Induced regulatory t cells with improved stability and suppressive function in inflammatory environment and preparation method and application thereof
Patent Information
- Application Number
- CN202611053498.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-15
- Publication Date
- 2026-10-27
Smart Images
Figure CN122979228A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to an inducible regulatory T cell with enhanced stability and inhibitory function in an inflammatory environment, its preparation method and application. Background Technology
[0002] Regulatory T cells (Tregs) are a key cellular subset for maintaining immune homeostasis, characterized by the expression of the transcription factor Foxp3 (Forkhead box P3). Tregs can suppress the activation and proliferation of effector T cells (Teffs) through contact-dependent mechanisms (such as CTLA-4 and LAG-3) and the secretion of repressive cytokines (such as IL-10 and TGFβ), thereby preventing autoimmune responses. Therefore, adoptive cell therapy (ACT) based on Treg cells is considered one of the most promising strategies for treating autoimmune diseases and graft-versus-host disease (GvHD). Depending on their origin, Tregs can be mainly divided into thymus-derived innate regulatory T cells (nTregs) and peripherally / in vitro induced regulatory T cells (iTregs). Although nTregs have good stability, their abundance in human peripheral blood is extremely low (accounting for only a small percentage of CD4+). + The fact that T cells account for only 5%-10% of the total T cells and that it is difficult to isolate and obtain a high-purity antigen-specific population, as well as the limited in vitro amplification, greatly restricts the accessibility of its clinical application.
[0003] In contrast, iTregs are Treg cells induced from naïve CD4+ T cells through in vitro antigen stimulation in the presence of cytokines such as TGFβ and IL-2. iTregs have enormous clinical application potential: they are abundant in source, and naïve T cells can be easily obtained in large quantities from the peripheral blood of patients or donors. However, despite the advantages of iTregs in preparation technology, they face a critical bottleneck in clinical translation—phenotypic instability and functional plasticity.
[0004] Studies have shown that Foxp3, as the "master regulator" of Treg cells, is essential for maintaining Treg function. Unlike nTregs, the conserved non-coding sequence 2 (CNS2 / TSDR) region of the Foxp3 gene in in vitro induced iTregs is often highly methylated, which means that the expression of Foxp3 is unstable at the epigenetic level.
[0005] Lesions in autoimmune diseases are typically located in a highly inflammatory environment, rich in pro-inflammatory cytokines such as IL-6, IL-1β, IL-21, and TNF-α. Numerous studies have confirmed that pro-inflammatory cytokines are key factors disrupting iTreg stability. In the presence of pro-inflammatory cytokines, the STAT3 signaling pathway is overactivated, which not only directly inhibits Foxp3 expression but also induces the expression of RORγt (Retinoic acid receptor-related orphan receptor gamma t), a key transcription factor in Th17 cells. This imbalance of transcription factors leads to iTreg transdifferentiation, where Tregs that originally had immunosuppressive functions lose Foxp3 and transform into Th17-like cells (Ex-Tregs) that secrete pro-inflammatory factors such as IL-17. This transformation means that reinfused iTregs, while losing their therapeutic effect, may also become pro-inflammatory immune cells, exacerbating the local inflammatory response. Therefore, how to enable iTreg to maintain Foxp3 expression and its inhibitory function stably in a strongly inflammatory microenvironment for a long period of time is a key technical problem that urgently needs to be solved in this field.
[0006] To address the instability of iTreg culture in vitro, existing technologies have mainly attempted the following strategies, but all have certain limitations: (1) Drug-assisted induction strategy: adding small molecule compounds such as rapamycin, all-trans retinoic acid (atRA), or vitamin C to the culture medium. Although these drugs can promote Foxp3 expression to some extent by regulating the mTOR pathway or TET enzyme activity, this effect is usually temporary and reversible. Once the cells leave the in vitro culture system and enter the complex inflammatory environment in vivo, the drug effect quickly subsides, and iTregs still face the risk of transformation; (2) Epigenetic modification strategy: treating cells with DNA methyltransferase inhibitors (such as 5-azacytidine) in an attempt to mimic the TSDR demethylation state of nTregs. However, these drugs have a general non-specific effect, which can lead to hypomethylation across the entire genome, causing genomic instability and potential tumorigenic risks and cytotoxicity; (3) Gene knockout strategy: using CRISPR / Cas9 technology to knock out certain negative regulatory factors. While knocking out certain genes can improve stability, it may also disrupt the normal physiological metabolism or proliferation of T cells, leading to a shortened cell survival time (reduced persistence); (4) Gene stable expression strategy: By forcibly expressing specific "stabilizing factors" or "transcriptional regulatory factors" in iTregs, a robust network against inflammatory signal interference can be established inside the cell. The most direct approach at present is to stably express Foxp3 itself. However, studies have shown that this strategy has not solved the dependence of iTregs on exogenous TGF-β supply. Once cells are removed from the culture environment containing TGF-β, the expression level of Foxp3 will rapidly decline over time.
[0007] Given the limitations of the existing technologies, especially the shortcomings of stable expression strategies represented by Foxp3 in terms of long-term stability, tolerance to inflammatory environments, and dependence on exogenous TGF-β, there is an urgent need in the field to develop an engineered modification scheme that does not depend on exogenous TGF-β and can stably maintain the iTreg lineage characteristics in an inflammatory environment for a long time. Summary of the Invention
[0008] The purpose of this invention is to overcome the limitations of existing technologies in enabling iTregs to maintain stable Foxp3 expression and its inhibitory function in an inflammatory environment for a long period of time, thereby providing a regulatory T cell with improved stability and inhibitory function in an inflammatory environment, as well as its preparation method and application.
