CN101126076A - TGF-beta induced regulatory T cells and methods of forming and using the same - Google Patents

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a TGF-β-induced regulatory T cell and its formation method and application. The regulatory T cell of the present invention is obtained by combining two cytokines, TGF_β and IL_2, and inducing CD4+ cells of patients with autoimmune diseases in vitro, and is recorded as CD4++CD25+Foxp3+ regulatory T cells, wherein FoxP3 is a FoxP3 gene or protein. The regulatory T cell of the present invention can be used as a pharmaceutical preparation for self-treatment of diseases of patients with autoimmunity (such as SLE).

Description

Regulatory T cells induced by TGF-β and methods and applications thereof

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a regulatory T cell related to the treatment of autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, etc.), as well as a formation method and application of the regulatory T cell.

Background technique

At present, the treatment of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis at home and abroad relies on hormone or non-hormonal immunosuppressants. Although the treatment of these drugs can control the symptoms of some patients, the long-term efficacy is limited. More importantly, long-term use of these drugs often produces serious complications, or secondary serious infections, and even induces the occurrence of tumors. In addition, the huge cost of medical resources is also a drawback.

In the middle and late 1990s, immunologists discovered that there are a small amount of CD4+CD25+ cells in normal animals and humans (accounting for about 2% of normal human CD4+ cells). Without such cells, animals and humans develop symptoms of various autoimmune diseases. Experiments have shown that this population of cells is derived from the thymus, since thymusectomized animals lack CD4+CD25+ cells and develop autoimmune disease. Injection of CD4+CD25+ cells into thymectomized mice prevents autoimmune disease in mice. This population of cells is now called "natural CD4+ regulatory T cells."

The relationship between lack of natural CD4+CD25+ cells and autoimmune diseases was first confirmed in experimental animal models of autoimmunity. For example, both BWF1 and SNF1 mice develop spontaneous lupus-like diseases around 22 weeks after birth, and current studies have shown that the number of CD4+CD25+ cells in the mice (peripheral blood and spleen) decreased. On the contrary, intravenous injection of CD4+CD25+ cells into this mouse can indeed delay the onset and progression of lupus disease.

The above-mentioned changes in the properties of CD4+ regulatory T cells are also closely related to the occurrence and development of human SLE. The first few research groups first found that the number of CD4+CD25+ cells in active SLE patients was significantly reduced. It was recently found that Foxp3 gene and protein can be specifically expressed in CD4+ regulatory T cells, and are closely related to the development and function of CD4+ regulatory T cells. In this way, identifying the number of Foxp3+ rather than CD25+ cells accurately reflects the level of natural CD4+ regulatory T cells. In fact, we recently found that the number of CD4+CD25+Foxp3+ cells in patients with active SLE is decreased, and this decrease is inversely proportional to the degree of disease activity. Others have also recently reported that the suppressive function of CD4+CD25+ cells in SLE patients is decreased.

Since natural CD4+regulatory T cells obviously affect the occurrence and development of SLE disease, people try to consider using CD4+regulatory T cells to treat SLE. Since the number of CD4+CD25+Foxp3+ positive cells is very limited, the researchers considered using expansion methods to increase the number of CD4+CD25+Foxp3+ cells. Although several research groups have demonstrated that expanded CD4+ regulatory T cells are still able to maintain immunomodulatory function and phenotype, and play a role in the prevention and treatment of acute graft-versus-host reactions to a certain extent, another European study The team found that repeatedly expanding natural CD4+ regulatory T cells lost their immunosuppressive effect. In addition, expanded CD4+ regulatory T cells are prone to activation-induced lymphocyte death, which also limits the therapeutic value of this cell.

Recently, only one research group (Prof. BLUESTONE, University of California, San Francisco, USA) used expanded CD4+CD25+ cells to treat LUPUS disease in animals (BWF1) mice. In any case, the effect is very limited. Also, they used expanded normal animal CD4+CD25+ cells rather than lupus disease-derived CD4+CD25+ cells, because CD4+CD25+ cells in lupus and SLE disease may be intrinsically defective. Therefore, whether these cells can be expanded to treat SLE and other autoimmune diseases remains unclear. It can be seen that this finding is still limited for regulatory T cell therapy in SLE patients.

Contents of the invention

The purpose of the present invention is to propose a TGF-β-induced regulatory T cell for autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, etc.) and its formation method and application.

