CN114958921A - Method for improving stability and function of regulatory T cells - Google Patents

Abstract

The invention provides a method for improving the stability and the immune regulation function of regulatory T cells based on the combination of a transcription factor inhibiting T-beta technology and cytokine overexpression. Specifically, the transcription factor T-beta inhibiting technology comprises hairpin RNA interference technology, and the cell factors comprise TGF-beta and IL-10. The technology provided by the invention can effectively maintain the stability of the regulatory T cells in the inflammatory environment for a long time, has a remarkable maintaining effect especially on the genetics and functional stability of the inducible regulatory T cells, has an important revelation value for solving the scientific problem of instability of the inflammatory environment function of the regulatory T cells, and has a wide application prospect in the fields of treating autoimmune diseases, intervening organ transplant rejection and the like.

Description

Method for improving stability and function of regulatory T cells

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to a method for improving stability and function of induced regulatory T cells.

Background

Regulatory T cells (tregs) are a group of lymphocytes that down-regulate the immune response of the body, maintain immune homeostasis by down-regulating the proliferation and function of immune effector cells, maintain the body's tolerance to self or foreign antigens, and prevent excessive immune responses. For a long time, systemic immunosuppressive drugs, hormones, and the like have been mainly used for clinical treatments of diseases caused by excessive immune response, such as allergic diseases, autoimmune diseases, and organ transplant rejection. Most of the drugs are taken for life, and are easy to induce liver injury, bone marrow suppression, metabolic disorder, increase the risk of bacterial and fungal infection and tumor. In recent years, clinical trials of Treg cell intervention are gradually developed at home and abroad aiming at the diseases, so as to replace or reduce the dosage of systemic immunosuppressive drugs or hormones and assist immune reconstitution.

The Treg Cell subpopulation selected by the current immunotherapy clinical test project is relatively single, and Natural Regulatory T cells (ntreglatory T cells, ntregs) with stable epigenetics from thymus are mostly adopted to ensure the lineage and functional stability of the Treg cells in the environment of rejection inflammation in vivo. Due to nTreg (CD 4) ⁺ CD25 ⁺ ) The proportion of mononuclear cells (PBMC) in the peripheral blood cells is very low (0.1-0.7%), the peripheral blood of a patient needs to be collected in a large amount, and the compliance and the safety of the patient are reduced. Furthermore, the technology of in vitro expansion of Treg cells by PBMCThe operation method is mainly used for promoting cell proliferation by adjusting the dosage of rapamycin and IL-2 cytokines in a culture system, and the maturity of an amplification technology still has a larger space for improvement.

Induced Regulatory T cells (iTreg), which have been discovered in recent years, have a strong ability to mediate recognition tolerance and mucosal and environmental tolerance of foreign antigens, and can exert a good immune negative Regulatory function in transplantation tolerance, allergic reactions and chronic inflammation. From Naive CD4 ⁺ The iTreg cells obtained by the T cell induced differentiation have sufficient initial cell number and short amplification period required by an in-vitro amplification culture system, and are expected to break through the restriction of in-vitro large-scale amplification of the nTreg cells. The TGF-beta signal pathway regulates and controls key molecules of iTreg cell differentiation, including expression and phosphorylation of transcription factor STAT3 and methylation of Foxp3 gene, so that an iTreg culture system is seriously dependent on exogenously added cell factor TGF-beta, a large amount of the latter increases the production cost of cells, and factors such as addition time phase, half-life period, functional stability and the like also need to be comprehensively considered so as to further optimize the controllability of an amplification system. The international clinical trial of first using ex vivo expanded iTreg cells to intervene in transplant rejection was conducted by the Margaret l. Mainly observe the safety and the effectiveness of the iTreg in negatively regulating the host rejection reaction resistance of the transplanted graft after the hematopoietic stem cell transplantation. Due to the large amount of blood collected from donors, only 14 out of 55 donors initially screened into the group eventually received such large amounts of blood collected into clinical trials. The iTreg cells account for about 44% at the end point of the in vitro amplification culture system, and the capability of in vitro negative regulation and control of the proliferation of effector cells is weaker, so that the iTreg cell amplification system is prompted to be further optimized. The validity of the clinical trial results has not been determined.

Due to the low epigenetic stability of iTreg, Foxp3 expression is easily lost in the context of high inflammation and is then polarized towards inflammatory effector T cells, leading to an increased risk of inducing inflammation. In addition, autoimmune diseases or organ transplant rejectants are in an inflammatory immune environment for a long time in vivo, the functions of self-separated Treg cells are low, and the clinical application has a larger challenge. Therefore, how to effectively maintain the phenotype and functional stability of the iTreg cells and further improve the immune negative regulation function of the iTreg cells is the key point for breaking through the technical bottleneck of the immune therapy of the Treg cells.

Disclosure of Invention

In order to solve the above scientific and technical problems, the present invention provides in a first aspect a method for increasing induction, phenotypic stability and negative immunoregulatory function of a regulatory T immune cell, said method comprising inhibiting expression of transcription factor T-beta or knocking out TBX21 gene.

In certain embodiments, the nucleic acid encoding the transcription factor T-beta comprises SEQ ID NO 1, a degenerate sequence thereof, or a spliceosome nucleotide sequence thereof.

In certain embodiments, the transcription factor T-beta protein comprises the amino acid sequence of SEQ ID NO 2, a spliceosome thereof, or a variant thereof.

In certain embodiments, the variant refers to a protein that is functionally identical or similar to SEQ ID No. 2 or a spliceosome thereof, resulting from substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence shown in SEQ ID No. 2 or a spliceosome thereof.

In certain embodiments, methods or products for the inhibition of the expression of transcription factor T-beta include, but are not limited to, small RNAs or small RNAs of multi-target multi-hairpin structures, ribozymes, gene targeting, protein inhibitors and compound inhibitors, and the like; preferably, siRNA interference through hairpin structure; more preferably, the siRNA has a sequence shown in SEQ ID NO 3-8.

In certain embodiments, the methods or products for knocking out the TBX21 gene are selected from CRISPR/Cas9 technology, zinc finger nucleases, TALENs, and the like.

In certain embodiments, the method of the first aspect of the invention further comprises the step of adding or overexpressing a combination of one or more cytokines.

In some embodiments, the combination of multiple cytokines may be achieved by adding multiple cytokines simultaneously, or by overexpressing multiple cytokine genes simultaneously, or by adding and overexpressing a combination of multiple cytokines or their fragments, or by adding fusion proteins of multiple cytokines or their fragments, or by overexpressing multiple cytokines or their fragments, to enhance induction, phenotypic stability, and immunomodulatory function of regulatory T-immune cells.

In certain embodiments, the cytokines include, but are not limited to, natural or recombinant interleukins, interferons, colony stimulating factors, erythropoietins, epidermal growth factors, nerve growth factors, basic fibroblast growth factors, tumor necrosis factors and antagonists thereof, thrombopoietin, platelet-derived growth factors, stem cell factors, vascular endothelial growth factors and antagonists thereof; preferably, the cytokine is TGF-beta, IL-10 or a combination thereof.

In certain embodiments, the TGF- β is selected from TGF- β 1, TGF- β 2, TGF- β 3, and their corresponding precursor molecules (per-pro-TGF- β), polypeptide chain precursors (pro-TGF- β), and mature forms (mTGF- β).