[0009] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide an inducible regulatory T cell expressing constitutively activated transforming growth factor β receptor 1 (caTGFBR1), wherein the inducible regulatory T cell expressing constitutively activated transforming growth factor β receptor 1 can improve its stability and inhibitory function in an inflammatory environment.
[0010] In some specific embodiments, the amino acid sequence of the constitutively activated transforming growth factor β receptor 1 is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.
[0011] In some specific embodiments, the constitutively activated transforming growth factor β receptor 1-inducible regulatory T cells also overexpress one or more cytokines, transcription factors and surface molecules, homing molecules, and targeting molecules that recognize disease site or transplant organ-specific targets. The cytokines are selected from any one or more of IL-2, IL-10, and IL-35; The transcription factors and surface marker molecules are selected from any one or more of Foxp3, CD25, CD39, PD-L1, and CTLA-4; The homing molecules are selected from any one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and CX3CR1; The target molecule is selected from chimeric antigen receptor molecules or ligands of tissue-specific receptors.
[0012] In this invention, the homing molecule promotes the chemotaxis of Tregs towards disease sites in vivo, thereby enhancing the therapeutic effect.
[0013] This invention provides a strategy for regulating the stability of iTreg cells based on constitutive activation of the stable expression of transforming growth factor-β type I receptor (caTGFBR1). This strategy establishes a persistent TGF-β signaling pathway in iTreg cells that is independent of exogenous ligands, activating the TGF-β / SMAD signaling axis upstream of Foxp3, thereby maintaining stable Foxp3 expression at the transcriptional level, rather than directly overexpressing the Foxp3 protein using traditional methods.
[0014] The second technical solution of the present invention is to provide a method for preparing induced regulatory T cells expressing constitutively activated transforming growth factor β receptor 1 (caTGFBR1) as described in one of the above technical solutions, wherein the encoding gene of constitutively activated transforming growth factor β receptor 1 is transfected into induced regulatory T cells by viral transfection.
[0015] In some specific embodiments, the virus transfection method includes the following steps: S1. The gene encoding constitutively activated transforming growth factor β receptor 1 as shown in SEQ ID NO.2 is recombinantly ligated with a viral vector to obtain a recombinant viral plasmid. S2. Transfect the recombinant viral plasmid obtained in step S1 into HEK293T cells and collect the supernatant containing the virus. S3. Co-culture the virus-containing supernatant obtained in step S2, induced regulatory T cells, and induction culture medium to induce the induced regulatory T cells to express constitutively activated transforming growth factor β receptor 1.
[0016] In some specific embodiments, in step S1, the viral vector is selected from any one of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, retrovirus vectors, herpesvirus vectors, RNA virus vectors, EB virus vectors, baculovirus vectors, bacteriophage vectors, and animal virus vectors.
[0017] In some specific embodiments, step S3, the induction culture medium includes IL-2, rapamycin, and TGFβ; After 72 h of co-culture, TGFβ was removed from the induction medium and culture continued.
[0018] The third technical solution of the present invention is to provide the application of inducible regulatory T cells expressing constitutively activated transforming growth factor β receptor 1 as described in one of the above technical solutions in the preparation of drugs for the treatment of autoimmune diseases and / or organ transplantation anti-rejection therapy and / or cell transplantation anti-rejection therapy.
[0019] In some specific embodiments, the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, inflammatory bowel disease, type I diabetes, autoimmune hepatitis, polyneuritis, primary thrombocytopenic purpura, and autoimmune hemolytic anemia.
[0020] The fourth technical solution of the present invention is to provide a drug for the treatment of autoimmune diseases and / or organ transplantation anti-rejection therapy and / or cell transplantation anti-rejection therapy, comprising, as described in one of the above technical solutions, inducible regulatory T cells expressing constitutively activated transforming growth factor β receptor 1.
[0021] In some specific embodiments, the drug further includes a pharmaceutically acceptable carrier and / or excipient and / or adjuvant.
[0022] The drug of this invention can be used alone or in combination with other drugs.
[0023] Compared with the prior art, the present invention has the following advantages: (1) This invention will express caTGFBR1 in Naive CD4 + T-induced iTregs effectively maintained the stability of the phenotype and function of induced caTGFBR1-iTreg cells (CAT-iTreg) after they were removed from the culture system. The induced CAT-iTregs had stronger in vivo immunosuppressive function compared with traditionally induced iTregs.
[0024] (2) This invention achieves sustained signal transduction independent of exogenous TGF-β by stably expressing caTGFBR1 in induced regulatory T cells (iTregs), thus maintaining the activation of the TGF-β signaling pathway in iTregs. It can maintain the expression of the characteristic transcription factor Foxp3 of iTregs in a long-term inflammatory environment lacking TGF-β in vivo and in vitro, significantly improving its phenotypic and functional stability, while greatly reducing the cost of in vitro expansion.
[0025] (3) The stable expression strategy of CAT-iTreg constructed in this invention significantly improves the stability and persistence of the Foxp3 lineage of iTreg cells compared with the traditional stable expression strategy of Foxp3. This is reflected in the fact that, during cell culture, under the condition of complete withdrawal of exogenous TGF-β, the Foxp3 positive rate of iTreg cells with stable expression of CAT-iTreg remained stable at over 60% after 28 days of expansion; while under the same conditions, the Foxp3 positive rate of the stable expression control group of Foxp3 had dropped to about 20% of the initial level.