The TGF_β-induced regulatory T cells for autoimmune diseases proposed by the present invention are obtained by combining two cytokines, TGF_β and IL_2, and inducing CD4+ cells of patients with autoimmune diseases in vitro, specifically recorded as CD4++CD25 +Foxp3+ regulates T cells, where FoxP3 is FoxP3 gene or protein.

The method for forming the above-mentioned CD4+CD25+Foxp3+ regulatory T cells is as follows:

(1) Collect peripheral blood from the veins of patients with autoimmune diseases (such as SLE), and use FICOLL technology to separate lymphocytes; then use monoclonal antibodies (such as anti-monocyte antibodies, B cell antibodies, CD8+ cell antibodies and memory cell antibodies) etc.) and negative technique to separate NAIVE_CD4+ cells;

(2) Stimulate the isolated NAIVE_CD4+ cells with microparticles coated with anti-CD3/CD28 antibodies; then add 3-10ng/ml TGF_β and 15-30unit/ml IL-2, and cultivate for 5-6 days;

3. Then, the CD25-positive cells in the CD4+ cells were separated with MACS microparticles. The specific steps are: use PE CD25 antibody to bind CD4+ cells, and then use anti-PE MACS microparticle beads for positive isolation to obtain the required CD4+CD25+Foxp3+ regulatory T cells.

The CD4+CD25+Foxp3+ regulatory T cells obtained by the above method can be used as a pharmaceutical preparation for treating diseases in patients with autoimmune diseases (such as SLE). The specific method is as follows:

The CD4+CD25+Foxp3+ regulatory T cells induced by the above method are injected into the patient, and the injection amount is 5-10 ²⁰ above-mentioned regulatory T cells.

In order to obtain a better therapeutic effect, patients in the active stage (such as SLE) can be treated with conventional drugs (such as conventional immunosuppressants, etc.) for a course of treatment, and then injected with the above-mentioned CD4+CD25+Foxp3+ regulatory T cells.

Since the survival period of the above-mentioned regulatory T cells in animals is about one month, the present invention can inject patients once a month.

In addition, in the present invention, the above-mentioned regulatory T cells can also be used in combination with conventional immunosuppressants and hormones. During this process, the dosage of hormones and immunosuppressants can be reduced.

The present invention has the following advantages:

1. CD4+CD25+FOXP3+ regulatory T cells can significantly inhibit the proliferation of T cells, the production of cytokines and cytotoxic activity;

2. CD4+CD25+FOXP3+regulatory T cells have the obvious ability to inhibit B cells from producing antibodies.

3. CD4+CD25+FOXP3+ regulatory T cells have the obvious ability to prevent the occurrence of lupus in vivo and control the development of lupus.

3. CD4+CD25+FOXP3+regulatory T cells have long-term protection ability in vivo.

4. CD4+CD25+FOXP3+ regulatory T cells themselves have no obvious toxic side effects.

5. CD4+CD25+FOXP3+ regulatory T cells can replace immunosuppressants and hormones or reduce the dosage of immunosuppressants and hormones, and use them in combination to improve the treatment of patients with autoimmune diseases such as SLE and determine the side effects.

Description of drawings

CD4+CD25阴性细胞发展成为CD4+CD25+Foxp3+细胞图示。来自正常C57BL/6小鼠的

CD4+CD25阴性细胞被抗CD3/CD28抗体贴附的微粒珠子(按每10个细胞一个微粒珠子浓度)进行刺激5天附图说明。IL-2(20u/ml)或/和TGF-β(2ng/ml)被加到刺激的细胞中。CD25，CD4和细胞内Foxp3的表达水平通过流式细胞仪进行检测。图示是5个分开的试验之代表。Figure 1. TGF-β is able to induce

CD4+CD25-negative cells develop into CD4+CD25+Foxp3+ cells. from normal C57BL/6 mice

CD4+CD25 negative cells were stimulated for 5 days by anti-CD3/CD28 antibody-attached microbeads (concentration of one microbead per 10 cells) for 5 days. IL-2 (20u/ml) or/and TGF-β (2ng/ml) was added to the stimulated cells. The expression levels of CD25, CD4 and intracellular Foxp3 were detected by flow cytometry. The graph is representative of 5 separate experiments.

