CN117813375A

CN117813375A - Method for selectively tolerating-selectively generating tolerogenic dendritic cells

Info

Publication number: CN117813375A
Application number: CN202280052116.2A
Authority: CN
Inventors: R·埃德尔森; K·亨柯; O·索伯列夫; D·汉隆; A·瓦萨尔; 辰野一树; P·韩
Original assignee: TRANSIMMUNE AG; Yale University
Current assignee: TRANSIMMUNE AG; Yale University
Priority date: 2021-07-29
Filing date: 2022-07-29
Publication date: 2024-04-02

Abstract

The present invention relates to a method for selectively producing tolerogenic dendritic cells. The invention also relates to patient-specific tolerogenic dendritic cells obtained by said method, which method reduces the immunogenicity of the graft when applied prior to transplantation. The invention also relates to patient-specific tolerogenic dendritic cells for reducing or preventing inflammatory conditions such as graft versus host disease. In particular, the method may be used to reduce graft versus host disease. The tolerogenic dendritic cells of the present invention are also useful for the treatment of autoimmune diseases.

Description

Method for selectively tolerating-selectively generating tolerogenic dendritic cells

Technical Field

The present invention relates to a method for selectively producing tolerogenic dendritic cells. The invention also relates to methods of reducing the immunogenicity of a graft prior to implantation by the generation of tolerogenic dendritic cells. The invention also relates to tolerogenic dendritic cells, including tolerogenic dendritic cells obtained by the method. Tolerogenic dendritic cells reduce the immunogenicity of the graft when administered prior to implantation. The invention also relates to tolerogenic dendritic cells for reducing or preventing inflammatory conditions such as graft versus host disease and/or autoimmune disease. In particular, tolerogenic dendritic cells can be used to reduce graft versus host disease.

Background

Transplantation of organs, tissues or cells from one genetically diverse person (donor) to another (recipient) remains the ultimate therapy for a variety of diseases, but is limited by the availability of organs and donors. Suitable donors are individuals having the same or nearly the same profile of cell surface antigens, known as major histocompatibility antigens (MHC) or HLA antigens. However, grafts from the same individual (autograft (autologous transplant) or autograft (autograft)) are not always available, and widespread use of grafts from different individuals (allograft (allogeneic transplant) or allograft (allograft)) is limited by MHC or HLA antigen differences. Each HLA antigen has many alternative forms (alleles) and therefore there is little chance that two unrelated individuals will be HLA matched closely. Adverse reactions that occur after transplanting organs or tissues of one genetically diverse individual into another individual can be very dangerous. The primary adverse effect is immune rejection of the transplanted organ or tissue. This may be due to the immune system of the recipient (organ, skin, etc.) attacking the graft. In addition, the graft may attack the recipient (GvHD). To prevent or limit rejection, patients typically receive a combination of immunosuppressive drugs. These drugs often have global immunosuppressive effects, greatly increasing the susceptibility of the receptor to severe infections. These adverse effects have prompted the search for therapies that more selectively inhibit rejection of transplanted tissue while leaving the remainder of the immune system intact and without damaging other vital organs. One method of reversing rejection of transplanted organs is to apply in vitro photopheresis (ECP), a procedure involving the treatment of blood with a DNA cross-linker (e.g., 8-MOP) and ultraviolet light. One possible mechanism that explains the positive role of ECP in the treatment of graft versus host disease (GvHD) is that monocytes contained in blood samples differentiate into immunosuppressive dendritic cells upon exposure to a combination of 8-MOP and uv light. These immunosuppressive dendritic cells are thought to promote immune tolerance. However, this is of great value for increasing the selective tolerance of allografts to increase the appropriate donor pool, advancing the treatment and/or prevention of autoimmune diseases, in particular graft versus host disease, and further breaking down the possible mechanisms behind the immunosuppressive effects of ECP and ECP-like processes.

Summary of The Invention

It is an object of the present invention to provide a method for selectively producing tolerogenic dendritic cells. It is another object to provide a method for selectively producing antigen-specific tolerogenic dendritic cells. It is another object to provide a method of selectively producing tolerogenic dendritic cells that reduce the immunogenicity of a graft prior to implantation.

It is another object of the invention to provide tolerogenic dendritic cells, including ex vivo tolerogenic dendritic cells.

It is a further object of the present invention to provide tolerogenic dendritic cells obtainable by the method of the present invention.

It is another object of the present invention to provide tolerogenic dendritic cells for preventing or reducing GvHD.

It is another object of the present invention to provide tolerogenic dendritic cells for preventing or reducing rejection of organ transplants (e.g. skin).

It is another object to provide a method of treating GvHD.

It is another object of the present invention to provide tolerogenic dendritic cells for the treatment of autoimmune diseases.

It is another object to provide a method of treating autoimmune diseases.

These and other objects will become apparent from the ensuing description which is addressed by the subject matter of the independent claims. Some preferred embodiments of the invention form the subject matter of the dependent claims. However, other embodiments of the invention may be derived from the following description.

The invention illustratively described below may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The invention is described with respect to the specific embodiments below and with reference to certain drawings but the invention is not limited thereto but only by the claims.

The present invention is based, in part, on the data presented below and clinical trials, which lead to an understanding of: if the apoptotic dendritic cells are taken up by healthy dendritic cells, the apoptotic dendritic cells can tolerate the immune system of future transplant recipients against allografts. Surprisingly, the inventors found that apoptotic dendritic cells can be derived from either the donor or the future recipient. Moreover, if apoptotic dendritic cells are derived from a donor, the donor may be HLA-matched or mismatched compared to future recipients.

Preferably, in the context of the present invention, healthy dendritic cells are produced in vitro during ECP derivatization. The inventors have observed that dendritic cells can be efficiently generated when a leukocyte-isolated blood sample containing monocytes and platelets is passed through the plate. However, the blood sample must contain at least monocytes. Monocytes have been found to mature into healthy dendritic cells upon application of shear stress. If platelets are present, the maturation process may be improved. Importantly, using this approach, monocytes can mature into healthy dendritic cells without the need to add expensive cytokine mixtures. Since the above process mimics some of the aspects that are supposed to occur in vivo (see Han et al 2020, "Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes," Science Advances), the transfer of generated dendritic cells through plates is hereinafter referred to as "physiological dendritic cells (physiological dendritic cells)" (phDC).

Thus, apoptotic dendritic cells derived from either the transplant donor or the future recipient are assumed to provide an antigen source for phDC from the future recipient, thereby initiating an effective tolerogenic immune response. The present invention facilitates and improves this process by directly contacting apoptotic dendritic cells with phDC. Direct incubation may improve future recipient tolerance to allografts after administration of selectively produced tolerogenic dendritic cells, thereby reducing or eliminating inflammatory conditions such as GvHD. phDC of the present invention are more effective than dendritic cells produced by other methods such as exposure to cytokine mixtures. Furthermore, phDC can be produced in a standardized and reproducible manner, thereby better controlling the process of generating well-tolerated dendritic cells. In addition, phDC can be used to treat autoimmune diseases.

The selective generation of tolerogenic dendritic cells described above may be developed in different ways, which are described below as first, second and third aspects.

First aspect: method for selectively producing tolerogenic dendritic cells, wherein donor dendritic cells exhibit apoptosis

In a first aspect, the invention relates to a method comprising the steps of:

a) Providing a dendritic cell from a donor;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic donor dendritic cells of step b) with physiological recipient dendritic cells from step c).

In one embodiment, the method is performed prior to implantation.

In one embodiment, the method is used to selectively reduce the immunogenicity of the graft or a portion thereof prior to implantation.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus phDC from the receptor will not be exposed to the apoptotic agent. In one embodiment, phDC from the recipient is not exposed to the apoptotic agent at any point during the method.

In one embodiment, the dendritic cells of step a) are obtained from a donor.

In one embodiment, the dendritic cells of step c) are obtained from a recipient.

In principle, after obtaining dendritic cells from a donor, the donor dendritic cells may or may not be living. The donor dendritic cells can, for example, be allowed to apoptosis and cryopreserved until they bind to the physiological recipient dendritic cells from step c).

In one embodiment, the donor is allogeneic. In one embodiment, the donor is a haploid donor.

In one embodiment, the present invention relates to a method comprising the steps of:

a) Providing a dendritic cell from a donor;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells of step c), and

d1 Co-incubating the mixture of step d).

In one embodiment, step d 1) of co-culturing the mixture is performed for at least 0.5h, 1h, 2h, 3h, 4h, 5h or 6h.

In one embodiment, step d) of combining apoptotic donor dendritic cells with physiological dendritic cells from a recipient is performed in the recipient.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus phDC from the receptor will not be exposed to the apoptotic agent.

In step b) of the method, the dendritic cells obtained from the donor in method step a) are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA and/or aminolevulinic acid, and light. Particularly preferred psoralens are 8-MOP and amotosalen. The most preferred psoralen is 8-MOP. In a most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA.

Embodiments should be preferentially selected such that substantially all dendritic cells from the donor are contacted with the apoptotic agent. In the case of 8-MOP/UVA, substantially all dendritic cells from the donor are contacted with 8-MOP and exposed to UVA light. Typical dosages of 8-MOP and UVA are 1J/cm ² To 3J/cm ² UVA was combined with 8-MOP at concentrations of 100ng/mL to 300 ng/mL.

In a preferred embodiment, the UVA dose is equal to or lower than 3J/cm ² Equal to or lower than 2J/cm ² Or 1J/cm or less ² . In other preferred embodiments, the dose of 8-MOP is equal to or lower than 300ng/mL, 250ng/mL, 200ng/mL, or 100ng/mL. In a particularly preferred embodiment, the dose of 8-MOP is 200ng/mL and the dose of UVA is 1J/cm ² 。

In the context of the present invention, dendritic cells can be obtained in particular by plate transfer of monocytes using an in vitro photopheresis (ECP) derivatization process.

Methods and devices for in vitro activation of monocytes and dendritic cells generated therefrom are described in WO2014/106629A1, WO2014/106631A1, WO2016/001405A1 and WO2017/005700A1, each of which is incorporated herein by reference in its entirety. ECP describes a process in which monocytes derived from a blood sample or fraction thereof are exposed to mechanical stress (e.g., shear force) and plasma components (e.g., platelets) or derivatives or mimics thereof, thereby activating the differentiation of the monocytes into healthy, physiological dendritic cells, also referred to herein as phdcs. ECP and ECP derivatization processes, including differentiation of monocytes into phdcs, can be performed in large-scale ECP devices (e.g., clinical ECP devices (e.g., Device) or miniaturized ECP devices (e.g. a trans-immune plate as described in WO2017/005700 A1); or a bag such as a plastic bag (e.g., a plastic bag for blood, blood components, cell therapies, etc.).

The inventors found that phDC obtained by the above method is advantageous compared to DCs obtained by other methods such as cytokines or direct isolation from the receptor, because phDC is physiologically generated (without the need for chemicals such as cytokines) with higher reproducibility and controllability under precise in vitro laboratory conditions.

Thus, in a particularly preferred embodiment, phDC of the recipient is obtained by passing the blood sample or a fraction thereof through a flow chamber of the device, subjecting monocytes contained in the blood sample to shear forces. Preferably, platelets are present in the flow chamber, which may be derived from a blood sample of the recipient or a fraction thereof or provided separately. Additionally or alternatively, a plasma component may be present in the flow chamber, which may be derived from a blood sample of the recipient or a fraction thereof or provided separately. However, phDC can also be produced without platelet and/or plasma components.

Monocytes of the recipient may be obtained by any suitable means, for example from a blood sample or fraction thereof. The fraction of the blood sample may be, for example, the buffy coat including white blood cells and platelets. Alternatively, the fraction of the blood sample may be isolated Peripheral Blood Mononuclear Cells (PBMCs). PBMCs may be isolated from blood samples using, for example, ficoll-Hypaque gradient centrifugation (Isolymph, CTL Scientific). In another example, the fraction of the blood sample may be a purified or enriched monocyte preparation. Plastic adhesion, for example, may be used; CD14 magnetic beads positive selection (e.g., from Miltenyi Biotec); and one, two or all three of the monocyte isolation kits II (Miltenyi Biotec) enrich monocytes from PBMCs.

Any suitable volume of blood may be used. The blood sample (e.g., a blood sample from which the fraction is derived) can be between about 1 μl and about 500mL, such as between about 1 μl and about 10mL, between about 1 μl and about 5mL, between about 1 μl and about 1mL, between about 1 μl and about 750 μl, between about 1 μl and about 500 μl, between about 1 μl and about 250 μl, between about 10mL and about 450mL, between about 20mL and about 400mL, between about 30mL and about 350mL, between about 40mL and about 300mL, between about 50mL and about 200mL, or between about 50mL and about 100mL. In some embodiments, the blood sample or fraction thereof, or additional blood sample or fraction thereof, is less than or equal to about 100mL (e.g., about 50mL to about 100 mL).

In some embodiments, the ECP device is a miniaturized ECP device, such as a trans-immune (TI) plate. In some embodiments, the ECP device is a plastic bag. The skilled artisan is familiar with methods of differentiating dendritic cells (including phDC) from monocytes, for example by assessing gene expression.

Without being bound by scientific theory, the inventors presently hypothesize that the significant effect of the invention is due to impaired dying DCs, particularly dying DCs that are impaired by apoptotic agents such as a combination of Psoralen and UVA (PUVA), particularly a combination of 8-MOP and UVA, which provide antigens to the physiological DCs of the recipient and tolerance signals to the immune system of the recipient. If phDC has received such tolerogenic signals from allogeneic PUVA-treated apoptotic DCs, phDC (in addition to the received tolerogenic signals) may display antigens from the allogeneic PUVA-treated apoptotic DCs on its surface, thereby being able to trigger antigen-specific tolerogenic responses. Similarly, the source of antigen may also be derived from immune cells, such as monocytes or lymphocytes. Thus, immune cells such as monocytes or lymphocytes can be apoptotic and bind to phDC to generate antigen-specific tolerance responses. However, damaged DCs or related precursor cells (e.g., monocytes) are preferred.

In one embodiment, the dendritic cells of step a) are derived from an extracorporeal blood sample of a donor. In another embodiment, the dendritic cells of step a) have been obtained by plate transfer of PBMCs from a donor. Thus, the dendritic cells of the donor may also be phDC. All embodiments described above in relation to the recipient relating to the provision of an extracorporeal blood sample and PBMCs are also applicable to the donor.

In one embodiment, the recipient and donor are mammals. Mammals include, for example, but are not limited to, humans, non-human primates, pigs, dogs, cats, horses, and rodents. In a preferred embodiment, the recipient and donor are humans.