In certain embodiments, the TGF- β is TGF- β 1, the nucleic acid encoding TGF- β 1 comprises SEQ ID NO 9, a degenerate sequence thereof, or a spliceosome nucleotide sequence thereof, and the TGF- β 1 protein comprises the amino acid sequence of SEQ ID NO 10, a spliceosome or a variant thereof;

in certain embodiments, the variant refers to a protein that is functionally identical or similar to SEQ ID NO. 10 or a spliceosome thereof, which is obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO. 10 or the spliceosome amino acid sequence thereof.

In certain embodiments, the TGF- β is mTGF- β 1, the mTGF- β 1 encoding nucleic acid comprises SEQ ID NO 11, a degenerate sequence thereof, or a splice nucleotide sequence thereof, and the mTGF- β 1 protein comprises SEQ ID NO 12, a splice thereof, or a variant amino acid sequence thereof.

In certain embodiments, the variant refers to a protein that is functionally identical or similar to SEQ ID No.12 or a spliceosome thereof, which is obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence shown in SEQ ID No.12 or a spliceosome thereof.

In certain embodiments, the IL-10 encoding nucleic acid has the nucleotide sequence of SEQ ID NO 13, a degenerate sequence thereof, or a splice thereof, and the mTGF- β 1 protein comprises the amino acid sequence set forth in SEQ ID NO 14, a splice thereof, or a variant thereof.

In certain embodiments, the variant refers to a protein that is functionally identical or similar to SEQ ID NO. 14 or a spliceosome thereof, which is obtained by substitution and/or deletion and/or addition of at least one amino acid in the amino acid sequence shown in SEQ ID NO. 14 or a spliceosome amino acid sequence thereof.

In certain embodiments, the methods of the first aspect of the invention further comprise the combination of overexpression, interference or knock-out of 1 or more other genes, including BCL-6, including GATA-3, ROR γ T, ROR α, etc., to induce Follicular Regulatory T cells (Tfr) or to further enhance the phenotype and function of tregs.

In certain embodiments, the methods further express the homing molecules CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, or a combination of homing molecules to promote chemotaxis of tregs in vivo to the disease site, enhancing therapeutic efficacy; preferably, the homing molecule is expressed on a viral vector or an mRNA vector.

In certain embodiments, the method further expresses or associates a molecule capable of recognizing a disease site or transplanted organ specific target, preferably, the molecule is a chimeric antigen receptor molecule, a ligand for a tissue specific receptor.

In certain embodiments, the interference or knock-out sequence, overexpressed gene, cytokine combination, may be expressed separately in combination after different vectors, or multiple genes or interference sequences may be expressed simultaneously in the same vector.

In certain embodiments, the vector comprises an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a retroviral vector, a herpes viral vector, an RNA viral vector, an EB viral vector, a baculovirus vector, a phage vector, an animal viral vector, a plant viral vector, a DNA plasmid vector, an RNA vector, preferably the vector is a DNA plasmid vector, more preferably the vector is a lentiviral vector.

In a second aspect, the invention provides the use of the regulatory T immune cells obtained by culturing according to the method of the first aspect of the invention in the preparation of a medicament for the treatment of autoimmune diseases, for inducing immune tolerance after organ transplantation.

In certain embodiments, the autoimmune disease comprises systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, type I diabetes, autoimmune hepatitis, polyneuritis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, or the like;

in certain embodiments, the organ transplantation comprises allogenic and xenogenic heart, liver, kidney transplantation, and the like; preferably, the xenotransplantation is organ transplantation from porcine origin to humans.

In certain embodiments, the regulatory T immune cells can be used alone or in combination with other drugs.

In a third aspect, the invention provides a vector or combination of vectors for use in improving the stability of a regulatory T immune cell.

In certain embodiments, the vector or combination of vectors is selected from the following: I) a vector comprising a T-beta shRNA; II) a vector comprising a TGF- β encoding nucleic acid; III) a vector comprising a nucleic acid encoding IL-10; IV) a vector comprising both T-beta shRNA and TGF-beta encoding nucleic acid; v) a vector comprising both T-beta shRNA and IL-10 encoding nucleic acid; VI) a vector comprising both a TGF-beta encoding nucleic acid and an IL-10 encoding nucleic acid; VII) vectors comprising both T-beta shRNA, TGF-beta encoding nucleic acid, and IL-10 encoding nucleic acid; VIII) a vector combination comprising a vector of T-beta shRNA and a TGF-beta encoding nucleic acid, a vector comprising an IL-10 encoding nucleic acid; IX) a vector combination comprising a vector of T-beta shRNA and IL-10 encoding nucleic acid, a vector comprising TGF-beta encoding nucleic acid; x) vector combinations comprising T-beta shRNA vectors, vectors comprising TGF-beta encoding nucleic acid and IL-10 encoding nucleic acid; XI) vector combinations comprising T-beta shRNA vectors, vectors comprising TGF-beta encoding nucleic acids, and vectors comprising IL-10 encoding nucleic acids.

In a fourth aspect, the invention provides the use of a vector or combination of vectors according to the third aspect of the invention for inducing iTreg cells.

The invention has the beneficial effects that:

1) the single vector or the combined vector containing shRNA, mTGF-beta 1 and IL-10 targeting transcription factor T-beta is used for the naive CD4 ⁺ T induced iTreg, fine maintenance induced iTreg leave the phenotype and the functional stability of culture system after, induced iTreg has stronger immunosuppressive function than traditional induced iTreg.

2) The induction method provided by the invention can improve the induction efficiency of iTregs to a greater extent.

3) By transfecting the cell factor coding gene by the virus and carrying out exogenous expression in a host, the use amount of expensive cell factors in an induction culture system is saved, and the culture cost is reduced to the maximum extent.

4) The method provided by the invention provides higher feasibility for industrial amplification production of iTregs as clinical therapeutic cell drugs, and lays a good foundation for better application of iTregs in clinical therapy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 characteristic phenotypic analysis of in vitro expanded iTregs. FIG. 1A shows the detection of IFN-gamma secretion in iTreg culture supernatant by ELISA method, FIG. 1B shows the iTreg expression transcription factor Foxp3 and T-beta level at day 5 of culture, FIG. 1B shows the expression of iTreg cell Tbet and Foxp3 at the upper right and lower left, respectively, and the co-expression of Tbet and Foxp3 at the lower right.

FIG. 2, shT-beta regulation iTreg effect verification. FIG. 2A is a schematic diagram of the construction of an interfering RNA (shT-beta) plasmid carrying T-beta molecules, the constructed plasmid and a plasmid expressing T-beta molecules (carrying GFP reporter genes) are co-transfected into HEK293T cells (FIG. 2B) in 6 different proportions, the left image is transfected cells under a fluorescence microscope, and the right image is the expression inhibition rate of T-beta molecules.

Flow analysis of the inhibitory effect of shT-beta lentivirus infection on transcription factor T-beta in vitro expanded iTregs is shown in FIG. 2C, where cells in mCherry positive gate in the left panel of FIG. 2C represent iTreg cells with shT-beta lentivirus infection, and cells in mCherry negative gate represent uninfected control group; FIG. 2C right shows T-beta expression in the two groups of cells. IFN-. gamma.expression in the supernatants of both groups was detected by ELISA (FIG. 2D). shT-beta regulated flow detection results of iTreg inhibiting CD8 effector T cell proliferation are shown in figure 2E, and the abscissa is iTreg and CD8 ⁺ Mixing ratio of T cells, ordinate iTreg to CD8 ⁺ Inhibition of proliferation of T cells; the expression of IFN-gamma in the supernatants of the suppressor cells detected by ELISA is shown in FIG. 2F, with the abscissa of iTreg and CD8 ⁺ T cells were mixed at different ratios.

