Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.     

Antigen‐specific regulatory T cells can induce tolerance to immunogenic grafts without the need for chronic immunosuppression

Regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) play an important role in the induction of allospecific tolerance. However tolerance in solid organ transplantation by mere transfer of Tregs has been difficult. Besides this the stability of the differentiation phenotype of Tregs has recently been questioned. We therefore aimed in generating large numbers of stable allospecific Tregs from naïve T cells by retroviral transduction with Foxp3. These were tested in an immunogenic skin transplantation model (C57BL/6→BALB/c). We established a system of transduction of mouse T cells with ecotropic retroviruses expressing Foxp3 and Thy1.1 as a surface marker to follow up transduced T cells. Alloantigen‐specific Tregs were generated by stimulating naïve recipient CD4⁺ T cells with irradiated donor splenocytes. CD25⁺ and/or CD69⁺ allospecific recipient CD4⁺ T cells were isolated and transduced with Foxp3. Alloantigen‐specific Foxp3 T cells (iTregs) showed high expression for the Treg markers Foxp3, CTLA4 and GITR. They could suppress a MLR in an alloantigen‐specific manner. Furthermore, they could be expanded up to 18 fold in vitro while maintaining their Treg phenotype and expression of lymph node homing markers like CCR7 and CD62L. iTregs prevented skin graft rejection without the need for chronic immunosuppression and recipients showed systemic allospecific allotolerance. Alloantigen‐specific Tregs were far more potent than polyspecific Tregs. Mechanisms of tolerance were graft specific homing, expansion and long‐term persistence of Tregs within the graft (>100 days, 90% of intragraft Tregs were alloantigen‐specific). In fact, tolerance could be transferred with re‐transplantation of the tolerant graft onto secondary recipients. Third party grafts were readily rejected demonstrating specificity of tolerance. Due to the Foxp3 transduction, iTregs did not lose their Treg phenotype. The results prove that large numbers of stable alloantigen‐specific Tregs can be generated from a polyclonal repertoire of naïve T cells. This is the first time that allotolerance was achieved in a non‐lymphopenic transplant model using skin grafts in an immunogenic strain combination. Therefore, antigen‐specific Tregs might have a huge therapeutic potential after solid organ transplantation.     

移植抗原特异性转基因CD8+T细胞对白细胞介素2的依赖性

目的研究白细胞介素2（IL-2）在调节移植抗原特异性转基因CD8^＋T细胞介导的免疫排斥反应中的作用。方法将经荧光染科CFSE标记后的C57BL／6小鼠和2CTg小鼠（CD4敲除鼠）淋巴细胞分别植入经致死剂量γ射线照射过的两组DBA／2J小鼠体内，检测CD4^＋与CD8^＋T细胞在体内分裂增殖的时相，并用胞浆内IL-2标志染色方法测定活化后T细胞表达IL-2的能力。以Balb／c小鼠为供者，糖尿病2CTg小鼠和2C Tg-IL-2KO小鼠（IL-2敲除鼠）为受者，进行胰岛细胞移植。观察CD8^＋ T细胞在介导移植排斥中的作用。结果DBA／2J小鼠输注了C57BL／6小鼠的淋巴细胞后，CD4^＋与CD8^＋T细胞分裂增殖均非常明显，前者表达大量IL-2，后者则不表达。DBA／2J小鼠输注了2CTg小鼠的淋巴细胞后。在完全没有CD4^＋T细胞存在的情况下，CD8^＋T细胞仍明显分裂增殖并大量表达IL-2。2CTg和2CTg—IL-2KO小鼠移植胰岛细胞后，前者迅速发生排斥反应，胰岛移植物的平均存活时间仅为8d，而后者胰岛移植物的平均存活时间〉50d。结论CD8^＋T细胞在产生和利用IL-2时有很大的可塑性。CD4^＋T细胞存在时，CD8^＋T细胞能有效利用CD4^＋T细来源的IL-2进行分裂增殖，在缺乏CD4^＋T细胞时，则利用自身来源的IL-2进行分裂增殖；移植抗原特异性CD8^＋T细胞的效应功能完全依赖于IL-2，排斥反应由CD8^＋T细胞介导时，阻断IL-2／IL-2受体通路可诱导移植物长期存活。     

Tolerance to rat heart grafts induced by intrathymic immunomodulation is mediated by indirect recognition primed CD4+CD25+ Treg cells

BACKGROUND: In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. METHODS: Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. RESULTS: Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. CONCLUSIONS: Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.     