CD4+CD25阴性细胞发展成为表达Foxp3信息RNA的CD4+CD25+细胞。来自正常C57BL/6小鼠的CD4+CD25阴性细胞被抗CD3/CD28抗体贴附的微粒珠子(按每lO个细胞一个微粒珠子浓度)进行刺激5天。IL-2(20u/ml)或/和TGF-β(2ng/ml)被加到刺激的细胞中。培养5天后的CD4+，被进一步通过流式细胞仪进行细胞分选，然后分离出CD25+和CD25阴性亚型，常规制备RNA和反转录成为cNDA，通过RT-PCR扩增Foxp3和看家基因HPRT。图示是5个分开的试验之代表。Figure 2. TGF-β can induce

CD4+CD25-negative cells develop into CD4+CD25+ cells expressing Foxp3 message RNA. from normal C57BL/6 mice CD4+CD25-negative cells were stimulated by anti-CD3/CD28 antibody-attached microbeads (at a concentration of one microbead per 10 cells) for 5 days. IL-2 (20u/ml) or/and TGF-β (2ng/ml) was added to the stimulated cells. After 5 days of culture, CD4+ cells were further sorted by flow cytometry, and then CD25+ and CD25-negative subtypes were isolated, RNA was routinely prepared and reverse-transcribed into cNDA, and Foxp3 and housekeeping gene HPRT were amplified by RT-PCR . The graph is representative of 5 separate experiments.

Figure 3. Schematic illustration of the critical role of IL-2 in the induction of Foxp3+ cells by TGF-β. CD4+CD25-negative cells were prepared from IL-2 knockout mice, and then stimulated with a method similar to that shown in Figure 1. In some culture wells, exogenous recombinant interleukin IL-7 (100ng/ml), or IL-15 (100ng/ml), or IL-2 (50u/ml) was added. Flow cytometry determines the expression of Foxp3+. This result was repeated three times with similar results. Note: In the absence of IL-2, the ability of TGF-β to induce Foxp3 expression is lost. To rule out that lack of IL-2 could not induce the expression of Foxp3 because of the lack of expression of CD25, the researchers also added IL-7, or IL-15, both of which could induce the expression of CD25, but still could not make TGF-β Induces Foxp3+. Only when exogenous IL-2 was added, the expression of Foxp3 could be restored.

Figure 4. Schematic illustration of the synergistic effect of IL-2 and TGF-β in the induction and expansion of Foxp3+. From normal C57BL/6 mice CD4+CD25-negative cells were labeled with the fluorescent dye CFSE, and then stimulated in the same way as in Figure 1. The difference is that in some culture wells, anti-IL-2 antibody (2ug/ml) which neutralizes IL-2 was added. To determine the specific effect of neutralizing IL-2 antibodies, isomeric control IgG was also added. The expression and amplification of FoxP3 were determined by the dilution of CFSE and the level of Foxp3 in cells at different times (see figure). Results are representative of 4 independent trials;

CD4+CD25阴性被图1相似的方法进行刺激，然后，对照CD4+(Tcon)以及IL-2和TGF-β诱导的CD4+细胞(Treg)中的CD25+和CD25阴性细胞被进一步用流式细胞仪进行细胞分选。这些细胞按1∶4的比例加到CFSE标记的T反应细胞和抗原呈递细胞中进行共培养(可溶性抗CD3抗体刺激)3天。CD4+细胞的增生比例通过CFSE稀释的程度进行计算。结果是4个分开的试验代表。Figure 5. The CD4+CD25+ cells induced by AIL-2 and TGF-β have obvious inhibitory effect in vitro.

CD4+CD25-negative cells were stimulated similarly to Figure 1, then CD25+ and CD25-negative cells in control CD4+ (Tcon) and IL-2 and TGF-β-induced CD4+ cells (Treg) were further analyzed by flow cytometry Cell sorting. These cells were added to CFSE-labeled T-reactive cells and antigen-presenting cells at a ratio of 1:4 for co-culture (stimulated with soluble anti-CD3 antibody) for 3 days. The proliferation ratio of CD4+ cells was calculated by the extent of CFSE dilution. Results are representative of 4 separate trials.

CD4+CD25阴性被图1相似的方法进行刺激，然后，对照CD4+(Tcon)以及IL-2和TGF-β诱导的CD4+细胞(Treg)中的CD25+和CD25阴性细胞被进一步用流式细胞仪进行细胞分选。这些细胞按一定的比例(见图示)加到CFSE标记的T反应细胞和抗原呈递细胞中进行共同培养(用可溶性抗CD3抗体刺激)3天。总的CD4+细胞增生情况通过增生百分比乘以培养细孔中的细胞总数来决定(B)。B是4个试验的均值加标准差。Figure 5. The CD4+CD25+ cells induced by BIL-2 and TGF-β have obvious inhibitory effect in vitro.

