 μl 1×PBS) and then this mixture was added to the top of the CAM. After staining for 20 min at 37.5 ℃ in a humidified incubator with 70%humidity, the egg was placed on a holder with the hole in direct contact with a cover glass, so that the weight of the egg held the CAM in tight contact with the cover glass. Last, the CAM was visualized under a Nikon A1 FV3000 confocal microscope and a Dragonfly Andor spinning disc confocal microscope.

Migrasome delivery by Matrigel or agarose

Fertilized chick eggs were incubated at 37.5 ℃ for 9 days. Then the E8d eggs were windowed. 5 μl (20 μg/μl, 100 μg) of migrasome sediment were embedded in 3 μl low-melting-point agarose or 3 μl Matrigel. After the mixture solidified, it was placed onto the CAM. 48 h later, images were captured by a Leica EZ4W stereomicroscope.

RNAi treatment of CAM and rescue by migrasome delivery

Fertilized chick eggs were incubated at 37.5 ℃ for 8 days. Then the E8d eggs were windowed and the CAM tissue was transfected with 2.5 μg siRNA for target genes (TSPAN4, VEGFA, CXCL12, GenePharma, Shanghai, China) with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, 100022052) and P3000 (Thermo Fisher Scientific, Waltham, MA, 100022058) according to the manufacturer’s instructions. A pair of platinum electrodes (Nepagene) was used for electroporation. Electroporation (five pulses of 50 ms duration at 20 mV) was used to improve the transfection efficiency. SiNS (GenePharma, Shanghai, China) was used for all control siRNA experiments. The knockdown efficiency of the target gene TSPAN4 was confirmed by quantitative real-time PCR (rtPCR) analysis. After the target gene was knocked down successfully, 5 μl (20 μg/μl, 100 μg) of migrasome sediment were embedded in 3 μl of low-melting-point agarose. After the mixture solidified, it was placed onto the CAM.

Flow cytometry

The mAbs used for flow cytometry are listed in STAR METHODS. Isolation of monocytes was performed by flow cytometry using anti-CD115 antibody. For isolation of WGA^high cells, briefly, CAMs were stained by WGA in vivo, then treated with collagenase II and trypsin. After removing the red blood cells, the residual cells (about 2×10⁷) were sorted by FACS (fluorescence activated cell sorting) . For isolation of CD115⁺ cells, cells were isolated from CAMs as described above, then incubated with anti-CD115 antibody (5×10⁶ cells in 600 μl 1×PBS, 1: 10, 37 ℃, 20 min) and sorted by FACS. To estimate the efficiency of TSPAN4 knockout in CAM9d, mCherry-positive cells were counted by flow cytometry.

TEM

CAMs isolated from 9d chick embryos were fixed with 2.5%glutaraldehyde + 2.0%paraformaldehyde diluted in 0.1 M Phosphate Buffer (0.1 M Na₂HPO₄. 12H₂O, 0.1 M NaH₂PO₄. 2H₂O, pH 7.2) . The CAMs were kept at room temperature for 2 h and then at 4 ℃ overnight. After three 10-min washes with 0.1M PB, the CAMs were treated with 1.5%K₃Fe (CN) ₆ + 1%OsO₄ (mixed before use) and kept at 4 ℃ for 1.5 h. The CAMs were washed three times with ddH₂O (10 min each wash) , and then treated with 1%uranyl acetate in water and kept at 4 ℃ overnight. After three 15 min washes in ddH₂O, the samples were dehydrated in ethanol (50, 70, 80, 90, 100, 100 and 100%; 15 min each) , then treated with 100%ethanol: 100%acetone at a 1: 1 ratio for 8 min, and finally with 100%acetone for 8 min. The CAMs were infiltrated with PON812 resin as follows: 1: 1 resin: acetone, 2 h at room temperature; 2: 1 resin : acetone, 2 h at room temperature; 3: 1 resin : acetone, 2 h at room temperature; resin alone, overnight; and resin alone, 2 h. Each CAM was then placed in the correct orientation on a 3.5-mm culture dish and a capsule filled with resin was placed over the CAM. The resin was polymerized at 37 ℃ for 8 h, 45 ℃ for 24 h and 60 ℃ for 12 h. Sections (70 nm) were cut with a Leica EM UC7 microtome and then stained with uranyl acetate and lead citrate. Images were obtained with a H-7650B TEM at 50-70 KV.

Imaging

To acquire two-dimensional images of vessel sprouting in CAMs in vivo, migrasomes or siRNA were added at the desired embryonic stage, then the CAMs were imaged by a Leica EZ4W stereomicroscope. Time-lapse multiple-view z-stack images (4D) of WGA^high cells were acquired for statistical analysis of migration and migrasome production. TSPAN4-KD, TSPAN4-KO or Cl-clodronate treatments were applied after windowing at the desired embryonic stage. Then the egg was placed on a holder with the window directly touching a cover glass, so that the weight of the egg kept the CAM in contact with the cover glass. The CAM was imaged by Olympus FV000 confocal microscopy, Nikon A1 confocal microscopy or spinning disk microscopy (Andor Dragonfly) .

Image processing

All of the time-lapse multiple-view z-stack embryo images (4D images) were processed using Imaris software 8.1.4 (Bitplane AG) . Images were processed by Image J to quantify the fluorescence intensity to assess the number of WGA^high cells, the number of sprouting capillaries or the density of sprouting capillaries. Anima software was used to reconstruct the 3D migrasome structure from FIB-SEM images. To determine the cell migration speed, time-lapse images were acquired by Nikon A1 and analyzed using Imaris software 8.1.4.

Tandem-Mass-Tag (TMT) quantitative mass spectrometry analysis

First, proteins either from migrasomes (case) or from cell bodies (control) were prepared using 8M urea in phosphate buffered saline (PBS) (Wisent, Nanjing, China) containing protease inhibitor cocktail. Second, all samples were sonicated for 2 min and centrifuged, and the supernatant was carefully separated. Protein concentrations were analyzed using the BCA protein assay kit. Third, in-solution digestion was performed. A total of 100 μg of protein extracted from each sample was reduced with 5 mM dithiothreitol (DTT) at room temperature and alkylated with 12.5 mM iodoacetamide (IAM) in the dark at room temperature. Then, the mixture was diluted to 1.5 M urea with PBS and the proteins were digested with trypsin (Promega, Madison, WI, USA) at 37 ℃ overnight. Fourth, the tryptic peptides were desalted using Sep-Pak desalting columns (Waters, Milford, MA, USA) and then the desalted peptides were labeled with 15 μl tandem mass tags (TMT) 10-PLEX reagents (Thermo Fisher Scientific, Waltham, MA, USA) . Three repeats in the control group were marked as 126, 126N and 127C. Three repeats in the case group were marked as 128C, 129N and 129C. These combined TMT-labeled peptides were desalted by Sep-Pak columns and separated with a UPLC 3000 system (Thermo Fisher Scientific, Waltham, MA, USA) with an XBridgeTM BEH300 C18 column (Waters, Milford, MA, USA) at a flow rate of 1 ml/min. The mobile phase A was H₂O (pH=10) and the mobile phase B was 98%acetonitrile (pH=10) . Peptides were separated with a gradient elution consisting of an increase from 8%to 18%phase B for 30 min, followed by an increase from 18%to 32%phase B for 22 min. Forty-eight fractions were dried by speedvac and recombined to 12 fractions. The fractions were dissolved in 20 μl of 0.1% (v/v) formic acid (FA) and analyzed by LC–MS/MS. Spectra from the mass spectrometer were searched against the UniProt Gallus gallus database using the SEQUEST search engine of Proteome Discoverer software (version 2.3) . The identified proteins were quality monitored, and each protein with more than 5 points and with a specific peptide segment number greater than 2 was judged to be credible and was carried forward for the subsequent quantitative analysis. For the results of the peptide segment search, X_corr needed to be higher than 2.5 for the peptide segment to be judged as credible.

Quantitative real-time PCR (qPCR)

To avoid contamination, all RNA-associated experiments were conducted in a molecular biology laboratory that is specifically designed for clinical diagnosis using molecular techniques, and which includes separate laboratories dedicated to performing each step of the procedure. Total RNA was isolated from CAM tissues with a TaKaRa MiniBEST Universal RNA Extraction Kit (Clontech TaKaRa, Cat#9796, USA) . cDNA was synthesized from 2 μg total RNA using a reverse transcription kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Total RNA was isolated from cells with Trizol reagent (Tiangen, Beijing, China) . qPCR was performed with the Roche LightCycler 480 II System (Roche, Basel, Switzerland) using SYBR green reaction mixture (GenStar, Beijing, China, Cat#A311-101) according to the manufacturer’s instructions. GAPDH and ACTB were used as internal controls for mRNA quantification. TSPAN4 primers were acquired from Primer-Blast and are listed in STAR METHODS.

Western Blotting

Whole cell extracts or migrasome extracts were isolated from E9.5 chick embryos using RIPA buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1%Triton X-100, 1%sodium deoxycholate, 0.1%SDS, 5 mM EDTA) or 2%SDS in 1×TBS complemented with protease inhibitors (complete protease inhibitor cocktail tablets, Roche) . After determining the protein concentration of each sample using the BCA kit (Biorad) , 40 μg of lysate was resolved on 10%SDS PAGE gels (Invitrogen) and transferred onto PVDF membranes (Amersham) . Blots were then blocked with 5%milk in 1×PBS for 1h at room temperature, followed by incubation with antibodies against Integrin α5/NDST1/CPQ/Calnexin/Tsg101/GAPDH/VEGFA/CXCL12/Alix at 4℃ overnight. Binding of HRP-conjugated secondary antibodies was subsequently visualized on the ChemiDoc MP Imaging System (BIO-RAD) .

