Novel method for promoting induction of regulatory T cells

Abstract

The invention provides a novel method for promoting induction of regulatory T cells. According to the method for preparing the pre-programmed T cell population with the Treg cell differentiation tendency, stem cells are cultured in the presence of an SCD1 inhibitor, and the obtained pre-programmed T cell population can be rapidly and efficiently differentiated into a large number of Treg cells in vivo or in vitro.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a new method for promoting the induction of regulatory T cells. Background Art

[0002] Since the discovery of the specific expression of transcription factor Foxp3 in Treg in 2003, scientists have used Foxp3 mutation or functional loss to confirm the important role of Foxp3 in Treg cell differentiation and function. Foxp3 mutant mice die from severe, systemic autoimmune / inflammatory diseases. Patients with familial CTLA-4 heterologous mutations also suffer from autoimmune / inflammatory diseases, which is closely related to the dysfunction of Treg inhibitory function caused by low expression of CTLA-4 in Treg, and CTLA-4 neutralizing antibodies significantly downregulate Foxp3 in tumors. + CD4 + The number of Tregs. Therefore, in recent years, immunotherapy has gradually focused on the regulatory role of Tregs in the occurrence and development of many diseases, and targeting Tregs to treat autoimmune diseases, infections, tumors, and cardiovascular diseases has gradually become a new direction for clinical cell therapy. Reducing pathogenic effector T cells and memory T cells while restoring peripheral immune tolerance is one of the treatment strategies for autoimmune diseases. At present, many programs based on the immune regulation function of Tregs have been developed. For example, targeting specific dysfunctional molecules or signaling pathways in the existing Treg cell pool in patients with autoimmune diseases has application potential for the treatment of autoimmune diseases. In addition, immunotherapy in which Tregs are expanded in vitro and then adopted and transferred to patients with autoimmune diseases has achieved certain results in the treatment of type 1 diabetes, lupus erythematosus, and Crohn's disease.

[0003] In this type of treatment, the source and method of cells used for Treg in vitro expansion have become the key to cell adoptive therapy. Unlike mouse studies that prepare Treg in large quantities from the spleen, the proportion of Treg in human peripheral blood is lower and more difficult to expand. Initial or resting Treg has become one of the first choice Treg subtypes for in vitro expansion. In order to ensure the rapid expansion of Treg in the in vitro system, researchers will add rapamycin, retinoic acid, histone deacetylase inhibitors or DNA methyltransferase inhibitors to the in vitro expansion system to stabilize the expression of Foxp3 and other Treg function-related molecules. However, the culture methods in the prior art have many shortcomings, such as low efficiency and small number of Tregs obtained.

[0004] Therefore, how to efficiently promote the formation of Tregs remains an urgent problem to be solved in this field. Summary of the invention

[0005] The object of the present invention is to provide a new method for promoting the induction of regulatory T cells.

[0006] In a first aspect of the present invention, a method for preparing a cell population with enhanced Treg cell differentiation potential is provided, comprising the steps of:

[0007] (S1) providing T cell precursors;

[0008] (S2) culturing T cell precursors at the double negative stage (i.e., DN stage) under conditions where stearoyl-CoA desaturase 1 (SCD1) is inhibited to obtain a cell population with enhanced Treg cell differentiation potential;

[0009] The expression of Atp2a2 is upregulated in the cell population.

[0010] In another preferred embodiment, the cell population expresses a gene marker selected from the following group:

[0011] Atp2a2, Chd9, Stt3b, Gm31814, Capn11, Gm32144, Nebl, Ifnlr1, Mtmr7, Ppp2r3a.

[0012] In another preferred embodiment, in step (S2), the cell population with improved Treg cell differentiation potential is cultured, and the percentage of Treg cells in CD4+ cells is recorded as A1, and in the control group cultured without inhibiting SCD1 and with the same other conditions, the percentage of Treg cells in CD4+ cells is recorded as A0,

[0013] The “increased Treg cell differentiation potential” refers to A1 / A0≥1; preferably ≥2.5; more preferably ≥10; and more preferably ≥20.

[0014] In another preferred embodiment, the T cell precursors in the double negative stage (ie, DN stage) include cells selected from the following group: T cell precursors in the DN3 stage and the DN4 stage.

[0015] In another preferred embodiment, the T cell precursors in the double negative stage (ie, DN stage) also include T cell precursors in the DN2 to DN3 period.

[0016] In another preferred embodiment, the T cell precursors in the double negative stage (ie, DN stage) include cells having markers selected from the group consisting of: CD4 - CD8 - CD44 + CD25 + (DN2), CD4-CD8-CD44-CD25+ (DN3) or CD4-CD8-CD44-CD25- (DN4).

[0017] In another preferred embodiment, the conditions for inhibiting SCD1 include culturing cells in the presence of a SCD1 inhibitor.

[0018] In another preferred embodiment, the SCD1 inhibitor is selected from the following group: small molecule compounds, antibodies, antisense nucleic acids, gene editing drugs, or a combination thereof.

[0019] In another preferred embodiment, the SCD1 inhibitor is a small molecule compound, preferably A939572, MF-438, SSI-4, MK-8245, or MK-8245 trifluoroacetate.

[0020] In another preferred embodiment, the SCD1 inhibitor is selected from small molecule compounds, antibodies, siRNA, shRNA, or an inhibitory complex mediated by CRISPR / Cas editing tools.

[0021] In another preferred embodiment, the "upregulated expression of Atp2a2" refers to the expression level M1 of Atp2a2 in the cell population with improved Treg cell differentiation potential, relative to the expression level M0 of Atp2a2 in the cell population obtained from T cell precursors cultured under conditions where SCD1 expression is not inhibited, and M1 / M0 is ≥2; preferably ≥3; more preferably ≥5.

[0022] In another preferred example, the activity of methyltransferase DOT1L in the cell population is increased.

[0023] In another preferred example, the activity of H3K79me2 in the cell population is increased.

[0024] In another preferred embodiment, the "increased activity of H3K79me2" refers to the activity N1 of H3K79me2 in the cell population with improved Treg cell differentiation potential relative to the activity N0 of H3K79me2 in the cell population obtained from T cell precursors cultured under conditions without inhibiting SCD1, N1 / N0 ≥ 1.5; preferably ≥ 2.5; more preferably ≥ 5.

[0025] In another preferred embodiment, 70%-90% (preferably 75%-85%) of the cells in the cell population with enhanced Treg cell differentiation potential express Atp2a2.

[0026] In another preferred embodiment, the cell population with enhanced Treg cell differentiation potential comprises DN3 stage thymocytes.

[0027] In another preferred embodiment, the T cell precursors are derived from induced pluripotent stem cells, hematopoietic stem cells, or hematopoietic precursor cells.

[0028] In another preferred embodiment, the autoimmune diseases include autoreactive encephalomyelitis, autoimmune gastritis, arthritis, mumps, colitis, allergic encephalitis, delayed hypersensitivity reaction, systemic lupus erythematosus, scleroderma, autoimmune hepatitis, ankylosing spondylitis, Hashimoto's thyroiditis, Sjögren's syndrome, psoriasis, vitiligo, systemic vasculitis and type I diabetes.

[0029] In another preferred embodiment, the cell population with enhanced Treg cell differentiation potential is further cultured in vitro or in vivo to obtain Treg cells.

[0030] In a second aspect of the present invention, a cell population with improved Treg cell differentiation potential is provided, wherein the cell population is prepared by the method described in the first aspect of the present invention.

[0031] The third aspect of the present invention provides a use of an SCD1 inhibitor for preparing a composition or a preparation for inducing Treg cell differentiation.

[0032] In another preferred embodiment, the composition or preparation is used for subjects with abnormally high expression of SCD1.

[0033] In another preferred embodiment, the composition or preparation is used to prevent and / or treat autoimmune diseases.

[0034] In another preferred embodiment, the composition or preparation is also used for one or more uses selected from the following group:

[0035] (c) promote the activity of DOT1L in thymocytes;

[0036] (d) promote H3K79me2 activity in thymocytes;

[0037] (e) Promote the expression of ATP2A2 in thymocytes;

[0038] (f) down-regulating the content of oleic acid in thymic tissue; and

[0039] (g) Upregulation of stearic acid content in thymus tissue.

[0040] In another preferred embodiment, the composition or preparation is also used for one or more uses selected from the following group:

[0041] (h) down-regulating the content of palmitoleic acid in thymic tissue; and

[0042] (i) Upregulate the content of palmitic acid in thymic tissue.

[0043] In another preferred embodiment, the "upregulated expression of Atp2a2" refers to the expression level M1 of Atp2a2 in the cell population with improved Treg cell differentiation potential relative to the expression level M0 of Atp2a2 in thymus tissue without the addition of SCD1 inhibitor, M1 / M0 ≥ 2; preferably ≥ 3; more preferably ≥ 5.