CD4+CD25-negative cells were stimulated similarly to Figure 1, then CD25+ and CD25-negative cells in control CD4+ (Tcon) and IL-2 and TGF-β-induced CD4+ cells (Treg) were further analyzed by flow cytometry Cell sorting. These cells were added to CFSE-labeled T-reactive cells and antigen-presenting cells in a certain ratio (see diagram) for co-culture (stimulated with soluble anti-CD3 antibody) for 3 days. Total CD4+ cell proliferation was determined by multiplying the percent proliferation by the total number of cells in the culture well (B). B is the mean plus standard deviation of 4 experiments.

Figure 6. Intravenous injection of IL-2 and TGF-β-induced BDA/2T cells into D2B6F1 mice can prevent D2T cell-induced lupus-like disease. 20x10 ⁶ , fresh T cells from DBA/2 mice (D2), or D2 cells plus 20x10 ⁶ T cells stimulated with xenoantigen and IL-2 (D2+T _con ), or D2 cells plus 20x10 ⁶ stimulated with xenoantigen, IL -2 and TGF-β-stimulated T cells (D2+T _TGFβ ) were injected into D2B6F1 mice, anti-IgG antibody, anti-dsDNA antibody (1-4 weeks after injection) and proteinuria (8 weeks) were detected by ELISA and Detection of proteinuria. NOTE: IL-2 and TGF-β-induced T cells, but not control cells, clearly prevented the ability of D2 fresh T cells to induce lupus disease.

Figure 7. BDA/2T cells induced by intravenous injection of IL-2 and TGF-β significantly improved the symptoms and survival of mice with lupus disease. 20x10 ⁶ , DBA/2 mouse fresh T cells (D2), were injected iv into D2B6F1 mice. Two weeks later (mice had developed disease), 5 to 20x10 ⁶ T cells (T _reg ) stimulated by xenogeneic antigens, IL-2 and TGF-β were injected into D2B6F1 mice respectively, and anti-ds-DNA antibodies (Fig. 7A , 2-12 weeks after injection) and proteinuria (Fig. 7B, 4-12 weeks) were detected by ELISA and proteinuria assay. Survival of mice was also monitored until death (Fig. 7C). Those who received D2 cells but did not receive T _reg cell injection were the positive control group, and D2B6F1 mice that did not receive D2 cells were the normal control group. Note: T cells induced by IL-2 and TGF-β can significantly reduce the levels of antibody ds-DNA and proteinuria in mice with disease, and prolong the survival of mice with lupus twice.

Figure 8. Intravenous injection of IL-2 and TGF-β-induced BDA/2T cells into D2B6F1 mice does not induce lupus-like disease. 20x10 ⁶ , fresh T cells from DBA2 mice (D2), or T cells stimulated by xenoantigen and IL-2 (D2-T _med ), or T cells stimulated by xenoantigen, IL-2 and TGF-β (D2 -T _TGFβ ) were injected into D2B6F1 mice respectively, anti-IgG antibody, anti-ds-DNA antibody (1-4 weeks after injection) and proteinuria (Proteinuria, 8 weeks) were detected by ELISA and proteinuria assay. Note: T cells treated with IL-2 and TGF-β lost the ability to induce lupus disease, and various indicators were similar to those of normal mice that did not receive injection of DBA/2 T cells.

Figure 9. DBA/2T cells survive longer in vivo induced by IL-2 and TGF-β. 10x10 ⁶ , CFSE-labeled DBA/2 mouse fresh T cells (marked as Fresh in the figure), or T cells stimulated by xenoantigen, IL-2 (Tcon), or stimulated by xenoantigen, IL-2 and TGF-β The T cells (T _TGFbeta ) were injected into D2 mice respectively, and the number of CFSE+CD3+T cells in the spleens of the mice in each group was examined on the 1st, 7th, and 14th days after the injection. The circles represent CFSE+CD3+ positive cells, the numbers on the upper right represent the intensity of CFSE, and the numbers on the lower left represent the total CFSE+CD3+ cells.