In one embodiment, the transplant is a kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, skin transplant, bone marrow transplant, or stem cell transplant.

In one embodiment, the stem cell transplantation is hematopoietic stem cell transplantation.

All of the above embodiments can be performed in vitro.

The methods of the invention may be used in combination with other therapies for treating immunodeficiency associated with hematopoietic stem cell transplantation.

With respect to the following embodiments of the first aspect, all of the above-described embodiments relating to the first aspect apply mutatis mutandis:

a) Exposing dendritic cells obtained from a donor to an apoptotic agent;

b) Combining the apoptotic donor dendritic cells of step a) with physiological dendritic cells obtained from a recipient.

In the above embodiment, co-incubation corresponding to step d 1) as described in the above embodiment may be performed.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus phDC obtained from the receptor will not be exposed to apoptotic agents. In one embodiment, phDC from the recipient is not exposed to the apoptotic agent at any point during the method.

a) Providing immune cells, preferably lymphocytes, from a donor;

b) Exposing the immune cells, preferably lymphocytes, of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic immune cells, preferably apoptotic lymphocytes, of step b) with the physiological receptor dendritic cells of step c).

All embodiments of the first aspect apply mutatis mutandis to the above embodiments (i.e. the dendritic cells obtained from the donor are replaced by immune cells, preferably lymphocytes, obtained from the donor).

The method may also be practiced based on apoptosis of cells associated with dendritic cells, such as monocytes. Accordingly, in another embodiment, the present invention relates to a method comprising the steps of:

a) Providing monocytes from a donor;

b) Exposing the monocytes of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic monocytes of step b) with the physiological receptor dendritic cells of step c).

All embodiments of the first aspect apply mutatis mutandis to the above embodiments (i.e. the dendritic cells obtained from the donor are replaced by monocytes obtained from the donor).

Second aspect: method for selectively producing tolerogenic dendritic cells, wherein complementing haploid donor dendritic filaments Cells exhibit apoptosis

In a second aspect, the invention relates to a method comprising the steps of:

a) Providing dendritic cells from a complementary haploid donor of the recipient;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing physiological dendritic cells from an acceptor haploid donor; and

d) Combining the apoptosis complementing haploid donor dendritic cells of step b) with the physiological haploid donor dendritic cells of step c).

In one embodiment, the method is performed prior to implantation.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus phDC from the recipient haploid donor will not be exposed to the apoptotic agent. In one embodiment, phDC from the recipient haploid donor is not exposed to the apoptotic agent at any point in the method.

In one embodiment, the dendritic cells of step a) are obtained from a complementary haploid donor of the recipient.

In one embodiment, the dendritic cells of step c) are obtained from a haploid donor of the recipient.

In principle, after obtaining dendritic cells from a complementing haploid donor, the dendritic cells may or may not be viable. The complementary haploid donor dendritic cells can for example be made apoptotic and cryopreserved until they bind to the physiological haploid donor dendritic cells from step c).

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing physiological dendritic cells from an acceptor haploid donor; and

d) Combining the apoptosis complementing haploid donor dendritic cells of step b) with the physiological haploid donor dendritic cells of step c); and

d1 Co-incubating the mixture of step d).

In one embodiment, step d) of combining the apoptosis-complementing haploid donor dendritic cells of step b) with physiological dendritic cells from a haploid donor is performed in the haploid donor.

In one embodiment, the above method steps are performed simultaneously.

In step b) of the method, the dendritic cells obtained in method step a) from the complementary haploid donor of the recipient are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA and/or aminolevulinic acid, and light. Particularly preferred psoralens are 8-MOP and amotosalen. The most preferred psoralen is 8-MOP. In a most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA.

The embodiments should preferably be selected such that substantially all dendritic cells from the complementary haploid donor of the recipient are contacted with the apoptotic agent. In the case of 8-MOP/UVA, substantially all dendritic cells from the recipient-complementing haploid donor should be contacted with 8-MOP and exposed to UVA light. Typical dosages of 8-MOP and UVA are 1J/cm ² To 3J/cm ² UVA and UVA8-MOP combinations at concentrations of 100ng/mL to 300 ng/mL.

In a preferred embodiment, the UVA dose is equal to or lower than 3J/cm ² Equal to or lower than 2J/cm ² Or 1J/cm or less ² . In other preferred embodiments, the dose of 8-MOP is equal to or lower than 300ng/mL, 250ng/mL, 200ng/mL, or 100ng/mL. In a preferred embodiment, the dose of 8-MOP is 200ng/mL and the dose of UVA is 1J/cm ² 。

As described in relation to the first aspect, dendritic cells in the context of the present invention may in particular be obtained by plate transfer of monocytes using an in vitro photopheresis (ECP) derivatization process, activating differentiation of monocytes into healthy phdcs. All embodiments as described for the first aspect relating to generation of phDC are also applicable to generation of phDC in the second aspect.

Thus, in a particularly preferred embodiment, the phDC of the haploid donor is obtained by passing the blood sample or a fraction thereof through a flow chamber of the device, subjecting monocytes contained in the blood sample to shear forces. Preferably, platelets are present in the flow chamber, which may be derived from a blood sample of a haploid donor or a fraction thereof or provided separately. Additionally or alternatively, a plasma component may be present in the flow chamber, which may be derived from a blood sample of a haploid donor or a portion thereof or provided separately. However, phDC can also be produced without platelet and/or plasma components.

The monocytes of the haploid donor may be obtained by any suitable means, for example from a blood sample or fraction thereof. The fraction of the blood sample may be, for example, the buffy coat including white blood cells and platelets. Alternatively, the fraction of the blood sample may be isolated Peripheral Blood Mononuclear Cells (PBMCs). PBMCs may be isolated from blood samples using, for example, ficoll-Hypaque gradient centrifugation (Isolymph, CTL Scientific). In another example, the fraction of the blood sample may be a purified or enriched monocyte preparation. Plastic adhesion, for example, may be used; CD14 magnetic beads positive selection (e.g., from Miltenyi Biotec); and one, two or all three of the monocyte isolation kits II (Miltenyi Biotec) enrich monocytes from PBMCs.

Without being bound by scientific theory, the inventors presently hypothesize that the significant effects of the invention are due to impaired dying DCs, in particular by apoptotic agents such as the combination of Psoralen and UVA (PUVA), in particular the combination of 8-MOP and UVA, which provide antigens to the physiological DCs of the haploid donor and tolerance signals to the immune system of the haploid donor. If phDC has received such tolerogenic signals from PUVA-treated apoptotic DCs from complementary haploid donors, phDC (in addition to the received tolerogenic signals) may display antigens from PUVA-treated apoptotic DCs on its surface, triggering antigen-specific tolerogenic responses in the haploid donor. Inflammatory conditions such as GvHD will be reduced or eliminated after transplantation from a haploid donor treated as described in the second aspect. Similarly, the source of antigen may also be derived from immune cells, such as lymphocytes or monocytes. Thus, immune cells, such as lymphocytes or monocytes, can be made apoptotic and combined with haploid donor phdcs to generate antigen specific tolerance responses. However, damaged DCs or related precursor cells, such as monocytes, are preferred.

In one embodiment, the dendritic cells of step a) are derived from an in vitro blood sample of a complementary haploid donor of the recipient. In another embodiment, the dendritic cells of step a) have been obtained by plate transfer of PBMCs from a complementary haploid donor of the recipient. Thus, the dendritic cells of the complementary haploid donor of the recipient may also be phDC.

In one embodiment, the complementary haploid donor and haploid donor are mammals. Mammals include, for example, but are not limited to, humans, non-human primates, pigs, dogs, cats, horses, and rodents. In a preferred embodiment, the complementing haploid donor and haploid donor are human.

In one embodiment, the transplant is a kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, bone marrow transplant, or stem cell transplant.

With respect to the following embodiments of the second aspect, all of the above-described embodiments relating to the first and second aspects apply mutatis mutandis:

a) Exposing dendritic cells obtained from a complementary haploid donor of the recipient to an apoptotic agent; and

b) Combining the complementary haploid donor dendritic cells of the apoptotic recipient of step a) with physiological dendritic cells that have been obtained from the haploid donor of the recipient.

It is to be understood that all method steps may be performed in vitro.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus, phDC obtained from haploid donors of recipients will not be exposed to apoptotic agents. In one embodiment, phDC from the recipient haploid donor is not exposed to the apoptotic agent at any point in the method.

a) Providing immune cells, preferably lymphocytes, from a complementary haploid donor of the recipient;

c) Providing physiological dendritic cells from an acceptor haploid donor; and

d) Combining the apoptosis complementing haploid donor immune cells of step b), preferably the apoptosis complementing haploid donor lymphocytes, with the physiological haploid donor dendritic cells of step c).

All embodiments of the second aspect apply mutatis mutandis to the above embodiments (i.e. wherein dendritic cells obtained from a complementary haploid donor of the recipient are replaced by immune cells, preferably lymphocytes, obtained from a complementary haploid donor of the recipient).

a) Providing monocytes from a complementary haploid donor of the recipient;

b) Exposing the monocytes of step a) to an apoptotic agent;

c) Providing physiological dendritic cells from an acceptor haploid donor; and

d) Combining the apoptotic complementary haploid donor monocytes of step b) with the physiological haploid donor dendritic cells of step c).

All embodiments of the first aspect apply mutatis mutandis to the above embodiments (i.e. wherein dendritic cells obtained from a complementary haploid donor of the recipient are replaced by monocytes obtained from a complementary haploid donor of the recipient).

Third aspect: method for selectively producing tolerogenic dendritic cells, wherein the recipient dendritic cells exhibit apoptosis

In a third aspect, the invention relates to a method comprising the steps of:

a) Providing a dendritic cell from a recipient;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c).

In one embodiment, the method is performed prior to implantation.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus phDC from the recipient (step c) is not exposed to the apoptotic agent. In one embodiment, phDC from the receptor of step c) is not exposed to the apoptotic agent at any point in the method.

In one embodiment, the dendritic cells of step a) are obtained from a recipient.

In principle, after obtaining dendritic cells from the recipient (step a), the dendritic cells may or may not be living. Dendritic cells can for example be apoptotic and cryopreserved until they bind to physiological receptor dendritic cells from step c).

a) Providing a dendritic cell from a recipient;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c); and

d1 Co-incubating the mixture of step d).

In one embodiment, step d) of combining apoptotic recipient dendritic cells with physiological dendritic cells from the recipient is performed within the recipient.

In one embodiment, the above method steps are performed simultaneously.

In step b) of the method, the dendritic cells obtained from the recipient in method step a) are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA and/or aminolevulinic acid, and light. Particularly preferred psoralens are 8-MOP and amotosalen. The most preferred psoralen is 8-MOP. In a most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA.

Embodiments should be preferentially selected such that substantially all dendritic cells from the recipient are contacted with the apoptotic agent. In the case of 8-MOP/UVA, substantially all dendritic cells from the receptor should be contacted with 8-MOP and exposed to UVA light. Typical dosages of 8-MOP and UVA are 1J/cm ² To 3J/cm ² UVA was combined with 8-MOP at concentrations of 100ng/mL to 300 ng/mL.

As described in relation to the first and second aspects, dendritic cells in the context of the present invention may in particular be obtained by monocyte plate delivery using an in vitro photopheresis (ECP) derivatization procedure, activating the differentiation of monocytes into healthy phdcs. All embodiments as described in the first aspect relating to the generation of phDC are also applicable to the generation of phDC of the third aspect.

Thus, in a particularly preferred embodiment, phDC of the recipient is obtained by passing the blood sample or a fraction thereof through a flow chamber of the device, subjecting monocytes contained in the blood sample to shear forces. Preferably, platelets are present in the flow chamber, which may be derived from a blood sample of the recipient or a fraction thereof or provided separately. Additionally or alternatively, a plasma component may be present in the flow chamber, which may be derived from a blood sample of the recipient or a fraction thereof or provided separately. However, phDC can also be generated without platelet and/or plasma components.

Without being bound by scientific theory, the inventors presently hypothesize that the significant effects of the invention are due to impaired dying DCs, particularly those damaged by the combination of Psoralen and UVA (PUVA), which provide antigens to the physiological DCs of the recipient and tolerance signals to the recipient's immune system. If phDC has received such tolerogenic signals from apoptotic autologous DCs from the PUVA treatment, phDC (in addition to the received tolerogenic signals) may exhibit apoptotic DCs from the autologous PUVA treatment on its surface, triggering antigen-specific tolerogenic responses. Similarly, the source of antigen may also be derived from immune cells, such as monocytes or lymphocytes. Thus, immune cells, such as monocytes or lymphocytes, can be made to apoptosis and combined with phDC to generate antigen specific tolerance responses. However, damaged DCs or related precursor cells, such as monocytes, are preferred.

In one embodiment, the dendritic cells of step a) are derived from an extracorporeal blood sample of the recipient. In another embodiment, the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the recipient. In one embodiment, the recipient is a mammal. Mammals include, for example, but are not limited to, humans, non-human primates, pigs, dogs, cats, horses, and rodents. In a preferred embodiment, the recipient is a human.

In one embodiment, the transplant is a kidney transplant, pancreas transplant, liver transplant, heart transplant, lung transplant, intestine transplant, bone marrow transplant, or stem cell transplant. In one embodiment, the stem cell transplantation is hematopoietic stem cell transplantation.

All of the above embodiments can be performed in vitro.

For the following embodiments of the third aspect, all of the above embodiments relating to the third aspect apply mutatis mutandis:

a) Exposing dendritic cells obtained from a recipient to an apoptotic agent;

b) Combining the apoptotic receptor dendritic cells of step a) with physiological dendritic cells that have been obtained from the receptor.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus phDC from the recipient (step b) is not exposed to the apoptotic agent. In one embodiment, phDC obtained from the receptor of step b) is not exposed to an apoptotic agent at any point in the method.

a) Providing immune cells, preferably lymphocytes, from the recipient;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic immune cells, preferably apoptotic lymphocytes, of step b) with the physiological dendritic cells of step c).

All embodiments of the third aspect apply mutatis mutandis to the above embodiments (i.e. the dendritic cells obtained from the recipient are replaced by immune cells, preferably lymphocytes, obtained from the recipient).

a) Providing monocytes from a recipient;

b) Exposing the monocytes of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

d) Combining the apoptotic monocytes of step b) with the physiological dendritic cells of step c).

All embodiments of the first aspect apply mutatis mutandis to the above embodiments (i.e. wherein the dendritic cells obtained from the recipient are replaced by monocytes obtained from the recipient).

