CD4+CD25+ regulatory T cell therapy for the induction of donor-specific clinical transplantation tolerance

Naturally occurring CD4⁺CD25⁺ regulatory T cells (Tregs) have been shown to play a key role in the control of autoimmunity. Interestingly, they are also capable of mediating transplantation tolerance and they can have indirect allospecificity for donor antigens. An increasing body of evidence in experimental studies has indicated that adoptive transfer of in vitro expanded CD4⁺CD25⁺ Tregs with indirect antidonor allospecificity can induce long-term donor-specific transplantation tolerance. Thus, adoptive cell therapy using patient-specific CD4⁺CD25⁺ Tregs as individualised medicine to promote clinical transplantation tolerance is promising.     

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.     

Role of IFN-gamma in induction of Foxp3 and conversion of CD4+ CD25- T cells to CD4+ Tregs

IFN-gamma is an important Th1 proinflammatory cytokine and has a paradoxical effect on EAE in which disease susceptibility is unexpectedly heightened in IFN-gamma-deficient mice. In this study, we provide what we believe is new evidence indicating that IFN-gamma is critically required for the conversion of CD4+ CD25- T cells to CD4+ Tregs during EAE. In our study, the added severity of EAE in IFN-gamma knockout mice was directly associated with altered encephalitogenic T cell responses, which correlated with reduced frequency and function of CD4+ CD25+ Foxp3+ Tregs when compared with those of WT mice. It was demonstrated in both human and mouse systems that in vitro IFN-gamma treatment of CD4+ CD25- T cells led to conversion of CD4+ Tregs as characterized by increased expression of Foxp3 and enhanced regulatory function. Mouse CD4+ CD25- T cells, when treated in vitro with IFN-gamma, acquired marked regulatory properties as evidenced by suppression of EAE by adoptive transfer. These findings have important implications for the understanding of the complex role of IFN-gamma in both induction and self regulation of inflammatory processes.     

T regulatory cells as an immunotherapy for transplantation

Advances in immunosuppressive therapies have made tissue and organ transplantation a common procedure in clinical medicine. However, true donor and recipient tolerance is not regularly achieved and almost all transplant recipients continue to require immunosuppressants throughout life, which is associated with side effects of the drugs. The identification and characterisation of regulatory T cells (Tregs) has recently opened up exciting opportunities for new ways of adoptive immunotherapy in transplantation. CD4+CD25+ Tregs of thymic origin have been shown to be key regulators of unseasoned immune responses in mice and in humans, preventing graft-versus-host disease and organ graft rejection in the transplantation setting. Although these cells can be found in the peripheral blood of healthy individuals, their isolation to a satisfying degree of purity is time-consuming and ineffective. Therefore, a variety of different methods to expand or induce regulatory T cells ex vivo have been advocated. Antigen-specific activation of Tregs is a prerequisite for their optimal function, making the design of new strategies to create and expand antigen-specific Tregs highly desirable. This review will focus on recent advances achieved in the field of transplantation tolerance using naturally occurring Tregs (CD4+CD25+), as well as other Tregs, and will discuss future applications of these cells in immunotherapy.     

CD4+ Tregs and immune control

Recent years have seen Tregs become a popular subject of immunological research. Abundant experimental data have now confirmed that naturally occurring CD25+CD4+ Tregs in particular play a key role in the maintenance of self tolerance, with their dysfunction leading to severe or even fatal immunopathology. The sphere of influence of Tregs is now known to extend well beyond just the maintenance of immunological tolerance and to impinge on a host of clinically important areas from cancer to infectious diseases. The identification of specific molecular markers in both human and murine immune systems has enabled the unprecedented investigation of these cells and should prove key to ultimately unlocking their clinical potential.     

Massive thymic deletion results in systemic autoimmunity through elimination of CD4+ CD25+ T regulatory cells

Incomplete deletion of KRN T cells that recognize the ubiquitously expressed self-antigen glucose-6-phosphate-isomerase (GPI) initiates an anti-GPI autoimmune cascade in K/BxN mice resulting in a humorally mediated arthritis. Transgenic (Tg) expression of a KRN T cell receptor (TCR) agonist under the major histocompatibility complex class II promoter resulted in thymic deletion with loss of anti-GPI T and B cell responses and attenuated arthritis course. However, double Tg mice succumbed to systemic autoimmunity with multiorgan inflammation and autoantibody production. Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery. Disease was transferred by these T cells and prevented by cotransfer of CD4+ CD25+ Tregs. Moreover, we extended our findings to another TCR system (anti-hen egg lysozyme [HEL] TCR/HEL mice) where similarly extensive thymic deletion also resulted in disease. Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion. Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.     

Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling

CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.     

T regulatory cells as an immunotherapy for transplantation

Advances in immunosuppressive therapies have made tissue and organ transplantation a common procedure in clinical medicine. However, true donor and recipient tolerance is not regularly achieved and almost all transplant recipients continue to require immunosuppressants throughout life, which is associated with side effects of the drugs. The identification and characterisation of regulatory T cells (Tregs) has recently opened up exciting opportunities for new ways of adoptive immunotherapy in transplantation. CD4⁺CD25⁺ Tregs of thymic origin have been shown to be key regulators of unseasoned immune responses in mice and in humans, preventing graft-versus-host disease and organ graft rejection in the transplantation setting. Although these cells can be found in the peripheral blood of healthy individuals, their isolation to a satisfying degree of purity is time-consuming and ineffective. Therefore, a variety of different methods to expand or induce regulatory T cells ex vivo have been advocated. Antigen-specific activation of Tregs is a prerequisite for their optimal function, making the design of new strategies to create and expand antigen-specific Tregs highly desirable. This review will focus on recent advances achieved in the field of transplantation tolerance using naturally occurring Tregs (CD4⁺CD25⁺), as well as other Tregs, and will discuss future applications of these cells in immunotherapy.     

肾移植患者外周血中的CD4+ CD25+ CD127low/-调节性T细胞

背景:越来越多的实验在分析免疫耐受标志,以期能够更好地辅助患者进行移植后免疫抑制治疗。目的:分析肾移植后患者外周血中CD4+CD25+CD127low/-调节性T细胞在肾移植免疫耐受中的作用。方法:采集62例肾移植后患者(急性排斥反应组22例,移植稳定组40例)及20例健康对照者的外周抗凝血,经免疫染色,应用流式细胞仪分析CD4+CD25+CD127low/-调节性T细胞所占CD4+T细胞百分含量,同时采用ELISA方法检测患者血清中白细胞介素2和白细胞介素10的质量浓度。结果与结论:移植稳定组中CD4+CD25+CD127low/-调节性T细胞所占CD4+T细胞百分含量显著高于健康对照组和急性反应排斥组(P<0.01);CD4+CD25+CD127low/-调节性T细胞百分含量与白细胞介素2呈显著负相关(P<0.05),与白细胞介素10呈显著正相关(P<0.01)。提示CD4+CD25+CD127low/-调节性T细胞在肾移植后免疫耐受的机制中发挥了一定作用。     

Large-scale depletion of CD25+ regulatory T cells from patient leukapheresis samples

The ability to selectively enrich or deplete T lymphocytes of specific phenotype and function holds significant promise for application in adoptive immunotherapy protocols. Although CD4+ T cells can have an impact on CD8+ T-cell effector function, memory, and maintenance, a subset of CD4+ T cells, CD25+ regulatory T cells (Treg), can regulate peripheral self tolerance and possess the ability to suppress antitumor responses. The authors report the ability to selectively deplete CD25+ Treg cells from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system. Using leukapheresis samples containing up to 1.3 x 10(10) white blood cells, efficient depletion of Treg cells was measured by flow cytometric analysis of CD25 expression and FOXP3 expression on post-depletion products. Remnant CD25+ cells could not be detected in CD25-depleted products after short-term culture in IL-2 or enriched following secondary immunomagnetic selection for CD25+ cells, confirming that efficient depletion had occurred. In parallel to efficient enrichment of CD25- cells, immunomagnetic selection resulted in the recovery of Treg cells, since CD25+ lymphocytes removed during depletion were primarily composed of CD4+ T cells that expressed high levels of FOXP3 and possessed suppressive activity against autologous TCR-stimulated CD4+ CD25- T cells in vitro. These results show that selective separation of functional CD25+ Treg cells from large-scale samples can be performed in large scale under clinical-grade conditions with sufficient selection, recovery, viability, ability to expand, and function for potential use in adoptive immunotherapy.     