[0044] In another preferred embodiment, the "downregulating the content of oleic acid in thymic tissue" refers to that the ratio of the oleic acid content E1 in the thymic tissue with the addition of SCD1 inhibitor to the oleic acid content E0 in the normal thymic tissue (i.e., E1 / E0) is ≤1 / 2, preferably ≤1 / 3, and more preferably ≤1 / 5; and / or the ratio of the activity A1 of stearic acid in the thymic tissue with the addition of SCD1 inhibitor to the oleic acid content A0 in the normal thymic tissue (i.e., A1 / A0) is ≥2, preferably ≥3, and more preferably ≥5.

[0045] In another preferred embodiment, the SCD1 inhibitor is used to inhibit or eliminate the enzyme activity of SCD1 and / or the expression of the SCD1 gene.

[0046] In another preferred embodiment, the SCD1 inhibitor is selected from the following group: small molecule compounds, antibodies, antisense nucleic acids, gene editing drugs, or a combination thereof; preferably A939572, MF-438, SSI-4, MK-8245, MK-8245 trifluoroacetate.

[0047] In another preferred embodiment, the inhibitor is selected from small molecule compound antibodies, siRNA, shRNA, or an inhibitory complex mediated by CRISPR / Cas editing tools.

[0048] The fourth aspect of the present invention provides a cell preparation, comprising: a cell population with enhanced Treg cell differentiation potential as described in the second aspect of the present invention; and a pharmaceutically acceptable carrier.

[0049] In another preferred embodiment, the cell preparation comprises a liquid preparation.

[0050] In another preferred embodiment, the cells in the cell preparation are substantially (≥90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or entirely composed of a cell population with enhanced Treg cell differentiation potential.

[0051] In another preferred embodiment, the carrier is selected from the following group: an infusion carrier and / or an injection carrier. Preferably, the carrier is one or more carriers selected from the following group: physiological saline, glucose saline, or a combination thereof.

[0052] In another preferred embodiment, in the cell preparation, the concentration of the cell population with enhanced Treg cell differentiation potential is 0.001-50×106 / kg body weight, preferably, 0.1-10×10 6 / kg body weight, preferably, 1-5×10 6 / kg body weight.

[0053] The fifth aspect of the present invention provides a method for inducing Treg cell differentiation, wherein the preprogrammed T cell population with enhanced Treg cell differentiation potential described in the second aspect of the present invention is infused into a subject in need thereof to obtain an induced differentiated Treg cell population.

[0054] In another preferred embodiment, the subject includes mice, rats or humans.

[0055] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 .CD4 cre SCD1 loxp Mice are not immune to EAE. (ab) In CD4 cre SCD1 loxp Mouse and SCD1 loxp EAE was induced in mice. (a) Clinical disease scores. (b) Treg ratios in the spleen, draining lymph nodes, and central nervous system of mice were measured at the Steady (day 0), Peak (day 18), and Recovery (day 24) stages of EAE induction. (c) Flow cytometry analysis of CD4 cre SCD1 loxp Mouse and SCD1 loxp The proportion of Treg cells in the spleen, blood, mesenteric lymph nodes, liver and lung of mice. (d) Isolation of CD4 cre SCD1 loxp Mouse and SCD1 loxp Mouse spleen CD4 + CD62L + T cells were induced to differentiate into Treg in vitro. After 3 days, the cells were collected and their Treg differentiation ratio was detected by flow cytometry.

[0057] Figure 2 .Construct a bone marrow transplant chimeric model. + Wild type (WT) mice and CD45.1 + CD45.2 + SCD1 - / -Bone marrow cells from (KO) mice (WT BM and KO BM) were mixed 1:1 and co-infused with irradiated (10 Gy) CD45.2 + WT recipient mice or CD45.2 + SCD1 - / - In recipient mice, a bone marrow transplant chimeric model was constructed. (a) Schematic diagram of the experimental process. (b) Bone marrow reconstruction efficiency. (c) Schematic diagram of flow cytometry analysis strategy. (d) Thymus reconstruction efficiency. (e) CD4 + T cell production ratio.

[0058] Figure 3 .The SCD1-deficient microenvironment affects T cell differentiation preference. Figure 2 The bone marrow chimeric mouse model was constructed as shown in a. (a) Flow cytometry was used to detect the proportion of Treg cells developed from bone marrow cells of different origins in the spleen, blood, mesenteric lymph nodes, liver, and lungs of bone marrow chimeric mice. (b) Sorting of spleen-derived CD4 + CD62L + T cells were induced to differentiate into Treg cells in vitro, and the proportion of differentiated Treg cells was detected after 3 days. (c) Statistical graph of the proportion of Treg cell differentiation in vitro.

[0059] Figure 4 .The loss of SCD1 in K14-positive cells is the reason that affects the differentiation preference of regulatory T cells. (a) Lyz2 in steady state cre SCD1 loxp and littermate controls SCD1 loxp The proportion of Treg in the blood, spleen, mesenteric lymph nodes, liver and lung of mice. (b) Dermo1 in steady state cre SCD1 loxp and littermate controls SCD1 loxp The proportion of Treg in the blood, spleen, mesenteric lymph nodes, liver and lung of mice. (c) K14 at steady state cre SCD1 loxp and littermate controls SCD1 loxp The proportion of Treg in the blood, spleen, mesenteric lymph nodes, liver and lung of mice. (d) Lyz2 cre SCD1 loxp and littermate controls SCD1 loxp Mouse spleen CD4 + CD62L + T cells were sorted and induced to differentiate into Treg in vitro, and the proportion of differentiated Treg was detected 3 days later. (e) Dermo1 cre SCD1 loxp and littermate controls SCD1loxp Mouse spleen CD4 + CD62L + T cells were sorted and induced to differentiate into Treg in vitro, and the proportion of differentiated Treg was detected 3 days later. (f) K14 cre SCD1 loxp and littermate controls SCD1 loxp Mouse spleen CD4 + CD62L + T cells were sorted and induced to differentiate into Treg in vitro, and the proportion of differentiated Treg was detected after 3 days.

[0060] Figure 5 .SCD1 deficiency in K14-positive cells promotes antigen-specific Treg cell differentiation and inhibits antigen-specific Th17 cells. (a) Schematic diagram. cre SCD1 loxp and SCD1 loxp The hematopoietic stem cells (HSCs) of OT-II transgenic mice were reconstructed, and CD62L + CD4 + TCRVβ5.1 + Cells, 1×10 7 / mice were transplanted with CD45.1 by i.v. + WT mice. In vivo and in vitro experiments were used to detect the expression of SCD1 in K14 + The effects of the lack of Treg cells on T cell differentiation and antigen-specific T cell immune response. Among them, the in vivo experiments included: oral administration of chicken ovalbumin (OVA) to recipient mice [antigen-specific Treg cell-mediated oral tolerance]; subcutaneous immunization of recipient mice with OVA + complete Freunds adjuvant (CFA) [inducing Th1 / Th17 cell-mediated antigen-specific immune response]; subcutaneous immunization of recipient mice with OVA + aluminum (Alum) [inducing Th2 cell-mediated antigen-specific immune response]. (b) Flow cytometry differentiation gate strategy. (c) CFSE was used to detect the proliferation of OVA-stimulated T cells. (d) After oral administration of OVA, the proportion and number of antigen-specific Treg cells in the spleen, mesenteric lymph nodes, and colon of recipient mice were detected. (ef) After subcutaneous immunization of recipient mice with OVA, the proportion and number of antigen-specific Th17 cells and Th1 cells in the draining lymph nodes and lung tissues were detected. (g) After subcutaneous immunization of recipient mice with OVA:Alum, the proportion and number of antigen-specific Th2 cells in the draining spleen and liver were detected.

[0061] Figure 6 .The SCD1-deficient thymic microenvironment preprograms T cells to differentiate into Tregs. (a) Schematic diagram of flow cytometry analysis strategy. (bc) K14 cre SCD1 loxp and SCD1 loxp The thymus was transplanted under the kidney capsule of nude mice. After wild-type mouse bone marrow cells were transplanted, the proportion of Treg in peripheral tissues and organs was observed (b), as well as the mature Treg cells from different thymus CD4 + The efficiency of T cells differentiating into Treg cells in vitro (c). (d) EAE was induced in recipient mice and the disease progression was observed.

[0062] Figure 7 The SCD1-deficient thymic microenvironment pre-programs T cells to differentiate into Tregs during the DN2-DN3 developmental period. + SCD1 - / - Mouse and CD45.1 + Thymocytes from the thymus of WT mice at various developmental stages (DN1, DN2, DN3, DN4, DP) were mixed at a 1:1 ratio and infused into irradiated (5 Gy) Rag2 - / - Recipient mice were used to detect and analyze the CD8 + The proportion of T cells (a) and the proportion of Treg cells generated by thymocytes at different stages from different donors in the spleen, liver, lung and colon of recipient mice (b).