Fourth aspect: tolerogenic dendritic cells obtained by the method according to the first aspect

In a fourth aspect, the present invention relates to a tolerogenic receptor dendritic cell obtained by a method according to the first aspect (including all embodiments described above).

In one embodiment, the tolerogenic recipient dendritic cells obtained by the method according to the first aspect reduce the immunogenicity of future grafts from the donor.

Fifth aspect: tolerogenic dendritic cells obtained by the method according to the second aspect

In a fifth aspect, the invention relates to a tolerogenic haploid donor dendritic cell obtained by a method according to the second aspect (including all embodiments described above).

In one embodiment, the tolerogenic haploid donor dendritic cells obtained by the method according to the second aspect reduce the immunogenicity of future grafts from the haploid donor.

Sixth aspect: tolerogenic dendritic cells obtained by the method according to the third aspect

In a sixth aspect, the invention relates to a tolerogenic receptor dendritic cell obtained by a method according to the third aspect (including all embodiments as described above).

In one embodiment, the tolerogenic recipient dendritic cells obtained by the method according to the third aspect reduce the immunogenicity of future grafts.

Seventh aspect, the tolerogenic dendritic cells according to the fourth aspect are used for preventing or reducing graft versus host disease Method

In a seventh aspect, the present invention relates to a tolerogenic dendritic cell according to the fourth aspect (including all embodiments of the first and fourth aspects as described above), for use in a method of preventing or reducing graft versus host disease.

In one embodiment, the tolerogenic dendritic cells obtained by the method of the first aspect are used for the treatment of an allogeneic or haploid mating recipient in need of transplantation. The risk of developing GvHD and, if developed, the severity of GvHD is significantly reduced after such treatment prior to transplantation, as compared to when tolerogenic dendritic cells were not administered prior to transplantation.

Eighth aspect: the tolerogenic dendritic cells according to the fifth aspect for preventing or reducing graft versus host disease In the method

In an eighth aspect, the present invention relates to a tolerogenic dendritic cell according to the fifth aspect (including all embodiments of the second and fifth aspects as described above), for use in a method of preventing or reducing graft versus host disease.

In one embodiment, the tolerogenic dendritic cells obtained by the method of the second aspect are used for treating haploid mating receptors in need of transplantation. The risk of developing GvHD and, if developed, the severity of GvHD is significantly reduced after such treatment prior to transplantation, as compared to when tolerogenic dendritic cells were not administered prior to transplantation.

Ninth aspect: the tolerogenic dendritic cells according to the sixth aspect for preventing or reducing graft versus host disease Method

In a ninth aspect, the present invention relates to a tolerogenic dendritic cell according to the sixth aspect (including all embodiments of the third and sixth aspects as described above), for use in a method of preventing or reducing graft versus host disease.

In one embodiment, the tolerogenic dendritic cells obtained by the method of the third aspect are used for the treatment of an allogeneic or haploid mating recipient in need of transplantation. The risk of developing GvHD and, if developed, the severity of GvHD is significantly reduced after such treatment prior to transplantation, as compared to when tolerogenic recipient dendritic cells were not administered prior to transplantation.

Tenth aspect: method for selectively producing tolerogenic dendritic cells

In a tenth aspect, the invention relates to a method comprising the steps of:

a) Providing a first sample of dendritic cells obtained from a subject;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a second sample of dendritic cells obtained from the subject; and

d) Combining the apoptotic dendritic cells of step b) with the dendritic cells of step c).

In one embodiment, the method involves selectively producing tolerogenic dendritic cells.

In one embodiment, step d) of combining apoptotic dendritic cells with dendritic cells is performed in the subject.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus, the DCs of step c) are not exposed to the apoptotic agent. In one embodiment, the DC of step c) is not exposed to an apoptotic agent at any point during the method.

a) Providing a first sample of dendritic cells obtained from a subject;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a second sample of dendritic cells obtained from the subject; and

d) Combining the apoptotic dendritic cells of step b) with the dendritic cells of step c); and

d1 Co-incubating the mixture of step d).

In one embodiment, both samples of dendritic cells (steps a) and c)) are obtained from the same subject. In one embodiment, step d 1) of co-culturing the mixture is performed for at least 0.5h, 1h, 2h, 3h, 4h, 5h or 6h.

In one embodiment, the above method steps are performed simultaneously.

In one embodiment, the above method steps are performed sequentially. Thus, the DCs of step c) are not exposed to the apoptotic agent.

In step b) of the method, the dendritic cells obtained from the subject in method step a) are exposed to an apoptotic agent. In one embodiment, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA and/or aminolevulinic acid, and light. Particularly preferred psoralens are 8-MOP and amotosalen. The most preferred psoralen is 8-MOP. In a most preferred embodiment, the apoptotic agent is a combination of 8-MOP and UVA.

Embodiments should be preferentially selected such that substantially all dendritic cells from a subject are contacted with an apoptotic agent. In the case of 8-MOP/UVA, substantially all dendritic cells from the subject should be contacted with 8-MOP and exposed to UVA light. Typical dosages of 8-MOP and UVA are 1J/cm ² To 3J/cm ² UVA was combined with 8-MOP at concentrations of 100ng/mL to 300 ng/mL.

As described in relation to the first and second aspects, in the context of the present invention, i.e. also in relation to the tenth aspect, dendritic cells may in particular be obtained by plate transfer of monocytes using an in vitro photopheresis (ECP) derivatization process, activating differentiation of monocytes into healthy phDC.

In one embodiment, the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the subject. In one embodiment, the dendritic cells of step c) have been obtained by plate transfer of PBMCs from the subject. Thus, the dendritic cells of step a) and/or step c) may be referred to as physiological DCs. All embodiments as described in the first aspect relating to the generation of phDC are also applicable to the generation of phDC of the tenth aspect. In one embodiment, the dendritic cells of step a) are obtained from an extracorporeal blood sample obtained from a subject. In one embodiment, the dendritic cells of step a) are obtained from an extracorporeal blood sample obtained from the subject, and the dendritic cells of step c) are obtained by plate transfer of PBMCs from the subject.

Thus, in a particularly preferred embodiment, phDC of the subject is obtained by passing a blood sample or fraction thereof through a flow chamber of the device, subjecting monocytes contained in the blood sample to shear forces. Preferably, platelets are present in the flow chamber, which may be derived from a blood sample of the subject or a fraction thereof or provided separately. Additionally or alternatively, a plasma component may be present in the flow chamber, which may be derived from a blood sample of the subject or a fraction thereof or provided separately. However, phDC can also be produced without platelet and/or plasma components.

Monocytes of the subject may be obtained by any suitable means, for example from a blood sample or fraction thereof. The fraction of the blood sample may be, for example, the buffy coat including white blood cells and platelets. Alternatively, the fraction of the blood sample may be isolated Peripheral Blood Mononuclear Cells (PBMCs). PBMCs may be isolated from blood samples using, for example, ficoll-Hypaque gradient centrifugation (Isolymph, CTL Scientific). In another example, the fraction of the blood sample may be a purified or enriched monocyte preparation. Plastic adhesion, for example, may be used; CD14 magnetic beads positive selection (e.g., from Miltenyi Biotec); and one, two or all three of the monocyte isolation kits II (Miltenyi Biotec) enrich monocytes from PBMCs.

Without being limited by scientific theory, the inventors presently hypothesize that the significant effects of the invention are due to impaired dying DCs from the subject, particularly those impaired by the combination of Psoralen and UVA (PUVA), which provide antigens to the physiological DCs of the subject and tolerance signals to the subject's immune system. If phDC has received such tolerogenic signals from apoptotic autologous PUVA-treated DC, phDC (in addition to the received tolerogenic signals) can display antigens, in particular autoantigens, from apoptotic DC of autologous PUVA-treated on its surface, triggering antigen-specific tolerogenic reactions. Similarly, the source of antigen may also be derived from immune cells, such as lymphocytes. Thus, immune cells, such as lymphocytes, can be made to apoptosis and bind to phdcs of a subject to generate antigen-specific tolerance responses.

In one embodiment, the dendritic cells of step a) are derived from an extracorporeal blood sample of the subject. In another embodiment, the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the subject.

In one embodiment, the invention relates to a process comprising the following steps (all of the embodiments described above apply mutatis mutandis to the following embodiments):

a) Providing a first sample of dendritic cells obtained from a subject;

a1 Incubating the dendritic cells with an antigen molecule;

b) Exposing the incubated dendritic cells of step a 1) to an apoptotic agent;

c) Providing a second sample of dendritic cells obtained from the subject; and

In one embodiment, the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the subject. In one embodiment, the dendritic cells of step c) have been obtained by plate transfer of PBMCs from the subject. In one embodiment, the dendritic cells of step a) are obtained from an extracorporeal blood sample of the subject. In one embodiment, the dendritic cells of step a) have been obtained from an extracorporeal blood sample of the subject, and the dendritic cells of step c) have been obtained by plate transfer of PBMCs from the subject.

In one embodiment, the antigenic molecule is a self-antigen. In one embodiment, the autoantigen is associated with one or more autoimmune disorders. In one embodiment, the antigenic molecule is derived from a natural source, chemically synthesized, or recombinantly produced. In one embodiment, the antigenic molecule is derived from a cell.

Table a below shows a list of exemplary autoantigens associated with autoimmune diseases, and an exemplary animal model system that can be used to evaluate improvement of autoimmune diseases using tolerizing phdcs (see also experiments 7 and 8).

TABLE A list of exemplary autoantigens, related autoimmune diseases and animal models

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In one embodiment, the autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70). In one embodiment, the autoantigen is selected from myelin basic protein and collagen.

In one embodiment, the subject is a mammal. Mammals include, for example, but are not limited to, humans, non-human primates, pigs, dogs, cats, horses, and rodents. In a preferred embodiment, the subject is a human.

All of the above embodiments can be performed in vitro.

The methods of the invention may be used in combination with other therapies for the treatment of autoimmune diseases. For example, the autoimmune disease may be any autoimmune disease described in table a above. Other autoimmune diseases are known in the art.

Eleventh aspect, a tolerogenic dendritic cell obtained by a method according to the tenth aspect

In an eleventh aspect, the invention relates to a tolerogenic dendritic cell obtained by a method according to the tenth aspect (including all embodiments described above).

Twelfth aspect, the tolerogenic dendritic cells according to the eleventh aspect are used for the treatment of autoimmune diseases

In a twelfth aspect, the present invention relates to a tolerogenic dendritic cell according to the eleventh aspect (including all embodiments of the tenth and eleventh aspects as described above), for use in the treatment of an autoimmune disease.

For the treatment of autoimmune diseases, it is expected that administration of tolerogenic dendritic cells will lead to some improved effects, such as an improvement in quality of life; reduction in severity of disease symptoms; a reduction in the number of autoimmune cells; prolonged survival, and the like.

The indicators of beneficial effects are well known in the art and the appropriate indicators for a particular application can be determined by the skilled artisan.

After such treatment, the severity of the autoimmune disease or symptoms associated therewith is significantly reduced as compared to the absence of administration of the tolerogenic dendritic cells.

In one embodiment, the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin Dependent Diabetes Mellitus (IDDM), addison's disease, diarrhea celiac disease, chronic fatigue syndrome, colitis, crohn's disease, fibromyalgia, hyperthyroidism, graves' disease, hypothyroidism, hashimoto disease, endometriosis, pernicious anemia, goodpasture's syndrome, wegener's disease and wind-damp-heat.

In another embodiment, provided herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the tolerogenic dendritic cells described herein.

In another embodiment, provided herein is a method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a tolerogenic dendritic cell, wherein the tolerogenic dendritic cell comprises a physiological dendritic cell comprising a material of apoptotic dendritic cells obtained from the subject, an autoantigen, a fragment thereof, or a combination thereof.

In any of the foregoing methods, the autoimmune disease may be selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin Dependent Diabetes Mellitus (IDDM), addison's disease, diarrhea celiac disease, chronic fatigue syndrome, colitis, crohn's disease, fibromyalgia, hyperthyroidism, graves' disease, hypothyroidism, hashimoto disease, endometriosis, pernicious anemia, goodpasture's syndrome, wegener's disease and wind-damp-heat.

In any of the foregoing methods, the autoantigen may be selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

Thirteenth aspect: ex vivo tolerogenic dendritic cells

In a thirteenth aspect, the invention relates to ex vivo tolerogenic dendritic cells comprising material of apoptotic dendritic cells obtained from a subject.

In some embodiments, the material of apoptotic dendritic cells obtained from a subject includes a polypeptide, a nucleic acid, an organelle or portion thereof, or any other cellular content.

In some embodiments, the ex vivo tolerogenic dendritic cells comprise autoantigens or fragments thereof. Any suitable autoantigen may be included, including any autoantigen described herein (e.g., in table a). For polypeptide antigens, the fragment can have any suitable size, such as, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 amino acids.

In some embodiments, the ex vivo tolerogenic dendritic cells comprise a type of autoantigen or fragment thereof. In other embodiments, the ex vivo tolerogenic dendritic cells can include two, three, four, five, ten or more different autoantigens or fragments thereof.

In some embodiments, the autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

The subject may have an autoimmune disease, including any of the autoimmune diseases disclosed herein (e.g., in table a).

Fourteenth aspect of: compositions comprising dendritic cell samples obtained from a subject

In a fourteenth aspect, the present invention relates to a composition comprising (a) a dendritic cell sample obtained from a subject; (b) an apoptotic agent; (c) an autoantigen or fragment thereof.

The composition may include any suitable apoptotic agent, including any apoptotic agent disclosed herein. In some embodiments, the apoptotic agent comprises psoralen, riboflavin phosphate, 5-aminolevulinic acid, or a combination thereof. In some embodiments, the apoptotic agent is a psoralen. In some embodiments, the psoralen is selected from the group consisting of 8-MOP and amotosalen. In some embodiments, the psoralen is 8-MOP.

In some embodiments, the composition comprises an autoantigen or fragment thereof. Any suitable autoantigen may be included, including any autoantigen described herein (e.g., in table a). For polypeptide antigens, the fragment may be of any suitable size, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 amino acids.

In some embodiments, the composition comprises one type of autoantigen or fragment thereof. In other embodiments, the composition may include two, three, four, five, ten or more different autoantigens or fragments thereof.

Dendritic cells can be obtained from a subject having an autoimmune disease, including any of the autoimmune diseases disclosed herein (e.g., in table a).

Drawings

FIG. 1 is a schematic representation of an ECP leukemia haploid phase assay.

FIG. 2 is a schematic representation of an ECP leukemia haploid phase assay.