[0026] (3) This invention effectively solves the technical problem that iTregs are prone to transdifferentiation and have unstable function in an inflammatory environment, and provides a more reliable cell preparation scheme for the treatment of autoimmune diseases and organ transplantation anti-rejection therapy, with outstanding technical advancement and clinical translational value.
[0027] (4) The method of the present invention is to transfect cytokine-encoding genes with viruses and express them exogenously in the host, which saves the amount of expensive cytokines used in the induction culture system and minimizes the culture cost. Attached Figure Description
[0028] Figure 1 Verification of the caTGFBR1-mediated iTreg signaling pathway. Among them, Figure 1 A: Schematic diagram of the construction of a stable expression plasmid carrying caTGFBR1. The constructed plasmid is used to package lentiviruses to infect iTreg cells. Figure 1 B: Schematic diagram of the culture of the positive control iTreg group, the negative control Mock-iTreg group, and the experimental group CAT-iTreg group. Figure 1 C: Flow cytometry plots of infection efficiency (positive rate of NGFR with truncation of labeled protein) in the positive control iTreg group, the negative control Mock-iTreg group, and the experimental CAT-iTreg group. Figure 1 D: Statistical graph of infection efficiency among positive control iTreg group, negative control Mock-iTreg group and experimental group CAT-iTreg group among different donors (data taken from 5 independent donors). Figure 1 E: In vitro amplification of CAT-iTreg stably expressed by caTGFBR1. Figure 1 F and 1G: Phosphorylation of smad2 / 3 proteins, downstream signaling proteins of TGF-β, and mRNA levels of smad7 in CAT-iTreg without the addition of TGF-β (data obtained from 3 independent donors). Statistical analysis was performed using one-way ANOVA, and Tukey's multiple comparisons method was used for post-hoc testing of differences between groups.
[0029] Figure 2 Phenotypic stability analysis of in vitro expanded CAT-iTreg. Among them, Figure 2 A: The in vitro culture procedure for CAT-iTreg. Figure 2 B: Flow cytometry plot of Foxp3 expression changes during 24 days of in vitro culture of CAT-iTreg. Figure 2 C: Line graph showing the proportion of Foxp3-positive cells in the iTreg, CAT-iTreg, and Mock-iTreg groups (data from 3 independent donors). Foxp3 was detected on days 9, 12, 15, 18, 21, and 24 of induction culture. Statistical analysis was performed using one-way ANOVA, and Tukey's multiple comparisons method was used for post-hoc testing of differences between groups. (p<0.05). Figure 2 D: Mean fluorescence intensity of Foxp3-positive cell populations in the iTreg, CAT-iTreg, and Mock-iTreg groups (data obtained from 3 independent donors). Foxp3 was detected on days 9, 12, 15, 18, 21, and 24 of induction culture. Figure 2 E: Radar graph of the relative amount of cytokine secretion in the cell supernatant of the iTreg group, CAT-iTreg group and Mock-iTreg group after 24 hours of stimulation by PMA / ionomycin on day 22 of culture. Figure 2 F: Figure 2 Statistical graph of corresponding cytokine secretion levels in E (data from 3 independent donors).
[0030] Figure 3 : Validation of the effect of stable expression of cAGFBR1 on regulating iTreg. Among them, Figure 3A: Three groups of iTreg cells were co-incubated with Tconv cells stained with eflour670 at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32, respectively, for 96 hours. Flow cytometry peaks were obtained showing the inhibition of Tconv cell proliferation by different groups of iTreg cells. Figure 3 B: Line graph showing the inhibition of Tconv cell proliferation by different groups of iTreg cells. The horizontal axis represents the effector-target ratio, and the vertical axis represents the proliferation inhibition rate. Figure 3 C: Flow cytometry overlay plots of the inhibition of CD8 effector T cell proliferation by three groups of iTreg cells with eflour670 at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32, respectively, after 96 hours of co-incubation with the iTreg cells. Figure 3 D: Line graph showing the inhibition of CD8 effector T cell proliferation by different groups of iTreg cells. The horizontal axis represents the effector-to-target ratio, and the vertical axis represents the proliferation inhibition rate.
[0031] Figure 4 : Validation of the effect of stable expression of cATGBR1 on regulating iTregs in an inflammatory environment. Among them, Figure 4 A: iTreg inflammatory environment culture flowchart. Figure 4 B: Flow cytometry peak overlay plots of Foxp3 expression in three groups of cells before inflammatory culture (day 0) and after inflammatory culture (day 10) (data from three independent donors). Figure 4 C: Statistical graphs of Foxp3 positivity rates in the three groups of cells before inflammatory culture (day 0) and after inflammatory culture (day 10). Statistical analysis was performed using two-way ANOVA, and post-hoc multiple comparisons were performed using Šídák's multiple test (ns: no statistically significant difference). :p<0.0001). Figure 4 D: After inflammatory culture, three groups of iTreg cells were co-incubated with Tconv cells stained with eflour670 at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32, respectively, for 96 hours. Flow cytometry peaks showing the inhibition of Tconv cell proliferation by different groups of iTreg cells. Figure 4 E: Flow cytometry overlay plots of the three groups of iTregs after inflammatory culture, which were co-incubated with CD8 effector T cells stained with eflour670 at ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32, respectively, after 96 hours. Figure 4F: Line graph showing the inhibition of Tconv cell proliferation by different groups of iTreg after inflammatory culture. The horizontal axis represents the effector-to-target ratio, and the vertical axis represents the proliferation inhibition rate. Statistical analysis was performed using two-way ANOVA, and Tukey's multiple test was used for post-hoc multiple comparisons. p<0.05 :p<0.01 (p<0.001). Figure 4 G: Line graph showing the inhibition of CD8 effector T cell proliferation by different groups of iTreg cells after inflammatory culture. The horizontal axis represents the effector-to-target ratio, and the vertical axis represents the proliferation inhibition rate.