 CD4+CD25阴性细胞被图1相似的技术进行5天刺激。流式细胞仪技术决定CD4+细胞的FoxP3表达。注：结合IL-2和TGF-β能够诱导已经发生了狼疮病的老龄BWF1小鼠的CD4+细胞表达Foxp3，虽然表达水平略低于幼龄小鼠。Figure 10. Combining IL-2 and TGF-β can induce the expression of Foxp3 in CD4+ cells of young and old BWF1 mice. from 8- and 22-week-old BWF1 mice

CD4+CD25-negative cells were stimulated for 5 days using a technique similar to that shown in Figure 1. FoxP3 expression in CD4+ cells determined by flow cytometry. Note: Combining IL-2 and TGF-β can induce the expression of Foxp3 in CD4+ cells of old BWF1 mice that have developed lupus, although the expression level is slightly lower than that of young mice.

Figure 11. CD4+CD25+ cells induced by IL-2 and TGF-β from 22-week BWF1 mice can inhibit the occurrence and development of lupus-like diseases. 1×10 ⁶ purified B cells from 22-week-old B/WF1 mice or/and 10×10 ⁶ purified CD4+ cells were injected into 8-week-old B/WF1 mice that received a dose of 350 rads of radiation. In some groups, ^5x106 purified CD4+CD25+ subtype cells from control cells (Tcon) and IL-2+TGF-β-induced (Treg) were also received. After 1 month, the concentration of serum anti-ds-DNA and the level of proteinuria after 4 months were determined according to the method similar to Fig. 5 . Each group contained 4-6 mice.

CD4+CD25阴性细胞从正常人和活动期SLE病人中常规制备，这些细胞被抗人的CD3/CD28抗体刺激。在有些组别，IL-2(20u/ml)或/和TGF-β被加入培养细胞中。FACS技术决定细胞内Foxp3的表达(A)，值代表10个试验的均值和标准差。(B).这些细胞按1∶6的比例加入到CFSE标记的新鲜T细胞中去检测抑制活性，具体方法与图4相似)。Figure 12. IL-2 and TGF-β can induce CD4+ in active SLE patients to develop into CD4+Foxp3+ regulatory T cells.

CD4+CD25-negative cells are routinely prepared from normal people and active SLE patients, and these cells are stimulated by anti-human CD3/CD28 antibody. In some groups, IL-2 (20u/ml) or/and TGF-β were added to cultured cells. Intracellular Foxp3 expression was determined by FACS technique (A), values represent mean and standard deviation of 10 experiments. (B). These cells were added to CFSE-labeled fresh T cells at a ratio of 1:6 to detect the inhibitory activity, and the specific method was similar to that in Figure 4).

Detailed ways

The invention uses the combination of cytokine TGF-β and IL-2 to induce peripheral blood to produce a group of regulatory T cells similar to natural CD4+ regulatory cells. The present invention establishes a brand-new method for inducing CD4+regulatory T cells, which replaces the method for expanding natural CD4+CD25+regulatory T cells, and is used for the treatment of SLE or other autoimmune diseases. The present invention finds that two cytokines, TGF-β and IL-2, can induce normal animal and human CD4+ cells to develop into CD4+Foxp3+ regulatory T cells, and these two cytokines also have the ability to induce lupus animals and SLE patients CD4+ cells develop into CD4+Foxp3+ regulatory T cells. Therefore, the present invention plans to obtain peripheral blood CD4+ cells from patients with autoimmune diseases such as SLE, and then isolate CD25+ cell populations after in vitro induction and culture with TGF-β and IL-2 to obtain CD4+CD25+Foxp3+ regulatory T cells. Finally, according to the calculation of the body surface area, about 10 ⁹ CD4+CD25+Foxp3+ regulatory T cells induced in vitro were injected into the patient. According to the in vivo kinetics and survival cycle of CD4+CD25+Foxp3+ regulatory T cells induced by TGF-β, the present invention will intravenously inject CD4+CD25+Foxp3+ regulatory T cells to patients once a month. In order to ensure the best effect, before CD4+CD25+Foxp3+regulatory T cell therapy, a course of immunosuppressive therapy should be used first.

The specific content is introduced as follows:

1. TGF-β can induce CD4+ cells to differentiate into CD4+CD25+FOXP3+ regulatory T cells.

In fact, regulatory T cells are heterogeneous. CD4+ regulatory T cells include thymus-derived natural CD4+ regulatory T cells and peripheral blood-induced acquired CD4+ regulatory T cells. We first found that TGF-β has the ability to induce CD4+CD25- cells in the peripheral blood of normal animals and normal humans to develop into CD4+regulatory T cells. These cells have similar phenotypic characteristics with natural CD4+ cells, such as expressing CD25, CD122, CTLA-4, GITR, etc. Since the recently identified Foxp3 gene and protein are only found in natural CD4+CD25+ regulatory T cells, and conventionally activated CD4+CD25+ cells do not express Foxp3, Foxp3 has become a specific marker of regulatory T cells.