Immunofluorescence

For immunofluorescent detection of VEGFA or CXCL12 in migrasomes, monocytes from CAM were isolated, and then cultured the monocytes on galectin-coated chambers for about 12 h. Then, the monocytes were stained by WGA488 (1: 2000) at 37 ℃ for 10 min. After that, monocytes were washed with PBS, fixed in 4%paraformaldehyde and permeabilized for 10 min with 0.3%Triton X-100 in 1×PBS. The monocytes were then blocked with 5%milk in 1×PBS for 1 h at room temperature and incubated with anti-VEGFA or CXCL12 antibody at 4℃ overnight. After that, the cells were washed three times with 1×TBST, then incubated with Alexa 561-conjugated IgG antibody (Sigma, USA) for 1 h at room temperature. The cells were finally washed with 1×TBST, and visualized under a Nikon A1 confocal microscope. For immunofluorescence of CSF1R and KULO1 in CAM, the CAM from E9d was isolated. Approximately 1 cm² of CAM was cut and incubated in diluted WGA buffer (1 μl WGA in 500 μl 1×PBS) at 37℃ for 20 min. Then the CAM was washed with 1×PBS, fixed in 4%paraformaldehyde for 2 h and permeabilized for 2 h with 0.3%Triton X-100 in 1×PBS. After permeabilization, the CAM was blocked with 5%BSA in 1×PBS for 6 h at room temperature and incubated with anti-CSF1R or KUL01 antibody at 4℃ overnight. After that, the CAM was washed three times with 1×TBST and incubated with Alexa 561-conjugated IgG antibody (Sigma, USA) for 1 h at room temperature. The CAM was finally washed with 1×TBST and coated on SuperFrost Plus microscope slides by ProLong TM Diamond Antifade Mountant (P36970, Thermo Fisher Scientific, USA) . Colocalization of WGA^high signal and CSF1R or KUL0l was visualized with a Nikon A1 confocal microscope.

Yeast phagocytosis by monocytes after GM-CSF stimulation

Monocytes from CAM9d were sorted by FACS after incubation with anti-CD115 antibody (5×10⁶ cells in 600 μl 1×PBS, 1: 10, 37℃, 20 min) . Then monocytes (1×10⁶) were cultured in chambers coated with 10%w/v gelatin solution. After that, monocytes were stimulated by GM-CSF (final concentration about 20 ng/μl, #315-03, PeproTech, USA) according to the manufacturer’s instructions and stained by WGA (1: 2000, 10 min, 37 ℃) . Yeast cells (strain BY4741, 1×10⁷) were stained by CellTracker^TM Red CMTPX (C34552, Thermo Fisher Scientific, USA, 1: 50000, 95 ℃, 30 min) and added to the chamber. 12 h later, images were acquired by spinning disk microscopy (Andor Dragonfly) .

Monocyte depletion by clodronate liposomes

When chick eggs had developed to embryonic day 6, they were windowed and 2 μl clodronate-liposomes or PBS-liposomes (Liposoma BV, The Netherlands) were microinjected into a vein in the CAM. After 48 h, 7.5 μl clodronate-liposomes or PBS-liposomes were microinjected into a vein of CAM. Then the windows were sealed by Parafilm and incubated at 37.5 ℃ with 60%humidity. The CAMs were imaged by a Leica EZ4W stereomicroscope.

Monocyte tracing

Chick eggs (E9d) were windowed and 20 μl CD115 (MCA5956GA488) were microinjected into the vein system. 20 min, 75 min and 120 min later, images were captured by FV3000 Olympus confocal microscopy.

Transwell chemotaxis assay

Transwell chemotaxis assays were performed with 12-well transwell plates. WGA^high or WGA^low cells were isolated by FACS after WGA staining. WGA^high or WGA^low cells were seeded in the upper chambers of the 12-well plate at 0.5×10⁶ cells/ml in RPMI 1640 medium (Gibco) supplemented with 10%v/v fetal calf serum (Sigma-Aldrich, UK) , 2 mM L-glutamine and 1%v/v (500 U/mL) penicillin/streptomycin. Generally, 12-well plates employed 1.5 ml of this RPMI 1640 medium in the lower chamber and 500 μl in the upper chamber. To identify the chemotaxis response of WGA^high or WGAl^ow cells to migrasomes, 15 μg migrasomes (1.5 μl of 10 μg/μl migrasomes in 1×PBS) were placed in the lower chambers as the case group and 1.5 μl 1×PBS were added in the lower chambers as the negative control group. The plates were then incubated for 6 h. The upper chambers were removed and the cells coating the top side of the polycarbonate membranes (Corning) were thoroughly removed with swabs. Then the polycarbonate membranes were cut off, washed with 1×PBS, fixed in 4%paraformaldehyde for 30 min, and stained by DAPI (1 μg/μl, 1: 1000) for 15 min. Lastly, the polycarbonate membranes were coated on SuperFrost Plus microscope slides with ProLong TM Diamond Antifade Mountant (P36970, Thermo Fisher Scientific, USA) . The number of migrated cells adhered to the underside of the polycarbonate membrane was visualized and counted by Nikon A1 confocal microscopy.

Generation of TSPAN4-KO-mCherry-KI chick embryos by the CRISPR-Cas9 system

A CRISPR/Cas9-based gene editing strategy was used to achieve TSPAN4 gene knockout in chick embryos. A guide RNA (gRNA) coding sequence was cloned into pUC57 vector (Addgene 55132) as the gRNA plasmid backbone, and the empty pUC57 vector was constructed as negative control (Scramble) which did not contain a sgRNA sequence. A sgRNA (Gallus-TSPAN4-gRNA1-Bsa1-F, 5’-TAGGGAAGGTTGAAGACAAACATT-3’ (SEQ ID NO: 1) ; Gallus-TSPAN4-gRNA1-Bsa1-R, 5’-AAACAATGTTTGTCTTCAACCTTC -3’ (SEQ ID NO: 2) ) was then designed to target exon 5 of chick TSPAN4. The sgRNA was inserted into the sgRNA expression cassettes of the pUC57 vector under control of the T7 promoter. Then the vector was introduced by chemical transformation into competent E. coli Top10 for cloning purposes using a kanamycin selectable marker.

To evaluate the TSPAN4 knockout efficiency, a knock-in plasmid was constructed to insert mCherry into the chick TSPAN4 gene under control of the original promoter. mCherry was inserted into TSPAN4 at the position targeted by the sgRNA. Thus, an mCherry-positive signal indicated that native TSPAN4 gene expression was silenced simultaneously. Follow this targeting strategy (FIG. 6) , the T4-Chick-KO-mcherry-KI plasmid (PM19040-A) was constructed by Biocytogen. The integration detection primers were as follows: PM19040-A-WT-F, 5’-GGTCCAGCACTGATGAGTCCACCTA-3’ (SEQ ID NO: 3) ; PM19040-A-Mut-R, 5’-GGGGAAGGACAGCTTCAAGTAGTCG-3’ (SEQ ID NO: 4) ; PM19040-A-WT-F, 5’-GGTCCAGCACTGATGAGTCCACCTA-3’ (SEQ ID NO: 3) ; PM19040-A-WT-R, 5’-ACCATCTTGCCCAACTTTCGAGTTCA-3’ (SEQ ID NO: 5) .

To generate the TSPAN4-KO chick embryos, a square hole (0.6 cm × 0.6 cm) was cut in the eggshell of gastrulating chick embryos. 1.2 μg of the guide RNAs (target sequences are listed in STAR METHODS) were co-injected with 1 μg Cas9 and 1.6 μg mCherry-KI plasmid into chick embryos at Hamburger Hamilton stage 4 (HH4, embryonic 18 h) using a glass capillary with a tip diameter of 0.1 mm. Then chick embryos were then electroporated using previously described techniques (Sauka‐Spengler and Barembaum, 2008) . The hole was covered by Parafilm and the chick embryos were incubated at 37.5 ℃ in the air under 70%humidity. Knockouts were confirmed by direct FACS for mCherry-positive signal sorting and by in vivo imaging for mCherry-positive signal detection.

Single-cell RNA-seq and data analysis

WGA^high CAM cells were sorted by FACS into PCR tubes. Single-cell RNA-seq experiments were performed according to the Smart-seq2 protocol with 20 cDNA pre-amplification cycles. Samples were sequenced by Illumina Hiseq 4000 with 150-bp paired-end reads. The transcriptome was quantified by Salmon with the chicken genome reference GRCg6a. Data from cells with more than 1500 genes detected were considered as high quality and were used for subsequent analysis. Further data analysis and visualization used Seurat.

Identification of monocyte-like cells

Annotated homologs of many classical cell-type marker genes could not be found in the chicken reference genome. Therefore, all reported classical and non-classical marker genes for the cell types suspected based on morphology data were collected. The collection of classical and non-classical marker genes contained experimental results and single-cell RNA sequencing results from http: //biocc. hrbmu. edu. cn/CellMarker/. 317 high-expression genes were found in the monocyte-like cell group (FIG. 2E) .

Statistical analysis

Data are expressed as the mean ± standard error of the mean from at least three separate experiments performed in triplicate. Statistical analysis was performed using one-way/two-way ANOVA or two-tailed Student’s t-test. A value of P<0.05 was considered statistically significant. All experiments were performed at least three independent times with similar results.

Examples 7-13

Reagents and antibodies

Fibronectin (PHE0023) , WGA (W7024) , Puromycin (A1113803) , Prolong Live Antifade Reagent (P36975) and Lipofectamine 3000 (L3000001) were purchased from ThermoFisher. BAPTA-AM (A1076) , Diaminobenzidine (D12384) , Propidium Iodide (81845) , LPS (L2630) and Proteinase K (P8811) were purchased from Sigma-Aldrich. Z-VAD-FMK (S7023) was purchased from Selleck. Vigofect (T001) was purchased from Vigorous. G418 (E859) was purchased from Amresco. Hygromycin B (10843555001) was purchased from Roche.

Anti-Rab8a (ab188574) , anti-SNAP23 (ab4114) and anti-TACE (ab2051) antibodies were from Abcam. Anti-Myosin5a (#3402) , anti-Rab8a (#6975) , anti-VAMP2 (#13508) , anti-TNF-α (#11948) , anti-IL-6 (#12912) , anti-Integrin α5 (#4705) , anti-Integrin β1 (#4706) , anti-Atp1α1 (#3010) , and anti-Slc1a5 (#8057) antibodies were from Cell Signaling Technology. Anti-GAPDH (60004-1-Ig) and anti-VAMP7 (22268-1-AP) antibodies were from Proteintech Group. Anti-CPQ (HPA023235-100UL) antibody was from Sigma-Aldrich. PE anti-mouse CCR2 (150609) , APC anti-mouse CCR2 (150627) , PE anti-mouse F4/80 (123109) , APC anti-mouse F4/80 (123115) , PE anti-mouse CD115 (135505) , APC anti-mouse CD115 (135509) , APC anti-mouse Ly6C (128015) and Biotin anti-mouse CD115 (135507) antibodies were from BioLegend. Goat anti-Rabbit IgG (111-035-003) and Goat anti-Mouse IgG (115-035-003) were from Jackson.