[0032] Figure 5 : Validation of the effect of stable expression of caTGFBR1 regulating iTreg on intervention of GvHD. Among them, Figure 5 A: A schematic diagram of the GvHD model construction process and the iTreg treatment method. Figure 5 B: A line graph showing the percentage decrease in body weight of mice in each group over 28 days. Figure 5 C: Survival curves reflecting the survival status of mice in each group over 28 days. Figure 5 D: Line graph reflecting the GvHD scores of mice in each group over 28 days. Figure 5 E: Pathological findings of mice in each group on day 14. Tissue samples taken included skin, lungs, and small intestine.
[0033] Figure 6 Assessment of the in vivo lineage stability of iTreg cells stably expressing caTGFBR1. Among them, Figure 6 A: Schematic diagram of in vivo lineage stability verification. Figure 6 B: Changes in Foxp3 expression in iTreg and CAT-iTreg cells before and after reinfusion (day 0) and after reinfusion (day 5). The left side shows the Foxp3 peak diagram before reinfusion, and the right side shows the Foxp3 expression peak diagram, positive rate, and average fluorescence intensity of iTreg and CAT-iTreg cells in peripheral blood, spleen, liver, and lungs of mice. An independent samples t-test was used. p<0.05 :p<0.01).
[0034] Figure 7 The ability of caTGFBR1 stable expression to mediate the maintenance of iTreg lineage stability. Among other things, Figure 7 A: Flow cytometry plot of the expression changes of the marker gene NGFR in the CAT-iTreg group and the Foxp3-iTreg group during in vitro culture over 28 days. Figure 7 B: Line graph showing the proportion of NGFR-positive cells in the CAT-iTreg group and the Foxp3-iTreg group (data from 3 independent donors). NGFR was detected on days 7, 14, 21, and 28 of induction culture. Statistical analysis was performed using one-way ANOVA, and Tukey's multiple comparisons method was used for post-hoc testing of differences between groups. :p<0.001 :p<0.0001). Figure 7 C: Flow cytometry plot of Foxp3 expression changes during in vitro culture over 28 days in the CAT-iTreg and Foxp3-iTreg groups. Figure 7 D: Line graph showing the proportion of Foxp3-positive cells in the CAT-iTreg group and the Foxp3-iTreg group (data from 3 independent donors). Foxp3 was detected on days 7, 14, 21, and 28 of induction culture. Statistical analysis was performed using one-way ANOVA, and Tukey's multiple comparisons method was used for post-hoc testing of differences between groups. p<0.05 :p<0.01 :p<0.0001). Detailed Implementation
[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.
[0036] Unless otherwise defined, 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
[0037] As used in this article, the term "caTGFBR1" stands for Constitutively Activated Transforming Growth Factor β Receptor 1, which means that after a mutation occurs in the TGFBR1 gene, the receptor can continuously activate downstream signaling pathways without binding to a ligand (TGF-β) or type II receptor (TGFBR2).
[0038] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.
[0039] Example 1: This embodiment provides a method for preparing induced regulatory T cells stably expressing caTGFBR1, comprising the following steps: (1) Constructing the recombinant plasmid pHAGE-SFFV-caTGFBR-P2A-NGFR containing caTGFBR1 The gene sequence for stable expression of caTGFBR1 was designed, as shown in SEQ ID NO.2, and its corresponding amino acid sequence is shown in SEQ ID NO.1. The synthesized... caTGFBR1 Using gene sequences as templates, design and amplify target genes. caTGFBR1 The upstream and downstream primers, with nucleotide sequences shown in SEQ ID NO. 3 and 4, respectively, were used to amplify the target gene by PCR. caTGFBR1 Finally, the target gene was recovered by 1% agarose gel electrophoresis. caTGFBR1 DNA band (SEQ ID NO.2, 1521 bp).
[0040] caTGFBR1 (SEQ ID NO.1): MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTGPFSVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGAALLVQRTIARDIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM caTGFBR1 Gene (SEQ ID NO.2): Upstream primer (SEQ ID NO.3): AACTTACGCGTCACCATGGAGGCGGCGGTCG; Downstream primer (SEQ ID NO.4): AGTAGCTCCGCTTCCCATTTTGATGCCTTCC.
[0041] The recombinant plasmid pHAGE-SFFV-P2A-NGFR was independently constructed and preserved in our laboratory. Using a commercially available pHAGE lentiviral vector as its backbone, the plasmid inserted an SFFV promoter, a P2A self-cleaving peptide sequence, and an NGFR gene expression element. After sequence verification, it was cryopreserved. Using this plasmid as a template, a linearized vector was obtained through PCR amplification. The PCR reaction system is shown below. The amplification product was separated by 1% agarose gel electrophoresis and then recovered by gel excision to obtain the purified linearized vector. The nucleotide sequences of primer 1 and primer 2 are shown in SEQ ID NO. 5 and 6, respectively: Primer 1 (SEQ ID NO.5): GGAAGCGGAGCTACTAACTT Primer 2 (SEQ ID NO. 6): GGTGACGCGTAAGTTTAGCT The target gene was homologously ligated to the linearized vector. The ligation reaction was carried out at 37°C for 30 min. The ligation reaction system is as follows: The homologous recombination ligation product was transformed as follows: 10 μL of the ligation product was mixed with 100 μL of DH5α competent bacteria and incubated on ice for 30 min. After heat shock at 42℃ for 45 s, the mixture was immediately placed on ice for 2 min. 900 μL of preheated LLB medium was added and cultured in a shaker at 37℃ for 1 h. After centrifugation at 5000 rpm for 5 min, the 900 μL culture supernatant was discarded. The remaining 100 μL was mixed with a pipette and evenly spread on an LB plate containing 100 ug / mL Ampicillin resistance. The plate was incubated upside down in a 37℃ incubator overnight.