CD4+CD25阴性细胞表达Foxp3蛋白和mRNA(郑颂国等，JImmunol.2002 169：4183-4189)。所有的Foxp3蛋白(图1)和信息RNA(mRNA，蛋白质合成的调节者，图2)定位于CD25+细胞群。另外，TGF-β诱导Foxp3也需要有IL-2的存在。如果内源性IL-2被抗体中和之后，TGF-β并不能够诱导CD4+细胞表达Foxp3。如果用IL-2基因敲除小鼠的CD4+细胞，TGF-β诱导Foxp3表达的能力也消失了(图3)。更多的试验证明IL-2和TGF-β在诱导和扩增CD4+Foxp3+调节T细胞中起了一个协同作用(图4)。We found that the cytokine TGF-β can induce

CD4+CD25 negative cells express Foxp3 protein and mRNA (Zheng Songguo et al., J Immunol. 2002 169: 4183-4189). All Foxp3 protein (Fig. 1) and messenger RNA (mRNA, regulator of protein synthesis, Fig. 2) localized to the CD25+ cell population. In addition, the induction of Foxp3 by TGF-β also requires the presence of IL-2. If endogenous IL-2 was neutralized by antibodies, TGF-β could not induce CD4+ cells to express Foxp3. The ability of TGF-β to induce Foxp3 expression also disappeared if CD4+ cells from IL-2 knockout mice were used (Fig. 3). More experiments proved that IL-2 and TGF-β played a synergistic role in the induction and expansion of CD4+Foxp3+ regulatory T cells (Figure 4).

2. Regulatory T cells induced by TGF-β can inhibit the proliferation of T cells, the production of cytokines and the pathogenesis of lupus.

TGF-β and IL-2 not only induce the expression of Foxp3, but also the transformation of newly induced CD4+CD25+ cells into immunosuppressive cells. In a standard in vitro immunosuppressive test, the results of the present invention show that CD4+ cells induced by TGF-β and IL-2 have a strong immunosuppressive function. This group of CD4+ cells induced in vitro can significantly inhibit the proliferation of T cells, including the inhibition of T cell proliferation rate (Figure 5A) and the total number of T cell proliferation (Figure 5B), in addition, they can also inhibit the proliferation of T cells Production of factors, including IFN-γ and IL-2 (results not shown).

Because in vitro immunosuppression does not absolutely mean that there is a similar immunosuppressive function in vivo. In order to clarify this problem, the present invention further analyzed whether the CD4+ cells induced by TGF-β and IL-2 in vitro have immunosuppressive function in vivo. The present invention employs a chronic lupus disease model. After intravenous injection of 2 billion spleen T cells from DBA/2 mice into DBA/2xC57BL/6F1 hybrid mice, after two weeks, F1 mice showed typical high titers of anti-IgG and anti-nuclear antibodies, 6 to 8 weeks Proteinuria and lupus nephritis began to develop, and mice began to die after 16 weeks. The present invention shows that if regulatory T cells induced by TGF-β and IL-2 are injected simultaneously with disease induction, the occurrence of the disease can be significantly prevented ( FIG. 6 ). In addition, the present invention has further observed the protective effect of these regulatory T cells on the already-onset lupus model. The research shows that if 5 to 2 billion cells are injected into the already-onset F1 mice, the disease can be significantly controlled. Progression, in particular, was able to significantly prolong the survival of lupus mice (Fig. 7A, B and C).

3. Regulatory T cells induced by TGF-β itself have no toxic side effects.

Injection of spleen cells or T cells from DBA/2 mice into F1 mice induces a lupus-like syndrome in F1 mice. The present invention also investigated whether this group of cells (IL-2 and TGF-β-induced T cells) had a pathogenic effect on F1. The results showed that T cells from this group of DBA/2 mice stimulated with C57BL/6 non-T cells were still able to induce lupus-like disease without TGF-β treatment. However, if treated with cytokines such as IL-2 and TGF-β, this group of T cells no longer induced lupus-like disease in F1 mice (Fig. 8). In addition, we also injected IL-2 and TGF-β-induced CD4+CD25+ cells stimulated by anti-CD3/CD28 antibodies into allogenic DBA/2 mice, and found that these mice receiving CD4+CD25+ cells did not have any side effects, Important organs, such as lung, liver, kidney, heart, and brain, were biopsied and no pathological changes were found (results not shown).