Indirectly primed CD8+ T cells are a prominent component of the allogeneic T-cell repertoire after skin graft rejection in mice

BACKGROUND: Alloreactive CD4 and CD8 T lymphocytes recognize antigen through both the direct and the indirect pathways. Although indirect priming of CD4+ T cells has been well described, little is known about the frequency and cytokine profile of indirectly primed CD8+ T cells during fully allogeneic graft rejection. METHODS: We used a cytokine enzyme-linked immunosorbent spot assay to characterize indirect priming of alloreactive CD8 and CD4 cells in mice. RESULTS: Interferon-gamma-producing CD8+ T cells specific for indirectly presented alloantigen were detectable in allograft-primed mice at a frequency of 50-100 per million cells (compared with 3000 per million for responses through the direct pathway) and were similar in frequency to indirectly primed CD4 cells. CONCLUSION: CD8+ T cells primed through the indirect pathway are a prominent component of the alloreactive T-cell repertoire induced after skin graft placement in mice, raising the possibility that these cells may play a significant role in the rejection process itself.     

Acute rejection after liver transplantation: Is there a specific immunological pattern?

The aim of the study was to assess various T-cell subsets and cytokine secretion patterns both in liver tissue and in the peripheral blood of 24 liver transplant patients to assess possible specific immunological involvement in early acute rejection episodes after liver transplantation. Particularly, we studied CD4+ CD7+, CD8+ CD38+, and CD4+ CD25+ T cells by flow cytometry, as well as contemporaneously, interleukin (IL)-2 and IL-10 secretion by ELISpot to determine possible Th1-like immune responses and the immunomodulation expressed by Treg cells in acute liver rejection, respectively. As a control group we included patients transplanted without acute rejection. Early acute rejection within the first 4 weeks was proven histologically in 42% of patients. It was associated with a greater expression of CD4+ CD7+ and CD8+ CD38+ T cells in the liver than in the blood (P < .001). A contemporaneous reduced expansion of liver Treg cells was evident in patients with acute rejection (P < .001). Our data suggested that a preferential Th1-like immune mechanism operated in local fashion as characterized by a decreased presence in the liver and blood of Treg cells.     

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.     

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.     

CD28 superagonist antibody treatment attenuated obliterative bronchiolitis in rat allo-orthotopic tracheal transplantation by preferentially expanding Foxp3-expressing regulatory T cells

Obliterative airway disease (OAD) due to chronic alloantigen rejection remains a major challenge for long-term graft survival in lung transplantation. It is known that superagonistic CD28-specific monoclonal antibody JJ316 (supCD28 MAb) has the ability to induce regulatory T cells (Tregs) efficiently. Here we used a rat orthotopic tracheal transplantation model to investigate the effects of supCD28 MAb on expanding Tregs in vivo and its application in suppression of acute and chronic airway allograft rejection. SupCD28 MAb administration revealed a significant increase in the CD4+CD25+ T cells, CD4+FoxP3+ T cells, and CD4+CD25+ FoxP3+ T cells population among CD4+ T cells in spleen, peripheral blood, as well as cervical lymph nodes. The allografts from animals treated with supCD28 MAb showed significantly less airway obliteration and rejection of the respiratory epithelium compared with allografts of the mouse immunoglobulin G-treated group on the 5th day and the 60th day after transplantation. Overall, our data demonstrated that an intraperitoneally administrated low dose of supCD28 MAb was sufficient to induce Treg cell expansion in vivo and was effective in protecting the airway graft from early rejection and chronic OAD development. These findings provide the basis for new therapies to prevent OAD and perhaps rejection of allografts in other human transplantations.     

Different mechanisms of cardiac allograft rejection in wildtype and CD28-deficient mice.