FIG. 3 shows the effect of combined regulation of iTreg by overexpression of mTGF-beta 1 and shT-beta. The mature TGF-. beta.sequence and the shT-beta sequence were ligated in tandem on the same vector (FIG. 3A), and the group of cells transduced with the constructed plasmid was designated SH-TGF. The expression levels of the transcription factors Foxp3 (FIG. 3B) and T-beta (FIG. 3C) in cells, as well as TGF- β in cell culture supernatant (FIG. 3D), were examined at various times during iTreg culture. The time of detection of Foxp3 was day 5, day 10 and day 30 of induction culture, both day 5. The inhibitory effect of induced iTreg cells on CD8 effector T cell proliferation is shown in fig. 3E, with non-induced iTreg cells as a control.

FIG. 4 validation of the effect of over-expressing IL-10 in modulating iTreg. The construction scheme of the IL-10 overexpression plasmid is shown in FIG. 4A; on the fifth day after IL-10-carrying lentiviruses infect iTregs, the flow analysis result of the intracellular IL-10 factor expression is shown in figure 4B, and the ELISA detection result of IL-10 secretion in cell supernatant is shown in figure 4C. The results of the RTCA experiment of killing a small cell lung cancer cell line overexpressing CD19 (NCI-H292-CD19) by iTreg suppressor T cells (CD19-CART) cultured on the tenth day are shown in FIG. 4D; the inhibition of CD8 effector T cell proliferation results are shown in fig. 4E, with the abscissa representing the effective target ratio and the abscissa representing the proliferation inhibition rate.

FIG. 5 shows that the effect of the TGF-beta and IL-10 dual factor combined regulation iTreg is verified. iTreg (TGF-beta) jointly regulated by TGF-beta and IL-10 double factors&IL-10-iTreg) on CD19-CART killing NCI-H292-CD19 cells as shown in FIG. 5A, the abscissa is time and the ordinate is NCI-292 cell proliferation parameters. FIG. 5B is TGF-. beta.&The result of the experiment that the IL-10-iTreg inhibits the proliferation of CD8 effector T cells, the abscissa is the effective target ratio, and the ordinate is the proliferation inhibition rate. FIG. 5C shows TGF-. beta.&Supernatant and CD8 secreted by IL-10-iTreg ⁺ Flow detection results of T cell co-incubation and further induction of apoptosis, abscissa co-incubation time, cell proportion of mid-stage apoptosis shown on the left of FIG. 5C, and cell proportion of late-stage apoptosis shown on the right of FIG. 5C.

FIG. 6 shows that the effect of shRNA, TGF-beta and IL-10 combined regulation iTreg on GVHD intervention is verified. The GVHD model construction and iTreg treatment are shown in fig. 6A, 2.0GY indicates irradiation of mice with gamma rays of 2.0 intensity, the experiment was divided into four groups, and the four groups of mice were irradiated with the same intensity for the same time, wherein: (1) irradiation control group: a control group which was irradiated with gamma rays only and did not receive cell return; (2) model group: human PBMC (3X 10) were returned alone ⁶ Control group of/only) (3) iTreg control group: human PBMC (3X 10) were reinfused ⁶ /+ iTreg cells (6X 10) carrying empty control lentivirus infection ⁶ Control group of/only) (4) SH-iTreg group: human PBMC (3X 10) were reinfused ⁶ (6X 10) + iTreg cells carrying sh-T-beta, TGF beta and IL-10 lentivirus infection ⁶ Experimental group/only). Fig. 6B reflects weight loss and fig. 6C reflects survival of mice in each group. FIG. 6D shows the pathology of the day 14 mice in each group, with tissues including skin, lung, liver and small intestine.

Detailed Description

The present invention is further described below, and the embodiments described in the present description are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and it is intended that all such changes and modifications be considered as within the scope of the invention.

Definition of terms

As used herein, the term "Treg" is a regulatory T cell, a subset of T cells that control autoimmune reactivity in vivo, and can be divided into naturally occurring regulatory T cells (ntregs) and inducible regulatory T cells (itrregs), such as Th3, Tr1, and further, CD8 tregs, NKT cells, etc., that are closely related to the development of autoimmune diseases.

As used herein, the term "T-beta", also known as "TBX 21(T-box transcription factor 21)" is a novel transcription factor of the T-box gene family, whose T-box binding domain contains 189 amino acids. T-beta selection is specifically expressed in Th1 cell, regulates its development and differentiation by initiating Th1 genetic program, and inhibits synthesis of Th2 type cell factor.

As used herein, the term "shRNA" is a "short hairpin RNA" comprising two short inverted repeats. The shRNA carried in the shRNA expression vector comprises two short inverted repeat sequences, the middle of the two short inverted repeat sequences is separated by a stem-loop sequence to form a hairpin structure, and the hairpin structure is controlled by a pol III promoter. Followed by 5-6 Ts as transcription terminators for RNA polymerase III. As used herein, "shT-beta" refers to a T-beta targeted shRNA.

As used herein, the term "TGF- β 1" cytokine is a member of the TGF- β family, and forms of TGF- β 1 existence include precursor molecules (per-pro-TGF- β 1), polypeptide chain precursors (pro-TGF- β 1, sometimes also referred to as LAP-TGF- β 1), and mature forms (mTGF- β 1), with the non-exclusive indication in the examples of this patent that TGF- β 1 is its mature form mTGF- β 1.

As used herein, the term "IL-10" is interleukin 10, a multi-cellular source, multifunctional cytokine that regulates the growth and differentiation of cells, participates in inflammatory and immune responses, and is a recognized inflammatory and immunosuppressive factor.

As used herein, the term "lentiviral vector" is a gene delivery vehicle based on human immunodeficiency virus type I (HIV-1), which is effective in infecting a variety of cell types, including neuronal, hepatic, cardiac, tumor, endothelial, stem cells, and the like, and has the ability to infect both dividing and non-dividing cells. The vector can effectively integrate the exogenous gene into the host chromosome, thereby achieving persistent expression.

As used herein, the term "CD 4 ⁺ T cells "are a type of T lymphocytes that can be divided into different classes or subpopulations based on the presence of a specific protein molecule on their surface. CD4+ T cells are surface CD4 ⁺ T lymphocytes of T molecules. As used herein, "naive CD4+ T cells" refers to naive CD4 ⁺ T cells, also called naive CD4+ T cells, mature in the thymus and migrate to peripheral lymphoid tissues such as spleen, lymph nodes, and the like.

As used herein, the term "ELISA" is an enzyme linked immunosorbent assay (enzyme linked immunosorbent assay) which refers to a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid support such as polystyrene, and an immune reaction is carried out using the specific binding of antigen to antibody.