[0063] Figure 8 .K14 + SCD1 deficiency in cells pre-programs T cells to differentiate into Tregs during the DN2-DN3 developmental period. + K14 + SCD1 loxp and CD45.1 + WT mouse thymus DN2 and DN3 cells were mixed at a ratio of 1:1 and injected into the irradiated (5 Gy) Rag2 - / - In recipient mice, the proportion of Treg cells developed from thymocytes of different donors was detected in the spleen, liver, lung, colon mesenteric lymph nodes and blood of recipient mice after 8 weeks.

[0064] Fig. 9 .SCD1's regulation of T cell differentiation into Treg depends on its enzyme activity. (ac)SCD1 mut / mut The proportion of Treg cells in mouse blood (a), liver (b), and spleen (c).

[0065] Fig.10 .SCD1 - / - Oleic acid in the mouse thymus was significantly reduced. Detection of SCD1 by mass spectrometry - / - Mouse and SCD1 + / - The ratio of various fatty acid components in mouse thymus.

[0066] Fig.11 .Inhibition of SCD1 activity during in vitro T cell development significantly downregulated the proportion of oleic acid and increased Treg levels. + WT mice and CD45.2 + SCD1 - / - Mouse hematopoietic stem cells (HSC) were used to induce thymocyte development using the OP9-DL1 cell in vitro culture system with or without the addition of SCD1 inhibitor. DN3 thymocytes were isolated and infused into Rag2 - / - In mice, the mice were euthanized after 8 weeks and analyzed. (a) Schematic diagram of the experimental process. (b) Mass spectrometry was used to detect the ratio of 18:0 and 18:1n9 fatty acids in DN3 cells developed in vitro. (c) Receptor Rag2 - / - The proportion of Tregs produced in the spleen, liver, lung and colon after mice were transplanted with DN3 thymocytes from different treatment groups.

[0067] Fig.12 .Replenishment of oleic acid during in vitro T cell development significantly reversed the increase in Treg levels caused by SCD1 inhibitors. + WT mice, CD45.2 + WT mice and CD45.1 + CD45.2 + HSCs from WT mice were used to induce thymocyte development using the OP9-DL1 cell in vitro culture system, with or without the addition of SCD1 inhibitors or oleic acid (OA). DN3 thymocytes were obtained by flow cytometry and infused into Rag2 - / - In mice, the analysis was performed after 8 weeks. (a) Schematic diagram of the experimental process. (b) Flow cytometry analysis strategy. (c) Receptor Rag2 - / - The proportion of Treg in the spleen, blood, mesenteric lymph nodes, liver, lung and colon of mice.

[0068] Fig.13 .SCD1 - / - and SCD1 + / - Mouse DN3 thymocytes and CD4+ Differential analysis of chromatin accessibility in T cells. Detection of SCD1 using ATAC-seq - / - Mouse and SCD1 + / - Mouse-derived DN3 thymocytes and CD4 + (a) Heat map of differential peaks in T cell chromatin openness; (b) differentially expressed genes in DN3 thymocytes and CD4 + Venn diagram of the intersection of T cell differentially expressed genes.

[0069] Fig.14 .SCD1 - / - Mouse CD4 + ATP2A2 expression level in T cells was significantly upregulated. (a) SCD1 - / - Mouse and SCD1 + / - Mouse DN3 thymocytes and CD4 + ATAC-seq peak plot of Atp2a2 gene locus in T cells. (b) SCD1 - / - Mouse and SCD1 + / - Mouse CD4 + Heat map of histone modification profile analysis of T cells and DN3 thymocytes. (cd) ChIP-qPCR detection of SCD1 - / - Mouse and SCD1 + / - Mouse DN3 thymocytes (c) and CD4 + (d) H3K79me2, H3K9me3, H3K4me2, and H3K27me3 modification levels at the Atp2a2 gene locus in T cells. (e) SCD1 - / - Mouse and SCD1 + / - Mouse CD4 + ATP2A2 mRNA expression level in T cells. (f) SCD1 - / - Mouse and SCD1 + / - Mouse CD4 + ATP2A2 protein expression level in T cells.

[0070] Fig.15.Inhibition of SCD1 activity during in vitro T cell development leads to upregulation of ATP2A2 expression in DN3 thymocytes. (a) HSCs from WT mice were sorted by magnetic beads, and thymocyte development was induced using the OP9-DL1 cell in vitro culture system with or without the addition of SCD1 inhibitors. DN3 cells were obtained by flow cytometry sorting and their ATP2A2 protein expression levels were detected. (b) CD4 + CD62L + T cells were induced to differentiate into Treg cells in vitro with or without SCD1 inhibitor. Cells were collected 3 days later and their ATP2A2 protein expression levels were detected. (c) SCD1 was knocked down using siRNA - / - Mouse CD4 + ATP2A2 in T cells and detected its differentiation ability in the Treg cell in vitro differentiation system.

[0071] Fig.16 .SCD1 inhibitors increase Dot1L enzyme activity and H3K79me2 modification at the Atp2a2 locus in developing T cells. (ab) SCD1 inhibitors A939572 (40nM) and oleic acid (20μM) were added to the T cell in vitro development system. DN3 thymocytes were collected to detect the H3K79me2 level at the Atp2a2 gene locus (a) and the ATP2A2 protein level (b). (cd) SCD1 inhibitors A939572 (40nM) and DOT1L inhibitor EPZ004777 (10μM) were added to the T cell in vitro development system. DN3 thymocytes were collected to detect the H3K79me2 level at the Atp2a2 gene locus (c) and the ATP2A2 protein level (d). (e) The effects of oleic acid and stearic acid on the DOT1L enzyme activity were detected. (f) The effects of palmitic acid and palmitoleic acid on the DOT1L enzyme activity were detected.

[0072] Fig.17 .SCD1 - / - Mouse CD4 + CD45Rb high T cells significantly tend to differentiate into Tregs and slow down the occurrence of IBD. - / - and SCD1 + / - Mouse spleen-derived CD4 + CD45Rb high T cells and injected into Rag2 - / - In vivo in recipient mice. (a) Receptor Rag2 was detected weekly - / - Changes in mouse body weight. (b) Detection of receptor Rag2 in the eighth week after cell infusion - / -The proportion of Treg in the spleen, blood, mesenteric lymph nodes and colon of mice.

[0073] Fig.18 .Markers for each stage of thymus development in mice and humans respectively. DETAILED DESCRIPTION

[0074] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that oleic acid production mediated by SCD1 in thymic epithelial cells is crucial for the differentiation potential of regulatory T cells (Treg). Specifically, the oleic acid level in the thymus can regulate the histone methylation state of the Atp2a2 locus of thymocytes in the early DN2-DN3 transition stage, so that the initial CD 4+ T cells ( CD4+T cells) are more likely to differentiate into Treg cells, and the present invention was completed on this basis.

[0075] the term

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0077] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0078] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0079] Treg cells and cell populations with a tendency to differentiate into Treg cells

[0080] The main function of regulatory T cells (Treg) characterized by Foxp3 is to establish and maintain the body's immune tolerance. Immune tolerance is an active process. On the one hand, Treg directly acts on T cells or regulates antigen-presenting cells through various mechanisms to achieve immune tolerance; on the other hand, Treg can also induce the production of other inhibitory cells to shape the inhibitory immune microenvironment by regulating the surrounding immune microenvironment. As an indispensable part of the immune system, Treg maintains the self-tolerance and homeostatic regulation of the immune system, and therefore plays an important role in the occurrence and development of various diseases such as autoimmune diseases, infections, tumors and even cardiovascular diseases. Treg mainly achieves its immune regulatory function through cell contact mechanisms targeting specific cell surface receptors (such as CTLA4, TIGIT, etc.) or by secreting inhibitory cytokines (such as TGFβ, IL-10, etc.) [1-7]. According to the different organs in which they mature, Treg cells are divided into tTreg, pTreg, and iTreg. Among them, tTreg matures in the thymus and is then transported to peripheral tissues and organs, while pTreg and iTreg are generated by naive T cells that mature in the thymus and differentiate through antigen stimulation in the presence of cytokines such as IL-2 and TGFβ. iTreg is generated by naive CD4+T cells activated in vitro. Although pTreg and iTreg are considered to be two different cell subsets, they are both generated through Differentiated from CD4+T cells.

[0081] T lymphocytes belong to the acquired immune system and play an indispensable role in executing and controlling humoral and cell-mediated immune responses. T lymphocytes are distinguished from other lymphocytes (B cells and NK cells) by the expression of T cell receptors (TCR) on their cell surface. T lymphocytes are derived from bone marrow precursor cells (T cell precursors: TCP), which differentiate in the thymus through four stages, all of which are characterized by a CD4-CD8- phenotype, the so-called double negative (DN) cells. Different stages of DN differentiation (DN1-DN4) can be distinguished based on the differential expression of CD44, CD25, and CD117. DN3 expresses preTCR and enters the DN4 stage, followed by the expression of CD8 (ISP: intermediate single positive), followed by the expression of CD4, thus progressing to the CD4+CD8+ double positive (DP) stage. Finally, positive selection results in the generation of CD4 or CD8 single positive T cells. Mature CD4 and CD8 single positive cells leave the thymus and reach secondary lymphoid organs through the peripheral circulation to become initial T cells ( CD4+T cells), each of which has a unique antigen specificity. The corresponding markers for each stage of thymus development in mice and humans are shown in Fig.18 .