[0042] Three single colonies were picked from LB plates and inoculated into 5 mL of LB medium containing 100 μg / mL Ampicillin resistance. The cultures were incubated overnight at 37°C with a constant temperature shaker at 250 rpm. The plasmid was extracted using a small-volume plasmid extraction kit to obtain the pHAGE-SFFV-caTGFBR1-P2A-NGFR recombinant plasmid, which was then sequenced for verification.
[0043] (2) Lentiviral preparation and packaging Take 3 μg of the successfully verified pHAGE-SFFV-caTGFBR1-P2A-NGFR recombinant plasmid in step (1), mix it with the packaging plasmid psPAX2 and the envelope plasmid pVSVG at a mass ratio of 1:3:1, and co-transfect it into HEK293T cells in a 10cm cell culture dish using Mirus high-efficiency transfection reagent. After transfection, the cells were cultured for 48 hours, and the supernatant containing the virus was collected. Cell debris and impurities were removed by centrifugation and filtration, and the contents were aliquoted and stored at -80℃ for later use.
[0044] like Figure 1 As shown in Figure A, the control group iTreg and Mock-iTreg do not contain caTGFBR1 The gene, namely the plasmid pHAGE-SFFV-P2A-NGFR, the packaging plasmid psPAX2, and the envelope plasmid pVSVG, was co-transfected into cultured HEK293T cells.
[0045] (3) Separate CD4 + CD45RA + Naive CD4 + T cells Naive CD4 cells were isolated from human peripheral blood mononuclear cells (PBMCs) using a Stemcell sorting kit with magnetic beads. + T cells, Naive CD4 was evaluated by flow cytometry. + The purity of the T cells exceeds 95%.
[0046] (4) Activation-induced Naive CD4 + T cells In a 96-hole U-shaped base plate, add 2×10 ohms per hole. 5 Naive CD4 at a cell density + T cells, induction culture medium (500 U / mL recombinant human IL-2, 50 nM rapamycin, 5 ng / mL TGFβ), and 0.5 μL of T cell activator anti-CD3 / CD28 magnetic beads (concentration 4 × 10⁻⁶). 5 The magnetic beads were co-cultured in 200 μL of X-vivo 15 cell culture medium (04-418Q, Lonza) at a ratio of approximately 1:1 to 1 μL.
[0047] (5) Lentiviral transduction of Naive CD4 + T cells were used to prepare caTGFBR1-iTreg cells (CAT-iTreg cells for short). Naive CD4 +After culturing T cells for 48 hours, the magnetic beads were removed, and 0.5 μL of 100x NATE (nucleic acid transfection enhancer) was added for 30 min. Then, 200 μL of viral supernatant and 2.5 μL of 100x LVTE (lentiviral transduction enhancer) were added, and the mixture was centrifuged at 1000g for 1 hour and 30 minutes. Subsequently, the viral supernatant was removed, and the induction medium (500 U / mL recombinant human IL-2, 50 nM rapamycin, 5 ng / mL TGFβ) was added.
[0048] (6) According to Figure 1 B is subjected to grouped induction culture. The specific grouped cell culture procedure is as follows: Based on steps (4) and (5), Naive CD4 + After 48 h of T cell activation induction culture, lentivirus was used to activate Naive CD4 cells. + T cells were transduced; 72 h after transduction, TGF-β was removed from the culture medium of both the CAT-iTreg group (transduced with caTGBFR1 lentivirus) and the Mock-iTreg group (transduced with NGFR control lentivirus); the iTreg control group (transduced with NGFR control lentivirus) was cultured in induction medium throughout the process.
[0049] The transduction efficiency (NGFR positivity rate) of CAT-iTreg cells was detected using flow cytometry fluorescence sorting (FACS). Figure 1 C and Figure 1 As shown in D, the transduction efficiency of the CAT-iTreg group was approximately 70%, and the in vitro expansion rate of transduced cells was not affected. Figure 1 E), the counting time points are 0 days, 6 days, 12 days, 8 days, 24 days and 30 days.
[0050] The phosphorylation level of TGFβ downstream signal Smad 2 / 3 was detected by Western blot. Figure 1 F) and the transcriptional level of smad7 was detected by qPCR ( Figure 1 The results showed that the phosphorylation levels of smad2 / 3 and the transcription levels of smad7 in the CAT-iTreg group were significantly higher than those in the Mock-iTreg group, indicating that caTGFBR1 can still spontaneously activate downstream signals of TGFβ without the addition of TGFβ.
[0051] Example 2 This example demonstrates the phenotypic stability analysis of in vitro amplified CAT-iTreg.