4. Regulatory T cells induced by TGF-β have a longer survival period in vivo than conventionally activated CD4+ cells.

In the present invention, T cells induced by TGF-β and IL-2 are labeled with carboxy fluoresceindiacetate succinimidyl ester (CFSE), and then injected into the same animal body, and through tracking analysis, it is found that they are It can survive at least half a month in vivo (Figure 9). In another experimental study, using the same animal species with different (congenic) markers, it was found that the injected regulatory T cells could survive for at least one month (results not shown).

5. TGF-β can induce CD4+ cells of B/WF1 mice to develop into regulatory T cells.

CD4+细胞发展成为Foxp3+细胞，TGF-β诱导22周小鼠的CD4+细胞发展成为Foxp3+细胞的能力稍微弱于8周小鼠(图10)。进一步研究发现，TGF-β和IL-2诱导的22周小鼠的CD4+细胞在注射到BWF1小鼠中之后，能够明显地降低B/WF1小鼠的疾病的进展(图11)。Although TGF-β and IL-2 can induce normal animal and human CD4+ cells to develop into CD4+ regulatory T cells, the present invention further studies whether these two cytokines can induce individuals with autoimmune diseases such as: lupus mice CD4+ cells develop into regulatory T cells. Because B/WF1 mice can spontaneously develop lupus, Naive CD4+ cells were prepared from B/WF1 mice at 8 weeks (not yet onset) and 22 weeks (already onset), we observed that TGF-β and IL -2 can induce two kinds of mice

CD4+ cells developed into Foxp3+ cells, and the ability of TGF-β to induce CD4+ cells in 22-week mice to develop into Foxp3+ cells was slightly weaker than that in 8-week-old mice ( FIG. 10 ). Further studies found that TGF-β and IL-2-induced CD4+ cells from 22-week-old mice could significantly reduce the disease progression of B/WF1 mice after injection into BWF1 mice ( FIG. 11 ).

6. TGF-β can induce CD4+ cells of SLE patients to develop into regulatory T cells.

Because the ultimate goal of the present invention is to use TGF-β and IL-2, two cytokines, to induce CD4+ cells in patients with autoimmune diseases such as SLE to develop into regulatory T cells, and then reinfuse them back to the patient. Or regulatory T cells with less toxic side effects can replace the hormonal and non-hormonal immunosuppressants that are being used clinically to treat autoimmune diseases such as SLE.

CD4+CD25-细胞被制备后，用抗CD3/CD28抗体进行刺激，试验组加TGF-β和IL-2，对照组仅仅加IL-2。5到6天后，对照组和试验组细胞的Foxp3蛋白的表达被检测到，如图2显示。TGF-β和IL-2能够明显地诱导激活的活动期SLE病人的

CD4+细胞表达Foxp3蛋白，并且TGF-β和IL-2诱导的CD4+细胞也能够抑制SLE病人的T细胞的增生。In this invention study, the peripheral blood of patients with active SLE

After CD4+CD25- cells were prepared, they were stimulated with anti-CD3/CD28 antibody, TGF-β and IL-2 were added to the test group, and IL-2 was only added to the control group. After 5 to 6 days, the Foxp3 of cells in the control group and the test group Protein expression was detected, as shown in Figure 2. TGF-β and IL-2 can obviously induce activation of active SLE patients

CD4+ cells express Foxp3 protein, and CD4+ cells induced by TGF-β and IL-2 can also inhibit the proliferation of T cells in SLE patients.

The specific operation steps are summarized as follows:

CD4+细胞。1. Collect peripheral blood from SLE patients, and use Ficoll technology to separate lymphocytes. Use monoclonal antibodies (such as: anti-monocyte antibodies, B cell antibodies, CD8+ cell antibodies, memory cell antibodies, etc.) plus negative screening techniques to isolate

CD4+ cells.