[0082] According to the expression of CD4 and CD8, the development of thymic T cells is divided into three main stages, namely double negative (DN), double positive (DP) and single positive (SP). Based on the expression of CD117, CD44 and CD25, DN cells are divided into four groups, DN1 to DN4, representing different development stages.

[0083] Among them, the molecular markers expressed by CD4 and CD8 in DN1 to DN4 are as follows: CD4 - CD8 - CD44 + CD25 - (DN1), CD4 - CD8 - CD44 + CD25 + (DN2), CD4 - CD8 - CD44 - CD25 + (DN3), CD4 - CD8 - CD44 - CD25 - (DN4).

[0084] The present invention unexpectedly discovered for the first time that, during the stage of T cell precursor differentiation (preferably the DN2-DN3 period), regulating the oleic acid or palmitoleic acid content in the environment (for example, inhibiting the activity of the SCD1 enzyme) can significantly affect the differentiation of T cell precursors, thereby obtaining a cell population (initial T cell subpopulation) with improved Treg cell differentiation potential, wherein the expression of Atp2a2 in the cell population is upregulated, making it easier to differentiate into Treg cells, and the proportion of Treg cells obtained can be 1-10 times higher than that of the control group.

[0085] As used herein, "T cell precursors" are cells at the DN2, DN3, DN4 or DP stages; or T cells at any stage from DN2 to DP; preferably T cells at the DN3 stage.

[0086] As used herein, thymic T cells are derived from mice, rats or humans. It is also understood that thymic T cells at different stages, especially T cells at the DN2-DN3 stage, can obtain a cell population with improved Treg cell differentiation potential under conditions of SCD1 inhibition or oleic acid deficiency.

[0087] As used herein, "a cell population with improved Treg cell differentiation potential", "a cell population with improved Treg cell differentiation tendency", "preprogrammed T cells with Treg differentiation tendency", and "preprogrammed T cell population of the present invention" can be used interchangeably and refer to a group of T cells with improved ability to differentiate into Tregs, wherein the expression of Atp2a2 in the cell population is upregulated relative to the expression level of Atp2a2 in the cell population obtained from T cell precursors cultured under conditions where SCD1 expression is not inhibited.

[0088] SCD1

[0089] SCD1 (stearoyl-CoA desaturase-1) is a rate-limiting enzyme that exists in the endoplasmic reticulum and catalyzes the formation of monounsaturated fatty acids. Studies have shown that SCD1 can affect the content and proportion of fatty acid components in cells through its enzymatic activity, plays an important role in cell membrane fluidity and intracellular signal transduction, and affects cell growth, differentiation and functional regulation.

[0090] The enzymatic function of SCD1 is mainly to catalyze the desaturation reaction of fatty acids to generate monounsaturated fatty acids such as oleic acid and palmitoleic acid. Monounsaturated fatty acids can be used as substrates to synthesize a series of lipid molecules, including phospholipids and triglycerides, providing a material basis for intracellular life activities. In addition, monounsaturated fatty acids can also act as signal molecules and participate in intracellular signal transduction. Therefore, the regulation of T cell differentiation preference by SCD1 is likely to be mediated by the fatty acid components it affects. For example, the mouse thymus mainly contains fatty acids with 16 carbons and 18 carbons as the basic skeleton, such as palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid.

[0091] In another preferred example, the accession number of the SCD1 gene is NC_000010.11.

[0092] In another preferred embodiment, the accession number of the SCD1 mRNA is NM_005063.5.

[0093] In another preferred example, the accession number of the SCD1 protein is NP_005054.3.

[0094] As used herein, the SCD1 inhibitor is selected from the group consisting of small molecule compounds, antibodies, antisense nucleic acids, gene editing drugs, or a combination thereof; preferably A939572 (CAS No. 1032229-33-6), MF-438 (CAS No. 921605-87-0), SSI-4 (CAS No. 1875084-68-6), MK-8245 (CAS No. HY-13070), and MK-8245 trifluoroacetate.

[0095] Atp2a2

[0096] Atp2a2 is closely related to the activation and differentiation of T cells. The protein encoded by Atp2a2 is ATP2A2, also known as sarcoplasmic / endoplasmic reticulum calcium ATPase 2, which is a Ca2+-ATPase present in the endoplasmic reticulum of cells (or sarcoplasmic reticulum of muscle cells). It can catalyze ATP hydrolysis and transport Ca2+ from the cytoplasm to the endoplasmic reticulum. As an important molecule regulating intracellular Ca2+ homeostasis, ATP2A2 plays a key role in the survival, activation and differentiation of T cells. In mice, SCD1 deficiency leads to ATP2A2 expression was upregulated in CD4+T cells.

[0097] Pharmaceutical composition (cell preparation)

[0098] The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: an SCD1 inhibitor.

[0099] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals. In a preferred embodiment of the present invention, the effective amount is: 1.0×10 3 -1.0×10 9 / ml, preferably 1.0×10 4 -1.0×10 8 / ml, preferably 1.0×10 5 -1.0×10 7 Preferably, the effective amount of cells is injected once.

[0100] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0101] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration. The dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0102] The effective amount of the active ingredient of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, a satisfactory effect can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally.

[0103] The pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0104] Treatment

[0105] The present invention also provides the use of the active ingredients of the present invention or the corresponding drugs for preventing and / or treating autoimmune diseases, such as inflammatory diseases, comprising administering an effective amount of the active ingredients to a mammal, culturing stem cells treated with an SCD1 inhibitor in the presence of an SCD1 inhibitor, obtaining a preprogrammed T cell population with a tendency to differentiate into Treg cells, or administering a pharmaceutical composition containing a preprogrammed T cell population or Treg cells.

[0106] When the active ingredient of the present invention is used for the above purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in isotonic media). For example, these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% (weight) of the active ingredient mixed with a carrier.

[0107] The active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration. Preferred routes of administration include oral administration, intramuscular administration or intravenous administration.

[0108] From the standpoint of ease of administration, the preferred pharmaceutical composition is a liquid composition, especially an injection.

[0109] In addition, the active ingredient or drug of the present invention can also be used in combination with other (a) drugs for preventing and / or treating autoimmune diseases, such as inflammatory diseases.

[0110] The main advantages of the present invention include

[0111] (1) The present invention suggests for the first time that fatty acids can continuously affect the T cell process from early thymic development to peripheral Treg cell differentiation.

[0112] (2) The present invention reveals for the first time that the oleic acid level in the thymus can regulate the histone methylation state of the Atp2a2 locus of thymocytes in the early stage of DN2-DN3 conversion, making the generated initial CD4+ T cells more likely to differentiate into Treg cells, revealing the origin of the theoretical intrinsic heterogeneity of initial T cells.

[0113] (3) The present study found that the signal provided by oleic acid in the thymus can be regarded as a "No. 0" signal, which acts on developing T cells and affects their responsiveness before TCR assembly and stimulation. Therefore, in-depth research on the transmission of this "No. 0" signal during T cell development will help understand the heterogeneity of T cells in the same pathological microenvironment and provide new strategies for regulating T cell responses in infection, autoimmune diseases and cancer situations.

[0114] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0115] Experimental Materials and Methods

[0116] Experimental cells

[0117] The OP9-DL1 cell line was donated by a professor at the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. OP9-DL1 cells were cultured in αMEM medium containing 20% ​​FBS (v / v), 2mM glutamine, 100U / mL penicillin and 100μg / mL streptomycin. During subculturing, the culture medium was removed and the residual culture medium was washed with PBS, and then digested with 0.1% trypsin at 37°C for 2-3min, and then the culture medium was added in a 1:1 ratio to terminate the digestion and collect the cell suspension. After the cell suspension was centrifuged at 200g for 5min, the cell pellet was subcultured at a ratio of 1:5.

[0118] In vitro T cell development

[0119] Prepare mouse bone marrow cell suspension. Then use lineage cell depletion kit (130-090-858, Miltenyi Biotec) to sort Lineage- cells. Then use Sca1-PE antibody and anti-PE MultiSortKit (130-090-757, Miltenyi Biotec) to sort Sca1+ cells. The sorted Lineage-Sca1+ hematopoietic precursor cells (HSC) were plated into a 6-well plate with half-confluent OP9-DL1 cells as the nutrient layer at a density of 1×105 cells / well. The development medium is αMEM medium with 20% FBS (v / v), 2mM glutamine, 100U / mL penicillin, 100μg / mL streptomycin, 10ng / mL IL-7, 10ng / mL Flt3L and 50ng / mL SCF added. The cells were passaged every 4 days until the final sample was collected.