FIG. 3 shows a schematic diagram of a Balb/C.fwdarw.B6 all-mismatching GVHD system; results are also described.

The ex vivo psoralen UVA treatment (PUVA) of the graft of fig. 4 improves GVHD in the full MHC mismatch model. With 2X10 before the-4 day or on the day of the 0 day transplantation ⁵ MC38 tumor cells were subcutaneously injected into mice. Mice were subjected to 950cGy lethal irradiation on day-1. On day 0, mice underwent Balb/c→B6 transplantation. It received intravenous injection 5x 10 ⁶ Bone Marrow (BM) cells depleted of individual allogeneic T cells and 10x10 ⁶ Individual non-manipulated spleen cells, or injected after ex vivo PUVA treatment of the allograft. As a control, one group of mice received syngeneic BM and spleen cells after tumor inoculation. (a, figures 1-3) summary data of average body weight, gvHD score, and survival for all groups. (B) average tumor volume.

FIG. 5 survival curve of cardiac transplant Kaplan-Meier.

FIG. 6 subject characteristics

FIG. 7GVHD level and stage

Figure 8 cumulative incidence of acute GVHD. The unrelated donor assay included a patient with a 5/6HLA matched related donor.

Figure 9 is a graph of the cumulative incidence of chronic GVHD.

Figure 10 overall survival. The unrelated donor assay included a patient with a 5/6HLA matched related donor.

Figure 11 cumulative incidence of graft-related mortality. The unrelated donor assay included a patient with a 5/6HLA matched related donor.

Selection criteria for the history control of fig. 12.

Figure 13 features of study and historical control subjects.

The relative risk and 95% confidence interval for the graft results in the multivariate analysis of fig. 14 (control group as reference, relative risk of 1.0 was assigned).

Figure 15 cumulative incidence of grade II-IV acute GVHD.

Fig. 16 adjusted probability of disease-free survival.

Fig. 17 shows the survival probability after adjustment.

FIG. 18 PD-L1 expression from fresh PBMC alone compared to phDC incubated with 8-MOP/UVA-damaged homologs PBMC (PUVAsyn PBMC) or 8-MOP/UVA-damaged isoforms PBMC (PUVA allo PBMC).

Figure 19 CFSE labeled responder T cells (T cells) from one donor were co-incubated with gamma irradiated stimulator PBMCs from the same donor (syn. Culture) or from unrelated donors (MLR). To inhibit the MLR response, some cultures were additionally supplemented with syngeneic 8-MOP/UVA treated PBMC and syngeneic TI plate passing phDC (MLR+PUVA syn. PBMC+phDC). CFSE dilutions were measured by flow cytometry (FACS) to determine proliferation of responder CD8 and CD 4T cells (a, B). Activation status of responder CD 8T cells and CD 4T cells was additionally assessed by FACS, using CD44 and PD1 expression to detect activated T cells (C, D). N = number of blood donors analyzed; p-value = unpaired t-test corrected with Welch.

Detailed Description

The following general definitions are provided.

When the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of. If in the following a group is defined to contain at least a certain number of embodiments, this should also be understood as disclosing groups preferably consisting of only these embodiments.

For the purposes of the present invention, the term "obtained" is considered to be the preferred embodiment of the term "obtainable". If, hereinafter, for example, an antibody is defined as being obtainable from a particular source, this is also to be understood as disclosing antibodies obtained from that source.

Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated. In the context of the present invention, the term "about" or "about" means the interval of accuracy that will be understood by those skilled in the art, in order to still ensure the technical effect of the feature in question. The term generally denotes a deviation from the indicated values of + -20%, preferably + -15%, more preferably + -10%, even more preferably + -5%.

Furthermore, the terms "first," "second," "third," or "(a)", "(b)", "(c)", "(d)", or "(i)", "(ii)", "(iii)", "(iv)", and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

If the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", or "(i)", "(ii)", "(iii)", "(iv)", etc. refer to steps of a method or use or assay, unless otherwise indicated there is no time or interval of consistency between the steps, i.e., the steps may be conducted simultaneously or may be an interval of seconds, minutes, hours, days, weeks, months or even years between the steps, unless otherwise indicated in the application set forth above or below.

Technical terms are used in accordance with their common sense. If certain terms express a specific meaning, definitions of terms will be given below in the context of using these terms.

As used herein, "graft" refers to any sample of cells taken from a mammalian subject ("donor") and suitable for total or partial reintroduction into the same ("autologous") or a different ("allogeneic") mammalian subject ("recipient"). The grafts may be freshly obtained, cultured or frozen, but have been preserved under conditions suitable for maintaining sterility and promoting viability. The term graft (transplant) may be used interchangeably with the term graft (graft).

The methods of the invention can be practiced on individuals who are HLA closely matched, share all or nearly all of the class I and class II HLA antigens; haploid concordance, e.g., siblings share half of the HLA antigen; or irrelevant, and therefore HLA-mismatched. In the context of the present invention, a "haploid donor" refers to one genetic parent of a future recipient, while the term "complementary haploid donor" refers to another genetic parent. Thus, the future recipient is the offspring. In other words, if the female parent is a haploid donor, the male parent is a complementary haploid donor (future recipient) of the offspring, and vice versa. In a preferred embodiment of the first aspect, the acceptor and the donor are unrelated. In a preferred embodiment of the second aspect, the haploid donor and the complementary haploid donor each have half of the HLA antigen of the future recipient.

The extent of HLA identity between individuals can be readily demonstrated by methods known in the art, including polymerase chain reaction, mixed Lymphocyte Reaction (MLR), and serological measurements.

As used herein, the term "antigen" refers to a compound, composition or substance that can stimulate antibody production or a T cell response in an animal, including compositions that are injected or absorbed into the animal. The antigen reacts with the products of a particular humoral or cellular immunity, including products induced by heterologous immunogens. The term is used interchangeably with the terms "immunogen" or "antigenic molecule". The term "antigen" includes all relevant epitopes. The term "antigen", "antigenic molecule" or "immunogen" includes fragments thereof which are still capable of functioning as antigens. An "epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. In one example, the receptor antigen includes an antigen from a dendritic cell, such as a dendritic cell obtained from plate transfer of peripheral blood leukocytes (including monocytes or monocyte-derived cells).

As used herein, the term "immunogenicity" refers to the ability of a substance, cell or portion thereof, e.g., an antigen, to elicit an immune response in a human or animal body.

As used herein, the term "autoantigen" refers to a host antigen (or microbial superantigen) that is believed by those of skill in the art to be associated with an autoimmune disease, such that the presence of autoantigen-specific activated T cells is associated with the occurrence or progression of the disease.

The autoantigen may be a defined autoimmune target antigen, e.g., in multiple sclerosis, identified as Myelin Basic Protein (MBP) MBP 84-102 or MBP 143-168; islet cell antigen; in uveitis, it is the S antigen; or in rheumatoid arthritis, type II or other types of collagen; in SLE, cytoplasmic linker protein 170 (CLIP-170); sjogren's syndrome antigen A (SS-A/Ro), sjogren's syndrome antigen B (SS-B/se:Sup>A), sjogren's lupus antigen (S); scleroderma antigen 70 (Scl-70); in Grave's disease, thyroid receptors; among myasthenia gravis, acetylcholine receptors are identified. The autoantigens of the present invention also comprise peptide mixtures eluted from MHC molecules known to be associated with autoimmunity, such as HLA-DQ and-DR molecules, which confer susceptibility to several common autoimmune diseases (e.g. type 1 diabetes, rheumatoid arthritis and multiple sclerosis), or HLA-B27 molecules known to confer susceptibility to reactive arthritis and ankylosing spondylitis. The autoantigens of the present invention may also be synthetic peptides predicted to bind to WIC molecules associated with autoimmune diseases. For other autoimmune diseases in which the individual's autoantigen has not been characterized, an autoantigen suitable for carrying out the method of the invention may be a cell or cell extract from the affected tissue (e.g. synovial cells of rheumatoid arthritis, skin lesions of psoriasis, etc.). The term autoantigen also includes fragments thereof which act as autoantigens.

As used herein, "immune cells" broadly refer to cells that are of hematopoietic origin and play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; white blood cells; natural killer cells; and bone marrow cells such as monocytes, dendritic cells, macrophages, eosinophils, mast cells, basophils and granulocytes.

"dendritic cells", also referred to herein as "DCs", are antigen presenting immune cells that process and present antigenic material to other cells of the immune system, most notably T cells. The function of DCs is to capture and process antigens. When DCs endocytose antigens, they process the antigens into smaller fragments (typically peptides) that are displayed on the surface of the DCs and presented to, for example, antigen-specific T cells via MHC molecules. After antigen absorption, DCs migrate to the lymph nodes. During maturation, DCs can be promoted by a variety of signals, including signals sent through Toll-like receptors (TLRs), to express induced cognate effector T cells (T _eff ) A co-stimulatory signal that is activated and proliferated, thereby initiating a T cell mediated immune response against the antigen. Alternatively, the DC may present antigen to antigen-specific T cells without providing a costimulatory signal (or simultaneously providing a costimulatory signal), resulting in T _eff And cannot be activated properly. Such presentation may cause death or anergy of, for example, antigen-recognizing T cells, or may induce regulatory T cells (T _reg ) And/or amplification of the same. The term "dendritic cells" includes differentiated dendritic cells, immature and mature dendritic cells. These cells are characterized by the expression of certain cell surface markers (e.g., CD11c, MHC class II, and at least low levels of CD80 and CD 86), CD11b, CD304 (BDCA 4)). In some embodiments, the DCs express CD8, CD103, CD1d, etc. Other DCs can be identified by the absence of lineage markers such as CD3, CD14, CD19, CD56, and the like. In addition, dendritic cells can be usedFunctional characterization was performed by its ability to stimulate allogeneic and Mixed Lymphocyte Responses (MLR).

"tolerogenic DC" refers to dendritic cells capable of suppressing an immune response or generating a tolerogenic immune response, such as an antigen-specific T-cell mediated immune response, e.g., by reducing the response of effector T-cells to a particular antigen, by affecting an increase in the number of antigen-specific regulatory T-cells, etc. Tolerogenic DCs are characterized by induction of antigen-specific tolerogenic immune responses ex vivo and/or in vivo. Such induction refers to the induction of a tolerogenic immune response to one or more antigens of interest presented by the induced tolerogenic dendritic cells. A tolerogenic dendritic cell having a tolerogenic phenotype characterized by at least one of the following properties i) being capable of transforming a naive T cell into a foxp3+ T regulatory cell ex vivo and/or in vivo (e.g. inducing expression of Foxp3 in a naive T cell); ii) capable of deleting effector T cells ex vivo and/or in vivo; iii) Retaining its tolerogenic phenotype after ex vivo stimulation with at least one TLR agonist (and, in some embodiments, increasing expression of the co-stimulatory molecule as a response to such stimulation); iv) after ex vivo stimulation with at least one TLR agonist, does not temporarily increase its oxygen consumption rate; v) increased expression of the expression marker PDL1 and/or vi) increased expression of the expression marker GILZ. Clauses v) and vi) can be assessed by comparison with monocytes or PBMCs.

By "tolerogenic immune response" is meant any immune response that can result in immunosuppression specific to an antigen or cells, tissues, organs, etc. expressing such an antigen. Such immune responses include any reduction, delay or inhibition in an undesired immune response specific to an antigen or to a cell, tissue, organ, etc. expressing such an antigen. Such immune responses also include any stimulation, generation, induction, promotion, or recruitment of an antigen or a desired immune response specific to a cell, tissue, organ, etc. expressing such an antigen. Thus, a tolerogenic immune response includes the absence or reduction of an undesired immune response to an antigen that may be mediated by an antigen-reactive cell, as well as the presence or promotion of an inhibitory cell. The tolerogenic immune responses provided herein include immunological tolerance. "generating a tolerogenic immune response" refers to generating any of the aforementioned immune responses specific for an antigen or cells, tissues, organs, etc. expressing such an antigen.

A tolerogenic immune response includes any reduction, delay or inhibition of cd4+ T cell, cd8+ T cell or B cell proliferation and/or activity. The tolerogenic immune response also includes a reduction in antigen-specific antibody production. Tolerizing an immune response may also include causing regulatory cells (e.g., CD4+T _reg Cells, CD8+T _reg Cell, B _reg Cells, etc.), stimulation, induction, production, or recruitment. In some embodiments, a tolerogenic immune response is a response that results in a transformation to a regulatory phenotype characterized by the production, induction, stimulation or recruitment of regulatory cells.

Tolerogenic immune responses also include the induction of CD4+T _reg Cells and/or cd8+ T _reg Any response to stimulation, production or recruitment of cells. CD4+T _reg The cell can express the transcription factor FoxP3 and inhibit inflammatory response and autoimmune inflammatory disease (Human regulatory T cells in autoimmune diseases. Cvetanovich G L, hafler D A. Curr Opin immunol.2010December;22 (6): 753-60.Regulatory T cells and autoimmunity.Vila J,Isaacs J D,Anderson AE.Curr Opin Hematol.2009July;16 (4): 274-9). These cells also inhibit T cell helper to B cells and induce tolerance to self and foreign antigens (Therapeutic approaches to Allergy and autoimmunity based on FoxP3+regulatory T-cell activation and expansion. Miyara M, wing K, sakaguchi S.J Allergy Clin immunol.2009april;123 (4): 749-55). CD4+T _reg Cells recognize antigens when presented by class II proteins on APCs. CD8+T _reg The cells recognize class I presented antigens and can also inhibit T cell helper to B cells and result in activation of antigen specific inhibition, thereby inducing tolerance to self and foreign antigens. In some embodiments, the provided tolerogenic dendritic cells can effectively result in two types of responses (cd4+t _reg And CD8+T _reg ). In other embodiments, foxP3 can be induced in other immune cells such as macrophages, iNKT cells, etc., the tolerogenic dendrites provided hereinThe cells may also cause one or more of these responses.