Cells

L929 cells were grown in DMEM (C11995500BT, Gibco) supplemented with 10%FBS (04-001-1A, Biological Industries) , 2 mM GlutaMAX (35050-061, Gibco) and 100 U/mL penicillin-streptomycin (GNM15140, GENOM) . Cells were cultured at 37 ℃ in an incubator with 5%CO₂.

Mouse bone marrow monocytes (BMMs) were acquired from WT or T9 KO C57BL/6 mice as previously described (Beguier et al., 2020) using the EasySep mouse monocyte isolation kit (#19861, STEMCELL Technologies) . Briefly, bone marrow cell suspensions (1 x 108 cells/mL) were incubated sequentially with isolation cocktail components and dextran rapidspheres. Unwanted cells (T cells, B cells, NK cells, DCs, etc. ) were labeled with antibodies coupled to magnetic particles, and separated from unlabeled cells (monocytes) by a magnet. For purity assessment, cells stained with CD115 and Ly6C antibodies were analyzed by flow cytometry. BMMs were grown in RPMI 1640 (C11875500BT, Gibco) supplemented with 10%FBS, 2 mM GlutaMAX and 100 U/mL penicillin-streptomycin in 5%CO2 at 37℃. BMMs were activated with 500 ng/mL LPS for 12-24 hr.

Mouse bone marrow monocytes (BMMs) were acquired from WT or T9 KO C57BL/6 mice as previously described (Beguier et al., 2020) using the EasySep mouse monocyte isolation kit (#19861, STEMCELL Technologies) . Briefly, bone marrow cell suspensions (1 x 108 cells/mL) were incubated sequentially with isolation cocktail components and dextran rapidspheres. Unwanted cells (T cells, B cells, NK cells, DCs, etc. ) were labeled with antibodies coupled to magnetic particles, and separated from unlabeled cells (monocytes) by a magnet. For purity assessment, cells stained with CD115 and Ly6C antibodies were analyzed by flow cytometry. BMMs were grown in RPMI 1640 (C11875500BT, Gibco) supplemented with 10%FBS, 2 mM GlutaMAX and 100 U/mL penicillin-streptomycin in 5%CO₂ at 37℃. BMMs were activated with 500 ng/mL LPS for 12-24 hr.

Mice

CRISPR/Cas9 was used to generate Tspan9 KO C57BL/6 mice as previously described (Jiao et al., 2021) . The sgRNA sequence designed to target exon 4 of mouse Tspan9 was 5’-GAAGGTGGCGAAGTTGCCTT-3’ (SEQ ID NO: 6) . Gender-and age-matched WT C57BL/6 mice were used as controls for T9 KO animals. WT C57BL/6 mouse were from the Animal Facility at Tsinghua University. Animals were maintained in a light-and temperature-regulated room under specific pathogen-free (SPF) conditions. Animal studies and experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee at Tsinghua University, and were performed following the guidelines of the Laboratory Animal Research Center of Tsinghua University.

Cell transfection and virus infection

Cell transfection was conducted using Vigofect according to the manufacturer’s manual. The PiggyBac Transposon Vector System was used to generate stably expressing cell lines as previously described (Jiao et al., 2021) . Briefly, various proteins were cloned into pB-CAG (transposon vector) as the expressing plasmid backbone. The pB-CAG constructs combined with pBASE (transposase vector) were co-transfected into L929 cells at a ratio of 1: 3 using the above Vigofect transfection protocol. After 24 hr, the cells were treated with 600 μg/mL G418 or 200 μg/mL hygromycin B for selection (3-5 days) . Single cells were sorted into 96-well plates by flow cytometry. These single cell clones were cultured and expanded, followed by confocal analysis.

Gene knockdown was achieved with shRNA in the lentivirus-based vector pLKO. 1-puro. Lentiviral transduction and infection were performed as previously described (Jiao et al., 2015) . Briefly, for lentiviral production, lentiviral vectors (pLKO. 1, psPAX2 and pMD2. G) were co-transfected into L929 cells at a ratio of 4: 3: 1. After 48 hr, the supernatant was centrifuged at 600 g for 5 min to remove cell debris. Viruses were harvested and used in the following experiments. For virus infection, the indicated cells seeded to 40-60%confluence were co-cultured with virus containing 8 μg/mL polybrene for 24 hr. The cells were placed in fresh medium containing 5 μg/mL puromycin for selection until drug-resistant colonies become visible. The sequence of the siRNA to knock down mouse SNAP23 in pLKO. 1-puro was 5’-GAACAACTAAATCGCATAGAA-3’ (SEQ ID NO: 7) .

We used the CRISPR/Cas9-2hitKO system to generate the Myosin 5a gene knockout in L929 cells. Two guide RNA (sgRNA) coding sequences were cloned into PX458M (5’-GTGCCGGTATGCGCCAGGCA-3’ (SEQ ID NO: 8) and 5’-AGTTCGCTTCATCGATTCCA-3’ (SEQ ID NO: 9) ) . L929 cells were transfected with PX458M containing the Myosin5a targeting sequences. After single-cell sorting by flow cytometry, the single-cell clones were further analyzed by PCR and western blot.

Cell imaging and image analysis

10 μg/mL FN was used to pre-coat confocal dishes at 37 ℃ for at least 1 hour. For confocal snapshot images, cells were cultured in FN-precoated confocal dishes for 10-12 hr, and imaged by a NIKON A1RSiHD25 laser scanning confocal microscope at 1024 × 1024 pixels. Z-stack imaging of cells and migrasomes was performed with a NIKON A1 microscope. Structured illumination microscopy (SIM) images were acquired using a Nikon N-SIM Super Resolution Microscope.

For long-term time-lapse images, cells were grown in FN-precoated confocal dishes for 4-6 hr before imaging. Cells were then maintained in the living cell system (37 ℃, 5%CO₂) , and monitored by a NIKON A1 microscope. Ultra-fast super-resolution time-lapse images were collected using a Grazing Incidence-Structured Illumination Microscope (GI-SIM) . NIS-Elements analysis 5.4 software was used to deconvolute images acquired by the NIKON A1 microscope. Z-projection and 3D reconstruction were performed with NIS-Elements 5.4. Images were processed using Image J and Imaris software 8.1.4, and statistical analyses were conducted by Graphpad Prism 8.

Intravital imaging

Spinning disk microscopy (Perkin Elmer) was used to acquire time-lapse multiple-view z-stack intravital images as previously described (Jiao et al., 2021) . Briefly, to image circulating monocytes, LPS (12 mg/kg) was injected into mice by intraperitoneal (i. p. ) injection. C57BL6/J mice were injected with 5 mg WGA and 1 mg CCR2 antibody by intravenous (i. v. ) injection at 4-8 hr post LPS stimulation. WGA and CCR2 antibody were used to label vessels and monocytes, respectively. After 5 min, avertin (375 mg/kg) was i. p. injected into mice to induce anesthesia. Subsequently, the anesthetized mice were anatomized to expose the liver, and the blood vessels on the surface were monitored by spinning disk microscopy.

For the combined imaging of WT andT9 KO monocytes, monocytes were isolated from WT and T9 KO mice and incubated with PE anti-mouse CCR2 antibody and APC anti-mouse CCR2 antibody, respectively. After washing with PBS, the WT and T9 KO monocytes labelled with CCR2 antibodies conjugated to different colored fluroescent proteins were combined in equal amounts and injected into the spleen of C57BL6/J mice which had been i.v. injected with 5 mg WGA. After being anesthetized, the mice were anatomized to expose the liver, and the blood vessels on the surface were monitored by spinning disk microscopy. Images were processed using Image J and Imaris software 8.1.4, and statistical analyses were conducted by Graphpad Prism 8.

Isolation of migrasomes from culture cell

Crude migrasomes were collected by differential centrifugation as previously described (Zhao et al., 2019) . Briefly, cells and migrasomes in 15 cm dishes were gently harvested into 50 mL tubes after trypsin digestion. All subsequent manipulations were conducted at 4℃. After double centrifugation at 600 g for 10 min at 4 ℃, the supernatant was further centrifuged at 2000 g for 20 min at 4 ℃ to remove the cell bodies and large debris. Crude migrasomes were then acquired as the pellet by centrifugation at 18,000 g for 30 min at 4 ℃.

High-purity migrasome isolation was performed by iodixanol-sucrose density gradient centrifugation following the protocol we set up previously (Zhu et al., 2021) . Briefly, the crude migrasome pellet was resuspended in 800 μL extraction buffer, and then fractionated at 150,000g for 4 h at 4 ℃ in a multistep Optiprep dilution gradient. The step gradient was 50% (500 μL) , 40% (500 μL) , 35% (500 μL) , 30% (500 μL) , 25% (500 μL) , 20% (500 μL) , 15% (500 μL) , 10% (500 μL) , 5%(500 μL) and crude migrasomes (5%, 800 μL) . After centrifugation, samples were collected from top to bottom gently (500 μL per fraction) . Fractions 4, 5 and 6 were each mixed with 500 μL PBS and then centrifuged at 18,000g for 30 min at 4 ℃. The pellets were washed with PBS and centrifuged again at 18,000g for 30 min to pellet the migrasomes. The samples were immediately available for downstream applications such as western blot analysis and TEM.

Isolation of migrasome purification from blood

Blood was collected from mice by cardiac puncture and mixed with the same volume of blood collection buffer (10 mM EDTA in PBS) on ice. The blood mixture was centrifuged at 600 g, 4 ℃ for 10 min followed by 2000 g, 4 ℃ for 20 min to remove the blood cells. Crude migrasomes were then collected as the pellet by centrifugation at 18,000 g for 30 min. at 4 ℃.

To purify monocyte-derived migrasomes, crude migrasomes were negatively selected by magnetic sorting using the EasySep mouse monocyte isolation kit. Briefly, the pellet containing the crude migrasome fraction was resuspended and then incubated sequentially with isolation cocktail components and dextran rapidspheres. Unwanted components were labeled with antibodies coupled to magnetic particles, and separated from monocyte-derived migrasomes by a magnet.

For confocal imaging analysis, anti-CCR2 antibody was coated onto a cover glass. The crude migrasome preparation was incubated with the antibody-coated surface, following by washing and immunostaining. For western blot analysis, the crude migrasome preparation was incubated in antibody (CCR2) -precoated 6 cm dishes, following by washing and lysing by 2.5%SDS lysis buffer.

