Targeting acute allograft rejection by immunotherapy with ex vivo-expanded natural CD4+ CD25+ regulatory T cells

BACKGROUND: Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice. METHODS: Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry. RESULTS: By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection. CONCLUSIONS: Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.     

抗原特异性CD4+CD25+T细胞对同种异体胰岛移植影响及作用机制研究

目的:探讨抗原特异性CD4+CD25+Treg细胞免疫对同种异体胰岛急性移植排斥反应的影响和机制。方法:用MACS分选供体抗原特异性CD4+CD25+Treg细胞免疫糖尿病BALB/cByJ受体小鼠，以ICR小鼠胰岛为供体行同种异体胰岛移植。观察移植后小鼠的存活时间、移植前后外周血CD4+和CD8+T细胞亚群的变化和移植物中Th1/Th2细胞因子mRNA表达水平的变化。结果:抗原特异性Treg细胞联合胰岛移植组(C组)胰岛移植物平均生存期为(34.57±17.15)d，显著长于单纯胰岛移植组(B组)的(10.6±1.82) d (P<0.01)；移植后第3天，C组外周血CD4+/CD8+的值显著低于B组(P<0.01)；C组移植物中IL-10,TGF-β mRNA表达比B组显著增强。B组移植物中IL-1β，IL-2及IFN-γ mRNA表达明显强于C组。结论:抗原特异性CD4+CD25+ Treg细胞可通过调节Th2/Th1之间的反应平衡而延长同种异体胰岛移植物的存活时间。     

Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) regulatory T cells ameliorates rejection of DA-to-Lewis rat liver transplantation

BACKGROUND: Adoptive transfusion of splenocytes from long-term survivors of a tolerance model of rat orthotopic liver transplantation can induce acceptance of liver allografts in a rejection model preconditioned with donor gamma-irradiation before liver transplantation. Recent studies suggest that the regulatory T cells (Treg cells) in splenocytes from long-term survivors play an important role in the induction of liver graft tolerance, but this observation was made from a rejection model preconditioned with donor gamma-irradiation; little is known about the role of Treg cells in liver graft rejection using a naive rejection model. In this study, we examined the therapeutic potential of CD4(+)CD25(+) Treg cells in a naive rejection model of rat liver transplantation. METHODS: Freshly isolated or ex vivo alloantigen-stimulated CD4(+)CD25(+) Treg cells (1 x 10(6) cells) from naive Lewis RT(1) (LEW) rats were adoptively transferred into another LEW rat on days 1 and 7 after liver transplantation from a Dark Agouti RT1(a) (DA) rat. Recipients were treated with or without oral tacrolimus (FK506) (0.1 mg/kg/day) from days 1 to 7 after transplantation. For ex vivo alloantigen-stimulation, CD4(+)CD25(+) Treg cells from LEW rats were cocultured with mitomycin C-treated DA (donor alloantigen specific) or Brown Norway (BN)(RT1(n), third party) splenocytes for 72 hours. Ex vivo alloantigen-specific CD4(+)CD25(-) T-cell proliferation responses were assessed with fresh and stimulated CD4(+)CD25(+) Treg cells. RESULTS: Freshly isolated, donor alloantigen-stimulated and third-party alloantigen- stimulated CD4(+)CD25(+) Treg cells suppressed antigen-specific CD4(+)CD25(-) T-cell proliferation ex vivo, and adoptive transfusion of these 3 kinds of CD4(+)CD25(+) Treg cells prolonged survival of the liver allografts. The group transfused with the donor alloantigen-stimulated CD4(+)CD25(+) Treg cells had the greatest mean survival among the 3 groups (fresh Treg cells, 21 +/- 2 days, n = 6; third-party alloantigen-stimulated Treg cells, 20 +/- 2 days, n = 6; donor alloantigen-stimulated Treg cells, 30 +/- 2 days, n = 6). When combined with short-term tacrolimus administration, adoptive transfusion of donor antigen-stimulated Treg cells induced the greatest survival time in recipients (greater than 60 days; n = 6). CONCLUSION: Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4(+)CD25(+) Treg cells combined with short-term tacrolimus treatment may represent a new strategy for preventing rejection after liver transplantation.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