Although CD28 blockade results in long-term cardiac allograft survival in wildtype mice, CD28-deficient mice effectively reject heart allografts. This study compared the mechanisms of allogeneic responses in wildtype and CD28-deficient mice. Adoptive transfer of purified CD28-deficient T cells into transplanted nude mice resulted in graft rejection. However, this model demonstrated that the allogeneic T cell function was severely impaired when compared with wildtype T cells, despite similar survival kinetics. Cardiac allograft rejection depended on both CD4+ and CD8+ T cell subsets in CD28-deficient mice, whereas only CD4+ T cells were necessary in wildtype recipients. These results suggested that CD8+ T cells were more important in CD28-deficient than wildtype mice. In addition to the CD8+ T cell requirement, allograft rejection in CD28-deficient mice was dependent on a sustained presence of CD4+ T cells, whereas it only required the initial presence of CD4+ T cells in wildtype mice. Taken together, these data suggest that CD4+ T cells from CD28-deficient mice have impaired responses to alloantigen in vivo, thus requiring long-lasting cooperation with CD8+ T cell responses to facilitate graft rejection. These results may help to explain the failure to promote graft tolerance in some preclinical and clinical settings.     

Specific unresponsiveness in rats with prolonged cardiac allograft survival after treatment with cyclosporine. IV. Examination of T cell subsets in graft-versus-host assays

In DA rats grafted with PVG hearts a short course of cyclosporine induces a state of specific unresponsiveness. In animals with grafts surviving greater than 75 days, the W3/25+ (CD4+) subset loses its capacity to mediate rejection of PVG but not third-party heart grafts when transferred into irradiated DA hosts. In this study we examined whether there was an associated change in the capacity of peripheral lymphoid T cell subsets from unresponsive animals to induce graft versus host (GVH) reactivity. First we demonstrated that there is synergy between naive CD4+ and CD8+ cells in the popliteal lymph node PLN assay, but that alone, only CD4+ and not CD8+ cells proliferate. Unfractionated and CD4+ cells from unresponsive animals produced similar PLN enlargement in both donor-specific (DAxPVG)F1 hosts and third-party (W/FxDA)F1 hosts. This enlargement was comparable to that produced cells from naive and specifically sensitized hosts. MRC OX8+ cells from both unresponsive and naive hosts did not produce PLN enlargement unless large numbers were injected; small numbers of sensitized MRC OX8+ cells produced specific PLN enlargement CD4+ cells from CsA-treated DA did not respond to DA anti-PVG idiotype in an in vivo assay adapted from the host versus graft (HVG) PLN assay. As the PLN assay does not test cells capacity to effect tissue damage, cells from CsA-treated DA rats were tested in a lethal GVHD assay. These cells had the same capacity to induce lethal GVH in irradiated (DAxPVG)F1 and (DAxW/F)F1 hosts. The normal response of cells from unresponsive animals in both proliferative and effector GVH assays shows that cells with the potential to respond to PVG alloantigen and mediate tissue damage are present in unresponsive animals but are prevented from mediating rejection, possibly due to the relatively weak immune stimulus of an organ graft.     

Immune Exhaustion and Transplantation

Exhaustion of lymphocyte function through chronic exposure to a high load of foreign antigen is well established for chronic viral infection and antitumor immunity and has been found to be associated with a distinct molecular program and characteristic cell surface phenotype. Although exhaustion has most commonly been studied in the context of CD8 viral responses, recent studies indicate that chronic antigen exposure may affect B cells, NK cells and CD4 T cells in a parallel manner. Limited information is available regarding the extent of lymphocyte exhaustion development in the transplant setting and its impact on anti–graft alloreactivity. By analogy to the persistence of a foreign virus, the large mass of alloantigen presented by an allograft in chronic residence could provide an ideal setting for exhausting donor‐reactive T cells. The extent of T cell exhaustion occurring with various allografts, the kinetics of its development, whether exhaustion is influenced positively or negatively by different immunosuppressants, and the impact of exhaustion on graft survival and tolerance development remains a fertile area for investigation. Harnessing or encouraging the natural processes of exhaustion may provide a novel means to promote graft survival and transplantation tolerance.     

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.     