[0120] In vitro Treg cell differentiation

[0121] 1) CD4+CD62L+T cell isolation: The mice were euthanized by cervical dislocation, and their spleens were removed to prepare cell suspensions. CD4+CD62L+T cells were isolated from the cell suspension using a mouse CD4+CD62L+T cell isolation kit (130-106-643, Miltenyi Biotec). For specific operation steps, see the kit instruction manual.

[0122] 2) In vitro Treg cell differentiation: The obtained CD4+CD62L+T cells were placed in differentiation medium (RPMI-1640, 10% FBS (v / v), 2mM glutamine, 100U / mL penicillin, 100μg / mL streptomycin, 5μg / mL Anti-CD3 antibody, 2μg / mL Anti-CD28 antibody, 50ng / mL IL-2, 5ng / mL TGF-β) and plated in 96-well flat-bottom plates at a density of 1×106 cells per well. After 3 days of culture, the cells were collected and analyzed subsequently.

[0123] Fatty acid GC-MS determination

[0124] Take the same mass of tissue or the same number of cells and place them in pre-cooled PBS. The tissue samples need to be homogenized using a tissue homogenizer or an ultrasonic disruptor. Add 10μg of standard, 2mL of 0.88% saline, and 4mL of chloroform / methanol (1:1, v / v, HPLC) mixed solution to each sample, and then use a large Votex instrument to vortex and shake at 2500rpm for 10min. After the shaking, use a centrifuge to centrifuge at 2000rpm for 10min at 4℃. At this time, obvious stratification can be seen. Use a Pasteur tube to transfer the lower layer of liquid to a new glass tube, and connect it to a nitrogen blower to blow dry the liquid. Then add 2mL of methylation reagent (H2SO4 / MeOH, 1:25, v / v) to the glass tube, and use a large Votex instrument to vortex and shake at 2500rpm for 10min. After the shaking, place the glass tube in an 80℃ water bath and react for 2h. After the reaction was completed and cooled to room temperature, 2 mL of n-hexane and 1 mL of ddH2O were added to each sample, and the mixture was vortexed at 2500 rpm for 10 min. The upper layer of liquid was then transferred to a new glass tube, and a nitrogen blower was connected to dry the liquid. The corresponding volume of isooctane was used to dissolve the fatty acids precipitated at the bottom of the tube according to the fatty acid content, and the fatty acids were analyzed by mass spectrometry.

[0125] ATP2A2 knockdown experimental procedures

[0126] Using Mouse T Cell Nucleofector TM Kit (Lonza, VPA-1006) was used to transfer ATP2A2 siRNAs or scramble controls into primary WT and SCD1- / - T cells. For specific steps, please refer to the instructions for use of the kit. The cells were then rested for 24 hours. The obtained cells were collected and stained with Fluo-8 as described above. LIVE / DEAD was used before loading TMFixableNear-IR Dead Cell Stain was used to stain the cells to ensure the viability of the analyzed cells. ATP2A2 siRNAs and scramble controls were purchased from Gene Pharma, Shanghai.

[0127] Example 1CD4 cre SCD1 loxp Mice are not immune to EAE

[0128] Using the constructed CD4 cre SCD1 loxp mice, to construct and observe the pathological process of experimental autoimmune encephalomyelitis (EAE). loxp Mouse, CD4 + Cell-specific knockout of SCD1 did not significantly slow the progression of EAE ( Figure 1 a). By detecting the proportion of Treg cells infiltrating the spleen, axillary lymph nodes and central nervous system of mice during the stable, peak and recovery stages of EAE, the study found that CD4 cre SCD1 loxp Mice did not express SCD1 loxp Mice showed significant differences ( Figure 1 b).

[0129] These results suggest that the resistance of SCD1-deficient mice to EAE is not due to the loss of SCD1 function in T cells. cre SCD1 loxp Mice and their littermate controls SCD1 loxp The proportion of Treg cells in the blood, spleen, mesenteric lymph nodes, liver and lungs of mice showed no significant difference between the two groups ( Figure 1 c).

[0130] Similarly, separation of CD4 cre SCD1 loxp Mice and their littermate controls SCD1 loxp Mouse spleen CD4 + When T cells were induced to differentiate into Treg in vitro, there was no significant difference in the proportion of Treg ( Figure 1 d).

[0131] Therefore, the loss of SCD1 expression in T cells does not affect The potential of T cells to differentiate into Treg cells.

[0132] Example 2: SCD1 deficiency in K14-positive cells is the cause of the influence on Treg differentiation preference

[0133] 2.1. Loss of SCD1 in non-immune cells is the cause CD4 + Reasons for enhanced T cell Treg differentiation

[0134] CD45.1 + WT mice and CD45.1 + CD45.2 + SCD1 - / - Mouse bone marrow cells (WT BM and KO BM) were mixed 1:1 and co-injected into lethally irradiated CD45.2 + WT recipient mice or CD45.2 + SCD1 - / - In recipient mice, a bone marrow chimeric model was constructed ( Figure 2 a) It was verified that WT BM and KO BM had significant differences in WT or SCD1 - / - Reconstitution efficiency was similar in recipient mice ( Figure 2 b) In the thymus of recipient mice, the number of DN cells, DP cells and CD4 + SP or CD8 + There was no significant difference in the proportion of SP cells ( Figure 2 In addition, CD4 + There was no significant difference in the proportion of T cells among the groups ( Figure 2 e).

[0135] Based on the above-mentioned bone marrow chimera model, flow cytometry analysis was used to detect the proportion of Treg cells generated by bone marrow cells of different origins in various tissues and organs of recipient mice.

[0136] The results showed that whether derived from CD45.1 + WT mice or CD45.1 + CD45.2 + SCD1 - / - Mouse bone marrow cells, CD45.2 + SCD1 - / - The proportion of Treg cells in the blood, spleen, mesenteric lymph nodes, liver, and lung of recipient mice was significantly higher than that in CD45.2 + WT recipient mice ( Figure 3 a).

[0137] In the same recipient mice, CD45.1 + WT mice and CD45.1+ CD45.2 + SCD1 - / - There was no significant difference in the proportion of mouse-derived Treg cells ( Figure 3 a) In addition, the spleen of the bone marrow chimeric mice was isolated CD4 + T cells and induced Treg differentiation in vitro. The study found that compared with CD45.2 + WT recipient mice, CD45.2 + SCD1 - / - Isolated from spleen of recipient mice CD4 + T cells, whether derived from WT mouse bone marrow cells or SCD1 - / - Mouse bone marrow cells have more significant Treg cell differentiation ability ( Figure 3 bc).

[0138] These results suggest that the loss of SCD1 in non-immune cells of the tissue microenvironment is the cause of CD4 + The reasons for the enhanced differentiation of T cells Treg.

[0139] 2.2.K14 cre SCD1 loxp Mouse source CD4 + T cells have Treg differentiation preference

[0140] The expression level of SCD1 in non-immune cells of tissue microenvironment is a key factor shaping CD4 + The reason why T cells differentiate preferentially into Tregs. CD4 + During T cell maturation, they need to interact with and be regulated by many different types of cells in the tissue microenvironment, including epithelial cells, mesenchymal cells, and other non-T immune cells. CD4 + The cell populations that favor T cell differentiation into Treg cells were expressed by Lyz2-cre mice, Dermo1-cre mice, and K14-cre mice, respectively, with SCD1 loxp Mice were mated to obtain Lyz2 mice with SCD1 knockout specifically in myeloid cells. cre SCD1 loxp Mouse, Dermo1 with SCD1 knockout specifically in mesenchymal cells cre SCD1 loxp Mouse and K14 knockout of SCD1 specifically in epithelial cells cre SCD1loxp Mouse.Lyz2 cre SCD1 loxp and Dermo1 cre SCD1 loxp The proportion of Treg cells in the blood, spleen, mesenteric lymph nodes, liver, and lung of mice compared with their littermate controls SCD1 loxp There was no significant difference compared with mice ( Figure 4 ab).

[0141] Furthermore, by isolating spleen-derived CD4 + In vitro Treg cell differentiation experiments with T cells also showed that Lyz2 + Cell or Dermo1 + Specific knockout of SCD1 in cells did not affect CD4 + T cells differentiate into Tregs Figure 4 This suggests that changes in the expression level of SCD1 in myeloid immune cells and interstitial cells do not affect CD4 + The reasons for the preference of T cells to differentiate into Tregs.