阻断ICOS/B7h信号的供体特异性输血对移植受体CD4+CD25+调节性T细胞的影响

目的 观察阻断ICOS/B7h信号的供体特异性输血(DST)对异基因小鼠心脏移植术后体内CD4+CD25+调节性T细胞(Treg)的影响.方法 按陈氏方法建立小鼠颈部异位心脏移植模型,实验分3组,异基因组及同基因组:供心分别来源于BALB/C和C57BL/6小鼠,受体均为C57BL/6小鼠,未予治疗.治疗组:移植当天给予受体鼠(C57BL/6)尾静脉注射5×106 ICOS-Fc靶定的供体(BALB/C)脾B淋巴细胞,d0～6连续给予受体鼠尾静脉注射ICOS-Fc 200 μg/d.术后统计各组移植物的存活时间,通过流式细胞术检测受体鼠外周血中CD4+CD25+Treg的亚群比例,利用逆转录-聚合酶链反应(RT-PCR)检测移植物中FOXP3的mRNA表达,在混合淋巴细胞反应中检测CD4+CD25+Treg对CD4+CD25-效应T细胞(Teff)的增殖抑制效率.结果 与异基因组比较,治疗组心脏移植物存活时间明显延长[(84.38±29.14)d比(7.00±0.76)d,P＜0.01].各组中,治疗组受体外周血中CD4+CD25+Treg亚群比例显著上调[(15.60±5.69)%,P＜0.01].与其他两组比较,治疗组心脏移植物中FOXP3 mRNA表达显著上调.以正常鼠为对照,耐受鼠脾脏中获取的CD4+CD25+Treg能够更高效地抑制CD4+CD25-Teff在混合淋巴细胞培养中的增殖效应.结论 通过阻断ICOS/B7h信号的DST可以诱导异基因小鼠心脏移植耐受,CD4+CD25+Treg在耐受的形成与维持中均起着重要作用.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. Interleukin-4 does not maintain alloantigen specific CD4+CD25+ Treg

IL-4 is thought to promote induction of transplantation tolerance and alloantigen-specific CD4⁺CD25⁺ T regulatory cells (Treg). This study examined the effect of IL-4 on the induction and maintenance of the CD4⁺ T regulatory cells (Treg) that mediate transplantation tolerance. Tolerance was induced in DA rats with PVG heterotopic cardiac allografts by a short course of cyclosporine. Naïve and tolerant lymphocytes, including the CD4⁺ and CD4⁺CD25⁺ T cell subsets, were assayed in mixed lymphocyte cultures with or without recombinant (r)IL-4 or other cytokines. The proliferation, cell surface and cytokine phenotype of these cells was examined, as was their capacity to adoptively transfer tolerance. rIL-4 enhanced the proliferation of naïve and tolerant lymphoid cells, including CD4⁺ and CD4⁺CD25⁺ T cells, but this was not alloantigen specific. Naïve or tolerant CD4⁺ T cells cultured with rIL-4 and donor PVG antigen effected rapid graft rejection, even though before culture tolerant CD4⁺ T cells transferred antigen-specific tolerance. These rIL-4 cultured CD4⁺ T cells had a phenotype consistent with activated CD4⁺CD25⁺FoxP3⁻ Th2 cells. While naïve natural CD4⁺CD25⁺ T cells (nTreg) cultured with alloantigen and rIL-4 had enhanced proliferation and capacity to suppress rejection in vivo, the culture of tolerant CD4⁺CD25⁺ T cells with alloantigen and rIL-4 could not sustain their proliferation against specific donor, nor their capacity to transfer tolerance to specific donor allograft. Thus, IL-4 promotes both regulatory and effector T cells early in the immune response, but once alloimmune tolerance is established, IL-4 promoted the activation of effector cells to mediate rejection and did not support alloantigen-specific Treg that could transfer specific tolerance.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4⁺ T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4⁺ CD25⁺ FOXP3⁺ Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg.This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4⁺, especially CD4⁺ CD25⁺ T cells. CD4⁺ T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100 days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4⁺ T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4⁺ CD25⁺ T cells' response to rIL-5 and expression of IL-5Rα was also assessed.rIL-5 was sufficient to promote transplant tolerance mediating CD4⁺ T cells' survival in culture with specific-donor alloantigen. Tolerant CD4⁺ T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4⁺ T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4⁺ CD25⁺ T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4⁺ CD25⁺ T cells expressed IL-5Rα.This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4⁺ CD25⁺ T cells that mediate transplant tolerance.     

CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

BACKGROUND: Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. METHODS: Neonatal BALB/c mice were injected with 108 live CAF, splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using 51Cr release assays of A/J or third-party targets. RESULTS: We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 to CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4+/CD25+ cells, but not CD4+/CD25- cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2. Moreover, when added back into cultures, purified CD4+/CD25+ cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. CONCLUSION: These data indicate that CD8 anergy is associated with the state of tolerance, and that CD4+CD25+ cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.     

Infectious tolerance mediated by CD8+ T-suppresor cells after UV-B-irradiated donor-specific transfusion and rat heart transplantation

AIMS: CD8+CD28- human T-suppressor cells (Ts), which can be generated in vitro, act directly on APC rendering them tolerogenic to unprimed and primed CD4+ T cells. The aim of this study was to investigate the possibility that CD8+ T cells mediate the induction of tolerance in a heart transplantation model in rodents. MATERIALS AND METHODS: Blood from Lewis rats was UV-B-irradiated and transfused into ACI recipients on days -21, -14, and -7 before heart allograft transplantation on day 0. CD4(+) and CD8(+) T cells were positively selected from ACI rats, which had tolerated Lewis heart allografts for more than 100 days and were adoptively transferred to naive ACI rats pretreated (day -1) with gamma irradiation. These ACI rats underwent transplantation with Lewis hearts 24 hours after adoptive transfer of putative T-suppressor cells. RESULTS: Adoptive transfer of CD8(+) T cells from tolerant ACI to naive ACI rats significantly prolonged Lewis heart mean allograft survival time (MST +/- SD) to 69 +/- 13 days as compared with 15 +/- 1 and 14 +/- 1 days in animals adoptively transferred with CD4+ T cells or untreated controls, respectively (P < .001). Similarly, adoptive transfer of CD8(+) T cells from secondary ACI recipients to naive syngeneic animals also significantly prolonged survival of heart allografts to MST +/- SD of 72 +/- 4 for CD8(+) and 15 +/- 4 days for CD4(+) T cells (P < .001). CONCLUSIONS: These data demonstrate that allogeneic tolerance induced in ACI recipients by treatment with UV-B-irradiated blood from Lewis donors is mediated by CD8+ T-suppressor cells.     

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens

BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed na?ve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or na?ve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either na?ve or tolerant hosts effected rejection. In contrast neither tolerant nor na?ve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.     

Improvement of heart allograft acceptability associated with recruitment of CD4+CD25+ T cells in peripheral blood by recipient treatment with granulocyte colony-stimulating factor

BACKGROUND: Granulocyte colony-stimulating factor (G-CSF), an important hematopoietic growth factor of the myeloid lineage, exerts profound immunoregulatory effects in T-cell tolerance. The study objective was to investigate the potential mechanism of G-CSF's antirejection effects in a fully mismatched rat cardiac allograft model. METHODS: The allograft recipients were treated with subcutaneous injection of recombinant human G-CSF (rh-G-CSF) at a dose of 250 microg/kg/d for 6 days starting from the day of cardiac transplantation. The alloreactive T-cell response and rejection level of G-CSF-treated rats were compared with those of control rats using mixed lymphocyte reactions (MLR) and histological examinations. Cytokine and cellular profiles were determined using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The presence and suppressive functions of regulatory T cells were determined by adoptive cell transfer experiments. RESULTS: Posttransplantation treatment of recipients with rh-G-CSF alone prolonged allograft survival, improved allograft biopsy grading scores, and induced alloreactive T-cell hyporesponsiveness accompanied by high levels of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1) production in MLR. It also enhanced CD4+CD25+ T cells in peripheral blood. The splenocytes from rh-G-CSF-treated recipients transferred antirejection effects to secondary recipients. CONCLUSIONS: Posttransplantation treatment of cardiac allograft recipients with rh-G-CSF leads to alloreactive T-cell hyporesponsiveness in vivo and in vitro associated with recruitment of CD4+CD25+ T cells in the peripheral blood. This study may provide insight into the application of G-CSF to control acute rejection of solid organ transplantations.     