[0052] The iTreg cell expansion and culture protocol was optimized: for Naive CD4 cells... +T cells were activated and induced (500 U / mL recombinant human IL-2, 50 nM rapamycin, 5 ng / mL TGFβ, and 0.5 μL anti-CD3 / CD28 magnetic beads, with a bead-to-cell ratio of 1:1) and cultured for 48 h. Then, lentivirus was used to activate Naive CD4 cells. + T cells were transduced; 72 hours after infection, TGF-β was removed from the culture medium in the CAT-iTreg and Mock-iTreg groups, while the iTreg control group was cultured in induction medium throughout the process. Every 9 days, all iTreg groups received a new round of magnetic bead stimulation (the ratio of magnetic beads to cells was approximately 1:1). Results are detailed below. Figure 2 A.
[0053] Further investigation was conducted to examine the long-term maintenance effect of caTGFBR1 on stable Foxp3 protein expression in iTreg cells: On day 5 after infection with a stably expressed lentiviral vector, TGFβ cytokines were removed from the culture system, and the cells were cultured for 30 days. Results are as follows... Figure 2 B. Figure 2 C and Figure 2 As shown in Figure D, compared with the Mock-iTreg group, the CAT-iTreg group can significantly maintain stable Foxp3 expression, demonstrating a clear advantage in phenotypic maintenance.
[0054] Collect 1×10⁻⁶ cells cultured up to day 22. 5 iTreg cells were stimulated with phorbol ester PMA (50 ng / mL) combined with iomycin (1 μg / mL), and cell supernatant was collected. Cytokine secretion levels in each group were detected using the CBA method. The results are as follows: Figure 2 E and 2F showed that the levels of inflammatory cytokines such as IL-2, IL-4, IFN-γ, IL-17A, and TNF secreted by cells in the CAT-iTreg group were significantly lower than those in the conventional iTreg group and the Mock-iTreg group.
[0055] In summary, caTGFBR1 can significantly enhance the phenotypic stability of iTreg cells and effectively inhibit the abnormal secretion of inflammatory factors.
[0056] Example 3 This embodiment is for further evaluation of the in vitro immunosuppressive function of CAT-iTreg cells.
[0057] Tconv cells (conventional T cells) and CD8 effector T cells were isolated from human peripheral blood mononuclear cells (PBMCs) using magnetic bead sorting. The isolated Tconv cells and CD8 effector T cells were labeled and stained with efluor 670 proliferation dye (Thermofisher, catalog number: 65-0840-90). CD3 / CD28 stimulating magnetic beads were mixed with the T cells at a ratio of 1:5 for activation stimulation. The culture volume was 150 μL, and the cells were cultured in 5 × 10⁶ wells. 4 One cell was seeded in a 96-well U-shaped culture plate.
[0058] 50 μL of serially diluted iTreg cells were added to each well, with iTreg cell to T cell ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. A blank control group (Tconv cells or CD8 effector T cells without magnetic bead stimulation) and a single-stimulation control group (Tconv cells or CD8 effector T cells activated only by magnetic beads) were also set up, with a system volume of 200 μL / well. After 4 days of co-culture, the proliferation levels of Tconv cells and CD8 effector T cells were detected by flow cytometry.
[0059] like Figure 3 The experimental results of AD showed that, compared with the Mock-iTreg group, the CAT-iTreg group could significantly inhibit the abnormal proliferation of Tconv cells and CD8 effector T cells, and had a better in vitro immunosuppressive ability.
[0060] Example 4 This embodiment aims to further explore the regulatory effect of caTGFBR1 on iTreg cells under inflammatory stress.
[0061] In Example 2, a pro-inflammatory factor TNF-α with a final concentration of 100 ng / mL was added to the iTreg in vitro expansion culture system, and TGFβ and rapamycin were removed to simulate the in vivo inflammatory microenvironment. The experimental protocol is as follows: Figure 4 As shown in A: Collect 2 × 10n iTreg cells from each group at the end of the culture period. 5 Cells were placed in a culture medium containing 500 U / mL recombinant human IL-2, 100 ng / mL TNF-α, and 0.5 μL of anti-CD3 / CD28 magnetic beads (the ratio of magnetic beads to cells was approximately 1:1) for inflammatory stress culture. After 72 hours of culture, the magnetic beads were removed, and the cells were further cultured in the above-mentioned cytokine-containing system for 96 hours. Subsequently, 2 × 10⁶ cells were collected. 5Each iTreg cell was given a new round of stimulation (500 U / mL recombinant human IL-2 and 0.5 μL anti-CD3 / CD28 magnetic beads, with a bead-to-cell ratio of approximately 1:1). Cells were collected 72 hours later for subsequent testing.
[0062] The expression level of Foxp3 in iTreg cells of each group was detected by flow cytometry. Figure 4 The results of experiments B and 4C showed that after stimulation by inflammatory factors, the Foxp3 expression levels in cells of the iTreg group, Mock-iTreg group and CAT-iTreg group were downregulated to varying degrees; among them, the decrease in Foxp3 expression in the CAT-iTreg group was significantly lower than that in the iTreg group and the Mock-iTreg group.
[0063] The above results confirm that caTGFBR1 can effectively stabilize the expression of Foxp3 in iTreg cells under inflammatory stimulation and significantly improve the phenotypic stability of iTreg cells under inflammatory conditions.
[0064] To clarify the regulatory role of caTGFBR1 on the in vitro inhibitory function of iTreg cells under inflammatory environmental stimulation, iTreg cells induced by TNF-α inflammation were cultured in various ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32, and then compared with 5×10⁶ cells per cell. 4 One Tconv cell or 5 × 10 4 CD8 effector T cells were co-cultured; CD3 / CD28 stimulating magnetic beads were added simultaneously for activation stimulation, with the ratio of T cells to stimulating magnetic beads being 5:1.