The tolerogenic immune response also includes, but is not limited to, induction of regulatory cytokines, such as T _reg A cytokine; inducing an inhibitory cytokine; inhibiting inflammatory cytokines (e.g., IL-4, IL-1b, IL-5, TNF- α, IL-6, GM-CSF, IFN- γ, IL-2, IL-9, IL-12, IL-17, IL-18, IL-21, IL-22, IL-23, M-CSF, C-reactive protein, acute phase protein), chemokines (e.g., CCL-2, CXCL8, MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC, or IP-10), producing anti-inflammatory cytokines (e.g., IL-4, IL-13, IL-10, etc.), proteases (e.g., MMP-3, MMP-9), leukotrienes (e.g., cysLT-1, cysLT-2), prostaglandins (e.g., PGE 2), or histamine; inhibiting polarization of Th17, th1 or Th2 immune responses; inhibiting effector cell specific cytokines Th17 (e.g., IL-17, IL-25), th1 (IFN-gamma), th2 (e.g., IL-4, IL-13); inhibit Th1, th2 or Th17 specific transcription factors; inhibit proliferation of effector T cells; inducing apoptosis of effector T cells; inducing a tolerogenic dendritic cell-specific gene; inducing FoxP3 expression; inhibiting IgE-induced or IgE-mediated immune responses; inhibit antibody responses (e.g., production of antigen-specific antibodies); inhibiting a T helper cell response; TGF-beta and/or IL-10 production; inhibition of autoantibody effector function (e.g., inhibition of cell depletion, cell or tissue damage, or complement activation); etc.

Any of the foregoing may be measured in vivo in one or more animal models or may be measured in vitro. Those of ordinary skill in the art are familiar with such in vivo or in vitro measurements. Undesired immune responses or tolerogenic immune responses may be monitored using, for example, methods of assessing immune cell numbers and/or function, tetramer assays, ELISPOT, flow cytometry-based cytokine expression assays, cytokine secretion, cytokine expression profiling, gene expression profiling, protein expression profiling, cell surface marker assays, PCR-based immune cell receptor gene usage assays (see t.clay et al, "Assays for Monitoring Cellular Immune Response to Active Immunotherapy of Cancer" Clinical Cancer Research 7:1127-1135 (2001)), and the like. Methods such as assessing protein levels in plasma or serum, T cell or B cell proliferation and functional assays, etc. can also be used to monitor undesired immune responses or tolerogenic immune responses. In some embodiments, the tolerogenic immune response may be monitored by assessing the induction of FoxP 3.

Preferably, the tolerogenic immune response results in the inhibition of the occurrence, progression or pathology of a disease, disorder or condition described herein, in particular GvHD. In some embodiments, the reduction of an undesired immune response or the generation of a tolerogenic immune response may be assessed by determining clinical endpoints, clinical efficacy, clinical symptoms, disease biomarkers, and/or clinical scores.

As used herein, the term "animal" or "mammal" encompasses all mammals, including humans. Preferably, the mammal of the invention is a human subject.

As used herein, the term "exposed" refers to a state or condition that is brought into close proximity or direct contact.

The term "hematopoietic cell transplantation" (HCT) as used herein refers to blood and Bone Marrow Transplantation (BMT), a procedure involving the infusion of cells (hematopoietic stem cells; also known as hematopoietic progenitor cells) to reconstitute the patient's hematopoietic system.

The term "autoimmune disorder" or "autoimmune syndrome" as used herein refers to a condition that occurs when the immune system erroneously attacks and destroys the self-components of healthy body tissue. Autoimmune disorders may affect one or more organ or tissue types. Organs and tissues commonly affected by autoimmune disorders include blood vessels, connective tissue, endocrine glands (such as the thyroid or pancreas), joints, muscles, erythrocytes, and skin.

In any of the method steps of the invention using exposure to an apoptotic agent, the apoptotic agent comprises psoralen and UVA, riboflavin phosphate and UVA, and/or aminolevulinic acid and light. Particularly preferred psoralens are 8-MOP and amotosalen. The following numbers are used for instructional purposes. The skilled artisan can readily find the concentration and dosage administered to achieve an effect of apoptosis. The concentration of riboflavin phosphate may be 1 μm to 100 μm . The concentration of amotosalen may be 50 μm to 500 μm. The light dose associated with the above-mentioned riboflavin or amotosalen may be 1J/cm ² To 10J/cm ² . The corresponding light may be UVA or blue light. The concentration of 8-MOP may be 0.2. Mu.M to 2.5. Mu.M (or 43ng/mL to 540 ng/mL). The concomitant light dose may be 0.5J/cm ² To 5J/cm ² . The light may be UVA or blue light.

The method of the invention or specific steps of the method may be carried out in a bag, such as a plastic bag. If plastic materials are considered for use, bags made of the following plastic films may be used: polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymers, ethylene vinyl alcohol, polyvinylidene fluoride, or other plastic films approved for medical use. In a preferred embodiment of the invention, the bag is made of ethylene vinyl acetate copolymer. The bag may be made of a material that provides a degree of transparency such that the sample or cell mixture may be irradiated with visible or UV light.

The present invention will now be described with reference to a number of specific embodiments, however, these embodiments are for illustrative purposes and are not to be construed in a limiting manner.

Experiment

Experiment 1 production of phDC

All studies were performed using blood donated from healthy human volunteers. Peripheral blood was collected into 1:100,000U/mL heparin (McKesson Packaging Services) and platelet-containing PBMC were isolated by Isolyph (CTL Scientific Supply Corp.) density gradient centrifugation according to the manufacturer's protocol. Autologous plasma (also containing platelets) is collected and retained. Washed PBMCs and platelets were resuspended in autologous plasma and incubated for 1 hour in a trans-immune (TI) chamber or clinical ECP plate.

In the TI chamber, the cells were passed through at a rate of 0.09mL/min using a syringe pump. After plate transfer, cells were collected and the TI chamber was washed with 100% FBS at 0.49mL/min while physical agitation was performed by flicking or tapping the plate surface to help separate any adherent cells from the chamber. In clinical ECP plates, cells were passed at a flow rate of 24mL/min, then washed with human AB serum (Lonza BioWhittaker) at a flow rate of 100mL/min, and physically disturbed by flicking or tapping the plate surface to help separate any adherent cells. PBMCs passed through the TI chamber or ECP plate were collected, washed and cultured overnight under standard conditions in RPMI (Gibco, carlsbad, CA) without phenol red and supplemented with 15% human AB serum (Lonza BioWhittaker), 1% penicillin/streptomycin (Invitrogen, carlsbad, CA) and 1% L-glutamine (Invitrogen, carlsbad, CA). The following day, physiological dendritic cells were harvested (including any adherent cells harvested by scraping).

Experiment 2: ECP leukemia haploid transplantation test-protocol design and expected outcome

1. Mother (future donor) is made tolerant to father antigen by inputting father blood treated with ECP

2. Checking that the mother did have tolerance to the father antigen (reduced MLR response to father cells, no antibodies to father HLA type)

3. Transplanting bone marrow of the mother receiving treatment to leukemia children

4. Evaluation of bone marrow transplantation for children

5. The above treatments are shown in figure 1 to evaluate the endpoint of the trial-bone marrow transplantation, gvHD incidence and severity, and cancer recurrence rate.

After the treatment described above, children did not develop GvHD or exhibited reduced symptoms of GvHD as the grafts were pre-tolerated.

Experiment 3 ECP leukemia haploid transplantation test-protocol design and expected outcome

1. Recipients received tolerizing ECP treatment (following the complete matched transplantation trial design of Francine Foss, see experiment 1) prior to haplotype marrow transplantation or any immune ablation pretreatment

2. Transplantation was performed, but cyclophosphamide dose (PTCy) was reduced after transplantation.

3. Evaluation of bone marrow transplantation

4. The above treatments for assessing test endpoint-bone marrow transplantation, gvHD incidence and severity, and cancer recurrence rate are shown in fig. 2.

With the above treatment, PTCy can be reduced to maintain anti-tumor immunity. PTCy may be omitted entirely and replaced with ECP.

Experiment 4 prevention of GvHD by PUVA treated dendritic cells

Materials and methods

Future graft recipient mice (C57 BL/6, H2b MHC haplotypes) were inoculated subcutaneously (flank) with C57BL/6 tumor (MC 38 colon cancer) on day-4.

2 on day-1, future graft recipients (carrying tumors) received a lethal dose (950 cGy) of gamma radiation, thereby eliminating their own immune system.

3 on the day of transplantation (day 0), tissues were prepared for transplantation.

a. In the no-graft control, the irradiated C57BL/6 mice did not receive any grafts.

b. In syngeneic controls, the natural C57BL/6 immune system is reconstituted by reinfusion of C57BL/6 bone marrow and spleen cells

c. In the allograft, the recipient was reconstituted with fully mismatched bone marrow and spleen cells from a Balb/c donor mouse (H2 d MHC haplotype).

d. In allogeneic PUVA grafts, balb/C bone marrow and splenocytes (containing dendritic cells) were incubated with lethally irradiated C57BL/6 splenocytes for 5 hours, then placed in petri dishes with very low doses of PUVA (200 ng/mL 8-MOP, 0.1J/cm) ² UVA) treatment.

4 transplanting the prepared tissue into a subject carrying a tumor under irradiation.

5 the recipients were then monitored for bone marrow transplantation (survival, subsequently confirmed by blood analysis), gvHD and tumor growth.

Mice that received radiation but did not receive any grafts all died on day 14.

Syngeneic control mice transplanted well, did not develop GvHD (expected because the grafts matched perfectly), and developed large tumors.

Allogeneic control mice were transplanted well, developed severe GvHD at about day 25, all fatal (expected because of complete graft mismatch), and tumor growth was difficult to assess because it was relatively slow and fatal was rapid.

Allogeneic PUVA mice transplanted well, did not show any signs of GvHD (day 47), although the tumor grew, the growth rate appeared to be about 50% of that of syngeneic control mice, i.e., partially controlled by GvT effects.

The results are described in table 1 below and in fig. 3 and 4. Fig. 3 also schematically depicts the above method.

Results

TABLE 1 results for different treatment groups

The inventors have surprisingly found that grafts treated with low doses of 8-MOP/UVA (i.e. according to the methods of the invention) can prevent GvHD. The rationale for this is that healthy dendritic cells, which have absorbed dying dendritic cells, provide the recipient with tolerance to the graft.

Experiment 5 Effect of Pre-transplantation infusion of donor spleen cells treated with in vitro photopheresis (ECP) on heart transplant survival

Donor mice were BALB/c (H-2) ^d ) Male, 8-14 weeks old. Recipient mice were C57BL/C, male, 10-14 weeks old (n=24). Spleen cells from donor mice were collected and divided into two groups. The donor cells of group 1 were subjected to shear forces in the flow chamber ("untreated" in FIG. 5). The donor cells of group 2 were subjected to shear forces in the flow chamber and ECP ("ECP" in fig. 5). 10 recipient mice received untreated donor cells, while 14 recipient mice received ECP-treated donor cells.

Treatment of donor cells prior to infusion into recipient mice was as follows:

the flow cell was coated with Platelet Rich Plasma (PRP) by introducing 0.4mL of PRP fraction into the flow cell at 37 ℃ for 60 minutes.

Donor spleen cells were injected into the flow chamber with a 60mL syringe at a concentration of 4 billion cells in 13.33 mL.

The ECP is implemented as follows: mu.L of 8-MOP was added to 20mL of PBS (200 ng/mL) per 1 million cells. Exposing the cells to about 20-22mW/cm ² Is 200s under UVA. Recipient mice received treated donor cells, each recipient mouse receiving approximately 5000 tens of thousands of cells.

Heart transplantation was performed 7 days after donor cells were infused into the recipient mice. The results reported in fig. 5 are percent post-operative survival (in days). As can be seen in fig. 5, recipient mice that received untreated donor cells (i.e., group 1, donor cells received only shear force) died around day 9 post-surgery, while recipient mice that received ECP donor cells (i.e., group 2, donor cells received shear force and ECP) survived up to 29 days post-surgery. Thus, if the recipient receives ECP-treated donor cells, such as ECP-treated spleen cells (spleen cells including healthy dendritic cells), prior to transplantation, the recipient is significantly better tolerant to the allograft. It can be concluded that pretreatment of recipients with ECP-treated donor cells can lead to long-term survival and donor-specific tolerance of allografts.

Experiment 6 in vitro photopheresis to prevent acute GVHD in patients undergoing standard myeloablative modulation and allogeneic hematopoietic stem cell transplantation

Materials and methods

A subject

The study and consent were approved by the institutional review board or equivalent institution at all participating sites. All subjects signed an approved agreement prior to initiating study treatment. Qualification criteria include subjects 18-60 years old with hematological malignancy and organ function that do not interfere with myeloablative preparation regimens and allogeneic HCT treatment. Subjects were eligible to participate in the study if they were diagnosed with hematological malignancy and their treatment options were allogeneic bone marrow or PBSC transplantation. The subject may be enrolled in the group whether the subject's disease is alleviated or after a first or second recurrence of the disease. The subject is required to have a body weight of at least 40kg, a platelet count of greater than 20 000/cm, and no known sensitivity to psoralen or citrate products. The subject must possess an associated donor that is serologically or molecularly matched at all HLA-A, B and DR loci, or at one HLA-A or-B locus Mismatches, but with molecular matches at the HLA-DR locus, or with molecular matches at the HLA-A, -B and DR loci. Since the registration was performed during the period of 10 months 2002 to 1 month 2004, HLA-C matching was not performed regularly. The preparation regimen and prophylaxis were directed to GVHD subjects who received CY (60 mg/kg per day for 2 consecutive days) and TBI (10-13.5 Gy split dose delivery, 3 or 4 days). GVHD prevention was CSP 3-5mg/kg, starting intravenous injection from D-1 and adjusted to a trough level maintained between 200 and 600 ng/mL. When clinically tolerated, CSP is changed to oral administration and is tapered before D100, except for subjects who relapse or are intolerant to CSP. Subjects receiving HCT from matched relevant donors were given MTX 10mg/m on day 1 by intravenous injection ² And received 5mg/m on days 3, 6 and 11 ² Whereas subjects with mismatched related donors or matched unrelated donors received MTX 15mg/m on day 1 ² Day 3, 6 and 11 received 10mg/m ² . The dose of MTX is based on consensus among researchers to provide uniform prophylaxis for acute GVHD. Supporting care and prophylactic antimicrobial treatment were given according to institutional guidelines for each study site.

In vitro photopheresis

ECP was performed using UVAR XTS machine (Therakos, exton, PA, USA) as described previously (Miller et al 2004). Typically, at least 1500mL of buffy coat blood is collected per treatment prior to use of methoxsalen (methoxsalen) solution (UVADEX therapeutics), and after buffy coat collection is completed, it is injected into the recirculation bag of the ECP loop prior to the photoactivation process. After photoactivation is completed, the treated buffy coat is reinjected into the subject. Patients received ECP for 2 consecutive days within 4 days prior to starting the preparation regimen.