Transmission electron microscopy

Cells were grown in 35 mm dishes precoated with fibronectin (10 μg/mL) . After 10-12 hr, cells were pre-fixed using a 1: 1 ratio of growth medium to 2.5%glutaraldehyde for 5 min at room temperature. Cells were further fixed with 2.5%glutaraldehyde in PB buffer for 2 hr at room temperature, washed three times with PBS and dehydrated in an ascending gradual series of ethanol (50%, 70%, 90%, 95%, and 100%) for 8 min each. Samples were infiltrated with and embedded in SPON12 resin. After polymerizing for 48 h at 60 ℃, 70-nm-thick ultrathin sections were cut using a diamond knife, and then picked up with Formvar-coated copper grids (100 mesh) . The sections were double stained with uranyl acetate and lead citrate. After air drying, samples were examined with a transmission electron microscope H-7650B at an acceleration voltage of 80 kV.

Western blot analysis

Cells or migrasomes were lysed by 2.5%SDS lysis buffer and boiled for 10-20 min at 95℃. The protein concentration of each sample was determined using the BCA kit. Proteins were separated on SDS-PAGE gels of an appropriate percentage according to the MW of the target proteins, followed by electrophoretic transfer onto PVDF membranes. After blocking with 5%non-fat milk in TBST buffer, membranes were incubated with primary antibody overnight at 4℃. Membranes were then incubated with secondary antibody (HRP) for 1 hr at room temperature, and signals were detected with a WESTAR ηC 2.0 kit (CYANAGEN) .

The following primary antibodies were used for western blot analyses at the indicated dilution: anti-Myosin5a (1: 1000) , anti-SNAP23 (1: 1000) , anti-Slc1a5 (1: 2000) , anti-Atp1α1 (1: 5000) , anti-TNF-α (1: 1000) , anti-IL-6 (1: 1000) , anti-CPQ (1: 1000) , anti-Integrin α5 (1: 1000) , anti-Actin (1: 5000) and anti-GAPDH (1: 5000) .

Statistical analysis

Statistical analyses were conducted using the unpaired two-tailed t-test in Graphpad Prism 5 (or 8) software (Graphpad Software) . Error bars are the mean ± SEM. Significance is indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. Statistical parameters and significance are reported in the Figures and the Figure Legends.

Example 1 Detection of migrasomes in chick embryo chorioallantoic membrane (CAM)

To check whether migrasomes are formed in CAM during embryonic development, CAM from 9-day chicken embryos (CAM9d) was stained with WGA ex vivo. WGA staining revealed two types of cells: the majority of cells are large and flat with low WGA signal (WGA^low) ; the other cells are smaller but with a bright WGA signal (WGA^high) . It was found that the WGA^high cells form retraction fibers and migrasomes (FIG. 1A) . To further verify this observation, transmission electron microscopy (TEM) analysis was carried out on CAM9d, which revealed many large vesicles, with diameters up to 2 μm, in the extracellular space (FIG. 1B, 1C) . Consistent with the characteristic morphological features of migrasomes, these vesicles contain numerous intralumenal vesicles, and many of them were adjacent to fibers with diameters of about 50-100 nm (FIG. 1B) . To determine the 3D structure of this network of fibers and vesicles, FIB-SEM analysis was carried out. FIB-SEM showed that these vesicles were connected to fibers and, in many cases, the vesicles were localized on the tips of the fibers (FIG. 1D, 1E) , which is another morphological feature of migrasomes.

To directly observe the formation of migrasomes in vivo, an imaging protocol was designed. First, a hole was cut in the eggshell and stained the CAM with WGA. After staining, the egg was placed on a holder with the hole in direct contact with a cover glass, so that the weight of the egg white and yolk kept the CAM in tight contact with the cover glass. Using time-lapse microscopy, it was found that retraction fibers and migrasomes were indeed formed in CAM of living chicken embryos. Put together, these data suggest that migrasomes were formed in CAM by WGA^high cells (FIG. 1F) .

It was noticed that WGA^high cells were evenly distributed in the CAM, and most of them were outside the blood vessels (FIG. 1G) . WGA^high cells were extraordinarily mobile cells: in some cases, they can move as fast as 2 μm/min (FIG. 1H) , and they left a dense patch of migrasomes in these areas (FIG. 1I) .

Example 2 Migrasomes in CAM are generated by monocytes

To investigate the identity of WGA^high cells, WGA-stained CAM9d from chicken embryos was first isolated and then subjected to mechanical mincing. The chopped-up CAMs were then treated with collagenase II and trypsin, and the released cells were collected and subjected to fluorescence activated cell sorting (FACS) . The WGA^high and WGA^low cells were collected and cultured in vitro (FIG. 2A, 2B) . WGA^high and WGA^low cells had different morphologies (FIG. 2C, 2D) . TEM analysis showed that the migrasomes generated by WGA^high cells had a similar morphology to the migrasomes observed in vivo. Importantly, WGA^high cells generated much higher numbers of migrasomes than WGA^low cells (FIG. 2D) . Similar to the in vivo observations, cultured WGA^high cells moved much faster and were smaller than WGA^low cells (FIG. 2L, FIG. 3A-3C) . These data suggested that the WGA^high cells isolated were the migrasome-generating WGA^high cells observed in vivo.

Next, the WGA^high cells were subjected to single-cell RNA sequencing, which identified two subsets of cells in the WGA^high population (FIG. 2E) . The first group was enriched with markers for monocytes, while the second group was enriched with markers for endothelial cells (FIG. 2E, 2F) .

The RNA-seq results, and the fact that the migrasome-forming WGA^high cells were highly migratory, indicated that these cells could be monocytes. To verify the identity of WGA^high cells, immunostaining was carried out with KUL01, an anti-macrophage/monocyte monoclonal antibody, and an antibody against CD115, which was expressed by monocytes/macrophages. It was found that indeed the WGA^high cells stained positive for both antibodies, which indicated that WGA^high cells were monocytes (FIG. 2I) . To further verify this, CAM was labeled with anti-CD115 antibody, and the CD115-positive cells were sorted out (FIG. 2G) . It was found that these cells were identical to WGA^high cells in terms of morphology and their ability to form migrasomes (FIG. 2H) . Finally, it was found that WGA^high cells are highly phagocytic, and the phagocytosis can be further enhanced by treating WGA^high cells with GM-CSF (FIG. 2J, 2K) . Put together, these results indicate that WGA^high cells were monocytes.

Example 3 Monocyte migrasomes contain chemokines

Migrasomes from CAM9.5d were isolated (FIG. 4A) . The purity of the isolated migrasome was analyzed by TEM and by western blot for various migrasome markers. It was found that the isolated migrasomes had the characteristic morphological features of migrasomes (FIG. 4B) ; moreover, migrasome markers were highly enriched in the preparation (FIG. 4C) . Next, tandem-mass-tag (TMT) labelling was carried out followed by quantitative mass spectrometry (FIG. 4D) . The resulting volcano plot showed that the protein composition of migrasomes was markedly different from cell bodies (FIG. 4E) . Known migrasome-enriched proteins such as tetraspanins and integrinβ were enriched in CAM migrasomes, while nuclear proteins were depleted (FIG. 4F) , which suggested that the Q-MS analysis was reliable. Next, whether known chemokines were enriched in migrasomes was checked. Indeed, it was found that a host of these factors, including TGF-β3, VEGFA and CXCL12, were enriched in migrasomes (FIG. 4F) . It was worth noting that compared to epithelial cells (the majority of WGA^low cells) , CXCL12, TGF-β3 and VEGFA were highly expressed in monocytes (FIG. 4G and FIG. 8) .

Next, the enrichment of these chemokines was verified by western blotting and immunostaining. It was found that both proteins were enriched on migrasomes by both western blotting and immunostaining (FIG. 4H, 4I) . Next, cryo-sections of CAM were stained with anti-VEGFA and anti-CXCL12 antibodies. It was found that monocytes were the main VEGFA-and CXCL12-expressing cells (FIG. 4J and FIG. 9) .

Example 4 Migrasomes induce recruitment of monocytes

Next, the role of migrasomes was tested by adding isolated migrasomes on top of the CAM. To keep the migrasomes in place, they were mixed with Matrigel and added the mixture to the CAM of 9-day embryos. The migrasomes were also delivered by mixing them with low-melting-point agarose. In both cases, adding migrasomes significantly enhanced the recruitment of monocyte cells (FIG. 5A, 5B) , which suggested that migrasomes acted as a chemoattractant for monocytes.

Example 5 Migrasomes enhance monocyte chemotaxis in vitro

The role of migrasomes in recruitment of monocytes was also tested in vitro by transwell assay (FIG. 5C) . It was found that migrasomes markedly enhanced the chemotaxis of WGA^high monocytes; in contrast, migrasomes only slightly enhanced the chemotaxis of WGA^low cells (FIG. 5D, 5E) . Put together, these data supported a role for migrasomes in promoting monocyte recruitment.

Example 6 Migrasomes rescue monocyte recruitment defects in CAM with knockdown of CXCL12

The role of migrasomal CXCL12 in recruitment of monocytes was tested. It was found that CXCL12 was required for recruitment of monocytes and adding migrasomes can rescue the impaired monocyte recruitment in CXCL12 knockdown CAM (FIG. 7A, 7B) .

It was found that migrasomes were generated by monocytes or macrophages in the CAM during chicken embryonic development. It was also found that monocytes can recruit more monocytes via migrasomes through CXCL12-mediated chemotaxis.

In addition, it was found that monocytes and macrophages carried out a complicated set of functions in different biological settings including lymphangiogenesis, tissue remodeling, and inflammatory and immune responses. Many of these functions depended on secretion of cytokines, chemokines and growth factors; moreover, many of these biological processes require the spatial and temporal coordination and integration of a complex set of secreted ligands. The migrasomes also played important roles in these processes.

Example 7 Migrasome mediate localized secretion of cytokines in active monocyte

Monocyte was chosen to study the physiological roles of migrasome mediated secretion. Activated monocyte was highly migratory and secretory. It was found that activated monocyte generated large number of migrasome (FIG. 10A) , TEM analysis shown there were numerous intraluminal vesicles inside migrasome (FIG. 10B) .

Activated monocyte secreted TNF-α and IL-6, which played important roles in innate immune-response. TNF-α was secreted as membrane bound form, the soluble TNF-α was cleaved from membrane by metalloproteinase termed TNF-alpha-converting enzyme (TACE) . In contrast, IL-6 were secreted as soluble factor. Immunostaining monocyte with antibody against IL-6 and TNF- α revealed that both TNF-α and TACE are localized on the migrasome membrane, while IL-6 were localized in intraluminal vesicles inside migrasome (FIG. 10C and 10D, FIG. 17A) . Western blot confirmed TNF-α and IL-6 were not only present, but enriched in migrasome comparing to the cell body (FIG. 10E) .