GITR Ligation Blocks Allograft Protection by Induced CD25+CD4+ Regulatory T Cells without Enhancing Effector T-Cell Function

The induction of operational tolerance prior to transplant could provide a solution to the complications of current immunosuppression in transplantation. In rodents, operational tolerance frequently correlates with the presence of CD25(+)CD4(+) regulatory T cells (Tregs) but their function is usually demonstrated by adoptive transfer into lymphopenic hosts leading some to question their relevance to normal immunocompetent recipients. The role of these cells in primary transplant recipients has been explored using anti-CD25 antibody but specific targeting of Treg is not possible since CD25 is also up-regulated on activated effector T cells. To overcome this limitation we targeted the Treg associated molecule GITR in tolerized primary transplant recipients. This reverses regulation resulting in acute allograft rejection. This is not due to co-stimulation of effector cells since rejection mediated by isolated populations of CD4(+)CD25(-) or CD8(+)CD25(-) T cells transferred into Rag(-/-) mice was not enhanced by anti-GITR antibody. Furthermore, GITR cross-linking does not provide co-stimulation for in vitro proliferation of the same CD4(+)CD25(-) or CD8(+)CD25(-) T-cell populations in response donor-strain APC. Thus, CD4(+)CD25(+)GITR(+) Treg play an essential role in early graft protection in primary transplant recipients following tolerance induction providing further support for protocols that might generate similar populations in clinical transplantation.     

Superagonistic CD28 Antibody Induces Donor‐Specific Tolerance in Rat Renal Allografts

The ultimate goal of organ transplantation is to establish graft tolerance where CD4+CD25+FOXP3+ regulatory T (Treg) cells play an important role. We examined whether a superagonistic monoclonal antibody specific for CD28 (CD28 SA), which expands Treg cells in vivo, would prevent acute rejection and induce tolerance using our established rat acute renal allograft model (Wistar to Lewis). In the untreated or mouse IgG‐treated recipients, graft function significantly deteriorated with marked destruction of renal tissue, and all rats died by 13 days with severe azotemia. In contrast, 90% of recipients treated with CD28 SA survived over 100 days, and 70% survived with well‐preserved graft function until graft recovery at 180 days. Analysis by flow cytometry and immunohistochemistry demonstrated that CD28 SA induced marked infiltration of FOXP3+ Treg cells into the allografts. Furthermore, these long‐surviving recipients showed donor‐specific tolerance, accepting secondary (donor‐matched) Wistar cardiac allografts, but acutely rejecting third‐party BN allografts. We further demonstrated that adoptive transfer of CD4+CD25+ Treg cells, purified from CD28 SA‐treated Lewis rats, significantly prolonged allograft survival and succeeded in inducing donor‐specific tolerance. In conclusion, CD28 SA treatment successfully induces donor‐specific tolerance with the involvement of Treg cells, and thus the therapeutic value of this approach warrants further investigation and preclinical studies.     

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+ blood lymphocytes in kidney transplant recipients with good long-term graft outcome.

There is evidence that interferon-gamma (IFN-gamma)-dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28(-) (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T- and B-cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of < or =1.8 mg/dl and 32 recipients with a serum creatinine of > or =2.0 mg/dl more than 100 days post-transplant. Cell subsets were measured in whole blood using four-color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN-gamma+ PBL than patients with good graft function (P = 0.017). In patients with good graft function, CD3+CD4+CD25+IFN-gamma+ PBL were associated with high Foxp3+, IL-2+, IL-12+, IL-4+, and IL-10+ CD3+CD4+CD25+ T PBL (P < 0.001), low CD3+CD8+CD28(-)Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage(-)HLA-DR+CD11c+CD123(-) DC1 (P = 0.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co-expression of IFN-gamma and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN-gamma+ cells in a subgroup of transplant recipients with good graft acceptance.     