GVHD and adverse event grading

Adverse events were ranked according to established criteria (adverse event generic term criteria, version 3.0, 12 months 2003). The modified Seattle-Gluckberg standard is used for the staging of acute GVHD (Gluckberg et al, 1974), and a limited and broad diagnostic standard for chronic GVHD is described by Schulman et al, 1980. To ensure consistency of diagnosis of acute and chronic GVHD, researchers have been trained in diagnostic criteria prior to the start of the trial. Each researcher employs appropriate diagnostic methods at its study site to determine whether acute or chronic GVHD is present.

Statistics of

And (5) researching and analyzing. The primary analysis was the incidence of grade II-IV acute GVHD within the first 100 days after implantation and was calculated using a cumulative incidence function to accommodate the competing risk of death without acute GVHD. Likewise, cumulative morbidity methods were used to calculate the incidence of chronic GVHD, transplant Related Mortality (TRM), and relapse to accommodate competing risks. The probability of total survival (OS) and Disease Free Survival (DFS) is described using the Kaplan-Meier product limit method (95% confidence interval). Treatment failure (death or relapse) is an event used in DFS assessment. Descriptive statistics, such as median time to event occurrence, are also calculated.

Comparison with historical controls. Historical controls were identified using a database maintained by CIBMTR. CIBMTR is a research affiliate of the wisconsin medical institute International Bone Marrow Transplant Registry (IBMTR) and the national bone marrow donation program, which consists of voluntary work groups of more than 450 transplant centers worldwide, which provide detailed data on related allogeneic and autologous HCT to the coordination statistics center. The participating center is required to continuously report all transplants and monitor compliance through on-site auditing. All patients in the database were longitudinally followed and included annual assessments. Computerized error checking, doctor review of the submitted data, and on-site auditing of participating centers may ensure data quality. The observational study performed by CIBMTR during this period was performed with informed consent and was in compliance with HIPAA regulations established by institutional review board and privacy officials of the wisconsin medical college. CIBMTR collects data at two levels, enrollment and study. The enrollment data included disease type, age, sex, pre-transplant disease stage and chemotherapy response, date of diagnosis, type of transplant (bone marrow-derived and/or blood-derived stem cells), high dose pretreatment regimen, post-transplant disease progression and survival, development of new malignancy, and cause of death. Patient progress or death data was registered with requests every 6 months. All stations participating in CIBMTR provide registration data. Study data was collected using a subset of enrolled patients selected using a weighted randomization protocol to ensure representativeness and included detailed disease, pre-and post-transplant clinical information. Historical controls for this study were derived from the study database. ECP study subjects were transplanted between 2002 and 2004. The CIBMTR control was selected by applying the qualification criteria of the study to subjects transplanted between 1997 and 2004. The longer time frame of the control was used to ensure a reasonable statistical efficacy for the adjusted comparisons for a sufficient number of subjects. The outcome data for both study subjects and control subjects were reviewed over the course of a year to adjust for differences in the length of follow-up time. Since study subjects and controls were transplanted in two different time periods, the potential impact of the year of transplantation (1997-1999 and 2000-2004) on control outcome was examined. The control subjects transplanted in 1997-1999 did not differ from the 1 year results of the control subjects transplanted in 2000-2004. Subjects and controls were compared using multivariate Cox regression analysis. The ratio check is performed by adding a time-dependent covariate. These tests indicate that the ratio hypothesis is valid. A step-wise backward approach was used to identify significant covariates related to the results (except for the use of ECP). Variables considered in model construction include age, sex, race, donor relationship, HLA matching, graft type, disease state at the time of implantation, and CMV serological state. Therapeutic Effects (ECP) are included in each step of model construction. Testing for potential interactions between ECP treatment and other significant covariates showed no significant interactions. The overall probability and DFS after a year adjustment is estimated from the final Cox model, stratified according to the treatment received, and weighted according to the aggregate sample ratio value for each prognostic factor. These adjusted probabilities estimate the likelihood of outcome in populations with similar prognostic factors.

Results

Subject and donor characterization

66 subjects participated in the ECP study. Each of the 9 study centers received 1 to 16 subjects (an average of 7 subjects per study center). However, 3 subjects did not receive ECP study treatment; one patient had withdrawn the study consent, one patient had delayed transplantation and one central ECP machine had mechanical problems. One subject received ECP but was not transplanted due to rapid disease progression. After completion of the study, 62 subjects were considered to be able to evaluate in the modified intent-to-treat population dataset. Two of the subjects received only one ECP treatment before starting the preparation regimen, as mechanical problems with the ECP apparatus then occurred. Figure 6 summarizes subject, disease, donor characteristics and disease status information.

Transplantation

One subject was not transplanted and died of respiratory failure (unknown reason) on day 28. All other subjects had satisfactory neutrophil and platelet recovery and no late stage transplantation failure. Subjects receiving bone marrow transplantation reached a median (range) time of 21 (14-39) days for a neutrophil count of 4500/mL and 14 (13-28) days for peripheral blood transplantation. The corresponding times to reach platelets 420000/cm were 14 (11-43) days and 14 (6-56) days, respectively.

GVHD

Grade II-IV acute GVHD appeared in 22 of 62 subjects (36%) including 9 of 30 phase Guan Gongti HCT recipients (30%) and 13 of 32 matched unrelated or one HLA-antigen mismatched related donor HCT recipients (41%) (fig. 7). Skin is the most common and severely affected organ, 38% of subjects have stage 2 or higher skin involvement, while 16% and 13% of subjects have stage 2 or higher gastrointestinal or liver involvement, respectively (fig. 7). The 100 day cumulative incidence of grade II-IV acute GVHD was 35% (95% CI,23% -48%) (fig. 8). Forty (66%) patients had biopsy-confirmed acute GVHD. No patient was described as having early chronic GVHD before day 100 or advanced acute GVHD after day 100. The median time to the highest grade of both the first diagnosis and grade II-IV acute GVHD was 35 days, with the first diagnosis ranging from 18 to 52 days and the highest grade ranging from 22 to 96 days. 53 subjects can be assessed for chronic GVHD.7 patients died before day 100 (3 cases of acute GVHD, 1 case of idiopathic pneumonia, 3 cases of multiple organ system failure), and the data collected from both patients was insufficient to determine whether chronic GVHD occurred. Chronic GVHD occurred in 21 out of 53 subjects (40%) (limited: 8 (15%); broad: 13 (25%)). The 1 year cumulative incidence of limited and extensive chronic GVHD was 38% (95% CI,21% -47%) (fig. 9).

Toxicity of

The most common severe adverse events during the study were 8 (13%) fever, 4 (7%) febrile neutropenia, and 3 (5%) multiple organ system failure. Adverse events directly attributed to ECP included two subjects who developed hypotension when experiencing ECP. CMV reactivation occurred in 17 (27%) subjects, 2 of whom found CMV disease in lung tissue in bronchoscopy biopsies. Two (3%) subjects had systemic fungal infection when participating in the study, one occurring 19 days post-implantation and the other occurring 9 months post-implantation.

Survival of

Median follow-up time for surviving patients was 371 days (ranging from 366-643 days), with 48 out of 62 subjects surviving (77%). Kaplan Meier estimated survival at 100 days and 1 year post-implantation was 89% (95% CI,78% -97%) and 77% (95% CI,64% -86%), respectively. The annual OS probability for relevant and irrelevant donor HCT recipients were 89% (95% CI,70% -96%) and 66% (95% CI,46% -80%), respectively (fig. 10). The annual DFS probability was 69% (95% CI,64% -86%) for all patients, 79% (95% CI,59% -90%) after relevant donor HCT, 60% (95% CI,40% -75%) after irrelevant donor HCT. Recurrence occurred in 7 (11%) patients. 14 subjects (23%) died, 3 died after the relevant donor transplantation and 11 died after the irrelevant donor transplantation. The causes of death are recurrent (1), acute GVHD (3), chronic GVHD (1), infection (4), multiple organ system failure (3) and idiopathic pneumonia (2). The 1 year cumulative incidence of TRM was 21% (95% CI,11% -31%); cumulative incidence after relevant and irrelevant donor grafts was 10% (95% CI,1% -20%) and 31% (95% CI,18% -50%), respectively, fig. 11.

Comparison of ECP study subjects with CIBMTR control

The CIBMTR database was used to search for historical controls with similar characteristics to the study subjects. Control subjects were required to meet the qualification criteria defined in fig. 12. A total of 347 control subjects were determined. Their characteristics were compared with those of the study subjects in fig. 13. Subjects receiving ECP treatment are more likely to be older than 40 years of age than controls, caucasian, and have unrelated donors. There are also significant differences in the distribution of underlying diseases. The potential effects of these differences are considered in multivariate analysis. Multivariate analysis showed a significant reduction in the rate of incidence of grade II-IV acute GVHD in ECP treated subjects compared to historical controls (relative risk [ RR ],0.61;95% CI, 0.38-0.97) (p=0.04) (fig. 14). This lower rate was due to a significant delay in acute GVHD onset in ECP treated subjects, rather than an absolute decrease in incidence (fig. 15). The cumulative incidence of grade II-IV acute GVHD in study patients was 36% (95% CI,25% -48%) and 39% (95% CI,33% -44%) in the historical control cohort (fig. 15). There was also less TRM in subjects receiving ECP treatment compared to the historical control, but this was not statistically significant (RR, 0.55;95% CI, 0.29-1.04) (p=0.065). There was no difference in venous occlusive disease or interstitial pneumonia between groups. However, 85 (24%) control patients and 5 (8%) study patients developed opportunistic infections (p=0.008). Post-adjustment probabilities for DFS and OS were significantly higher for ECP treated subjects than for the historical controls (fig. 16 and 17). ECP treated subjects had a DFS rate of 74% (95% CI,62% -82%) after 1 year adjustment, a historical control cohort of 63% (95% CI,58% -67%) (RR of treatment failure [ relapse or death ]), 0.60;95% CI, 0.36-0.99) (p=0.045). ECP treated subjects had an OS rate of 83% (95% CI,72% -90%) after 1 year of adjustment, and a historical control of 67% (95% CI,62% -71%) (mortality RR,0.44;95% CI, 0.24-0.80) (p=0.007).

Discussion of the invention

After completion of the study, we have cooperated with CIBMTR to determine appropriate historical controls for comparison with ECP study subjects to properly view the results of this single phase II study. Controls were selected using the qualification criteria of the patients in the study. However, as shown in fig. 13, there was some difference in the profile between groups, ECP study subjects were more likely to be older and receive unrelated donor grafts, both of which were associated with increased risk of GVHD. Despite these key demographic differences in favor of the historical control group, acute GVHD occurred more slowly in the ECP treatment cohort. Multivariate analysis of differences in the adjusted prognostic factors showed significant differences in the ratio of acute GVHD (grade II-IV) between groups (fig. 15), with significant delays in acute GVHD onset time. Although the absolute incidence of acute GVHD for ECP was not reduced, multivariate analysis showed a trend of less TRM, less treatment failure (relapse and TRM), higher overall and DFS in the ECP study cohort compared to the historical control cohort. The toxicity associated with the regimen was similar between groups except that opportunistic infections were significantly reduced in ECP treated patients compared to the historical control group. The later-occurring acute GVHD may itself be beneficial because it may allow more recovery from preparation protocols and transplant surgery, as well as a higher degree of immune reconstitution, enabling these patients to better tolerate the side effects of corticosteroids, reduce end-organ damage, and overcome infections. ECP does not appear to suppress alloimmune-mediated graft-versus-malignancy effects, as there was no significant difference in recurrence rate between the two groups, although the follow-up time to evaluate recurrence was short. Experiment 7 use of tolerogenic phDC to ameliorate autoimmune disease

In this example, animal models were used to evaluate the improvement of autoimmune disease using tolerizing phdcs.

There are many well-known animal models of autoimmune diseases available; exemplary animal models are shown in table a above. Autoimmune disease is induced, if necessary, prior to treatment with tolerizing phDC or control. Animals were examined and scored according to relevant clinical criteria for animal models. In some embodiments, animals are divided into four treatment groups as follows:

a) Untreated animals

b) Treatment with healthy phDC presenting autoantigens

c) Treatment of apoptotic phDC with autoantigen-presenting 8-MOP/UVA lesions

d) Treatment with healthy phDC with internalized autoantigen-containing 8-MOP/UVA damaged apoptotic phDC

phDC of the above treatment group can be generated as follows:

b) Healthy phDC presenting autoantigens:

monocytes were taken from healthy syngeneic animals, passed through TI plates as described (Ventura et al J Vis Exp.2019May 17; 147), and incubated overnight with autoantigens to ensure antigen uptake.

c) Apoptosis phDC presenting autoantigen 8-MOP/UVA lesions:

monocytes were obtained from healthy syngeneic animals and passed through the TI plates as described above, then exposed to a dose of 8-MOP and UVA sufficient to induce cell damage, and incubated overnight with autoantigens.

d) Healthy phDC with internalized 8-MOP/UVA damage containing autoantigens apoptotic phDC:

cells from group c) were combined with an equal amount of fresh monocytes, passed through the TI plates as described above, and incubated overnight to ensure that healthy phDC absorbed 8-MOP/UVA-damaged phDC containing autoantigens.

phDC generated as described above is administered at an appropriate dose (e.g., at least 1x10 ⁶ Individual cells/animals), mice in the appropriate group are administered intravenously at appropriate time intervals, typically at least three times for animal models.

For a particular model, the animal is monitored (e.g., daily) for autoimmune disease over a suitable period of time. Furthermore, animals may be sacrificed at various time points during the course of the experiment:

(i) Prior to disease induction, i.e., healthy animals;

(ii) After disease induction, but untreated, i.e., immunized animals;

(iii) Between the second and third vaccination, i.e. the intermediate time point;

(iv) At the end of the experiment, i.e. end point

After euthanasia, animal samples (e.g., spleen and inguinal and axillary lymph nodes) were obtained, dissociated into single cell suspensions, and used to evaluate immune responses to immunization and treatment. Standard immune response assays include immune cell phenotyping by cell surface or intracellular flow cytometry, inflammatory or anti-inflammatory cytokine secretion assays (e.g., ELISA, luminex, ELISpot), and T cell proliferation in response to self antigen re-stimulation (e.g., carboxyfluorescein succinimidyl ester (CFSE) dilution).

Exemplary results will include:

a) Untreated animals:

progressive disease is consistent with expectations in the model.

b) Treatment with healthy phDC presenting autoantigens:

progressive disease, due to additional immune effects of healthy phDC, may be more severe than expected in the model.

c) Treatment with apoptotic phDC of 8-MOP/UVA lesions presenting autoantigens:

progressive disease, as some tolerance of apoptotic phDC of 8-MOP/UVA injury is absorbed by healthy DC in injected animals, may not be as severe as expected in the model.

d) Treatment with healthy phDC with internalized 8-MOP/UVA damage containing autoantigens apoptotic phDC:

significantly reduced disease, due to the tolerability of healthy phdcs, these phdcs absorb the autoantigen-carrying apoptotic phdcs of the 8-MOP/UVA lesions, thus enabling control and reduction of disease. Experiment 8 use of tolerogenic phDC to ameliorate autoimmune diseases including Multiple Sclerosis (MS)

In this example, a mouse model was used to assess whether tolerating phDC can ameliorate autoimmune diseases such as MS.