Next, whether migrasome can contribute to secretion of TNF-α and IL-6 was tested. Previously, it was reported that fibronectin can significantly enhance the formation of migrasome on NRK cells. Monocyte was first cultured on glass surface without coating fibronectin, it was found that on this surface, monocyte does not generate migrasome. Further, it was found that migrasome formation in monocyte from Tspan9-/-mouse were impaired (FIG. 10F) .

To test whether migrasome formation can promote the secretion, cell was cultured on FN coated or control surface for 12 hours, then measured the secreted TNF-α and IL-6 on the medium. As expected, it was found that the LPS treatment significantly enhanced the secretion of TNF-α and IL-6 (FIG. 10E) . Interestingly, compared to FN coated surface, secreted soluble TNF-α and IL-6 was significantly reduced on monocyte cultured on controlled surface, which does not support migrasome formation (FIG. 10G) . Similarly, the secreted TNF-α and IL-6 level in the medium were also measured from wild type and Tspan9-/-monocyte, which are cultured on FN coated surface. It was found that soluble TNF-α and IL-6 were markedly reduced in Tspan9-/-monocyte (FIG. 10G) . Put together, these data suggest migrasome not only mediated localized secretion, but also contributed to the amount of secretion by monocyte.

Example 8 Migrasome bound TNFα is functional

TNF-α and IL-6 can be located in detached migrasome. This observation raised the interesting possibility that after detachment, the migrasome can be the vesicular carrier for signaling ligands. Since TNF-α are present on the surface of migrasome, to test whether migrasome bound cytokines are functional, whether migrasome can transmit TNF-α signaling was tested. The combination of TNF-α and caspase 8 inhibitor zVAD can induce necroptosis in L929 cells, it was found indeed adding the isolated monocyte derived migrasome and zVAD in L929 cells could kill L929 cells effectively (FIG. 10H) , which suggested that the migrasome bound TNF-α is functional.

Example 9 Monocyte derived migrasomes specifically express CCR2

Using the in vivo migrasome imaging protocol, next, whether monocyte can generate migrasome in vivo was investigated. Using fluorophores conjugated anti-CCR2 antibody, monocyte was successfully labeled. It was found that on the control mouse, monocyte was very hard to found, however, after LPS stimulation, monocyte could be easily observed in blood vessel and robust migrasome formation in circulating monocyte was observed (FIG. 11A) . Free CCR2 positive migrasome was also observed in blood vessels (FIG. 11B) . Next, crude migrasome preparation from blood was isolated. For confocal imaging analysis, the anti-CCR2 was first coated on cover glass, then the crude migrasome preparation was incubated with the anti-CCR2 antibody coated surface, following by washing and immune-staining (FIG. 11C) . This isolation protocol yielded CCR2 positive vesicles with morphological hallmark of migrasome (FIG. 11D) , moreover, these vesicles contain VAMP2 positive intraluminal vesicles, indicating that these vesicles are indeed monocyte derived migrasome. Importantly, both TNF-α and IL-6 could be found in migrasome (FIG. 11E and 11F) .

Example 10 Monocyte release cytokine-containing migrasomes in vivo

Next, the monocyte derived migrasome was analyze by western blot. The blood was first centrifuged using lower speed to remove the cell, then the monocyte derived migrasome was isolated using a protocol base on negative selection from equal volume of blood. Next, the sample was analyzed using antibody against CPQ, a protein present on migrasome but not present in exosome, and antibody against TNF-α and IL-6. It was found LPS treatment enhanced the amount of CPQ, TNF-α and IL-6 in cell fraction, which was likely resulting from the enhanced recruitment of immune-cells into circulation, resulting in more immune cells in equal volume of blood. This observation was consistent with in vivo imaging data. Similarly, LPS treatment enhanced the amount of CPQ in migrasome fraction, suggesting that the monocyte derived migrasome formation was enhanced, which could result from more migrasome generating monocyte were recruited into circulation, as a consequence, LPS treatment also significantly enhanced the amount of TNF-α and IL-6 in migrasome fraction (FIG. 11G) .

To compare the relative abundance of CPQ, TNF-α and IL-6 in cell and in migrasome, equal amount of total protein was loaded from cell and from migrasome and analyzed the amount of CPQ, TNF-α and IL-6. It was found that CPQ, TNF-α and IL-6 were highly enriched in monocyte derived migrasome comparing to cell body, moreover, it is worth to note although LPS treatment increased the TNF-α and IL-6 expression level in monocyte, fold of increasing were much more dramatic in migrasome (FIG. 11H) . Put together, these data suggested that circulating monocyte released migrasome bound cytokines, and LPS treatment dramatically enhanced the release of cytokines enriched migrasomes into the bloodsteam.

Example 11 Monocyte derived migrasome are reduced in Tspan9^-/-mouse

To test the role of Tspan9 in monocyte migrasome formation in vivo, monocyte form both wild type and Tspan9-/-mouse was isolated, labeled with CCR2 antibody with different color, and then injected these cells into wild type mouse, it was found similar to what observed in vitro, monocyte from Tspan9-/-mouse produced less migrasome in vivo (FIG. 11I) . Next, equal volume of blood from wild type and Tspan9-/-mouse were taken, and monocyte derived migrasome was isolated as described above, it was found that the amount of CPQ were significantly reduced in the sample from Tspan9-/-mouse, suggesting monocyte derived migrasome were indeed reduced in Tspan9-/-mouse. Consistently, it was found that the amount of migrasome bound TNF-α and IL-6 were reduced in equal volume of blood from Tspan9-/-mouse (FIG. 11J) .

Example 12 Tspan9 knock out lead to reduced soluble cytokine level in blood

VAMP2 positive secretory vesicles could fuse with migrasome membrane, thus secreting the secretory proteins into the medium, moreover, it was found that in in vitro cultured active monocyte, the secretion of TNF-α and IL-6 were reduced, suggesting blocking migrasome formation could cause reduced secretion. The level of a panel of soluble cytokines in blood from wild type and Tspan9-/-mice was checked using cytokine array, consistent with what was found in in vitro cultured monocyte, it was found that TNF-α, IL-6, IL-10, IL-12 and IFN-γlevel were also significantly reduced in Tspan9-/-mouse (FIG. 11K) . Thus, both soluble and migrasome bound cytokines are reduced in Tspan9-/-mouse.

Example 13 Intraluminal vesicles of migrasome are secretory vesicles

Characterization of intraluminal vesicles of migrasome

Transmission electron microscopy study was carried out on migrasome forming cells. It was found that the size and the number of intraluminal vesicles was closely correlated with the distance between migrasome and cell body, the further away from cells, the larger the migrasomes and the lower the number of intraluminal vesicles were (FIG. 12A) . Besides migrasome, it was also found that individual or small cluster of intraluminal vesicles in retraction fiber, in many cases, actin filaments were clearly visible associated with these vesicles (FIG. 12B) . Moreover, large cluster of vesicles was observed on the base of retraction fibers and detached migrasome which contain intraluminal vesicles (FIG. 12C) . The positioning and the distribution of these vesicles suggested that the intraluminal vesicles of migrasome may be transported to the base of retraction fiber, then transported to migrasome via retraction fiber.

The fact that the distal migrasome had less intraluminal vesicles suggested that the intraluminal vesicles may fuse with migrasome membrane. Calcium was required for SNARE mediated vesicle fusion, it was found that treating cell with BAPTA-AM, a cell-permeant chelator for calcium, significantly increased the number of intraluminal vesicles in migrasome (FIG. 12D) , supporting that intraluminal vesicles fused with migrasome membrane in a calcium dependent manner.

Rabs mark migrasome intraluminal vesicles

It was also found that endogenous Rab8 were present inside migrasome and along the retraction fiber (FIG. 12E) . Structured illumination microscopy (SIM) images shown GFP-Rab8 signal were present as intraluminal puncta inside migrasome (FIG. 12F) . To check whether these puncta are indeed intraluminal vesicles, APEX2-based intracellular-specific protein imaging was carried out by electron microscopy. APEX2 catalyzed the local deposition of DAB, which enhancing the contrast in EM images by binding electron dense osmium. It was found that APEX2-GFP-Rab8 were localized on intraluminal vesicles of migrasome (FIG. 12G) , thus, intraluminal vesicles can be labeled by Rab8. Moreover, it was found that BAPTA-AM could significantly increase the number of GFP-Rab8-labeled vesicles in migrasome (FIG. 12H) , which was consistence with our TEM analysis.

Taken together, these results suggest that the intraluminal vesicles in the migrasomes were secretory vesicles.

Myosin5a mediate transporting of migrasome intraluminal vesicles

The motor proteins which may transport intraluminal vesicles to migrasome were checked. Since bundled actin was observed inside retraction fiber, the search was focused on actin-based motor myosin, such as Myosin5a. To check the localization of Myosin5a, a cell line in which GFP-Myosin5a were stably expressed was generated. It was found that GFP-Myosin5a formed bright puncta along retraction fibers and inside migrasome, which was much brighter than GFP-Myosin5a signal inside cells (FIG. 13A) . Moreover, GFP-Myosin5a motor domain were highly enriched in migrasome, in contrast, GFP-Myosin5a-tail domain were absent in migrasome, suggesting the motor domain was required for localization of Myosin5a in migrasome (FIG. 15A) . Moreover, GFP fused to the Myosin5a motor domain was highly enriched in migrasomes, while GFP fused to the Myosin5a tail domain was absent from migrasomes (FIG. 15A) . This suggested that the motor domain was required for localization of Myosin5a in migrasomes. APEX2-based intracellular-specific protein TEM imaging revealed APEX2-GFP-Myosin5a were indeed decorated around intraluminal vesicles and vesicles clustering around the base of retraction fibers, suggesting intraluminal vesicles were transported to the base of retraction fiber and into migrasome by Myosin5a (FIG. 13B) . To visualize the movement of intraluminal vesicles, time lapse imaging was carried out. Time lapse imaging shown GFP-Myosin5a signal on retraction fiber are getting brighter when cell migrating away, eventually become bright puncta, in many case, migrasome grew around GFP-Myosin5a puncta, eventually enclosed GFP-Myosin5a puncta in migrasome, this data indicate that GFP-Myosin5a are transported to the site of migrasome formation, moreover, the increasing GFP-Myosin5a on migrasome formation site suggested GFP-Myosin5a was gradually transported to migrasome formation site (FIG. 13C) . To better characterize the movement of Myosin5a, the ultra-fast super-resolution imaging was carried out using Grazing Incidence Structured Illumination Microscopy (GI-SIM) , which can reach 100nm resolution with a speed of 200 frame/second. GI-SIM imaging showed that small Myosin5a puncta were transported to the edge of cell and accumulated as bright cluster, from these cluster on the base of retraction fiber, a stream of small vesicles was rapidly moved into migrasome, in some case, the cluster of vesicles were left on retraction fiber when cells migrate away (FIG. 13D) . Put together, these data suggested that intraluminal vesicle were transported into migrasomes by Myosin5a.