As used herein, the term "RTCA" refers to a real-time label-free dynamic cell analysis technique by integrating a microelectronic cell sensor chip into the bottom of a cell detection plate to construct a cell impedance detection sensor system that tracks changes in cell morphology and proliferation and differentiation, dynamically and quantitatively in real time. When the adherent cells growing on the surface of the microelectrode cause the change of the interfacial impedance of the adherent electrodes, the change is related to the real-time functional state change of the cells, and biological information related to the physiological functions of the cells, including cell growth, extension, morphological change, death, adherence and the like, can be obtained through real-time dynamic electrode impedance detection.

The patent is described in further detail below with reference to the accompanying drawings and specific experiments. Unless otherwise indicated, all reagents, apparatus, devices and methods used in this patent are conventional and commercially available reagents, apparatus, devices and methods in the art.

The iTreg induction and the amplification culture in the embodiment and all the embodiments of the invention are carried out according to the following methods: naive CD4 was sorted from PBMC by magnetic bead negative selection kit (Stemcell, 17555) ⁺ T cells. Obtain naive CD4 ⁺ After T cells, induction culture of iTreg was performed. The induction culture medium comprises IL-2500 IU/ml (Peprotech, AF-200-02-1MG), TGF-beta 5ng/ml (Peprotech, 100-21-10UG), rapamycin 100nM (Selleck, S1039), a basic culture medium X-VIVO-15(Lonza, 04-418Q) is added, and the induction culture medium is used for culture five days before induction; amplification medium: IL-2500 IU/ml and rapamycin 100nM are added into a basic culture medium X-VIVO-15, and after lentivirus transduction, an amplification culture medium is supplemented in half every other day; naive CD4 separated from magnetic beads ⁺ T cells were counted and induction medium was used to adjust cell density to the desired density. The cell suspension was added to a prepared 96-well plate, 50. mu.l of cell suspension per well. Approximately 110,000 and 130,000 cells per well, in a total volume of 200. mu.l. The beads were added with CD3/28 beads at a 1:1 ratio to the cells, which were then replenished once a week. After 2-3 days of culture, the growth of the cells was observed under a microscope, at which time small cell masses were observed. When cultured to a suitable time point, samples can be taken for RNA extraction or flow cytometric assay, followed by lentiviral transduction according to different experimental objectives.

Lentiviral packaging and transduction in the embodiments of the invention, all examples, was performed as follows.

And (3) slow virus packaging: freshly resuscitated HEK293T (ATCC) cells in a 6cm cell culture dish, and the cells grown in the dish at a ratio of 1:4 (about 1X 10) 24h before transfection ⁷ ) Transfection was initiated at a growth density of about 60% for 293T cells passaged on 10cm cell culture dishes. Half-replaced 5ml of fresh cell culture medium 2h before transfection. A clean 1.5ml EP tube (labeled tube A) was taken and 30ul of MIRUS solution was added. Another clean EP tube (labeled as tube B) was added with 30ug of a DNA mixture (containing the desired plasmid, and the structure is shown in FIG. 2A, FIG. 3A, and FIG. 4B). Mixing the liquid of tube B and tube A, standing at room temperature for 15min, adding dropwise the mixture into 10cm cell culture dish, slightly mixing, standing at 37 deg.C and 5% CO ₂ Culturing in an incubator. After 6h of transfection, the medium was changed to 15ml fresh medium in a biosafety cabinet. Collecting cell culture medium containing virus 48h after transfection in 50ml centrifuge tube, centrifuging at 4 deg.C and 2500rpm for 5min, collecting supernatant, and collectingThe mixture was filtered through a 0.45 μm filter. The supernatant was centrifuged at 27000rpm for 2h at 4 ℃. After the end of the process, the supernatant was discarded, the centrifuge tube was placed upside down on a paper towel, dried in a biosafety cabinet for 5min, then the virus precipitate was dissolved in 50. mu.l of serum-free DMEM, and placed in a refrigerator at 4 ℃ overnight. HEK293T cells (12-well plate, 10) were used ⁵ Cells/well) for virus titer detection.

Lentivirus transduction: mixing 5E5 (5X 10) ⁵ ) Personal/well stimulation of day one naive CD4 ⁺ T is placed in a 96U bottom plate, and 50 ul/well of the concentrated virus solution is taken. Centrifuging for 2h at 1000g, removing supernatant in the holes, replacing new induction culture medium, and performing induction culture. The first induced iTreg, the control group infected with the unloaded plasmid lentivirus, and the experimental group infected with the shT-beta fragment plasmid (as shown in FIG. 2A, see SEQ ID NO:15) lentivirus. The second induction culture of iTreg, control infected with plasmid-free lentivirus, experimental infected with a lentivirus containing both shT-beta fragment and a plasmid containing mTGF-beta 1(SEQ ID NO:11) nucleic acid sequence (as shown in FIG. 3A). Wherein the sequence of shRNA obtained by transcription of the shT-beta fragment plasmid is shown in SEQ ID NO. 3-8. In the third batch of induced cultured iTregs, the control group was infected with lentivirus carrying plasmid, and the control group was simultaneously infected with two lentiviruses respectively containing shTbeta fragment and mTGF-beta 1 nucleic acid sequence (as shown in FIG. 3A) and plasmid carrying IL-10 nucleic acid sequence alone (as shown in FIG. 4A).

The experiments for inhibiting proliferation and killing of iTreg and the experiments for inhibiting killing are carried out according to the following methods.

Proliferation inhibition assay: separation of CD8 from human peripheral blood PBMC by magnetic bead sorting ⁺ A T cell; CD8 ⁺ T cells were stained with efluor670 (Thermofeisher, 65-0840-90) proliferation fuel; CD3/28 stimulation magnetic beads and CD8 ⁺ T cells are stimulated by mixing 1:10 in a system of 100 mu L in a 5E 4/hole and 96U bottom hole plate; 50 mu L of diluted iTreg with the same ratio as the iTreg and CD8 are added into each hole ⁺ T is 1:1, 1:2,1:4, 1:8, 1:16, 1:32, 1:64, 1: 128; in addition, a hole without magnetic bead stimulation CD8 is separated ⁺ T cells, and magnetic bead-alone stimulated CD8 ⁺ T cells, 150 ul/well; after 5 days of incubation, CD4 flow staining was performed, and flow staining was performedAnd (4) detecting.

Inhibition and killing experiment: the small cell lung cancer cell line NCI-H292 overexpressing CD19 was plated in RTCA well plates at 1E 4/well; effector cells chimeric to the CD19 antigen receptor (CD19-CART) were incubated with iTreg cells for 24 hours at a cell-to-cell ratio of: CD 19-CART: NCI-H292-CD19 cells were 2:1, the ratio of iTreg cells to CD19-CART cells was 1:2,1:4, 1: 8; the cells that completed the co-incubation were added together to the RTCA well plates plated with NCI-H292-CD19 cells and the data results were analyzed after 24 hours.

Example 1 characteristic phenotypic analysis of in vitro expanded iTregs

According to the characteristic that iTregs are unstable under the stimulation of inflammatory environment, the inflammatory environment is simulated by adding 50ng/ml IL-6 proinflammatory cytokines into an iTreg in-vitro amplification system, and IFN-gamma secreted by the iTregs is detected by an ELISA method, and the result is shown in figure 1A. One week after induction, the expression of transcription factors such as Foxp3, Tbet, etc. of iTreg was detected by flow assay, as shown in FIG. 1B. From the results in fig. 1, it can be seen that the conventionally induced iTreg can secrete a certain amount of IFN- γ at the initial stage of induction, and there are a large population of cells in which Foxp3 and Tbet are co-expressed, which further verifies the instability deep molecular mechanism of the iTreg that it is prone to polarization to Th1 pro-inflammatory cells in an inflammatory environment and further loses Foxp3 expression.