免疫调理治疗对脓毒症患者血CD4+CD25+T细胞的影响

目的 了解脓毒症患者外周血调节性T细胞(CD4+CD25+Tregs)比例水平及其与机体细胞免疫之间的关系,并探讨免疫调理治疗对其水平的影响.方法 选择滕州市中心人民医院住院治疗的脓毒症患者40例,随机分成治疗组和对照组,治疗组加用乌司他丁和胸腺肽α1作抗炎和免疫调节治疗,分别于治疗前和治疗后第3,第8天抽外周静脉血,检测T淋巴细胞亚群、CD4+CD25+Tregs,TNF-α,IL-6,IL-10水平,同时APACHEⅡ评分.结果 治疗前两组患者CD4+CD25+Tregs细胞水平明显升高,总淋巴细胞数、T细胞比例下降,其中以CD4+T细胞下降更明显,CD4+/CD8+明显下降,IL-6,TNF-αt水平升高;治疗后,两组患者CD4+C125+Tregs降低,总淋巴细胞数、CD4+/CD8+比值升高,APACHEⅡ评分和IL-6,TNF-αt水平均下降,治疗组改善更明显.结论 外周血CD4+CD25+Tregs细胞比例水平,可以作为评价机体免疫能力及预后新指标;联合应用胸腺肽α1和乌司他丁治疗脓毒症,可提高患者免疫力,显著降低APACHEⅡ评分、IL-6,TNF-α水平,改善患者病情.

Abstract：

Objective To investigate the percentage of CD4 + C125 +Tregs in peripheral blood of patients with sepsis and its effect on cell immunity so as to unravel the effect of immunomodulatory therapy on it. Method Fourty patients with sepsis in ICU were randomly (random number) divided into experimental group and control group . The patients of experimental group were treated with Ulinastatin and immunoregulation agent (Thymosin αl) as well. The blood specimens were collected just before treatment, 3 days and 8 days after treatment. The percentages of CD4 + CD25 + Tregs and lymphocyte subsets were detected by using FCM (flow cytometry), and TNF-α, IL-6 and IL-10 assayed by using ELISA, and APACHE Ⅱ scores were calculated. Results Before treatment, the percentage of CD4 + CD25 + Tregs increased, and the number of lymphocytes and the percentage of T lymphocytes decreased, especially the CD4 + T lymphocytes and CD4+/CD8+ decreased more markedly, and the levels of IL-6 and TNF-α increased. After treatment,the percentage of CD4+ CD25 + Tregs was decreased, the number of lymphocytes and CD4 +/CD8 + increased, and the levels of APACHE Ⅱ score, IL-6 and TNF-α decreased especially in the experimental group decreased more significantly (P ＜ 0. 05). Conclusions The percentage of CD4 + CD25+ Tregs in peripheral blood can reflect the immune status of patients with sepsis and become a novel indicator to estimate the progress of sepsis, and the immunity and prognosis of patients. Treating the patients with Thymosin αl and Ulinastatin can raise their immunity, decrease the levels of IL-6, TNF-α and APACHE Ⅱ score and improve their prognosis.     

血必净注射液对脂多糖刺激大鼠调节性T细胞凋亡及其介导效应T细胞免疫功能的影响

目的 探讨中药血必净注射液对体外脂多糖(LPS)刺激CD4+CD25+调节性T细胞(Treg)凋亡及其介导效应T细胞(Teff)增殖反应、分泌白细胞介素-2(IL-2)功能的影响.方法 40只雄性清洁级Wistar大鼠,实验前禁食12 h.免疫磁珠法分选大鼠脾脏CD4+CD25+Treg,分为对照组(n=8)、抗CD3/CD28组(n=8)、抗CXB/CD28+LPS组(/1,=8)、抗CD3/CD28+血必净组(n=8)和抗CD3/CD28+LPS+血必净组(n=8).培养3 d后流式细胞术检测CD4+CD25+Treg凋亡率、叉头翼状螺旋转录因子(Foxp3)和T淋巴细胞毒性相关抗原4(CTLA-4)表达,酶联免疫吸附试验检测IL-10分泌.同时,CD4+CD25+Treg与CD4+CD25-T细胞1:1培养,刀豆素A刺激68 h后分析Treg对Teff增殖的抑制效应,并检测上清中IL-2/可溶性IL-2受体水平.结果 抗CD3/CD28诱导Treg凋亡率为(33.70±3.06%),显著高于对照组(12.84±0.84)%;抗CD3/CD28+LPS+血必净组(45.13±2.70)%凋亡率明显高于抗CD3/CD28+LPS组[(29.41±1.63)%,P＜0.01].同时,CD4+CLY25+Treg roxp3和CTLA-4表达及IL-10分泌随凋亡增加而降低.抗CD3/CD28+血必净组(31.26%)Teff增殖反应平均抑制率显著低于对照组(54.48%,P＜0.05),抗CD3/CD28+LPS+血必净组抑制率与抗CD3/CD28+LPS组比较亦明显下降(P＜0.01).此外,抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组IL-2分泌水平均显著高于抗CD3/CD28+LPS组(P＜0.01).结论 LPS刺激可明显上调大鼠Treg对Teff的抑制功能,血必净注射液能通过促进Treg凋亡而有效改善其对Teff的抑制效应.     