To directly test this hypothesis, cell lines were established in which Myosin5a was stably overexpressed (Myosin5a OE, Uniprot Accession No. Q99104-1 for mouse) or knocked out (Myosin5a KO) , and the intraluminal vesicle number of TEM was checked. It was found that overexpressing Myosin5a enhanced, while knocking out Myosin5a decreased the number of intraluminal vesicles (FIG. 13E) , this suggest that intraluminal vesicles were indeed transported into migrasome by Myosin5a. Similarly, overexpressing Myosin5a enhanced the number of GFP-Rab8 puncta in migrasome, indicating that Rab8 positive vesicles were transported into migrasome by Myosin5a (FIG. 13F) .

VAMP2, VAMP7 and SNAP23 mediate the fusion of intraluminal vesicles with migrasome membrane

The SNAREs required for the fusion of intraluminal vesicles with migrasome membrane were investigated. It was found that VAMP2, a V-SNARE involved in constitutive exocytosis, was localized inside migrasome as small puncta (FIG. 14A) . APEX2-based TEM revealed that VAMP2 were localized on the membrane of intraluminal vesicles (FIG. 14B) . Overexpressing Myosin5a significantly enhanced the number of VAMP2 puncta, while knocking out myosin5a markedly reduced the number of VAMP2 puncta in migrasome, suggesting that these vesicles were transported by Myosin5a (FIG. 14C) . VAMP2 form SNARE complex with t-SNARE SNAP23, it was found that SNAP23 were highly enriched on migrasome (FIG. 14D and FIG. 16A) , to confirm this observation biochemically, the total membrane proteins was isolated from plasma membrane and from migrasome, it was found that indeed SNAP23 was markedly enriched in migrasome (FIG. 14E) . Finally, knocking down SNAP23 significantly enhanced the number of VAMP2 vesicles in migrasome, suggesting VAMP2 vesicles fused with migrasome membrane in a SNAP23 dependent manner (FIG. 14F) . Put together, these data suggested intraluminal vesicles fused with migrasome through SNARE.

To directly visualize the fusion of VAMP2 vesicles with migrasome membrane, time lapse imaging was carried out, it was found that VAMP2 signal started as a cluster of small puncta, as the migrasome grew, VAMP2 signal gradually moved to migrasome membrane, suggesting the fusion occurred (FIG. 14G) . To directly detect the fusion between VAMP2 vesicle and migrasome membrane, a VAMP2-pHlourin expressing cell line was generated. PHlourin is a pH-sensitive green fluorescent protein which have been widely used to visualized vesicle secretion. In contrast to VAMP2 antibody, VAMP2-pHlourin signal did not have the vesicular pool inside the cell or inside migrasome, as these vesicles were acidic, instead, all signal were on the plasma membrane or in membrane of migrasome, importantly, the VAMP2-pHlourin signal was more intense on migrasome membrane than on plasma membrane of cell body in an observation plate, suggesting in these cells, the migrasome was preferred secretion site comparing to the plasma membrane of cell body (FIG. 14H) . Put together, these data suggested that VAMP2 vesicles did fuse with migrasome, and migrasome appeared to be the preferred sites of fusion for VAMP2 vesicles. Besides VAMP2, it was also found the signal of VAMP7 in migrasome, suggesting that migrasome could release secretive vesicles from different origins (FIG. 16B) .

Taken together, these results demonstrated that the intraluminal vesicles in migrasomes were classical secretory vesicles.

Accordingly, the role of migrasomes in secretion was found. It was found that secretory vesicles were translocated into migrasome by actin based motor protein, once reaching migrasome, secretory vesicles can fuse with migrasome membrane and release its content. Moreover, it was shown that a package of cytokines can be released from immune cells as migrasome bound form, thus, migrasomes were the organelle for localized secretion and packaged release of secretory proteins in migrating cells.

It was found that migrasomes not only mediated localized secretion, but migrasomes served as the main site for secretion, as found both in vivo and in vitro.

The secretion of monocyte was coupled with its ability to generate migrasome.

It was found that after LPS treatment, monocyte released migrasomes enriched with cytokines such as TNF-α, IL-6 and IFN-γ into blood steam. Theoretically, packaged releasing of a set of cytokines by migrasome would have profound difference with releasing cytokines as soluble form. First, as μm scaled vesicles, the dynamic of cytokines enriched migrasome was vastly different from soluble cytokines in blood stream, which would result in different spatiotemporal distribution of cytokines in vivo. Secondly, since cytokines were enriched on migrasome, so on reach the site of action, migrasome bound cytokines could reach a much higher local concentration than soluble cytokine in blood stream. Third, since a set of cytokines could be packed in a single migrasome and released simultaneously, migrasome could deliver a combination of signals which is synergistic in nature, such as TNF-α and IL-6. Finally, release of cytokines from detached migrasome took time, thus, the migrasome could work as a sustained-release capsule to archive the latency of cytokine releasing. Accordingly, migrasome (such as migrasomes derived from or generated by immune cells) mediated packaged release of cytokines play important roles in immune response.