Example 2 shT-beta Regulation iTreg Effect verification

Aiming at the unstable factor that iTreg is easy to polarize towards Th1 or secrete Th 1-class related proinflammatory cytokines in an inflammatory environment, a hairpin-structure interference RNA (shT-beta) aiming at a Th1 characteristic transcription factor Tbeta gene is constructed, and the interference effect of shT-beta is indicated by using a reporter gene mCherry shown in figure 2A. shT-beta plasmid and T-beta plasmid with EGFP reporter gene are jointly transfected into HEK293T cells, if shT-beta has interference effect on T-beta target sequence, the EGFP protein expression strength of the T-beta expression level can be directly reported to indicate, so that the interference efficiency of shT-beta on T-beta is verified, and the constructed shT-beta has strong inhibition effect on exogenous over-expressed T-beta as shown in figure 2B.

For further verification of shT-beta internal sourceT-beta inhibitory Effect of sex, we will sort the negative CD4 ⁺ The following day of induction of T cells into iTreg and stimulation with magnetic beads, infecting the lentivirus with shT-beta vector by the lentivirus system, removing the magnetic beads on the third day of stimulation, and detecting the expression level of T-beta by flow-type after culturing for 5 days, the result is shown in FIG. 2C, wherein GFP is used as the reference ⁺ The subpopulation indicates cells successfully expressing shT-beta interfering RNA, GFP ^- The subgroup indicates cells which do not successfully express shT-beta interfering RNA, the result in the figure shows that shTbeta also has a strong inhibitory effect on endogenous T-beta, and the situation that IFN-gamma is secreted in iTreg supernatant is further detected by ELISA, as shown in figure 2D, the shT-beta interfering group hardly secretes IFN-gamma.

To further verify the functional superiority of T-beta interference, shT-beta-iTreg and WT-iTreg are subjected to inhibition tests, and the results show that the shT-beta-iTreg inhibition effect is superior (FIG. 2E), and the amount of IFN-gamma secreted by iTreg under the inflammatory environment is obviously reduced (FIG. 2F).

Example 3 verification of the Effect of over-expressing mTGF-beta 1 and shT-beta combined regulation iTreg

We designed a nucleic acid sequence expressing TGF-beta 1 mature peptide, which is composed of signal peptide and TGF-beta 1 mature peptide and named as mTGF-beta, and constructed the nucleic acid sequence on shT-beta expression vector (figure 3A), and tested the stable expression maintaining effect of mTGF-beta on Foxp3, the long-term stable expression of mTGF-beta on Foxp3, and after 5 days of infecting over-expression virus vector, remove TGF-beta 1 cell factor in culture medium system, and cultured for one month, compared with WT group, the mTGF-beta group has obvious maintaining advantage on Foxp3 stable expression, and the result is shown in figure 3B. Furthermore, the shTbet inhibition effect of the flow detection is more remarkable (fig. 4C). ELISA detected significant increase in mTGF- β overexpression levels (fig. 3D). And the mTGF-beta overexpression is improved on the iTreg inhibition function, and the result is shown in figure 3E.

Example 4 validation of the Effect of over-expressing IL-10 regulatory iTregs

To further improve the inhibitory ability of iTreg, we cloned the CDS region of IL-10 gene into a lentiviral vector and constructed as shown in fig. 4A. And after successful transduction and induction of iTreg cells for one week, IL-10 expression was detected by ELISA (fig. 4B) and flow assay (fig. 4C). The result shows that the IL-10 expression vector has a good overexpression effect, the functional effect of over-expression IL-10 generation is further verified, an RTCA inhibition and killing test is carried out, the result is shown in 4D, the traditional induced iTreg has almost no effect of inhibiting the specificity killing of allogenic CAR-T, and the iTreg group of over-expression IL-10 has a good inhibition and killing effect. Moreover, the ability of the iTreg cells overexpressing IL-10 to suppress the proliferation of effector cells was also significantly improved (FIG. 4E).

Example 5 Effect verification of TGF-beta and IL-10 double-factor combined regulation iTreg

In order to further improve the stability and the inhibition effect of iTreg, the CDS region of the IL-10 gene and the mTGF-beta gene are jointly cloned on a lentiviral vector, and lentiviral transduction is carried out to obtain TGF-beta of a co-expression double cell factor&IL-10-iTreg, RTCA inhibitory killing assay showed (FIG. 5A), TGF-beta specific killing effect of inhibiting allogeneic CAR-T compared to traditionally induced iTreg cells&The IL-10-iTreg group had significantly greater inhibitory potency. We performed proliferation inhibition experiments with the effective target ratios 1:64 and 1:128, as shown in FIG. 5B, from CD8 ⁺ According to the experimental result of T cell proliferation inhibition, the two-factor group has obvious inhibition function advantage, and meanwhile, in order to detect the iTreg paracrine function of over-expressing IL-10 and TGF-beta, the iTreg cells are cultured for 3 days, and supernatant and CD8 are obtained ⁺ T cells were co-incubated and tested for apoptosis, as shown in FIG. 5C, and we found TGF-. beta.&The IL-10-iTreg group entered metaphase apoptosis at the earliest day 3, TGF-. beta.at day 5&The late apoptosis of the IL-10-iTreg group is obviously higher than that of the control group, which shows that the combination of two factors greatly improves the lateral secretion inhibition function of the iTreg.

Example 6 validation of Effect of shRNA, TGF-beta and IL-10 combined regulation iTreg on intervention GVHD

The SH-iTreg induced by T-beta interference, TGF beta and IL-10 overexpression method and the traditional control iTreg in the patent are used for evaluating the treatment effect of the mouse graft-versus-host (GVHD), and fig. 6A shows the construction of a GVHD model and the iTreg treatment method.

The specific implementation steps are as follows: in the orbital veinIrradiating NSG mice with 2.0G gamma rays 1 day before injection, and weighing the first weight; the normal group is a group which only receives irradiation and does not return cells; PBMC (3X 10) from human peripheral blood mononuclear cells ⁶ Mice) and iTreg (6X 10) ⁶ Mice) mixed injection; weigh and record GVHD symptoms every other day until day 20; peripheral blood was drawn on days 7, 14 and one month for flow staining; 3 mice per group were sacrificed on day 14 and skin, lung, liver and small intestine were taken for immunohistochemical staining. The weight and survival rate of the model group only transfused with PBMC and the iTreg control group are in obvious descending trend, the GVHD mice treated by the SH-iTreg group induced by the method provided by the invention have lower weight descending rate (figure 6B), and the survival condition of the mice is obviously improved (figure 6C).