[0142] Interestingly, in K14 cre SCD1 loxp In mice, flow cytometry results showed that the proportion of Treg cells in various tissues and organs was significantly higher than that in the littermate control SCD1 loxp Mouse ( Figure 4 c), which is consistent with the - / - The phenomenon is consistent with that observed in mice. Crucially, in vitro Treg differentiation experiments also demonstrated that K14 cre SCD1 loxp Mouse origin CD4 + T cells have an intrinsic preference for Treg differentiation ( Figure 4 f).

[0143] These results show that K14 + Loss of SCD1 in epithelial cells shapes CD4 + The reasons for T cell Treg differentiation preference.

[0144] In addition, OT-II transgenic mice were given K14 cre SCD1 loxp Mice and SCD1 loxp Mice were reconstructed with HSCs from OT-II mice and K14 +Effects of SCD1 deficiency on the development and response of antigen-specific T cells Figure 5 ag). The results show that K14 + Cellular deletion of SCD1 promoted the generation of OVA-specific Treg cells and inhibited the differentiation of OVA-specific Th17 cells.

[0145] Example 3. Thymocytes are endowed with the potential to differentiate into Tregs during the DN2-DN3 developmental period

[0146] K14 is mainly expressed in epithelial cells, including skin and thymic epithelial cells. - / - Mouse origin CD4 + When T cells were isolated for in vitro and in vivo Treg differentiation experiments, they had left the SCD1-deficient microenvironment but still had an intrinsic preference for Treg differentiation, suggesting that CD4 + Before T cells mature, their Treg preference has been shaped. CD4 + The main site of T cell development is probably K14. + The thymic epithelium confers differentiation preference to thymocytes during their development.

[0147] To this end, nude mice were used to perform K14 cre -SCD1 loxp Thymus transplantation experiments from mice and control mice to verify the role of K14 in thymus development + Does the loss of SCD1 affect CD4 + The experimental results showed that when nude mice received K14 cre -SCD1 loxp When the thymus of mice was transplanted, the proportion of Treg in peripheral tissues and organs was significantly higher than that of nude mice receiving thymus transplantation in the control group ( Figure 6 ab).

[0148] At the same time, the spleen-derived receptor nude mice were isolated CD4 + When T cells were differentiated into Treg in vitro, they were treated with K14 cre -SCD1 loxp Recipient mice for mouse thymus transplantation CD4 + The efficiency of T cell differentiation into Treg was significantly higher than that in the control group ( Figure 6 c) This is similar to the cre-SCD1 loxp Mouse and SCD1 - / - This phenomenon is consistent with that observed in mice, demonstrating that K14 + The thymic epithelium confers Treg differentiation preference during thymocyte development. EAE was induced in two groups of recipient mice and it was found that compared with those receiving SCD1 loxp Mouse thymus transplant recipient mice, receiving K14 cre -SCD1 loxp Increased Treg levels in thymus transplant recipient mice can protect them from EAE ( Figure 6 d).

[0149] Isolation of CD45.2 + SCD1 - / - Mouse and its control CD45.1 + Thymocytes at various developmental stages in the thymus of WT mice, and CD45.2 + SCD1 - / - and CD45.1 + Cells from WT mice were mixed at a 1:1 ratio and injected into the irradiated Rag2 - / - In recipient mice, exogenously infused thymocytes of different developmental stages colonize Rag2 - / - Different T cell subsets were generated in recipient mice, and flow cytometry analysis confirmed that thymocytes from different sources matured and entered the periphery in the same manner. Figure 7 a).

[0150] The thymic developmental microenvironment experienced by cells from different sources can be divided into two stages: 1. The SCD1-deficient or wild-type thymic microenvironment from which they originate; 2. - / - The non-SCD1-deficient thymic developmental microenvironment in recipient mice completes development.

[0151] Therefore, by detecting the proportion of Treg cells produced by cells of different origins in various tissues of recipient mice infused with thymocytes of different developmental stages, it can be determined that the microenvironment of SCD1 deficiency specifically affects CD4 + Which stage of T cell development?

[0152] The results showed that when thymocytes at the DN1 or DN2 stage were transferred to Rag2 - / - When the recipient mice complete development and differentiation, regardless of the cell source, CD45.2 + SCD1 - / - Mouse or CD45.1 + There was no significant difference in the proportion of Tregs between WT mice and controls ( Figure 7b) This suggests that the absence of SCD1 in the thymic microenvironment during the DN1 or DN2 stage of thymocyte development does not confer CD4 + Treg differentiation preference of T cells.

[0153] However, in striking contrast, when CD45.2 + SCD1 - / - Mouse-derived DN3 or DN4, DP-stage thymocytes were transferred to Rag2 - / - In recipient mice, the proportion of Treg cells produced by their development and differentiation was significantly higher than that of CD45.1 + The proportion of Treg cells developed from WT ( Figure 7 b) The percentage of Treg cells obtained by developing T cells treated with SCD1 inhibitors can be 1-10 times higher than that of the control group.

[0154] The above experimental data suggest that during the DN2-DN3 period of thymocyte development, the absence of SCD1 in the thymic microenvironment confers the ability to become CD4 + The preference for differentiation into Treg at T.

[0155] Using CD45.2 + K14 + SCD1 loxp and CD45.1 + WT mice were also subjected to the transfer experiments of DN2 and DN3 cells.

[0156] The results showed that CD45.2 + K14 + SCD1 loxp and CD45.1 + DN2 thymocytes from WT mice express the receptor Rag2 - / - There was no significant difference in the proportion of Tregs generated in mice ( Figure 7 ). When the receptor Rag2 - / - When mice were given DN3 thymocytes, CD45.2 + K14 + SCD1 loxp The proportion of background Treg was significantly higher than that of CD45.1 + WT background ( Figure 8 , Table 1). This result is consistent with that in SCD1 - / - This is consistent with what was observed in mice, further demonstrating that K14 + The loss of SCD1 in thymocytes at the DN2-3 stage confers CD4+ T cells have a preference for differentiating into Tregs.

[0157] Table 1

[0158] The proportion of Treg cells in CD4+T cells

[0159]

[0160] Example 4. Oleic acid deficiency leads to CD4 + The reason why T cells differentiate into Tregs

[0161] 4.1SCD1 regulation of T cell differentiation into Treg depends on its enzyme activity

[0162] It has been proved in the previous experiments that K14 + The absence of SCD1 in cells gives thymocytes the potential to differentiate into Tregs after maturation during the DN2-3 period of development. SCD1 is a rate-limiting enzyme. By exerting its enzymatic activity, it can affect the content and proportion of fatty acid components in cells, affect cell membrane fluidity, intracellular signal transduction, cell growth, differentiation and functional regulation.

[0163] Therefore, it is necessary to first clarify the role of SCD1 in CD4 + Whether the regulation of T cell differentiation preference to Treg depends on its enzyme activity. To this end, SCD1 was introduced into mice with mutations in the SCD1 gene site. ab-Xyk (later marked as SCD1 mut / mut ). The mouse has a CCC trinucleotide insertion mutation in the exon 5 of the SCD1 gene locus, which results in the loss of its enzyme activity. The flow cytometry test of the proportion of Treg in various tissues and organs of the SCD1 gene mutant mouse showed that SCD1 mut / mut The proportion of Treg in the blood, spleen and liver of mice was significantly higher than that of the littermate control SCD1 mut / + Mouse ( Fig. 9 ac). This is consistent with SCD1 - / - The phenotype was consistent in mice.

[0164] These results suggest that SCD1's regulation of T cell differentiation preference depends on its enzymatic function.

[0165] 4.2SCD1 - / - The oleic acid content in the mouse thymus decreased significantly

[0166] To investigate which fatty acid components are affected by the loss of SCD1 in the mouse thymus development environment, we investigated the effects of SCD1 on the development of thymus in mice. - / - Mice and their littermate controls SCD1+ / - The fatty acid composition of mouse thymus tissue was detected and analyzed. The results showed that the mouse thymus mainly contained fatty acids with 16 carbon and 18 carbon as the basic skeleton, such as palmitic acid, stearic acid, oleic acid and linoleic acid ( Fig.10 ). Among them, compared with SCD1 + / - In mice, the proportion of oleic acid catalyzed by SCD1 is - / - The expression of stearic acid in the thymus of mice was significantly decreased, while the substrate for oleic acid production catalyzed by SCD1 was stearic acid. - / - Significantly upregulated in mice ( Fig.10 ).

[0167] Therefore, oleic acid deficiency is likely to be the cause of SCD1 - / - Causes of a range of phenotypes in mice.

[0168] Example 5. Inhibition of SCD1 during T cell development in vitro significantly increases Treg levels

[0169] In order to clarify the effect of oleic acid on the preference of thymocytes to differentiate into Tregs after maturation during the DN2-3 period of thymocyte development, a HSC-OP9-DL1 cell co-culture system was introduced. OP9 cells are stromal cells derived from the bone marrow of mice deficient in macrophage colony stimulating factor (MCSF). OP9 cells overexpressing DL1 molecules can provide Notch signals required for the directional development of T cell lineages and simulate the thymic microenvironment to support T cell development in vitro, which is an important tool for studying the development of T lymphocyte lineages.