CD25+CD4+ regulatory T cells develop in mice not only during spontaneous acceptance of liver allografts but also after acute allograft rejection

BACKGROUND: Liver grafts transplanted across a major histocompatibility barrier are accepted spontaneously and induce donor specific tolerance in some species. Here, we investigated whether liver allograft acceptance is characterized by, and depends upon, the presence of donor reactive CD25CD4 regulatory T cells. METHODS: CD25 and CD25CD4 T cells, isolated from CBA. Ca (H2) recipients of C57BL/10 (B10; H2) liver and heart allografts 10 days after transplantation, were transferred into CBA. Rag1 mice to investigate their influence on skin allograft rejection mediated by CD45RBCD4 effector T Cells. RESULTS: Fully allogeneic B10 liver allografts were spontaneously accepted by naive CBA.Ca recipient mice, whereas B10 cardiac allografts were acutely rejected (mean survival time=7 days). Strikingly, however, CD25CD4 T cells isolated from both liver and cardiac allograft recipients were able to prevent skin allograft rejection in this adoptive transfer model. Interestingly, CD25CD4 T cells isolated from liver graft recipients also showed suppressive potency upon adoptive transfer. Furthermore, depletion of CD25CD4 T cells in primary liver allograft recipients did not prevent the acceptance of a secondary donor-specific skin graft. CONCLUSIONS: Our data provide evidence that the presence of CD25CD4 regulatory T cells is not a unique feature of allograft acceptance and is more likely the result of sustained exposure to donor alloantigens in vivo.     

Presence of Functional Mouse Regulatory CD4+CD25+T Cells in Xenogeneic Neonatal Porcine Thymus-Grafted Athymic Mice

Xenotransplantation with porcine thymus is emerging as a possible means to reconstitute host cellular immunity and to induce immune tolerance in rodents and large animals. However, the presence of regulatory T cells (Treg cells) in this model needs to be determined. We herein demonstrated that efficient repopulation of mouse CD4+CD25+Treg cells was achieved in Balb/c nude mice by grafting neonatal porcine thymic tissue (NP THY). Mouse CD4+CD25+T cells expressed normal levels of Foxp3 in NP THY-grafted nude mice. Furthermore, these CD4+CD25+Treg cells showed significant inhibitory effects on the cell proliferation or interleukin-2 products of syngeneic T cells to alloantigens, Con A or a peptide antigen, although the potent immunosuppressive function might be lower than CD4+CD25+Treg cells in Balb/c mice. CD4+CD25+T cells in NP THY-grafted nude mice showed significantly stronger inhibition on the response to donor porcine antigens of CD4+CD25(-)T cells than CD4+CD25+Treg cells in Balb/c mice. Both CD4+CD25+Treg cells in NP THY-grafted nude and Balb/c mice prevented the development of autoimmune disease mediated by syngeneic CD4+CD25(-)T cells in a similar efficient way in the secondary recipients. These findings provide evidence for the potential involvement of CD4+CD25+Treg cells in keeping self-tolerance and transplant tolerance in this xeno-thymus transplantation model.     

Pretransplant donor peripheral blood mononuclear cells infusion induces transplantation tolerance by generating regulatory T cells

BACKGROUND: It was suggested that maintenance of tolerance to organ transplantation may depend on the formation of T regulatory cells. METHODS: Lewis (LW) rats were made tolerant to a Brown Norway kidney by pretransplant donor peripheral blood mononuclear cells (PBMC) infusion. At greater than 90 days after transplantation, lymph node cells (LN) and graft-infiltrating leukocytes (GIL) alloreactivity was tested in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype was analyzed by FACS. mRNA expression of cytokines and other markers was analyzed on CD4+ T cells from LN. The tolerogenic potential of tolerant cells in vivo was evaluated by adoptive transfer. RESULTS: Tolerant LN cells showed a reduced proliferation against donor stimulators but a normal anti-third-party alloreactivity. In coculture, these cells inhibited antidonor but not antithird-party reactivity of na?ve LN cells. Interleukin (IL)-10 and FasL mRNA expression was up-regulated in tolerant CD4+ T cells, but an anti-IL-10 monoclonal antibody (mAb) only partially reversed their inhibitory effect. Immunoregulatory activity was concentrated in the CD4+ CD25+ T-cell subset. In a transwell system, tolerant T cells inhibited a na?ve MLR to a lesser extent than in a standard coculture. Regulatory cells transferred tolerance after infusion into na?ve LW recipients. CD4+ T cells isolated from tolerized grafts were hyporesponsive to donor stimulators and suppressed a na?ve MLR against donor antigens. CONCLUSIONS: Donor-specific regulatory T cells play a role in tolerance induction by donor PBMC infusion. Regulatory activity is concentrated in the CD4+ CD25+ subset and requires cell-to-cell contact. Regulatory CD4+ T cells accumulate in tolerized kidney grafts where they could exert a protective function against host immune response.     