[0065] After co-culturing the cells in each group for 4 days, the proliferation levels of Tconv cells and CD8 effector T cells were detected by flow cytometry. The experimental results are as follows: Figure 4 As shown in D~4G: After intervention with an inflammatory environment, the CAT-iTreg group maintained stronger cell proliferation inhibition activity compared with the iTreg group and the Mock-iTreg group.
[0066] In summary, under inflammatory stimulation, caTGFBR1 can effectively preserve and maintain the immunosuppressive function of iTreg cells by continuously stabilizing the expression of Foxp3 in iTreg cells, thus preventing functional instability of iTreg cells in the inflammatory microenvironment.
[0067] Example 5 Using CAT-iTreg prepared in Example 1 as the research object, its in vivo therapeutic effects in a mouse graft-versus-host disease (GvHD) model were compared with those of conventional iTreg and Mock-iTreg. Figure 5A represents the overall experimental procedure for constructing the GvHD model and iTreg intervention in this embodiment.
[0068] The specific operational steps are as follows: One day before the orbital vein infusion of cells, female NSG mice aged 6-8 weeks and weighing 18-22g were irradiated with 2.0 Gy of γ-rays, and the initial weight of the mice was recorded. Human peripheral blood mononuclear cells (PBMCs, 4×10⁻⁶ cells / year) were then used for infusion. 6 (number / animal) and iTreg cells from each experimental group (8×10) 6 The mixture (4 × 10⁶ cells / mouse) was injected into mice via the orbital vein. A blank control group and a model group were set up, receiving only irradiation and PBS infusion without any cell infusion. The model group received only irradiation and PBMC infusion (4 × 10⁶ cells / mouse). 6 Processing (each individual). Set 5 individuals per group.
[0069] During the experiment, the mice were weighed every other day and their clinical symptoms of GvHD were continuously observed, scored, and recorded. The observation period was uniformly extended to day 28. On day 14, mice in each group (3 mice per group) were sacrificed, and skin, lung, liver, and small intestine tissues were isolated and obtained. Tissue sections were prepared and immunohistochemical staining analysis was performed.
[0070] like Figure 5 Results B and 5C showed that mice in the PBMC-only infusion group, the conventional iTreg intervention group, and the Mock-iTreg control group all experienced persistent weight loss and significantly decreased survival rate; however, intervention with the modified CAT-iTreg of this invention significantly alleviated the weight loss in GvHD model mice. Figure 5 B), long-term survival status was significantly improved, and survival rate was significantly increased ( Figure 5 C).
[0071] like Figure 5 Histopathological examination of mice at levels D and 5E showed that the model group mice receiving PBMC infusion alone exhibited typical and severe GvHD pathological damage, with extensive lymphocyte infiltration in the lung tissue, significant atrophy of intestinal villi, and destruction of the basal layer structure and loss of cell continuity in the skin. The conventional iTreg group and the Mock-iTreg group also showed severe organ pathological damage, with increased lymphocyte infiltration in the lung tissue, more pronounced atrophy and damage to intestinal villi, severe dissociation of skin tissue layers, destruction of the basal layer integrity, and significant consumption of the subcutaneous fat layer. In contrast, the CAT-iTreg intervention group showed the mildest inflammatory infiltration and histopathological damage in the lungs, intestines, and skin target organs, with significant improvement in GvHD-related pathological symptoms.
[0072] Example 6 This embodiment further systematically evaluates the Foxp3 expression stability in mice of CAT-iTreg cells prepared in Example 1 and conventional iTreg control cells. Figure 6 A represents the overall experimental procedure for constructing the GvHD model and detecting the stability of Foxp3 expression in this embodiment.
[0073] The specific operational steps are as follows: One day before the orbital vein infusion of cells, female NSG mice aged 6-8 weeks and weighing 18-22g were irradiated with 2.0 Gy of gamma rays, and the initial weight of the mice was recorded. Human peripheral blood mononuclear cells (PBMCs, 4×10⁻⁶) were then used. 6 (each individual) and CAT-iTreg cells or iTreg cells (8 × 10⁸) 6 (3 mice per group) were injected into mice via the orbital vein after being mixed with the sample. Peripheral blood, spleen, lung, and liver were collected on day 5 for Foxp3 expression stability assay.
[0074] like Figure 6 In vivo detection results in mice showed that in peripheral blood, the Foxp3 positive expression rate in the CAT-iTreg group was 3.48%, significantly higher than the 0.5% in the traditional iTreg group. Spleen tissue analysis showed that both groups maintained a certain level of Foxp3 positive expression, with the CAT-iTreg group showing a Foxp3 positive rate of 27.97%, significantly higher than the 16.50% in the iTreg control group. The most significant difference in Foxp3 expression was observed in lung tissue, with the CAT-iTreg group showing an average Foxp3 positive rate of 21.77%, far exceeding the 9.53% in the iTreg group, showing a statistically significant difference between the groups. Liver tissue analysis showed that the Foxp3 positive expression levels in both the CAT-iTreg group and the iTreg control group decreased to extremely low levels, with no significant difference. (See attached results for details.) Figure 6 B.
[0075] In summary, caTGFBR1 can significantly enhance the long-term stability of Foxp3 expression in iTreg cells in the microenvironments of key organs such as peripheral blood, spleen, and lungs in mice, effectively improving the problem of iTreg cell phenotypic loss in vivo.