[0170] Based on this in vitro development system, we first need to explore whether the decrease in oleic acid content will lead to an increase in the proportion of Treg cells generated. In order to reduce the oleic acid content, SCD1 inhibitors were added to the in vitro development system. CD45.1 was obtained by magnetic bead sorting + WT mice and CD45.2 + SCD1 - / - Mouse hematopoietic stem cells (HSCs) were co-cultured with OP9-DL1 cells in vitro with or without SCD1 inhibitor.

[0171] The study determined that SCD1 inhibitors did not affect the in vitro development of OP9-DL1 cells and T cells. It is worth noting that OP9-DL1 cells can support the development of thymocytes from the early DN stage to the DP stage and can successfully develop functional CD8 single positive T cells, but do not produce CD4 + T cells.

[0172] Therefore, in order to detect the proportion of Treg cells finally generated, it is necessary to infuse DN3 thymocytes obtained from the in vitro development system into immune-deficient Rag2- / - In order to complete the complete development and differentiation process from DN3 stage to Treg in mice, the cells treated with different methods were mixed in a 1:1 ratio according to the diagram and injected into the recipient mice together ( Fig.11 a).

[0173] By detecting the ratio of oleic acid to stearic acid in DN3 thymocytes developed in vitro, the experiment found that the addition of SCD1 inhibitor (A939572, CAS No.: 1032229-33-6) can significantly reduce the proportion of oleic acid content in cells, while significantly increasing the proportion of stearic acid content ( Fig.11 b). Correspondingly, in the group whose intracellular oleic acid content was reduced by SCD1 inhibitor, the proportion of Tregs produced in various tissues and organs of recipient mice increased significantly ( Fig.11 c).

[0174] The above results indicate that the intracellular content of oleic acid, a catalytic product of SCD1, during thymocyte development is related to the CD4 + The preference of T cells to differentiate into Tregs is relevant.

[0175] Interestingly, we found that SCD1 - / - There was no significant difference in the content of oleic acid and stearic acid in thymocytes at the DN3 stage of in vitro development between mice and WT mice ( Fig.11 b) In other words, the lack of SCD1 in thymocytes does not affect the content of intracellular oleic acid and stearic acid. - / - There was no significant difference in the proportion of Treg cells generated by cells derived from mice and WT mice in various tissues and organs of recipient mice ( Fig.11 c).

[0176] This is consistent with the phenomenon observed in the bone marrow transfer experiment, which once again proves that the loss of SCD1 in thymocytes does not affect CD4 + T cell differentiation preference towards Treg.

[0177] Example 6. Oleic acid supplementation during in vitro development can reverse Treg differentiation preference caused by inhibition of SCD1

[0178] In order to further clarify the effect of oleic acid on the preference of thymocytes to differentiate into Treg after maturation, an in vitro T cell development system was used to apply SCD1 inhibitors and conduct oleic acid replenishment experiments ( Fig.12 a), and its flow cytometry analysis strategy is shown in the figure ( Fig.12b). Consistent with previous studies, the addition of SCD1 inhibitor (A939572, CAS No.: 1032229-33-6) significantly increased the expression of Rag2 - / - The proportion of Treg cells in recipient mice ( Fig.12 c).

[0179] At the same time, the replenishment of oleic acid significantly reversed the upregulation of Treg ratio caused by SCD1 inhibitor, making Rag2 - / - The proportion of Treg cells in recipient mice returned to the level of WT group ( Fig.12 c).

[0180] Example 7. SCD1 deficiency affects DN3 and CD4 + T cell chromatin accessibility

[0181] Oleic acid regulates T cell differentiation fate in the thymocyte stage, but its influence lasts to the periphery CD4 + T cell differentiation period. This suggests that oleic acid is likely to play a role through epigenetic regulation. - / - Mice and their littermate controls SCD1 + / - DN3 thymocytes from the thymus and DN3 thymocytes from the spleen were isolated from mice. CD4 + T cells and used the ATAC-seq method to identify their open chromatin regions.

[0182] The results showed that in SCD1 - / - Mouse-derived DN3 thymocytes and CD4 + In T cells, its open chromatin regions are associated with SCD + / - Mouse-derived cells have significant differences ( Fig.13 a), suggesting that the effect of SCD1 deficiency on thymocyte epigenetics should last from DN3 to CD4 + T period.

[0183] By analyzing SCD1 - / - and SCD1 + / - Mouse DN3 thymocytes and CD4 + The intersection of T cell accessibility differentially expressed genes found that the chromatin accessibility of 10 genes, including Atp2a2, Chd9, and Stt3b, was significantly different in SCD1 - / - and SCD1 + / - Mouse DN3 thymocytes are different, and the differences are CD4 + Persistent in T cells ( Fig.13 b).

[0184] Fig.13 Among different gene markers: Atp2a2, Chd9, Stt3b, Gm31814, Capn11, Gm32144, Nebl, Ifnlr1, Mtmr7, and Ppp2r3a, except for the upregulated activity of Atp2a2, the chromatin accessibility of other marker genes was significantly increased.

[0185] Example 8. SCD1 deficiency upregulates ATP2A2 expression in DN3 thymocytes

[0186] It is worth noting that among these differentially expressed genes, Atp2a2 is closely related to the activation and differentiation of T cells. The protein encoded by Atp2a2 is ATP2A2, also known as sarcoplasmic / endoplasmic reticulum calcium ATPase 2, which is a Ca2+-ATPase present in the endoplasmic reticulum (or sarcoplasmic reticulum of muscle cells). 2+ -ATPase, which catalyzes the hydrolysis of ATP and converts Ca 2+ Transported from the cytoplasm to the endoplasmic reticulum. 2+ ATP2A2 is an important molecule in homeostasis, and plays a key role in T cell survival, activation, and differentiation. SCD1 was confirmed by ATAC-seq data - / - Mouse DN3 thymocytes and CD4 + Increased chromatin accessibility at the Atp2a2 locus in T cells Fig.14 a) The enhanced chromatin accessibility is likely caused by differences in epigenetic modifications, so SCD1 - / - Mice and SCD1 + / - Mouse DN3 thymocytes and CD4 + Global histone modification profiling of T cells ( Fig.14 b), and further verified by ChIP-qPCR at the Atp2a2 gene locus in cells. The study found that SCD1 - / - Mouse-derived DN3 thymocytes and CD4 + H3K79me2 modification (methylation mark of histone H3 lysine 79) at the Atp2a2 gene locus in T cells was significantly upregulated ( Fig.14 cd).

[0187] It has been reported that upregulation of H3K79me2 levels is associated with increased gene expression activity. + / - Mouse, SCD1- / - Mouse CD4 + The mRNA level of ATP2A2 in T cells was significantly increased ( Fig.14 e). Consistent with the mRNA level, the protein expression level of ATP2A2 was - / - Mouse CD4 + SCD1 was also significantly higher in T cells + / - Mouse ( Fig.14 f). The above results indicate that SCD1 deficiency in mice leads to CD4 + ATP2A2 expression is upregulated in T cells.

[0188] Using an in vitro T cell development system, we compared whether the addition of SCD1 inhibitors would affect the expression level of ATP2A2 in DN3 thymocytes during their development. The experiment showed that when SCD1 inhibitors were added, the expression level of ATP2A2 protein in DN3 thymocytes was significantly higher than that in the DMSO group ( Fig.15 a). In contrast, when When SCD1 inhibitor was added to the Treg development process of CD4+T cells in vitro, the expression of ATP2A2 was not affected ( Fig.15 b).

[0189] In summary, the loss of SCD1 regulates the expression of ATP2A2 at the DN2-DN3 stage of thymocyte development. Inhibition of ATP2A2 expression also reversed the expression of SCD1 - / - Mouse source CD4 + The proportion of T cells that differentiate into Tregs during in vitro Treg induction differentiation ( Fig.15 c).

[0190] Example 9. Oleic acid produced by SCD1 inhibits DOT1L enzymatic activity and H3K79me2 modification of ATP2A2.

[0191] Using an in vitro T cell development system, we studied the effects of SCD1 inhibitor addition and oleic acid replenishment on H3K79me2 modification at the ATP2A2 gene locus and ATP2A2 protein levels in DN3 thymocytes.

[0192] Using ChIP-qPCR and western blot, the study found that the addition of SCD1 inhibitor significantly increased the modification of H3K79me2 at the ATP2A2 gene locus in DN3 thymocytes ( Fig.16 a) and ATP2A2 protein levels ( Fig.16b), oleic acid supplementation significantly inhibited this effect ( Fig.16 ab).