骨髓间充质干细胞上调再生障碍性贫血患者CD4~+ CD25~+ Foxp3~+调节性T细胞的临床研究

目的:初步探讨再生障碍性贫血(AA)患者外周血CD4+CD25+Foxp3+调节性T细胞(Treg)的表达及临床意义;探讨临床使用异体骨髓间充质干细胞(MSC)治疗对CD4+CD25+Foxp3+Treg表达的影响。方法:(1)流式细胞仪检测13例重型再生障碍性贫血(SAA),17例慢性再生障碍性贫血(CAA)及10例健康对照者外周血CD4+CD25+Foxp3+Treg表达;(2)检测7例AA患者MSC治疗前后外周血CD4+CD25+Foxp3+Treg表达。结果:(1)AA患者外周血CD4+CD25+Foxp3+Treg表达明显低于健康对照者(P<0.01);(2)SAA患者CD4+CD25+Foxp3+Treg表达较CAA者低(P<0.05);(3)MSC治疗后AA患者外周血CD4+CD25+Foxp3+Treg表达升高(P<0.05)。结论:(1)AA患者存在外周血CD4+CD25+Foxp3+Treg表达减低,其表达异常可能参与AA的发病;(2)CD4+CD25+Foxp3+Treg表达在SAA患者更低,提示其表达水平可能可以作为判断AA病情轻重的指标;(3)MSC治疗可上调AA患者CD4+CD25+Foxp3+Treg表达,为MSC纠正AA患者的免疫异常提供了临床依据。     

PTEN inhibits IL-2 receptor-mediated expansion of CD4+ CD25+ Tregs

One of the greatest barriers against harnessing the potential of CD4+ CD25+ Tregs as a cellular immunotherapy is their hypoproliferative phenotype. We have previously shown that the hypoproliferative response of Tregs to IL-2 is associated with defective downstream PI3K signaling. Here, we demonstrate that targeted deletion of the lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) regulates the peripheral homeostasis of Tregs in vivo and allows their expansion ex vivo in response to IL-2 alone. PTEN deficiency does not adversely affect either the thymic development or the function of Tregs, which retain their ability to suppress responder T cells in vitro and prevent colitis in vivo. Conversely, reexpression of PTEN in PTEN-deficient Tregs as well as in activated CD4+ T cells inhibits IL-2-dependent proliferation, confirming PTEN as a negative regulator of IL-2 receptor signaling. These data demonstrate that PTEN regulates the "anergic" response of Tregs to IL-2 in vitro and Treg homeostasis in vivo and indicate that inhibition of PTEN activity may facilitate the expansion of these cells for potential use in cellular immunotherapy.     

Effects of cryopreservation on CD4+ CD25+ T cells of HIV-1 infected individuals

The role of T regulatory cells (Tregs) in human immunodeficiency virus (HIV)-1 infection, although not entirely clear, has recently been highlighted. Despite their lack of specificity, fluorochrome-labeled CD4 and CD25 antibodies are common flow cytometric reagents used to identify these cells with immunosuppressive potential. Cryopreservation has previously been shown to alter the proportions of lymphocytes with certain phenotypes expressed in peripheral blood mononuclear cells (PBMCs). The aim of this study was to assess the effect of cryopreservation on CD4+ CD25+ T cells in PBMCs from HIV-1+ individuals to guide the design of future studies on Tregs. We recruited 30 HIV-1+ individuals and nine healthy controls. CD25 expression in CD4+ T cells was compared between fresh and frozen/thawed PBMC samples from the same time point. In this study, cryopreservation significantly decreased the proportion of CD4+ CD25+ T cells in PBMC samples from HIV-1 infected subjects. This finding suggests that studies of CD4+ CD25+ T cells should be carried out on fresh samples to avoid bias introduced by cryopreservation.     

Antigen-dependent proliferation of CD4+ CD25+ regulatory T cells in vivo

The failure of CD25+ regulatory T cells (Tregs) to proliferate after T cell receptor (TCR) stimulation in vitro has lead to their classification as naturally anergic. Here we use Tregs expressing a transgenic TCR to show that despite anergy in vitro, Tregs proliferate in response to immunization in vivo. Tregs also proliferate and accumulate locally in response to transgenically expressed tissue antigen whereas their CD25- counterparts are depleted at such sites. Collectively, these data suggest that the anergic state that characterizes CD25+ Tregs in vitro may not accurately reflect their responsiveness in vivo. These observations support a model in which Treg population dynamics are shaped by the local antigenic environment.