[0076] Example 7 To further evaluate the effect of stable caTGFBR1 expression on the lineage stability of iTreg, this example compares the difference in the ability of CAT-iTreg and Foxp3-iTreg cells prepared in Example 1 to maintain stable Foxp3 expression during in vitro expansion.
[0077] Foxp3-iTreg cells were obtained through the following preparation method: Naive CD4 + After culturing for 48 h with T cell activation induction (500 U / mL recombinant human IL-2, 50 nM rapamycin, 5 ng / mL LTGFβ, and anti-CD3 / CD28 magnetic beads in a 1:1 ratio), naive CD4 cells were activated using Foxp3 lentivirus. + T cells were transduced; 72 h after transduction, TGF-β was removed from the culture medium of the Foxp3-iTreg group to obtain Foxp3-iTreg cells.
[0078] The construction process of the Foxp3 lentivirus in this embodiment is the same as that in Example 1, the only difference being the gene. The gene used in this embodiment has a nucleotide sequence as shown in SEQ ID NO.7. FoxP3 The gene, whose corresponding amino acid sequence is shown in SEQ ID NO.8.
[0079] FoxP3 Gene (SEQ ID NO.7): Foxp3 (SEQ ID NO.8): MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP Flow cytometry was used to detect Naive CD4. + T cell activation induces the positive rate of the marker gene NGFR and the expression level of Foxp3 in CAT-iTreg and Foxp3-iTreg cells during the expansion cycle. Figure 7 The results in A and 7B indicate that the NGFR positivity rate in the CAT-iTreg group can be maintained above 70%, suggesting that the stable expression protocol of caTGFBR1 does not affect the in vitro expansion rate of iTreg cells or the purity of the target gene-stable cell population; while the proportion of NGFR-positive cells in the Foxp3-iTreg group gradually decreases with prolonged expansion time, suggesting that its stable expression cell population is easily lost during in vitro expansion. Figure 7 The results of Foxp3 expression level detection at C and 7D showed that the CAT-iTreg group could maintain stable high expression of Foxp3 throughout the in vitro amplification process; while the Foxp3-iTreg group showed a continuous downward trend in Foxp3 expression level, which had dropped to 20% of the initial level by 28 days of amplification.
[0080] In summary, the stability of the iTreg lineage mediated by stable caTGFBR1 expression is superior to that mediated by stable Foxp3 expression.
[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
[0082] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.
Claims
1. An inducible regulatory T cell expressing constitutively activated transforming growth factor β receptor 1, characterized in that, The expression of constitutively activated transforming growth factor β receptor 1-induced regulatory T cells can enhance their stability and inhibitory function in an inflammatory environment.
2. The inducible regulatory T cells expressing constitutively activated transforming growth factor β receptor 1 according to claim 1, characterized in that, The amino acid sequence of the constitutively activated transforming growth factor β receptor 1 is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.
2.
3. The inducible regulatory T cells expressing constitutively activated transforming growth factor β receptor 1 according to claim 1, characterized in that, The constitutively activated transforming growth factor β receptor 1-inducible regulatory T cells also overexpress one or more cytokines, transcription factors, surface molecules, homing molecules, and targeting molecules that recognize disease site or transplant organ-specific targets. The cytokines are selected from any one or more of IL-2, IL-10, and IL-35; The transcription factors and surface marker molecules are selected from any one or more of Foxp3, CD25, CD39, PD-L1, and CTLA-4; The homing molecules are selected from any one or more of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and CX3CR1; The target molecule is selected from chimeric antigen receptor molecules or ligands of tissue-specific receptors.
4. A method for preparing inducible regulatory T cells expressing constitutively activated transforming growth factor β receptor 1 as described in any one of claims 1 to 3, characterized in that, The gene encoding constitutively activated transforming growth factor β receptor 1 was obtained by transfecting it into inducible regulatory T cells using a viral transfection method.
5. The preparation method according to claim 4, characterized in that, The virus transfection method includes the following steps: S1. The gene encoding constitutively activated transforming growth factor β receptor 1 as shown in SEQ ID NO.2 is recombinantly ligated with a viral vector to obtain a recombinant viral plasmid. S2. Transfect the recombinant viral plasmid obtained in step S1 into HEK293T cells and collect the supernatant containing the virus. S3. Co-culture the virus-containing supernatant obtained in step S2, induced regulatory T cells, and induction culture medium to induce the induced regulatory T cells to express constitutively activated transforming growth factor β receptor 1.
6. The preparation method according to claim 5, characterized in that, In step S1, the viral vector is selected from any one of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, retrovirus vectors, herpesvirus vectors, RNA virus vectors, EB virus vectors, baculovirus vectors, bacteriophage vectors, and animal virus vectors.
7. The preparation method according to claim 5, characterized in that, In step S3, the induction medium includes IL-2, rapamycin, and TGFβ; After 72 h of co-culture, TGFβ was removed from the induction medium and culture continued.
8. The use of an inducible regulatory T cell expressing constitutively activated transforming growth factor β receptor 1 as described in any one of claims 1 to 3 in the preparation of a medicament for the treatment of autoimmune diseases and / or organ transplantation anti-rejection therapy and / or cell transplantation anti-rejection therapy.
9. A medicament for the treatment of autoimmune diseases and / or organ transplant anti-rejection therapy and / or cell transplant anti-rejection therapy, characterized in that, Including the induced regulatory T cells expressing constitutively activated transforming growth factor β receptor 1 as described in any one of claims 1 to 3.
10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients and / or adjuvants.