Experimental Autoimmune Encephalomyelitis (EAE) is a widely accepted mouse model of human MS, with many similarities to human clinical disease. The mouse EAE model is characterized by progressive paralysis, central nervous system inflammation and demyelination, mediated primarily by myelin-specific cd4+ cells, although cd8+ cells and B cells also function. Thus, EAE mice can be used to model tolerance induction of dendritic cells in an autoimmune disease setting.

1. EAE was induced in 11-13 week old female C57BL/6 mice by immunization with Myelin Oligodendrocyte Glycoprotein (MOG) peptide MOG35-55 or MOG1-125 in freund's complete adjuvant (CFA) emulsion, followed by Pertussis Toxin (PTX) administration in PBS, following standard protocols known in the art.

Eae were expected to occur 8 to 18 days after immunization. After disease induction, all animals were checked daily for health and scored according to the following criteria EAE clinical criteria, 0, asymptomatic; 0.5, the tail distal half loses tension; 1, the whole tail loses tension; 1.5, hind limb weakness; 2, hind limb paralysis; 2.5, hind limb paraplegia; 3, weakness of the forelimbs; 4, quadriplegia; 4.5, severe quadriplegia; 5, quadriplegia; and 6, death. Treatment began on the first day with an average clinical score of over 1.0, reflecting the onset of clinically relevant disease in most mice.

3. Mice were divided into 4 treatment groups of at least 10 animals each, as follows:

a) Untreated mice

b) Treatment with healthy phDC presenting MOG antigen

c) Apoptotic phDC treatment with 8-MOP/UVA lesions presenting MOG antigen

d) Treatment with healthy phDC with internalized MOG antigen containing 8-MOP/UVA damaged apoptotic phDC

4. phDC for the above treatment group will be generated as follows:

b) Healthy phDC presenting MOG antigen:

monocytes were taken from healthy syngeneic mice as described (Ventura et al J Vis exp.2019May17; 147) through the TI plate and incubated overnight with MOG antigen to ensure antigen uptake.

c) Apoptosis phDC with 8-MOP/UVA lesions presenting MOG antigen:

monocytes were obtained from healthy syngeneic mice, passed through the TI plate as described above, then exposed to a dose of 8-MOP and UVA sufficient to induce cell damage, and incubated overnight with MOG antigen.

d) Healthy phDC with internalized 8-MOP/UVA-damaged apoptotic phDC containing MOG antigen:

cells from group c) were combined with an equal amount of fresh monocytes, passed through the TI plates as described above, and incubated overnight to ensure that healthy phDCs absorbed 8-MOP/UVA-damaged phDCs containing MOG antigen.

5. phDC generated as described above (see 4.) was run at least 1x 10 ⁶ The individual cells/mice are dosed intravenously to mice in the appropriate group (see 3.), at least 3 times, e.g., as follows:

the first day with an average clinical score exceeding 1.0, reflecting the onset of clinically relevant disease in most mice (e.g., day 13 post immunization)

Four days after the first treatment dose (e.g., day 17 after immunization)

Four days after the second treatment dose (e.g., day 21 after immunization)

6. Mice were monitored daily for EAE clinical scores until at least 4 weeks post immunization.

7. In addition, mice may be sacrificed at different time points during the course of the experiment:

(i) Before EAE induction, i.e. healthy mice;

(ii) After EAE induction, but untreated, mice were immunized;

(iv) At the end of the experiment, i.e. end point

After euthanasia, spleen and inguinal and axillary lymph nodes were obtained, dissociated into single cell suspensions and used to evaluate immune responses to immunization and treatment. Standard immune response assays include immune cell phenotyping by cell surface or intracellular flow cytometry, inflammatory or anti-inflammatory cytokine secretion assays (e.g., ELISA, luminex, ELISpot), and T cell proliferation in response to MOG peptide restimulation (e.g., carboxyfluorescein succinimidyl ester (CFSE) dilution).

Spinal cords were obtained at euthanasia, fixed in 4% Paraformaldehyde (PFA), and used for additional assessment of disease by histology and immunohistochemistry. Typical histological analysis includes inflammatory foci counts, apoptotic cell counts and degree of demyelination (immunohistochemistry for myelin basic protein).

Exemplary results will include:

a) Untreated mice:

progressive EAE disease is consistent with expectations in this model.

-detecting inflammatory immune cells that react to MOG antigens after immunological examination.

Upon histological examination, signs of axonal and inflammatory lesions were consistent with the model.

b) Treatment with healthy phDC presenting MOG antigen:

progressive EAE disease, due to additional immune effects of healthy phdcs, may be more severe than expected in this model.

-increasing detection of inflammatory immune cells in response to MOG antigens after immunological examination.

After histological examination, there is evidence of more severe axonal and inflammatory lesions.

c) Apoptotic phDC treatment with 8-MOP/UVA lesions presenting MOG antigen:

progressive EAE disease, probably less severe than expected in this model, due to some tolerance of apoptotic phdcs of 8-MOP/UVA injury, is absorbed by healthy DCs in injected mice.

After immunological examination, the detection of inflammatory immune cells in response to MOG antigens is reduced.

After histological examination, there was a sign of minor axonal and inflammatory lesions.

d) Treatment with healthy phDC with internalized 8-MOP/UVA-damaged apoptotic phDC containing MOG antigen:

Significantly reduced EAE disease, due to the tolerability of healthy phDC that absorbs apoptotic phDC of 8-MOP/UVA lesions carrying MOG antigen, thereby enabling control and reduction of EAE disease.

After immunological examination, tolerogenic immune cells, such as tregs, are detected, as well as inflammatory immune cells that react to MOG antigens with a significantly reduced response.

After histological examination, axonal and inflammatory lesions were significantly reduced.

Experiment 9 use of tolerogenic phDC to ameliorate other autoimmune diseases

In this example, a suitable animal model (e.g., the animal model described in table a above) is used to evaluate whether tolerizing phDC ameliorates autoimmune disease.

For example, non-obese diabetic (NOD) mice are a well-known in the art model of Insulin Dependent Diabetes (IDDM). In this example, NOD mice were evaluated using a similar method as described in experiments 7 and 8.

Animals were examined and scored according to relevant clinical criteria for NOD mice. In some embodiments, animals are divided into four treatment groups as follows:

a) Untreated animals

b) Treatment with healthy phDC presenting autoantigens (e.g., pancreatic beta cell antigens)

c) Treatment of apoptotic phDC with 8-MOP/UVA lesions presenting autoantigens (e.g., pancreatic beta cell antigens)

d) Treatment with healthy phDC with internalized 8-MOP/UVA damaged apoptotic phDC containing autoantigens (e.g., pancreatic beta cell antigens)

phDC of the above treatment group can be generated as follows:

b) Healthy phDC presenting autoantigens (e.g., pancreatic beta cell antigens):

monocytes were taken from healthy syngeneic animals as described (Ventura et al J Vis exp.2019May17; 147) through the TI plate and incubated overnight with autoantigens to ensure antigen uptake.

c) Apoptosis phDC of 8-MOP/UVA lesions presenting autoantigens (e.g., pancreatic beta cell antigens):

monocytes were taken from healthy syngeneic animals and passed through the TI plates as described above, then exposed to a dose of 8-MOP and UVA sufficient to induce cell damage, and incubated overnight with autoantigens (e.g., pancreatic beta cell antigens).

d) Healthy phDC with internalized 8-MOP/UVA damaged apoptotic phDC containing autoantigens (e.g., pancreatic beta cell antigens):

cells from group c) were combined with an equal amount of fresh monocytes, passed through the TI plates as described above, and incubated overnight to ensure that healthy phdcs absorbed phdcs containing 8-MOP/UVA lesions of autoantigens (e.g., pancreatic β cell antigens).

phDC generated as described above is administered at an appropriate dose (e.g., at least 1x 10 ⁶ Individual cells/animals), are administered intravenously at appropriate time intervals to mice in the appropriate group, typically at least three times, for animal models.

The diabetic phenotype of the animals was monitored. Furthermore, animals may be sacrificed at various time points during the course of the experiment:

(i) Before disease induction, i.e. healthy animals;

(ii) After disease induction, but untreated, i.e., immunized animals;

(iv) At the end of the experiment, i.e. end point

After euthanasia, animal samples (e.g., spleen and inguinal and axillary lymph nodes) were obtained and isolated as single cell suspensions for evaluation of immune responses to immunization and treatment. Standard immune response assays include immune cell phenotyping by cell surface or intracellular flow cytometry, inflammatory or anti-inflammatory cytokine secretion assays (e.g., ELISA, luminex, ELISpot), and T cell proliferation in response to self antigen re-stimulation (e.g., carboxyfluorescein succinimidyl ester (CFSE) dilution).

Exemplary results will include:

a) Untreated animals:

progressive disease consistent with what is expected in NOD models.

b) Treatment with healthy phDC presenting autoantigens:

Progressive disease, possibly more severe than expected in the NOD model, due to the additional immune effects of healthy phdcs.

c) Treatment with apoptotic phDC of 8-MOP/UVA lesions presenting autoantigens:

progressive disease, probably less severe than expected in the NOD model, due to some tolerance of apoptotic phdcs of 8-MOP/UVA injury is absorbed by healthy DCs in injected animals.

-significantly reduced disease, due to the tolerability of healthy phDC that absorbs apoptotic phDC carrying autoantigen 8-MOP/UVA damage, disease can be controlled and reduced.

Other autoimmune diseases can be assessed using similar methods as described above.

Experiment 10 treatment of autoimmune diseases with tolerogenic phDC

Tolerogenic phDC produced as described herein is used to treat human patients suffering from autoimmune diseases.

For example, a dendritic cell sample is obtained from a patient suffering from an autoimmune disease (e.g., MS). Dendritic cells are exposed to an apoptotic agent (e.g., a combination of Psoralen and UVA (PUVA), particularly a combination of 8-MOP and UVA). In some examples, autoantigens are also added to dendritic cells exposed to the apoptotic agent. For example, for MS, any suitable autoantigen described in table a, such as MBP and/or MOG, may be added. The cells are exposed to an apoptotic agent for a period of time under conditions in which the cells undergo apoptosis.

The resulting apoptotic dendritic cells are then combined with physiological dendritic cells produced as described herein and may be co-incubated, for example, for at least 0.5h, 1h, 2h, 3h, 4h, 5h, or 6h prior to administration to a subject. Alternatively, the combination may be administered directly to the subject without co-incubation. phDC treatment is expected to improve autoimmune diseases.

Experiment 11 increased PD-L1 expression in human phDC incubated with PUVA-damaged PBMC

Human phDC was generated from blood of healthy volunteers using TI plates and incubated overnight with equal amounts of 8-MOP/UVA treated homologs PBMC (PUVA syn PBMC) or allografts PBMC (PUVA allo PBMC). PD-L1 expression (reported as mean fluorescence intensity of the fraction of viable cd14+phdc, MFI) was measured by flow cytometry at 18 hours and found to be significantly higher in healthy phDC than precursor monocytes after co-incubation with allogeneic or syngeneic PUVA-treated PBMC (fig. 18). N = number of blood donors analyzed; p-value = Welch corrected unpaired t-test.

Experiment 12 PD1 expression reduction in responsive T cells in the MLR assay

MLR (mixed lymphocyte reaction) assays were performed using blood from healthy volunteer donors. Briefly, 2×10 from one donor ⁵ Individual purified, CFSE-labeled responder T cells (T cells) were 4 x 10 from the same donor (syngeneic culture) or from unrelated donors (MLR) ⁵ Gamma radiation (3000 rad) stimulatory PBMC co-cultures. To inhibit the MLR response, some cultures were additionally supplemented with 1×10 ⁵ 8-MOP/UVA-treated syngeneic PBMC and 1 x 10 ⁵ phDC (MLR+PUVAsyn. PBMC+phDC) from the individual homologs passed through the TI plate. After 5 days of culture, CFSE dilutions were measured by flow cytometry (FACS) to determine proliferation of responding CD 8T cells and CD 4T cells (A, B). Activation status of responding CD 8T cells and CD 4T cells was also assessed by FACS, using CD44 and PD1 expression to detect activated T cells (C, D). For CD 8T cells and CD 4T cells, proliferation and activation were significantly inhibited by addition of healthy phDC and PUVA treated PBMC (fig. 19). N = number of blood donors analyzed; p-value = Welch corrected unpaired t-test.

In addition, the present invention relates to the following embodiments.

1. A method of selectively producing a tolerogenic dendritic cell, the method comprising the steps of:

a) Providing a dendritic cell from a donor;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

2. The method according to claim 1, wherein after step d), a step of incubating the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells of step c) is performed.

3. The method according to claim 2, wherein the co-incubation step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

4. The method according to claim 1, wherein step d) of combining said apoptotic donor dendritic cells with said physiological dendritic cells from said recipient is performed within said recipient.

5. The method according to claim 1, wherein the dendritic cells of step a) are derived from an in vitro blood sample of the donor.

6. The method according to claim 1, wherein the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the donor.

7. The method according to claim 1, wherein the apoptotic agent in step b) comprises psoralen and UVA, riboflavin phosphate and UVA, and/or 5-aminolevulinic acid and light.

8. The method according to claim 7, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

9. The method according to 8, wherein said psoralen is 8-MOP.

10. The method according to claim 1, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from said recipient.

11. The method according to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the donor and/or recipient is a mammal, preferably a human.

12. A method of selectively producing a tolerogenic dendritic cell, the method comprising the steps of:

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing physiological dendritic cells from the recipient haploid donor; and

13. The method according to claim 12, wherein after step d) a step of incubating the apoptotic complementary haploid donor dendritic cells of step b) with the physiological haploid donor dendritic cells of step c) is performed.

14. The method according to claim 13, wherein the co-incubation step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

15. The method according to claim 12, wherein step d) of combining the apoptosis complementing haploid donor dendritic cells of step b) with the physiological haploid donor dendritic cells of step c) is performed in said haploid donor.

16. The method according to claim 12, wherein said dendritic cells of step a) are derived from an in vitro blood sample of a complementary haploid donor of said recipient.