Model group mice infused with PBMC showed significant GVHD, with lymphocyte infiltration in the liver and lung, severe atrophy of small intestine villi, severe discontinuation of skin basal layer cells, lymphocyte infiltration in the liver and lung of iTreg control group, even more severe small intestine villi, severe separation of skin layers, severe discontinuation of basal layer cells, depletion of adipose layer, SH-iTreg showed the lightest pathological symptoms of GVHD, as shown in fig. 6D.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Zhongshan Hospital affiliated to Fudan university

<120> a method for improving the stability and function of regulatory T cells

<130> 202203

<160> 15

<170> PatentIn version 3.3

<210> 1

<211> 2583

<212> DNA

<213> Homo sapiens

<400> 1

agtgacagcg gcccgctgga gaggaagccc gagagctgcc gcgcgcctgc cggacgaggg 60

cgtagaagcc aggcgtcaga gcccgggctc cggtggggtc ccccacccgg ccctcgggtc 120

ccccgccccc tgctccctgc ccatcccagc ccacgcgacc ctctcgcgcg cggaggggcg 180

ggtcctcgac ggctacggga aggtgccagc ccgccccgga tgggcatcgt ggagccgggt 240

tgcggagaca tgctgacggg caccgagccg atgccgggga gcgacgaggg ccgggcgcct 300

ggcgccgacc cgcagcaccg ctacttctac ccggagccgg gcgcgcagga cgcggacgag 360

cgtcgcgggg gcggcagcct ggggtctccc tacccggggg gcgccttggt gcccgccccg 420

ccgagccgct tccttggagc ctacgcctac ccgccgcgac cccaggcggc cggcttcccc 480

ggcgcgggcg agtccttccc gccgcccgcg gacgccgagg gctaccagcc gggcgagggc 540

tacgccgccc cggacccgcg cgccgggctc tacccggggc cgcgtgagga ctacgcgcta 600

cccgcgggac tggaggtgtc ggggaaactg agggtcgcgc tcaacaacca cctgttgtgg 660

tccaagttta atcagcacca gacagagatg atcatcacca agcagggacg gcggatgttc 720

ccattcctgt catttactgt ggccgggctg gagcccacca gccactacag gatgtttgtg 780

gacgtggtct tggtggacca gcaccactgg cggtaccaga gcggcaagtg ggtgcagtgt 840

ggaaaggccg agggcagcat gccaggaaac cgcctgtacg tccacccgga ctcccccaac 900

acaggagcgc actggatgcg ccaggaagtt tcatttggga aactaaagct cacaaacaac 960

aagggggcgt ccaacaatgt gacccagatg attgtgctcc agtccctcca taagtaccag 1020

ccccggctgc atatcgttga ggtgaacgac ggagagccag aggcagcctg caacgcttcc 1080

aacacgcata tctttacttt ccaagaaacc cagttcattg ccgtgactgc ctaccagaat 1140

gccgagatta ctcagctgaa aattgataat aacccctttg ccaaaggatt ccgggagaac 1200

tttgagtcca tgtacacatc tgttgacacc agcatcccct ccccgcctgg acccaactgt 1260

caattccttg ggggagatca ctactctcct ctcctaccca accagtatcc tgttcccagc 1320

cgcttctacc ccgaccttcc tggccaggcg aaggatgtgg ttccccaggc ttactggctg 1380

ggggcccccc gggaccacag ctatgaggct gagtttcgag cagtcagcat gaagcctgca 1440

ttcttgccct ctgcccctgg gcccaccatg tcctactacc gaggccagga ggtcctggca 1500

cctggagctg gctggcctgt ggcaccccag taccctccca agatgggccc ggccagctgg 1560

ttccgcccta tgcggactct gcccatggaa cccggccctg gaggctcaga gggacgggga 1620

ccagaggacc agggtccccc cttggtgtgg actgagattg cccccatccg gccggaatcc 1680

agtgattcag gactgggcga aggagactct aagaggaggc gcgtgtcccc ctatccttcc 1740

agtggtgaca gctcctcccc tgctggggcc ccttctcctt ttgataagga agctgaagga 1800

cagttttata actattttcc caactgagca gatgacatga tgaaaggaac agaaacagtg 1860

ttattaggtt ggaggacacc gactaatttg ggaaacggat gaaggactga gaaggccccc 1920

gctccctctg gcccttctct gtttagtagt tggttgggga agtggggctc aagaaggatt 1980

ttggggttca ccagatgctt cctggcccac gatgaaacct gagaggggtg tccccttgcc 2040

ccatcctctg ccctaactac agtcgtttac ctggtgctgc gtcttgcttt tggtttccag 2100

ctggagaaaa gaagacaaga aagtcttggg catgaaggag ctttttgcat ctagtgggtg 2160

ggaggggtca ggtgtgggac atgggagcag gagactccac tttcttcctt tgtacagtaa 2220

ctttcaacct tttcgttggc atgtgtgtta atccctgatc caaaaagaac aaatacacgt 2280

atgttataac catcagcccg ccagggtcag ggaaaggact cacctgactt tggacagctg 2340

gcctgggctc cccctgctca aacacagtgg ggatcagaga aaaggggctg gaaagggggg 2400

aatggcccac atctcaagaa gcaagatatt gtttgtggtg gttgtgtgtg ggtgtgtgtt 2460

ttttcttttt ctttcttttt attttttttg aatgggggag gctatttatt gtactgagag 2520

tggtgtctgg atatattcct tttgtcttca tcactttctg aaaataaaca taaaactgtt 2580

gaa 2583

<210> 2

<211> 535

<212> PRT

<213> Homo sapiens

<400> 2

Met Gly Ile Val Glu Pro Gly Cys Gly Asp Met Leu Thr Gly Thr Glu

1 5 10 15

Pro Met Pro Gly Ser Asp Glu Gly Arg Ala Pro Gly Ala Asp Pro Gln

20 25 30

His Arg Tyr Phe Tyr Pro Glu Pro Gly Ala Gln Asp Ala Asp Glu Arg

35 40 45

Arg Gly Gly Gly Ser Leu Gly Ser Pro Tyr Pro Gly Gly Ala Leu Val

50 55 60

Pro Ala Pro Pro Ser Arg Phe Leu Gly Ala Tyr Ala Tyr Pro Pro Arg

65 70 75 80

Pro Gln Ala Ala Gly Phe Pro Gly Ala Gly Glu Ser Phe Pro Pro Pro

85 90 95

Ala Asp Ala Glu Gly Tyr Gln Pro Gly Glu Gly Tyr Ala Ala Pro Asp

100 105 110

Pro Arg Ala Gly Leu Tyr Pro Gly Pro Arg Glu Asp Tyr Ala Leu Pro

115 120 125

Ala Gly Leu Glu Val Ser Gly Lys Leu Arg Val Ala Leu Asn Asn His

130 135 140

Leu Leu Trp Ser Lys Phe Asn Gln His Gln Thr Glu Met Ile Ile Thr

145 150 155 160

Lys Gln Gly Arg Arg Met Phe Pro Phe Leu Ser Phe Thr Val Ala Gly

165 170 175

Leu Glu Pro Thr Ser His Tyr Arg Met Phe Val Asp Val Val Leu Val

180 185 190

Asp Gln His His Trp Arg Tyr Gln Ser Gly Lys Trp Val Gln Cys Gly

195 200 205

Lys Ala Glu Gly Ser Met Pro Gly Asn Arg Leu Tyr Val His Pro Asp

210 215 220

Ser Pro Asn Thr Gly Ala His Trp Met Arg Gln Glu Val Ser Phe Gly

225 230 235 240

Lys Leu Lys Leu Thr Asn Asn Lys Gly Ala Ser Asn Asn Val Thr Gln

245 250 255

Met Ile Val Leu Gln Ser Leu His Lys Tyr Gln Pro Arg Leu His Ile

260 265 270

Val Glu Val Asn Asp Gly Glu Pro Glu Ala Ala Cys Asn Ala Ser Asn

275 280 285

Thr His Ile Phe Thr Phe Gln Glu Thr Gln Phe Ile Ala Val Thr Ala

290 295 300

Tyr Gln Asn Ala Glu Ile Thr Gln Leu Lys Ile Asp Asn Asn Pro Phe

305 310 315 320

Ala Lys Gly Phe Arg Glu Asn Phe Glu Ser Met Tyr Thr Ser Val Asp

325 330 335

Thr Ser Ile Pro Ser Pro Pro Gly Pro Asn Cys Gln Phe Leu Gly Gly

340 345 350

Asp His Tyr Ser Pro Leu Leu Pro Asn Gln Tyr Pro Val Pro Ser Arg

355 360 365

Phe Tyr Pro Asp Leu Pro Gly Gln Ala Lys Asp Val Val Pro Gln Ala

370 375 380

Tyr Trp Leu Gly Ala Pro Arg Asp His Ser Tyr Glu Ala Glu Phe Arg

385 390 395 400

Ala Val Ser Met Lys Pro Ala Phe Leu Pro Ser Ala Pro Gly Pro Thr

405 410 415

Met Ser Tyr Tyr Arg Gly Gln Glu Val Leu Ala Pro Gly Ala Gly Trp

420 425 430

Pro Val Ala Pro Gln Tyr Pro Pro Lys Met Gly Pro Ala Ser Trp Phe

435 440 445

Arg Pro Met Arg Thr Leu Pro Met Glu Pro Gly Pro Gly Gly Ser Glu

450 455 460

Gly Arg Gly Pro Glu Asp Gln Gly Pro Pro Leu Val Trp Thr Glu Ile

465 470 475 480

Ala Pro Ile Arg Pro Glu Ser Ser Asp Ser Gly Leu Gly Glu Gly Asp

485 490 495

Ser Lys Arg Arg Arg Val Ser Pro Tyr Pro Ser Ser Gly Asp Ser Ser

500 505 510

Ser Pro Ala Gly Ala Pro Ser Pro Phe Asp Lys Glu Ala Glu Gly Gln

515 520 525

Phe Tyr Asn Tyr Phe Pro Asn

530 535

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