The protective effect of CD8+CD28- T suppressor cells on the acute rejection responses in rat liver transplantation

Regulatory T cells (Tr) or T-suppressor cells (Ts), which include CD4+CD25+ T cells and CD8+CD28- T cells respectively, have been shown to be essential for the induction and maintenance of immune tolerance. We have investigated the effect of CD8+CD28- Ts and CD4+CD25+ Tr on acute rejection responses in rat liver transplantation (OLT). METHODS: CD8+CD28- Ts/CD4+CD25+ Tr were obtained from inbred and na?ve rats that show spontaneous tolerance to OLT. Adoptive transfers were performed in acute rejection models of various strain combinations with survival times observed to evaluate suppressive effects. Donor-specific blood transfusion (DST) was used to induce CD8+CD28- Ts in na?ve rats, which were assayed in vitro using carboxyfluorescein diacetate succinimidyl easter-labeled one-way mixed lymphocyte reactions. Secondary adoptive transfers of DST-induced CD8+CD28- Ts were also performed in an acute OLT rejection model. RESULTS: CD8+CD28- Ts from tolerant OLT model rats possessed immunosuppressive activity in allogeneic recipients; adoptive transfers of these cells alleviated the acute rejection responses. However, CD4+CD25+ Tr derived from tolerant or na?ve rats failed to do so. In vitro DST-induced CD8+CD28- Ts inhibited alloantigen T-cell responses in na?ve syngeneic rats in an antigen-specific manner. Secondary adoptive transfer of DST-induced CD8+CD28-Ts further reduced acute rejection but not chronic rejection responses. CONCLUSIONS: CD8+CD28- Ts cells protected allogeneic recipients from acute rejection in rat OLT. Furthermore, this activity was not present in CD4+CD25+Tr. DST was observed to be an effective method to generate functional CD8+CD28-Ts in na?ve rats.     

Regulation of transplant arteriosclerosis by CD25+CD4+ T cells generated to alloantigen in vivo

BACKGROUND: CD25+CD4+ regulatory T cells have been shown to suppress alloimmunity in various experimental settings. Here, we hypothesized that alloantigen-reactive regulatory T cells would reduce the severity of transplant arteriosclerosis. METHODS: CD25+CD4+ T cells from CBA mice that were pretreated with C57BL/6 (B.6) blood (donor-specific transfusion, DST) and nondepleting anti-CD4 Ab (YTS 177) were cotransferred with na?ve CBA CD25-CD4+"effector" T cells into CBA-rag-/- mice. These animals received aorta transplants from B.6 CD31-/- donors. CBA wild-type recipients of B.6 aorta grafts were pretreated with 177/DST directly. Some animals received 6x10(5) CD25+CD4+ T cells from pretreated mice to augment regulation on day -1. Grafts were harvested on day 30. RESULTS: Luminal occlusion of the graft caused by neointima formation was 29.3+/-19.4% (n=5) after transfer of effector T cells only. Co-transfer of CD25+CD4+ regulators reduced occlusion significantly (2.4+/-3.3%, n=3; P=0.009). This effect was partially abrogated in the presence of a CTLA4 blocking Ab (11.1+/-4.7%, n=4; P=0.008). Pretreating immunocompetent CBA recipients of B.6 aortic allografts with 177/DST did not reduce transplant arteriosclerosis significantly (43.0+/-15.7%, n=5 vs. 56.6+/-16.8%, n=5; 177/DST vs. controls; P=0.22). However, when pretreated primary CBA recipients received an additional transfer of 6 x 10(5) CD25+CD4+ T cells procured from other mice pretreated with 177/DST before transplantation, luminal occlusion of the graft was markedly reduced (33.0+/-7.6%, n=5; P=0.002). CONCLUSION: Regulatory T cells generated in vivo to alloantigen can prevent CD25-CD4+ T-cell-mediated transplant arteriosclerosis. In immunocompetent recipients, these cells have potential to be used as cellular immunotherapy to control transplant arteriosclerosis.