CD4+细胞，2x10⁵个抗CD3/CD28抗体包裹的微粒珠子和重组的细胞因子白介素IL-2(20u/ml)共一周。在诱导调节T细胞的过程中，我们加0.5-10ng/ml的TGF-β。根据我们的预初实验结果，我们发现5ng/ml的TGF-β是最理想的条件。为了排除抗原呈递细胞对体外诱导调节T细胞和随后的体内注射所产生的副作用，本发明不用抗原呈递细胞。2. For SLE patients CD4+ cells, stimulated with anti-CD3/CD28 antibody-coated microparticle beads. In a Petri dish of a 24-well plate, add 2x10 ⁶

CD4+ cells, ^2x105 microparticle beads coated with anti-CD3/CD28 antibody and recombinant cytokine IL-2 (20u/ml) for one week. In the process of inducing regulatory T cells, we added 0.5-10ng/ml TGF-β. According to our preliminary experimental results, we found that 5ng/ml TGF-β is the most ideal condition. In order to exclude the side effects of antigen-presenting cells on the in vitro induction of regulatory T cells and subsequent in vivo injection, the present invention does not use antigen-presenting cells.

3. After culturing for 5 to 6 days, the CD25+ cells in the CD4+ cells were separated with micro-magnetic beads from the German Miltenyi company that complied with GMP standards. Specifically, after the cultured cells are collected, they are stained with CD25 antibody, and after 20 minutes at 4°C, CD25 will combine with the CD25+ subpopulation in the CD4+ cells, and then, add micro-magnetic beads from Miltenyi Company (according to 10ul and 1x10 ⁶ The ratio of cells plus the amount of magnetic beads), because the CD25 antibody has a phycoerythrin (Phycoerythrin, PE), and Miltenyi’s tiny magnetic beads have an anti-phycoerythrin antibody, so that all CD25+ cells are Form complexes with magnetic beads. Then, through a cell elution column with a magnetic field, the positively isolated cells are CD4+CD25+FOXP3+ regulatory T cells.

4. According to the calculation of body surface area, the present invention plans to inject 10 ⁹ CD4+CD25+FOXP3+ regulatory T cells to SLE patients. In order to ensure maximum effect, we plan to give active SLE patients a course of conventional drug therapy first, and then start injecting CD4+CD25+ regulatory T cells.

5. Since the survival cycle of CD4+CD25+FOXP3+regulatory T cells in animals is about one month, the present invention plans to repeat the injection once every month.

6. In the process of regulating T cell therapy, in addition to observing the progress of the patient's clinical symptoms, the present invention also needs to closely detect the concentration of autoantibodies and other indicators reflecting the degree and activity of the disease.

7. The present invention also plans to use CD4+CD25+FOXP3+ regulatory T cells in combination with immunosuppressants and hormones to treat SLE, and reduce the doses of hormones and immunosuppressants in the process.

Claims (5)

1. one kind at the beta induced adjusting T cell of the TGF_ of autoimmune disorder, it is characterized in that it being to combine by TGF_ β and two cytokines of IL_2, CD4+ cell by external evoked autoimmune disorder patient obtains, specifically be designated as CD4++CD25+Foxp3+ and regulate the T cell, FoxP3 is FoxP3 gene or albumen here.

2. formation method at the beta induced adjusting T cell of the TGF_ of autoimmune disorder is characterized in that concrete steps are as follows:

(1) from autoimmune disorder patient vein, extracts peripheral blood, with lymphocyte separation medium technology isolated lymphocytes; Use monoclonal antibody and negative technical point from the NAIVE_CD4+ cell again;

(2) to isolated NAIVE_CD4+ cell, the particulate pearl that wraps up with anti-CD3/CD28 antibody stimulates; Add the TGF_ β of 3-10ng/ml and the IL_2 of 15-30unit/ml then, cultivated 5-6 days;

(3) then the CD25 positive cell in the CD4+ cell is separated with MACS particulate pearl, promptly obtain required CD4+CD25+Foxp3+ and regulate the T cell.

One kind by the beta induced adjusting T cell of TGF_ as claimed in claim 1 as the application in preparation treatment autoimmune disorder patient's the pharmaceutical preparation.

4. application according to claim 3 is characterized in that the beta induced adjusting T cell ampoule of TGF_ is 5-1020, and injection in every month once.

5. application according to claim 3, it is characterized in that the autoimmune disorder patient is earlier with immunosuppressor or treat a course of treatment, use the beta induced adjusting T cell of TGF_ then, perhaps that TGF_ is beta induced adjusting T cell and immunosuppressor or hormone combine use, and reduce the using dosage of hormone or immunosuppressor mutually.

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