gtggaggtgt gtgaagtta 19

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

taacttcaca cacctccac 19

<210> 5

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

tgctcgaatt gacagaaaa 19

<210> 6

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

ttttctgtca attcgagca 19

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

catccttcga gttgaatat 19

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

atattcaact cgaaggatg 19

<210> 9

<211> 1173

<212> DNA

<213> Homo sapiens

<400> 9

atgccgccct ccgggctgcg gctgctgccg ctgctgctac cgctgctgtg gctactggtg 60

ctgacgcctg gccggccggc cgcgggacta tccacctgca agactatcga catggagctg 120

gtgaagcgga agcgcatcga ggccatccgc ggccagatcc tgtccaagct gcggctcgcc 180

agccccccga gccaggggga ggtgccgccc ggcccgctgc ccgaggccgt gctcgccctg 240

tacaacagca cccgcgaccg ggtggccggg gagagtgcag aaccggagcc cgagcctgag 300

gccgactact acgccaagga ggtcacccgc gtgctaatgg tggaaaccca caacgaaatc 360

tatgacaagt tcaagcagag tacacacagc atatatatgt tcttcaacac atcagagctc 420

cgagaagcgg tacctgaacc cgtgttgctc tcccgggcag agctgcgtct gctgaggctc 480

aagttaaaag tggagcagca cgtggagctg taccagaaat acagcaacaa ttcctggcga 540

tacctcagca accggctgct ggcacccagc gactcgccag agtggttatc ttttgatgtc 600

accggagttg tgcggcagtg gttgagccgt ggaggggaaa ttgagggctt tcgccttagc 660

gcccactgct cctgtgacag cagggataac acactgcaag tggacatcaa cgggttcact 720

accggccgcc gaggtgacct ggccaccatt catggcatga accggccttt cctgcttctc 780

atggccaccc cgctggagag ggcccagcat ctgcaaagct cccggcaccg ccgagccctg 840

gacaccaact attgcttcag ctccacggag aagaactgct gcgtgcggca gctgtacatt 900

gacttccgca aggacctcgg ctggaagtgg atccacgagc ccaagggcta ccatgccaac 960

ttctgcctcg ggccctgccc ctacatttgg agcctggaca cgcagtacag caaggtcctg 1020

gccctgtaca accagcataa cccgggcgcc tcggcggcgc cgtgctgcgt gccgcaggcg 1080

ctggagccgc tgcccatcgt gtactacgtg ggccgcaagc ccaaggtgga gcagctgtcc 1140

aacatgatcg tgcgctcctg caagtgcagc tga 1173

<210> 10

<211> 390

<212> PRT

<213> Homo sapiens

<400> 10

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu

1 5 10 15

Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr

20 25 30

Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala

35 40 45

Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser

50 55 60

Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu

65 70 75 80

Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu

85 90 95

Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu

100 105 110

Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr

115 120 125

His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val

130 135 140

Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu

145 150 155 160

Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn

165 170 175

Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser

180 185 190

Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu

195 200 205

Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys Ser

210 215 220

Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe Thr

225 230 235 240

Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro

245 250 255

Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln

260 265 270

Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser

275 280 285

Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys

290 295 300

Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn

305 310 315 320

Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr

325 330 335

Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala

340 345 350

Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr

355 360 365

Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val

370 375 380

Arg Ser Cys Lys Cys Ser

385 390

<210> 11

<211> 423

<212> DNA

<213> Homo sapiens

<400> 11

atgccaccta gcggcctgag actgctccca ctgctcctcc ctctcctctg gctcctggtg 60

ctcacacccg gcagaccagc cgccggcctc gacacaaact actgcttctc tagcaccgag 120

aagaactgct gcgtgagaca gctgtacatt gacttccgca aggacctggg ctggaagtgg 180

attcacgagc ctaagggcta ccacgccaac ttctgcctgg gcccttgccc atacatttgg 240

agcctcgaca cacagtactc taaggtgctc gccctgtaca accagcacaa ccccggcgcc 300

agcgccgccc cttgctgcgt gcctcaggcc ctcgaaccac tgcctatcgt gtactacgtg 360

ggcaggaagc ctaaggtgga gcagctgtct aacatgatcg tgcggtcttg caagtgctct 420

tga 423

<210> 12

<211> 141

<212> PRT

<213> Homo sapiens

<400> 12

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu

1 5 10 15

Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Ala Leu Asp

20 25 30

Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg Gln

35 40 45

Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu

50 55 60

Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile

65 70 75 80

Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln

85 90 95

His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro Gln Ala Leu

100 105 110

Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu

115 120 125

Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser

130 135 140

<210> 13

<211> 537

<212> DNA

<213> Homo sapiens

<400> 13

atgcacagct cagcactgct ctgttgcctg gtcctcctga ctggggtgag ggccagccca 60

ggccagggca cccagtctga gaacagctgc acccacttcc caggcaacct gcctaacatg 120

cttcgagatc tccgagatgc cttcagcaga gtgaagactt tctttcaaat gaaggatcag 180

ctggacaact tgttgttaaa ggagtccttg ctggaggact ttaagggtta cctgggttgc 240

caagccttgt ctgagatgat ccagttttac ctggaggagg tgatgcccca agctgagaac 300

caagacccag acatcaaggc gcatgtgaac tccctggggg agaacctgaa gaccctcagg 360

ctgaggctac ggcgctgtca tcgatttctt ccctgtgaaa acaagagcaa ggccgtggag 420

caggtgaaga atgcctttaa taagctccaa gagaaaggca tctacaaagc catgagtgag 480

tttgacatct tcatcaacta catagaagcc tacatgacaa tgaagatacg aaactga 537

<210> 14

<211> 178

<212> PRT

<213> Homo sapiens

<400> 14

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn

<210> 15

<211> 911

<212> DNA

<213> Homo sapiens

<400> 15

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

ccgcgcgatc cgtggaggtg tgtgaagtta ttcaagagat aacttcacac acctccactt 300

ttttgggagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 360

agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 420

cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 480

tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 540

aaaggaccca gcgatcctgc tcgaattgac agaaaattca agagattttc tgtcaattcg 600

agcatttttt ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 660

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 720

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 780

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 840

tgtggaaagg acgatcccat ccttcgagtt gaatatttca agagaatatt caactcgaag 900

gatgtttttt a 911