While exemplary embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method for regulating the recruitment of a second immune cell by a first immune cell, comprising regulating the formation and/or function of a migrasome generated by said first immune cell.
The method of claim 1, which increases said recruitment of the second immune cell and comprises promoting the formation and/or function of said migrasome.
The method of claim 2, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said first immune cell and/or in said migrasome.
The method of claim 3, wherein said promoting the formation and/or function of said migrasome comprises overexpressing said tetraspanin protein, the functional fragment thereof, and/or the functional variant thereof in said first immune cell.
The method of any one of claims 3-4, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The method of any one of claims 2-5, wherein said promoting the formation and/or function of said migrasome comprises increasing the number of intraluminal vesicles in said migrasome.
The method of any one of claims 2-6, wherein said promoting the formation and/or function of said migrasome comprises increasing the transportation of intraluminal vesicles into said migrasome.
The method of any one of claims 2-7, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a motor protein in said first immune cell.
The method of claim 8, wherein said motor protein comprises a Myosin.
The method of any one of claims 8-9, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 2-10, wherein said promoting the formation and/or function of said migrasome comprises increasing fusion of the membrane of said migrasome with an intraluminal vesicle therein.
The method of claim 11, wherein increasing said fusion comprises increasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 11-12, wherein increasing said fusion comprises increasing calcium in said migrasome.
The method of any one of claims 2-13, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a chemokine in said first immune cell and/or in said migrasome.
The method of claim 14, wherein said chemokine comprises CXCL12.
The method of claim 1, which decreases said recruitment of the second immune cell and comprises inhibiting the formation and/or function of said migrasome.
The method of claim 16, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting the expression and/or function of a tetraspanin in said first immune cell and/or in said migrasome.
The method of claim 17, wherein said inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said first immune cell.
The method of any one of claims 17-18, wherein said tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.
The method of any one of claims 16-19, wherein said inhibiting the formation and/or function of said migrasome comprises decreasing the number of intraluminal vesicles in said migrasome.
The method of any one of claims 16-20, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting transportation of intraluminal vesicles into the migrasome.
The method of any one of claims 16-21, wherein said inhibiting the formation and/or function of said migrasome comprises decreasing the amount and/or function of a motor protein in said first immune cell.
The method of claim 22, wherein said motor protein comprises a Myosin.
The method of any one of claims 22-23, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 16-24, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting fusion of the membrane of said migrasome with an intraluminal vesicle therein.
The method of claim 25, wherein inhibiting said fusion comprises decreasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 25-26, wherein inhibiting said fusion comprises decreasing calcium in said migrasome.
The method of claim 27, wherein decreasing calcium in said migrasome comprises administering a calcium chelator.
The method of claim 28, wherein said calcium chelator comprises BAPTA-AM.
The method of any one of claims 6-29, wherein said intraluminal vesicle comprises one or more immune signaling molecules.
The method of claim 30, wherein said immune signaling molecule comprises a cytokine and/or a flavonoid.
The method of claim 31, wherein said cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.
The method of claim 32, wherein said interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.
The method of any one of claims 32-33, wherein said interferon comprises IFN-γ.
The method of any one of claims 32-34, wherein said chemokine comprises CCL2 and/or CXCL12.
The method of any one of claims 32-35, wherein said Tumor Necrosis Factor comprises TNF-α.
The method of any one of claims 31-36, wherein said flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.
The method of any one of claims 16-37, wherein said inhibiting the formation and/or function of said migrasome comprises decreasing the amount and/or function of a chemokine in said first immune cell and/or in said migrasome.
The method of claim 38, wherein said decreasing the amount and/or function of the chemokine comprises knocking out or knocking down the expression of a gene encoding for said chemokine in said first immune cell.
The method of any one of claims 38-39, wherein said decreasing the amount and/or function of the chemokine comprises treating said migrasome with an agent capable of inhibiting the function of said chemokine.
The method of claim 40, wherein said agent capable of inhibiting the function of said chemokine comprises a protease, a small molecule, and/or an antibody capable of inhibiting the activity of said chemokine.
The method of any one of claims 38-41, wherein said chemokine comprises CXCL12.
The method of any one of claims 1-42, wherein said first immune cell comprises a monocyte and/or a macrophage.
The method of any one of claims 1-43, wherein said first immune cell consists essentially of a monocyte and/or macrophage.
The method of any one of claims 1-44, wherein said second immune cell comprises a monocyte and/or a macrophage.
The method of any one of claims 1-45, wherein said second immune cell consists essentially of a monocyte and/or a macrophage.
The method of any one of claims 1-46, wherein said first immune cell is of the same type as the second immune cell.
The method of any one of claims 1-47, wherein said first immune cell is a monocyte and/or a macrophage, and said second immune cell is a monocyte and/or a macrophage.
The method of any one of claims 1-48, wherein said migrasome comprises and/or expresses a chemokine.
The method of claim 49, wherein said chemokine comprises CXCL12.
The method of any one of claims 49-50, wherein said second immune cell comprises and/or expresses a molecule capable of specifically recognizing said chemokine comprised and/or expressed by said migrasome.
The method of any one of claims 49-51, wherein said second immune cell comprises and/or expresses CXCR7 and/or CXCR4.
A method for regulating the migration of an immune cell towards a location, comprising regulating the amount and/or function of a migrasome present at or near said location.
The method of claim 53, which increases the migration of said immune cell and comprises increasing the amount and/or function of said migrasome.
The method of claim 54, wherein increasing the amount of said migrasome comprises administering said migrasome to said location.
The method of claim 54, wherein said increasing the amount and/or function of said migrasome comprises promoting the formation and/or function of said migrasome generated by a local immune cell, the local immune cell is an immune cell at or near said location.
The method of claim 56, wherein said promoting the formation and/or function of said migrasome generated by the local immune cell comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said local immune cell.
The method of claim 57, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The method of any one of claims 56-58, wherein said promoting the formation and/or function of said migrasome generated by the local immune cell comprises increasing the number of intraluminal vesicles in said migrasome.
The method of any one of claims 56-59, wherein said promoting the formation and/or function of said migrasome generated by the local immune cell comprises increasing the transportation of intraluminal vesicles into said migrasome.
The method of any one of claims 56-60, wherein said promoting the formation and/or function of said migrasome generated by the local immune cell comprises increasing the amount and/or function of a motor protein in said local immune cell.
The method of claim 61, wherein said motor protein comprises a Myosin.
The method of any one of claims 61-62, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 56-63, wherein said promoting the formation and/or function of said migrasome generated by the local immune cell comprises increasing fusion of the membrane of said migrasome with an intraluminal vesicle therein.
The method of claim 64, wherein increasing said fusion comprises increasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 64-65, wherein increasing said fusion comprises increasing calcium in said migrasome.
The method of any one of claims 56-66, wherein said promoting the formation and/or function of said migrasome generated by the local immune cell comprises increasing the amount and/or function of a chemokine in said local immune cell and/or in said migrasome.
The method of claim 67, wherein said chemokine comprises CXCL12.
The method of claim 53, which decreases the migration of said immune cell and comprises decreasing the amount and/or function of said migrasome.
The method of claim 69, wherein said decreasing the amount and/or function of said migrasome comprises inhibiting the formation and/or function of said migrasome generated by a local immune cell, the local immune cell is an immune cell at or near said location.
The method of claim 70, wherein said inhibiting the formation and/or function of said migrasome generated by the local immune cell comprises inhibiting the expression and/or function of a tetraspanin in said local immune cell and/or in said migrasome.
The method of claim 71, wherein said inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said local immune cell.
The method of any one of claims 71-72, wherein said tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.
The method of any one of claims 70-73, wherein said inhibiting the formation and/or function of said migrasome generated by the local immune cell comprises decreasing the number of intraluminal vesicles in said migrasome.
The method of any one of claims 70-74, wherein said inhibiting the formation and/or function of said migrasome generated by the local immune cell comprises inhibiting transportation of intraluminal vesicles into the migrasome.
The method of any one of claims 70-75, wherein said inhibiting the formation and/or function of said migrasome generated by the local immune cell comprises decreasing the amount and/or function of a motor protein in said local immune cell.
The method of claim 76, wherein said motor protein comprises a Myosin.
The method of any one of claims 76-77, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 70-78, wherein said inhibiting the formation and/or function of said migrasome generated by the local immune cell comprises inhibiting fusion of the membrane of said migrasome with an intraluminal vesicle therein.
The method of claim 79, wherein inhibiting said fusion comprises decreasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 79-80, wherein inhibiting said fusion comprises decreasing calcium in said migrasome.
The method of claim 81, wherein decreasing calcium in said migrasome comprises administering a calcium chelator.
The method of claim 82, wherein said calcium chelator comprises BAPTA-AM.
The method of any one of claims 6-52 and 59-83, wherein said intraluminal vesicle comprises one or more immune signaling molecules.
The method of claim 84, wherein said immune signaling molecule comprises a cytokine and/or a flavonoid.
The method of claim 85, wherein said cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.
The method of claim 86, wherein said interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.
The method of any one of claims 86-87, wherein said interferon comprises IFN-γ.
The method of any one of claims 86-88, wherein said chemokine comprises CCL2 and/or CXCL12.
The method of any one of claims 86-89, wherein said Tumor Necrosis Factor comprises TNF-α.
The method of any one of claims 85-90, wherein said flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.
The method of any one of claims 70-91, wherein said inhibiting the formation and/or function of said migrasome generated by the local immune cell comprises decreasing the amount and/or function of a chemokine in said local immune cell and/or in said migrasome.
The method of claim 92, wherein said decreasing the amount and/or function of the chemokine comprises knocking out or knocking down the expression of a gene encoding for said chemokine in said local immune cell.
The method of any one of claims 92-93, wherein said decreasing the amount and/or function of the chemokine comprises treating said migrasome with an agent capable of inhibiting the function of said chemokine.
The method of claim 94, wherein said agent capable of inhibiting the function of said chemokine comprises a protease, a small molecule, and/or an antibody capable of inhibiting the activity of said chemokine.
The method of any one of claims 92-95, wherein said chemokine comprises CXCL12.
The method of any one of claims 56-96, wherein said local immune cell comprises a monocyte and/or a macrophage.
The method of any one of claims 56-97, wherein said local immune cell consists essentially of a monocyte and/or macrophage.
The method of any one of claims 53-98, wherein said immune cell that migrates towards said location comprises a monocyte and/or a macrophage.
The method of any one of claims 53-99, wherein said immune cell that migrates towards said location consists essentially of a monocyte and/or a macrophage.
The method of any one of claims 56-100, wherein said local immune cell is of the same type as said immune cell that migrates towards said location.
The method of any one of claims 56-101, wherein said local immune cell is a monocyte and/or a macrophage, and said immune cell that migrates towards said location is a monocyte and/or a macrophage.
The method of any one of claims 53-102, wherein said migrasome comprises and/or expresses a chemokine.
The method of claim 103, wherein said chemokine comprises CXCL12.
The method of any one of claims 103-104, wherein said immune cell that migrates towards said location comprises and/or expresses a molecule capable of specifically recognizing said chemokine comprised and/or expressed by said migrasome.
The method of any one of claims 103-105, wherein said immune cell that migrates towards said location comprises and/or expresses CXCR7 and/or CXCR4.
A method for regulating an immune response and/or an immune response mediated biological process, comprising regulating the formation and/or function of a migrasome generated by an immune cell mediating said immune response.
The method of claim 107, wherein said immune response and/or immune response mediated biological process comprises secretion of a substance by said immune cell.
The method of any one of claims 107-108, wherein said immune response and/or immune response mediated biological process is regulated by the secretion of a substance by said immune cell.
The method of any one of claims 107-109, wherein said immune response and/or immune response mediated biological process comprises inflammatory response, and/or other diseases, conditions or disorders affected by the function of an immune cell.
The method of any one of claims 107-110, comprising promoting the formation and/or function of said migrasome.
The method of any one of claims 107-110, comprising inhibiting the formation and/or function of said migrasome.
A method for regulating the secretion of a substance by an immune cell, comprising regulating the formation and/or function of a migrasome generated by said immune cell.
The method of any of claims 107-113, wherein said immune cell is a migrating immune cell.
The method of any of claims 107-114, wherein said immune cell is a migrating immune cell in or from the blood.
The method of any one of claims 107-115, wherein said immune cell comprises a monocyte and/or a macrophage.
The method of any one of claims 108-116, wherein said substance comprises one or more immune signaling molecules.
The method of claim 117, wherein said one or more immune signaling molecules comprise a cytokine and/or a flavonoid.
The method of claim 118, wherein said cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.
The method of claim 119, wherein said interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.
The method of any one of claims 119-120, wherein said interferon comprises IFN-γ.
The method of any one of claims 119-121, wherein said chemokine comprises CCL2 and/or CXCL12.
The method of any one of claims 119-122, wherein said Tumor Necrosis Factor comprises TNF-α.
The method of any one of claims 118-123, wherein said flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.
The method of any one of claims 113-124, which increases the secretion of said substance, and comprises promoting the formation and/or function of said migrasome.
The method of claim 125, wherein said promoting the formation and/or function of said migrasome comprises promoting the generation of said migrasome by said immune cell.
The method of any one of claims 125-126, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said immune cell and/or in said migrasome.
The method of claim 127, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The method of any one of claims 125-128, wherein said promoting the formation and/or function of said migrasome comprises increasing the number of intraluminal vesicles in said migrasome.
The method of any one of claims 125-129, wherein said promoting the formation and/or function of said migrasome comprises increasing the transportation of intraluminal vesicles into said migrasome.
The method of any one of claims 125-130, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a motor protein in said immune cell.
The method of claim 131, wherein said motor protein comprises a Myosin.
The method of any one of claims 131-132, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 125-133, wherein said promoting the formation and/or function of said migrasome comprises increasing fusion of the membrane of said migrasome with an intraluminal vesicle therein.
The method of claim 134, wherein increasing said fusion comprises increasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 134-135, wherein increasing said fusion comprises increasing calcium in said migrasome.
The method of any one of claims 113-124, which decreases the secretion of said substance, and comprises inhibiting the formation and/or function of said migrasome.
The method of claim 137, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting the expression and/or function of a tetraspanin in said immune cell and/or in said migrasome.
The method of claim 138, wherein said inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said immune cell.
The method of any one of claims 138-139, wherein said tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.
The method of any one of claims 137-140, wherein inhibiting the formation and/or function of said migrasome comprises decreasing the number of intraluminal vesicles in said migrasome.
The method of any one of claims 137-141, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting transportation of intraluminal vesicles into the migrasome.
The method of any one of claims 137-142, wherein said inhibiting the formation and/or function of said migrasome comprises decreasing the amount and/or function of a motor protein in said immune cell.
The method of claim 143, wherein said motor protein comprises a Myosin.
The method of any one of claims 143-144, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The method of any one of claims 137-145, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting fusion of the membrane of said migrasome with an intraluminal vesicle therein.
The method of claim 146, wherein inhibiting said fusion comprises decreasing the amount and/or function of a SNARE complex in said migrasome.
The method of any one of claims 146-147, wherein inhibiting said fusion comprises decreasing calcium in said migrasome.
The method of claim 148, wherein decreasing calcium in said migrasome comprises administering a calcium chelator.
The method of claim 149, wherein said calcium chelator comprises BAPTA-AM.
The method of any one of claims 129-150, wherein said intraluminal vesicle comprises said substance to be secreted by said immune cell.
A method for regulating an immune response and/or an immune response mediated biological process in a subject in need thereof, comprising administering to said subject an effective amount of an immune cell derived migrasome.
The method of claim 152, wherein said immune response and/or immune response mediated biological process is regulated by a secreted substance of said immune cell.
The method of any one of claims 152-153, wherein said immune response and/or immune response mediated biological process comprises inflammatory response and/or other diseases, conditions or disorders affected by the function of the immune cell.
The method of any one of claims 152-154, wherein said immune cell is a migrating immune cell.
The method of any of claims 152-155, wherein said immune cell is a migrating immune cell in or from the blood.
The method of any one of claims 152-156, wherein said immune cell comprises a monocyte and/or a macrophage.
The method of any one of claims 153-157, wherein said secreted substance of said immune cell comprises one or more immune signaling molecules.
The method of claim 158, wherein said one or more immune signaling molecules comprise a cytokine and/or a flavonoid.
The method of claim 159, wherein said cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.
The method of claim 160, wherein said interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.
The method of any one of claims 160-161, wherein said interferon comprises IFN-γ.
The method of any one of claims 160-162, wherein said chemokine comprises CCL2 and/or CXCL12.
The method of any one of claims 160-163, wherein said Tumor Necrosis Factor comprises TNF-α.
The method of any one of claims 159-164, wherein said flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.
The method of any one of claims 152-165, further comprising administering to said subject an additional active molecule.
The method of claim 166, wherein said additional active molecule comprises a caspase inhibitor.
The method of claim 167, wherein said caspase inhibitor is a caspase 8 inhibitor.
The method of any one of claims 166-168, wherein said additional active molecule comprises z-VAD-FMK or a functional derivative thereof.
A method for monitoring an immune response and/or an immune response mediated biological process in a subject, comprising analyzing the presence, amount and/or function of a migrasome obtained from a biological sample of said subject.
The method of claim 170, wherein said immune response and/or an immune response mediated biological process comprises inflammatory response and/or other diseases, conditions or disorders affected by the function of an immune cell.
The method of any one of claims 170-171, wherein said biological sample comprises a body fluid sample of said subject.
The method of any one of claims 170-172, wherein said biological sample comprises a blood sample of said subject.
The method of any one of claims 170-173, wherein an increase of the amount of said migrasome indicates an increase of said immune response.
The method of any one of claims 170-174, wherein analyzing the presence, amount and/or function of said migrasome comprises analyzing the presence and/or amount of a marker molecule of said migrasome.
The method of any one of claims 170-175, wherein analyzing the presence, amount and/or function of said migrasome comprises determining the presence and/or amount of Tspan4⁺, Integrin⁺, Pleckstrin Homology (PH) domain⁺, NDST1⁺, PIGK⁺, CPQ⁺, EOGT⁺, KUL01⁺, CD115⁺, and/or CCR2⁺ vesicles in said biological sample.
The method of any one of claims 170-176, wherein analyzing the presence, amount and/or function of said migrasome comprises staining said biological sample with wheatgerm agglutinin (WGA) .
The method of any one of claims 170-177, wherein said migrasome is KUL01⁺, CD115⁺, and/or CCR2⁺.
A method for regulating an immune response and/or an immune response mediated biological process in a subject, comprising:

i) monitoring the immune response and/or the immune response mediated biological process in said subject according to any one of claims 170-178; and

ii) administering a regulating agent according to the result of step i) .
The method of any one of claims 1-179, which is an in vitro or ex vivo method.
The method of any one of claims 1-179, which is an in vivo method.
An agent capable of regulating the formation and/or function of a migrasome generated by a first immune cell, for use in recruiting a second immune cell to said first immune cell.
An agent capable of regulating the formation and/or function of a migrasome present at or near a location, for use in regulating the migration of an immune cell towards said location.
An agent capable of regulating the formation and/or function of a migrasome generated by an immune cell mediating an immune response, for use in regulating the immune response and/or the immune response mediated biological process.
An agent capable of regulating the formation and/or function of a migrasome generated by an immune cell, for use in regulating the secretion of a substance by said immune cell.
An agent capable of detecting the presence, amount and/or function of a migrasome obtained from a biological sample of a subject, for use in monitoring an immune response and/or an immune response mediated biological process in said subject.
An isolated migrasome derived from an immune cell.
The isolated migrasome of claim 187, for use in regulating an immune response and/or an immune response mediated biological process.
An engineered immune cell with altered ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell, said engineered immune cell has been modified to alter its migrasome generation ability.
The engineered immune cell of claim 189, which has increased ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell.
The engineered immune cell of claim 189, which has decreased ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell.
An engineered immune cell with altered ability for regulating an immune response and/or an immune response mediated biological process comparing to a corresponding unmodified immune cell, said engineered immune cell has been modified to alter its migrasome generation ability.
The engineered immune cell of claim 192, which has increased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell.
The engineered immune cell of claim 192, which has decreased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell.
An engineered immune cell with altered secretion ability comparing to a corresponding unmodified immune cell, said engineered immune cell has been modified to alter its migrasome generation ability.
The engineered immune cell of claim 195, which has increased secretion ability comparing to a corresponding unmodified immune cell.
The engineered immune cell of claim 195, which has decreased secretion ability comparing to a corresponding unmodified immune cell.
The engineered immune cell of any one of claims 190, 193 and 196, which has been modified to have increased ability for generating migrasomes.
The engineered immune cell of claim 198, which has been modified to increase the amount and/or function of a tetraspanin therein.
The engineered immune cell of claim 199, which has been modified to overexpress a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
The engineered immune cell of any one of claims 199-200, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The engineered immune cell of any one of claims 191, 194 and 197, which has been modified to have decreased ability for generating migrasomes.
The engineered immune cell of claim 202, which has been modified to decrease the amount and/or function of a tetraspanin therein.
The engineered immune cell of claim 203, wherein the expression of a gene encoding for a tetraspanin has been knocked out or knocked down.
The engineered immune cell of any one of claims 202-203, wherein said tetraspanin comprises tetraspanin 4 and/or tetraspanin 9.
The engineered immune cell of any one of claims 190, 193, 196, and 198-201, which has been modified to generate a migrasome with increased number of intraluminal vesicles comparing to the corresponding unmodified immune cell.
The engineered immune cell of claim 206, which has been modified to have increased ability to transport an intraluminal vesicle into the migrasome comparing to the corresponding unmodified immune cell.
The engineered immune cell of any one of claims 206-207, which has been modified to increase the amount and/or function of a motor protein therein.
The engineered immune cell of any one of claims 191, 194, 197, and 202-205, which has been modified to generate a migrasome with decreased number of intraluminal vesicles comparing to the corresponding unmodified immune cell.
The engineered immune cell of claim 209, which has been modified to have decreased ability to transport an intraluminal vesicle into the migrasome comparing to the corresponding unmodified immune cell.
The engineered immune cell of any one of claims 209-210, which has been modified to decrease the amount and/or function of a motor protein therein.
The engineered immune cell of any one of claims 208 and 211, wherein said motor protein comprises a Myosin.
The engineered immune cell of any one of claims 208 and 211-212, wherein said motor protein is Myosin1c, Myosin5a or comprises a motor domain thereof.
The engineered immune cell of any one of claims 190, 193, 196, 198-201, 206-208 and 212-213, which has been modified to generate a migrasome with increased fusion ability of the migrasome membrane with an intraluminal vesicle therein comparing to the corresponding unmodified immune cell.
The engineered immune cell of claim 214, which has been modified to increase the amount and/or function of a SNARE complex in a migrasome generated by said engineered immune cell.
The engineered immune cell of any one of claims 214-215, which has been modified to increase calcium in a migrasome generated by said engineered immune cell.
The engineered immune cell of any one of claims 191, 194, 197, 202-205 and 209-213, which has been modified to generate a migrasome with decreased fusion ability of the migrasome membrane with an intraluminal vesicle therein comparing to the corresponding unmodified immune cell.
The engineered immune cell of claim 217, which has been modified to decrease the amount and/or function of a SNARE complex in a migrasome generated by said engineered immune cell.
The engineered immune cell of any one of claims 217-218, which has been modified to decrease calcium in a migrasome generated by said engineered immune cell.
The engineered immune cell of any one of claims 206-219, wherein said intraluminal vesicle comprises one or more immune signaling molecules.
The engineered immune cell of claim 220, wherein said immune signaling molecule comprises a cytokine and/or a flavonoid.
The engineered immune cell of claim 221, wherein said cytokine comprises an interleukin, an interferon, a chemokine, and/or a Tumor Necrosis Factor.
The engineered immune cell of claim 222, wherein said interleukin comprises IL-6, IL-10, IL-12, and/or a functional subunit thereof.
The engineered immune cell of any one of claims 222-223, wherein said interferon comprises IFN-γ.
The engineered immune cell of any one of claims 222-224, wherein said chemokine comprises CCL2 and/or CXCL12.
The engineered immune cell of any one of claims 222-225, wherein said Tumor Necrosis Factor comprises TNF-α.
The engineered immune cell of any one of claims 221-226, wherein said flavonoid comprises 3’-O- (3-chloropivaloyl) quercetin.
The engineered immune cell of any one of claims 189-227, wherein said immune cell comprises a monocyte and/or a macrophage.
Use of the agent according to claim 182, the isolated migrasome according to any one of claims 187-188 and/or the engineered immune cell according to any one of claims 189-191 and 198-228 in the preparation of a regulator for the recruitment of a second immune cell to said first immune cell.
Use of the agent according to claim 183 and/or the isolated migrasome according to any one of claims 187-188 in the preparation of a regulator for the migration of an immune cell towards said location.
Use of the agent according to claim 184, the isolated migrasome according to any one of claims 187-188 and/or the engineered immune cell according to any one of claims 192-194 and 198-228 in the preparation of a regulator of an immune response and/or an immune response mediated biological process.
Use of the agent according to claim 185, the isolated migrasome according to any one of claims 187-188 and/or the engineered immune cell according to any one of claims 195-228 in the preparation of a regulator for the secretion of a substance by said immune cell.
Use of the agent according to claim 186 in the preparation of an indicator for an immune response and/or an immune response mediated biological process in said subject.
A composition, comprising the agent according to any one of claims 182-186, the isolated migrasome according to any one of claims 187-188, and/or the engineered cell according to any one of claims 189-228.
The composition of claim 234, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.
A kit, comprising the agent according to any one of claims 182-186, the isolated migrasome according to any one of claims 187-188, the engineered cell according to any one of claims 189-228, and/or the composition according to any one of claims 234-235.