17. The method according to claim 12, wherein said dendritic cells of step a) have been obtained by plate transfer of PBMCs from a complementary haploid donor of said recipient.

18. The method according to claim 12, wherein said apoptotic agent of step b) comprises psoralen and UVA, riboflavin phosphate and UVA and/or 5-aminolevulinic acid and light.

19. The method according to claim 18, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

20. The method according to claim 19, wherein said psoralen is 8-MOP.

21. The method according to claim 12, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from a complementary haploid donor of said recipient.

22. The method according to any one of claims 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, wherein said complementary haploid donor, said haploid donor and/or said recipient is a mammal, preferably a human.

23. A method of selectively producing a tolerogenic dendritic cell, the method comprising the steps of:

a) Providing a dendritic cell from a recipient;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from the receptor; and

24. The method according to claim 23, wherein after step d), a step of incubating the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c) is performed.

25. The method according to 24, wherein the co-incubation step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

26. The method according to claim 23, wherein step c) of combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c) is performed within the recipient.

27. The method according to claim 23, wherein said dendritic cells of step a) are derived from an extracorporeal blood sample of said subject.

28. The method according to claim 23, wherein said dendritic cells of step a) have been obtained by plate transfer of PBMCs from said recipient.

29. The method according to claim 23, wherein said apoptotic agent of step b) comprises psoralen and UVA, riboflavin phosphate and UVA and/or 5-aminolevulinic acid and light.

30. The method according to claim 29, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

31. The method according to claim 30, wherein said psoralen is 8-MOP.

32. The method according to claim 23, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from said recipient.

33. A method according to any one of claims 23 to 32, wherein the donor and acceptor are mammals, preferably humans.

34. The method according to any one of claims 1 to 33, wherein the graft is an organ or stem cell graft.

35. A tolerogenic dendritic cell obtainable by a method according to any one of claims 1 to 11.

36. A tolerogenic dendritic cell obtainable by a method according to any one of claims 12 to 22.

37. A tolerogenic dendritic cell obtainable by a method according to any one of claims 23 to 34.

38. A tolerogenic dendritic cell according to 35 to 37 for use in a method of preventing or reducing graft versus host disease.

39. A method of selectively producing a tolerogenic dendritic cell, the method comprising the steps of:

a) Providing a first sample of dendritic cells obtained from a subject;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a second sample of physiological dendritic cells obtained from the subject; and

40. The method according to 39, wherein after step d), a step of incubating the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c) is performed.

41. The method according to 40, wherein the co-incubation step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

42. The method according to claim 39, wherein step c) of combining the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c) is performed in the subject.

43. The method according to any one of claims 39 to 42, wherein the dendritic cells of step a) are derived from an extracorporeal blood sample of the subject.

44. The method according to 43, wherein said dendritic cells of step a) have been obtained by plate transfer of PBMCs from said subject.

45. The method according to any one of claims 39 to 44, wherein the method further comprises a step a 1) of incubating the dendritic cells with an antigen molecule.

46. The method according to claim 45, wherein the antigenic molecule is a self-antigen.

47. The method according to claim 45 or 46, wherein the antigenic molecule is derived from natural sources, chemically synthesized or recombinantly produced.

48. The method according to 45 or 46, wherein the antigenic molecule is derived from a cell.

49. The method according to 46, wherein the autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

50. The method according to any one of claims 39 to 49, wherein said apoptotic agent in step b) comprises psoralen and UVA, riboflavin phosphate and UVA and/or 5-aminolevulinic acid and light.

51. The method according to 50, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

52. The method according to 50, wherein said psoralen is 8-MOP.

53. The method according to any one of claims 39 to 52, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from said subject.

54. The method according to any one of claims 39 to 53, wherein the subject is a mammal, preferably a human.

55. A tolerogenic dendritic cell obtainable by a method according to any one of claims 39 to 54.

56. A tolerogenic dendritic cell according to 55 for use in the treatment of an autoimmune disease.

57. A tolerogenic dendritic cell for use according to 56, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin Dependent Diabetes Mellitus (IDDM), addison's disease, diarrhea celiac disease, chronic fatigue syndrome, colitis, crohn's disease, fibromyalgia, hyperthyroidism, graves' disease, hypothyroidism, hashimoto disease, endometriosis, pernicious anemia, goodpasture's syndrome, wegener's disease and wind-damp-heat.

58. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of 55 tolerogenic dendritic cells.

59. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a tolerogenic dendritic cell, wherein the tolerogenic dendritic cell comprises a physiological dendritic cell comprising material from an apoptotic dendritic cell of the subject, autoantigen, fragment thereof, or combination thereof.

60.58 or 59, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin Dependent Diabetes Mellitus (IDDM), addison's disease, diarrhea celiac disease, chronic fatigue syndrome, colitis, crohn's disease, fibromyalgia, hyperthyroidism, graves' disease, hypothyroidism, hashimoto disease, endometriosis, pernicious anemia, goodpasture's syndrome, wegener's disease and wind-damp-heat.

61.59 or 60, wherein the autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

62. An ex vivo resistant dendritic cell comprising material from an apoptotic dendritic cell obtained from a subject.

63.62, which further comprises an autoantigen or fragment thereof.

64. A composition comprising:

(a) A dendritic cell sample obtained from a subject;

(b) An apoptosis agent; and

(c) Autoantigens or fragments thereof.

65.64 wherein the apoptosis agent comprises psoralen, riboflavin phosphate, or 5-aminolevulinic acid.

66.65, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

67.66, wherein said psoralen is 8-MOP.

68.62 or 63, or a composition of any one of 64-67, wherein said autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

Claims

a) Providing a dendritic cell from a donor;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from a recipient; and

2. The method according to claim 1, wherein after step d) a step of incubating the apoptotic donor dendritic cells of step b) with the physiological recipient dendritic cells of step c) is performed.

3. The method according to claim 2, wherein the co-incubation step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours.

4. The method according to any one of the preceding claims, wherein step d) of combining said apoptotic donor dendritic cells with said physiological dendritic cells from said recipient is performed within said recipient.

5. The method according to any of the preceding claims, wherein the dendritic cells of step a) are derived from an extracorporeal blood sample of the donor.

6. The method according to any one of the preceding claims, wherein the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the donor.

7. The method according to any one of the preceding claims, wherein the apoptotic agent of step b) comprises psoralen and UVA, riboflavin phosphate and UVA, and/or 5-aminolevulinic acid and light.

9. The method according to claim 7 or 8, wherein said psoralen is 8-MOP.

10. The method according to any one of the preceding claims, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from said recipient.

11. A method according to any one of the preceding claims, wherein the donor and/or recipient is a mammal, preferably a human.

b) Exposing the dendritic cells of step a) to an apoptotic agent;

13. Method according to claim 12, wherein after step d) a step of co-incubating the apoptotic complementary haploid donor dendritic cells according to step b) with the physiological haploid donor dendritic cells according to step c) is performed.

14. The method according to claim 13, wherein the co-incubating step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

15. The method according to claim 12, wherein step d) of combining said apoptosis complementing haploid donor dendritic cells of step b) with said physiological haploid donor dendritic cells of step c) is performed in said haploid donor.

16. The method according to any one of claims 12 to 15, wherein the dendritic cells of step a) are derived from an in vitro blood sample of a complementary haploid donor of said recipient.

17. The method according to any one of claims 12 to 15, wherein the dendritic cells of step a) have been obtained by plate transfer of PBMCs from a complementary haploid donor of said recipient.

18. The method according to any one of claims 12 to 17, wherein the apoptotic agent according to step b) comprises psoralen and UVA, riboflavin phosphate and UVA and/or 5-aminolevulinic acid and light.

20. The method according to claim 18 or 19, wherein said psoralen is 8-MOP.

21. The method according to any one of claims 12 to 20, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from a haploid donor of said recipient.

22. Method according to any one of claims 12 to 21, wherein said complementary haploid donor, said haploid donor and/or said recipient is a mammal, preferably a human.

a) Providing a dendritic cell from a recipient;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a physiological dendritic cell from the receptor; and

24. The method according to claim 23, wherein after step d) a step of co-incubating the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c) is performed.

25. The method according to claim 24, wherein the co-incubating step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

26. The method according to claim 23, wherein step c) of combining said apoptotic dendritic cells of step b) with said physiological dendritic cells of step c) is performed within said recipient.

27. The method according to any one of claims 23 to 26, wherein the dendritic cells of step a) are derived from an extracorporeal blood sample of the recipient.

28. The method according to any one of claims 23 to 27, wherein the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the recipient.

29. The method according to any one of claims 23 to 28, wherein said apoptotic agent of step b) comprises psoralen and UVA, riboflavin phosphate and UVA and/or 5-aminolevulinic acid and light.

31. The method according to claim 29 or 30, wherein said psoralen is 8-MOP.

32. The method according to any one of claims 23 to 31, wherein said physiological dendritic cells of step c) have been obtained by plate transfer of PBMCs from said recipient.

34. A tolerogenic dendritic cell obtained by a method according to any one of claims 1 to 11.

35. A tolerogenic dendritic cell obtainable by a method according to any one of claims 12 to 22.

36. A tolerogenic dendritic cell obtainable by a method according to any one of claims 23 to 33.

37. A tolerogenic dendritic cell according to claims 34 to 36 for use in a method of preventing or reducing graft versus host disease.

38. A method of selectively producing a tolerogenic dendritic cell, the method comprising the steps of:

a) Providing a first sample of dendritic cells obtained from a subject;

b) Exposing the dendritic cells of step a) to an apoptotic agent;

c) Providing a second sample of dendritic cells obtained from the subject; and

39. The method according to claim 38, wherein after step d) a step of co-incubating the apoptotic dendritic cells of step b) with the physiological dendritic cells of step c) is performed.

40. The method according to claim 39, wherein the co-incubating step is performed for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours.

41. The method according to any one of claims 38 to 40, wherein step c) of combining the apoptotic dendritic cells of step b) with the dendritic cells of step c) is performed in the subject.

42. The method according to any one of claims 38 to 41, wherein the dendritic cells of step a) are derived from an extracorporeal blood sample of the subject.

43. The method according to any one of claims 38 to 42, wherein the dendritic cells of step a) have been obtained by plate transfer of PBMCs from the subject.

44. The method according to any one of claims 38 to 43, wherein the method further comprises a step a 1) of incubating the dendritic cells with an antigen molecule.

45. The method of claim 44, wherein the antigenic molecule is a self-antigen.

46. The method of claim 44 or 45, wherein the antigenic molecule is derived from a natural source, chemically synthesized, or recombinantly produced.

47. The method of claim 44 or 45, wherein the antigenic molecule is derived from a cell.

48. The method of claim 45, wherein the autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

49. The method according to any one of claims 38 to 48, wherein said apoptotic agent according to step b) comprises psoralen and UVA, riboflavin phosphate and UVA and/or 5-aminolevulinic acid and light.

50. The method according to claim 49, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

51. The method according to claim 49 or 50, wherein said psoralen is 8-MOP.

52. The method according to any one of claims 38 to 51, wherein the dendritic cells of step c) have been obtained by plate transfer of PBMCs from the subject.

53. The method according to any one of claims 38 to 52, wherein the subject is a mammal, preferably a human.

54. A tolerogenic dendritic cell obtainable by a method according to any one of claims 38 to 53.

55. A tolerogenic dendritic cell according to claim 54 for use in the treatment of an autoimmune disease.

56. The tolerogenic dendritic cell for use according to claim 55, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin Dependent Diabetes Mellitus (IDDM), addison's disease, diarrhea celiac disease, chronic fatigue syndrome, colitis, crohn's disease, fibromyalgia, hyperthyroidism, graves' disease, hypothyroidism, hashimoto disease, endometriosis, pernicious anemia, goodpasture's syndrome, wegener's disease and wind-damp-heat.

57. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of the tolerogenic dendritic cells of claim 54.

58. A method of treating an autoimmune disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a tolerogenic dendritic cell, wherein the tolerogenic dendritic cell comprises a physiological dendritic cell comprising material from an apoptotic dendritic cell obtained from the subject, autoantigen, fragment thereof, or combination thereof.

59. The method of claim 57 or 58, wherein said autoimmune disease is selected from the group consisting of: multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, amyotrophic lateral sclerosis, pemphigus vulgaris, psoriasis, myasthenia gravis, thyroiditis, scleroderma, sjogren's syndrome, thrombocytopenic purpura, cryoglobulinemia, autoimmune hemolytic anemia, insulin Dependent Diabetes Mellitus (IDDM), addison's disease, diarrhea celiac disease, chronic fatigue syndrome, colitis, crohn's disease, fibromyalgia, hyperthyroidism, graves' disease, hypothyroidism, hashimoto disease, endometriosis, pernicious anemia, goodpasture's syndrome, wegener's disease and wind-damp-heat.

60. The method of claim 58 or 69, wherein said autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).

61. An ex vivo resistant dendritic cell comprising material from an apoptotic dendritic cell obtained from a subject.

62. The ex vivo resistant dendritic cell of claim 61, further comprising an autoantigen or fragment thereof.

63. A composition comprising:

(a) A dendritic cell sample obtained from a subject;

(b) An apoptosis agent; and

(c) Autoantigens or fragments thereof.

64. The composition of claim 63, wherein said apoptosis agent comprises psoralen, riboflavin phosphate, or 5-aminolevulinic acid.

65. The composition of claim 64, wherein said psoralen is selected from the group consisting of 8-MOP and amotosalen.

66. The composition of claim 65, wherein said psoralen is 8-MOP.

67. The ex vivo resistant dendritic cell of claim 61 or 62, or the composition of any one of claims 63-66, wherein said autoantigen is selected from the group consisting of: rh blood group antigens, platelet integrins GpIIb: IIIse:Sup>A, non-collagenous domain of basement membrane type IV collagen, epidermal cadherin, streptococcal cell wall antigen, rheumatoid factor IgG complex (with or without hepatitis C antigen), pancreatic betse:Sup>A cell antigen, myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein, desmoglein 3, glutamate decarboxylase, acetylcholine receptor, carboxypeptidase H, chromogranin A, glutamate decarboxylase, imagen-38, insulin, insulinomse:Sup>A antigen-2 and 2 betse:Sup>A, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, alphse:Sup>A-enolase, aquaporin-4, betse:Sup>A-inhibitor protein, S100-betse:Sup>A, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein 39, se:Sup>A antigen, nucleosome histone and nucleoprotein (snRNP), phospholipid-betse:Sup>A-2 glycoprotein I complex, poly (ADP-ribose) polymerase, U-1 microglobulin complex, sm-170, sjog antigen (sjog-70).