[0193] H3k79me2 is mainly regulated by histone methyltransferase DOT1L (disruptor of telomericsilencing1-like). In order to prove whether the SCD1 inhibitor enhances the H3K79me2 modification in developing T cells and whether it enhances the activity of DOT1L, we used an in vitro T cell development system, added SCD1 inhibitor and DOT1L inhibitor EPZ004777, obtained DN3 thymocytes, and used ChIP-qPCR and western blot to detect the modification of H3K79me2 at the ATP2A2 gene locus ( Fig.16 c) and ATP2A2 protein levels ( Fig.16 d).

[0194] The results showed that inhibition of DOT1L activity could significantly reverse the H3K79me2 modification at the ATP2A2 gene locus and the protein level of ATP2A2 enhanced by SCD1 inhibitor ( Fig.16 cd).

[0195] In order to prove whether the fatty acid metabolites produced by SCD1 affect the activity of DOT1L, the study used different concentrations of oleic acid / stearic acid and palmitoleic acid / palmitic acid to detect their effects on the activity of DOT1L. The results showed that oleic acid significantly inhibited the activity of DOT1L, while stearic acid had no such effect ( Fig.16 e). In addition, palmitoleic acid also showed a certain degree of influence on the enzyme activity of DOT1L ( Fig.16 f).

[0196] In summary, oleic acid and palmitoleic acid produced by SCD1 can inhibit DOT1L enzymatic activity and thus inhibit H3K79me2 modification.

[0197] Example 10. SCD1 - / - Mouse CD4 + CD45Rb high T cells significantly tend to differentiate into Treg and slow down the occurrence of colitis

[0198] Recombination activating gene 2 protein (Rag2) is an important protein that makes up VJ recombinase. When it is missing, the generation of T and B cells in mice is blocked. + CD45Rb high T cells mainly contain activated T cells, in Rag2 - / -In recipient mice, these cells spontaneously differentiate into Th1, Th17, or Treg cell subsets and induce IBD, leading to Rag2 - / - The mice lost weight. T cells are a classic model for inducing colitis. SCD1 - / - and SCD1 + / - Mouse spleen-derived CD4 + CD45Rb high T cells and injected into Rag2 - / - In mice, when Treg cell differentiation is enhanced, inflammation in the recipient mice is suppressed, and the incidence of chronic enteritis is alleviated, as shown by slower weight loss.

[0199] The results showed that compared with the control group, the - / - Mouse CD4 + CD45Rb high Rag2 in T cells - / - The recipient mice showed a slower weight loss ( Fig.17 a), and the proportion of Treg cells in the blood, spleen, mesenteric lymph nodes and colon increased significantly ( Fig.17 b).

[0200] The results showed that SCD1 - / - Mouse source T cells differentiate into Tregs and resist the progression of colitis.

[0201] discuss

[0202] Thymic epithelial cells provide a crucial tissue microenvironment for the development of T cells. Studies have found that in the thymic microenvironment, the SCD1 activity of thymic epithelial cells plays a key role in regulating the ability of T cells to differentiate. This is done by adjusting the availability of oleic acid in DN3 thymocytes, thereby presetting the response characteristics of initial CD4+T cells at an early stage. Lack of oleic acid during thymic development causes mature T cells to tend to differentiate into Treg cells and reduces signs of autoimmune diseases. The study also pointed out that in SCD1 - / - Mouse and K14 Cre SCD1 oxp In mice, Th17 cell differentiation was affected, but Th1 and Th2 cell differentiation did not change significantly. Since Treg and Th17 cells are interrelated in the development of autoimmunity and tolerance induction, oleic acid in the thymic microenvironment is essential for balancing Treg and Th17 cell differentiation in peripheral mature T cells. - / -Changes in the transcriptional levels of certain genes, such as Ccl19, Serpina1a and Serpina1b, were found in the cortical and medullary epithelial cells of mice, but thymus transplantation in nude mice and experiments with oleic acid supplementation in vivo and in vitro confirmed that thymic epithelial cells mainly affect the tendency of Treg cell differentiation by producing oleic acid rather than expressing specific genes.

[0203] Fatty acids play an important role in cellular energy supply, and Treg cells rely on fatty acid β-oxidation to obtain energy. Studies have shown that supplementation of fatty acids, such as oleic acid, can enhance the immunosuppressive ability of Treg cells. However, by inhibiting ACC1 to block the synthesis of new fatty acids, the level of Treg cells in the body can be increased. At present, studies on the regulation of fatty acids and T cells mainly focus on peripheral mature T cells. However, we found that the differentiation trend of Treg cells caused by SCD1 deficiency is not related to the effect of oleic acid on mature T cells. In fact, the regulatory effect of oleic acid on the differentiation trend of Treg is to affect early thymocytes by regulating the activity of the only H3K79 methyltransferase Dot1L. This finding reveals a new function of oleic acid in inhibiting the activity of H3K79 methyltransferases. Interestingly, we also observed that palmitic acid also had a certain degree of inhibitory effect on the enzymatic activity of Dot1L, although the effect was not as obvious as oleic acid. This suggests that other monounsaturated fatty acids may also affect the development of T cells through epigenetic modifications.

[0204] Calcium signaling is a key step in regulating T cell activation and differentiation, but its regulatory effects on the fate of CD4+ T cells are complex and diverse. For example, in mice lacking specific calcium signaling molecules, different cell subsets such as Th17 and Treg cells showed different responses. For example, mice lacking Stim1 / Stim2 had decreased Treg cell levels due to defects in calcium signaling, while Orai1 - / - Mice have reduced Th17 cell differentiation without affecting Treg cells. Recent studies have also found that there is a naive T cell subset that is inherently inclined to Treg cell differentiation, which exhibits this tendency due to the increase of calcium-NFAT signaling pathway. In addition, the NFAT-Foxp3 signaling pathway plays a key role in the induction of peripherally derived Treg (pTreg) cells driven by high-affinity antigen stimulation. Studies have found that in SCD1 - / - Mouse CD4 + In T cells, enhanced ATP2A2-related calcium signals regulated the expression of Foxp3 and directed its developmental potential to Treg cells, discovering a new mode of calcium ion signal regulation of T cell differentiation fate.

[0205] In adults, the rate of T cell lymphopoiesis under physiological conditions is relatively low. However, lymphopenia caused by chemotherapy, radiotherapy or infection stimulates the body to produce new T cells. Studies have found that by manipulating oleic acid availability or SCD enzyme activity, we can develop a new means of immune regulation. In addition, we can use an in vitro T cell development system to obtain more T cell precursors with Treg cell tendencies by adjusting the oleic acid content of developing T cells, and then transplant them into recipients with autoimmune diseases. In summary, the study found that oleic acid in the thymic microenvironment has a regulatory role in the development of T cells at the DN2-DN3 stage, providing new opportunities for regulating T cell immune responses.

[0206] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing a cell population with enhanced Treg cell differentiation potential, comprising the steps of: (S1) providing T cell precursors; (S2) culturing T cell precursors at the double negative stage (i.e., DN stage) under conditions where stearoyl-CoA desaturase 1 (SCD1) is inhibited to obtain a cell population with enhanced Treg cell differentiation potential; The expression of Atp2a2 is upregulated in the cell population.

2. The method according to claim 1, characterized in that In step (S2), the cell population with improved Treg cell differentiation potential is cultured, and the percentage of Treg cells in CD4+ cells is recorded as A1. In the control group cultured without inhibiting SCD1 and with the same other conditions, the percentage of Treg cells in CD4+ cells is recorded as A0. The “increased Treg cell differentiation potential” refers to A1 / A0≥1; preferably ≥2.5; more preferably ≥10; and more preferably ≥20.

3. The method according to claim 1, characterized in that The T cell precursors in the double negative stage (ie, DN stage) include cells selected from the following group: T cell precursors in the DN3 stage and the DN4 stage.

4. The method according to claim 1, characterized in that The T cell precursors are derived from induced pluripotent stem cells, hematopoietic stem cells, or hematopoietic precursor cells.

5. A cell population with improved Treg cell differentiation potential, characterized in that: The cell population is prepared by the method of claim 1.

6. A use of a SCD1 inhibitor, characterized in that: Used to prepare a composition or preparation, wherein the composition or preparation is used to induce Treg cell differentiation.

7. The use according to claim 6, characterized in that The composition or preparation is used for preventing and / or treating autoimmune diseases.

8. The use according to claim 6, characterized in that The composition or preparation is also used for one or more uses selected from the following group: (c) promote the activity of DOT1L in thymocytes; (d) promote H3K79me2 activity in thymocytes; (e) Promote the expression of ATP2A2 in thymocytes; (f) down-regulating the content of oleic acid in thymic tissue; and (g) Upregulation of stearic acid content in thymus tissue.

9. A cell preparation comprising: A cell population with improved Treg cell differentiation potential as claimed in claim 5; and a pharmaceutically acceptable carrier.

10. A method for inducing Treg cell differentiation, characterized in that: The preprogrammed T cell population with enhanced Treg cell differentiation potential according to claim 5 is infused into a subject in need thereof to obtain an induced differentiated Treg cell population.

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