Claims (16)

1. A method for increasing induction, phenotypic stability and immunomodulatory function of a regulatory T immune cell comprising inhibiting expression of transcription factor T-beta or knocking out TBX21 gene.

2. The method of claim 1, wherein the nucleic acid encoding the transcription factor T-beta comprises SEQ ID NO 1, a degenerate sequence thereof, or a spliceosome nucleotide sequence thereof.

3. The method of claims 1-2, wherein the transcription factor T-beta protein comprises the amino acid sequence of SEQ ID No. 2, a spliceosome thereof, or a variant thereof.

4. The method of claim 1, wherein the method or product for inhibiting the expression of the transcription factor T-beta is selected from the group consisting of small RNAs or small RNAs of multi-target multi-hairpin structures, ribozymes, gene targeting, protein inhibitors and compound inhibitors; preferably, siRNA interference through hairpin structure; more preferably, the siRNA has a sequence shown in SEQ ID NO 3-8.

5. The method according to claim 1, wherein the method or product for knocking out the TBX21 gene is selected from CRISPR/Cas9 technology, zinc finger nucleases, TALENs.

6. The method of claims 1-5, further comprising the step of adding or overexpressing a combination of one or more cytokines.

7. The method of claim 6, wherein the cytokine is selected from the group consisting of natural or recombinant interleukins, interferons, colony stimulating factors, erythropoietins, epidermal growth factors, nerve growth factors, basic fibroblast growth factors, tumor necrosis factors, thrombopoietin, platelet-derived growth factors, stem cell factors, vascular endothelial growth factors; preferably, the additional or overexpressed cytokine is TGF- β, IL-10, or a combination thereof.

8. The method according to claim 7, wherein the TGF- β is selected from TGF- β 1, TGF- β 2, TGF- β 3 and their corresponding precursor molecules (per-pro-TGF- β), polypeptide chain precursors (pro-TGF- β) and mature forms (mTGF- β); preferably, the TGF-beta is TGF-beta 1, the encoding nucleic acid of the TGF-beta 1 comprises SEQ ID NO 9, a degenerate sequence thereof or a spliceosome nucleotide sequence thereof, and the TGF-beta 1 protein comprises an amino acid sequence of SEQ ID NO 10, a spliceosome or a variant thereof; more preferably, the TGF- β is mTGF- β 1, the mTGF- β 1 encoding nucleic acid comprises SEQ ID NO 11, a degenerate sequence thereof, or a splice nucleotide sequence thereof, and the mTGF- β 1 protein comprises SEQ ID NO 12, a splice thereof, or a variant amino acid sequence thereof.

9. The method according to claim 7, characterized in that said IL-10 encoding nucleic acid has the nucleotide sequence of SEQ ID NO 13, a degenerate sequence thereof or a spliceosome thereof, and said IL-10 protein comprises the amino acid sequence shown in SEQ ID NO 14, a spliceosome thereof or a variant thereof.

10. The method of any one of claims 1-8, further comprising overexpression, interference, or knock-out of 1 or more additional genes in combination, wherein the overexpressed genes comprise BCL-6 and the interfered or knocked-out genes comprise GATA-3, ROR γ t, ROR α.

11. The method of any one of claims 1 to 10, wherein the method further expresses the homing molecule CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1 or a combination of homing molecules to promote Treg chemotaxis in vivo to the disease site and to increase the therapeutic effect; preferably, the homing molecule is expressed on a viral vector or an mRNA vector.

12. The method according to any one of claims 1 to 11, wherein the method further expresses or links a molecule capable of recognizing a disease site or transplanted organ specific target, preferably wherein the molecule is a chimeric antigen receptor molecule, a ligand for a tissue specific receptor.

13. The method of any one of claims 1-12, wherein the interfering or knockout sequences, overexpressed genes, cytokine combinations, can be expressed separately in combination after different vectors, or multiple genes or interfering sequences can be expressed simultaneously in the same vector; the vector comprises an adenovirus vector, an adeno-associated virus vector, a pox virus vector, a retrovirus vector, a herpes virus vector, an RNA virus vector, an EB virus vector, a baculovirus vector, a phage vector, an animal virus vector, a plant virus vector, a DNA plasmid vector and an RNA vector, preferably the vector is a DNA plasmid vector, and more preferably the vector is a lentivirus vector.

14. Use of the regulatory T immune cells cultured according to the method of claims 1-13 for the preparation of a medicament for the treatment of autoimmune diseases, inducing immune tolerance after organ transplantation, wherein the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, type I diabetes, autoimmune hepatitis, polyneuritis, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia; the organ transplantation comprises allogenic and xenogenic heart, liver and kidney transplantation; the regulatory T immune cells can be used alone or in combination with other drugs.

15. A vector or combination of vectors for use in increasing the stability of a regulatory T immune cell, wherein said vector or combination of vectors is selected from the group consisting of: I) a vector comprising a T-beta shRNA; II) a vector comprising a TGF- β encoding nucleic acid; III) a vector comprising a nucleic acid encoding IL-10; IV) a vector comprising both T-beta shRNA and TGF-beta encoding nucleic acid; v) a vector comprising both T-beta shRNA and IL-10 encoding nucleic acid; VI) a vector comprising both a TGF-beta encoding nucleic acid and an IL-10 encoding nucleic acid; VII) vectors comprising both T-beta shRNA, TGF-beta encoding nucleic acid, and IL-10 encoding nucleic acid; VIII) a vector combination comprising a vector of T-beta shRNA and a TGF-beta encoding nucleic acid, a vector comprising an IL-10 encoding nucleic acid; IX) a vector combination comprising a vector of T-beta shRNA and IL-10 encoding nucleic acid, a vector comprising TGF-beta encoding nucleic acid; x) vector combinations comprising first a T-beta shRNA vector, a vector comprising a TGF-beta encoding nucleic acid and an IL-10 encoding nucleic acid; XI) vector combinations comprising T-beta shRNA vectors, vectors comprising TGF-beta encoding nucleic acids, and vectors comprising IL-10 encoding nucleic acids.

16. Use of the vector or vector combination of claim 15 for inducing T immune cells of the regulatory type.

Images