人CD8+CD28-抑制性T淋巴细胞的体外扩增及其抗原特异性调节作用

目的 探讨在体外大量扩增CD8+CD28-抑制性T淋巴细胞(Ts细胞)的方法,并检验其免疫调节作用.方法 分离健康志愿者全血中CD8+T淋巴细胞,在含不同细胞因子和异体抗原提呈细胞(APC)的培养条件下进行体外扩增.应用流式细胞术对扩增过程中CD28 -细胞亚群的比例进行监测.分为3组进行混合淋巴细胞培养,反应细胞均为CD4+T淋巴细胞:B-APc组以来源于原致敏供者的APC作为刺激细胞,I-APC组以HLA-A、B、DR全错配的无关供者的APC作为刺激细胞,Dynabeads组以包被有抗CD3和CD28单克隆抗体的免疫微球Dynabeads作为刺激细胞;扩增后的Ts细胞作为第三方调节细胞加入混合淋巴细胞培养中,测定其对CD4+T淋巴细胞增殖的抑制作用.结果 在含白细胞介素2(IL-2)+ IL-7+ IL-15的培养条件下,CD8+T淋巴细胞培养后CD8+CD28-T淋巴细胞亚群的比例最高(P＜0.05).B-APC组不加入Ts细胞时,增殖的CD4+T淋巴细胞比例为66.7％,当加入的Ts细胞与反应细胞的比例分别为0.5∶1、0.1∶1和0.02∶1时,增殖的CD4+T淋巴细胞比例分别为16.5％、34.1％和62.6％.Ts细胞对CD4+T淋巴细胞的增殖有明显抑制作用,而在I-APC组和Dynabeads组中,此抑制作用不明显.结论 应用含IL-2+ IL-7+ IL-15的培养条件联合异体APC刺激可以在体外大量扩增Ts细胞,扩增所得Ts细胞在体外对供者CD4+T淋巴细胞的增殖有明显抗原特异性抑制作用.     

The roles of CD25+CD4+ regulatory T cells in operational tolerance after living donor liver transplantation

Recent evidence suggests that CD4+CD25+ regulatory T cells (Tregs) affect immune responses, including those to alloantigens in organ transplants. We have followed a group of liver allograft recipients with good liver graft function who have been weaned off immunosuppression (IS). The purpose of this study was to determine whether Tregs contributed functionally to the mechanisms of graft acceptance. MATERIAL AND METHODS: The functional assay used peripheral blood obtained from LTx recipients free of immunosuppression. The Whole population of CD4+ T cells and the CD4+ T cells depleted of CD4+CD25 high cells were tested for proliferation against donor versus third party stimulators. Moreover to determine the antigen-specificity of the Tregs, serially diluted numbering of CD4+CD25+ T cells were co-cultured with CD4+CD25- T cells. The proliferation responses were examined toward donor versus third party stimulators. RESULT: CD4+ T cells from all LTx recipients off immunosuppression showed hyporesponsiveness to the donor but not to third party stimulators. However, even after depletion of the CD4+CD25 high population, the cells continued to be hyporesponsive toward the donor. In four out of five cases, the suppression exhibited by CD4+CD25+ T cells was more specific for the donor. DISCUSSION: These findings suggest that donor alloantigen specific regulation by Tregs is one of multiple mechanisms that may contribute to the maintenance of liver graft survival in the absence of immunosuppression.     

Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance

Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to na?ve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.     

Suppression of anti-skin-allograft response by photodamaged effector cells--the modulating effects of prednisolone and cyclophosphamide.

Using a murine model of skin allotransplantation, we have demonstrated previously that inhibition of specific response to alloantigen is inducible by immunization of the host with intravenously administered photoinactivated antigraft effector T cells. This hyporesponsiveness, which was demonstrated by specific inhibition of mixed leukocyte culture (MLC), inhibition of cytotoxic T lympholysis (CTL), specific suppression of the delayed type hypersensitivity (DTH) response, and prolongation of specific skin allograft survival, was adoptively transferable by CD8+ radiosensitive T lymphocytes. In this study, we extend those results to evaluate the effects of an immunosuppressive agent (prednisolone) and an alkylating drug (cyclophosphamide) on the induction of this specific suppressive cellular response. Our results reveal that the administration of prednisolone reduces the induction of the specific hyporesponsiveness to alloantigen, as demonstrated by maintenance of the DTH response to alloantigen and continued accelerated rejection of skin allografts. In contrast, the administration of cyclophosphamide augmented this specific suppressive response to alloantigen in the DTH assay and in prolongation of specific skin allograft survival. These results indicate that adjuvant immunomodulating chemotherapy alters the immune response to photoinactivated effector T cells.     

CD154 on the surface of CD4+CD25+ regulatory T cells contributes to skin transplant tolerance

BACKGROUND: It is known that the infusion of whole blood from donors (donor-specific transfusion) into recipients combined with anti-CD154 therapy can prolong allograft survival. It has generally been agreed that the effectiveness of anti-CD154 therapy is caused by the inactivation of alloreactive CD4+ and CD8+ effector T cells. The recent literature has implicated CD4+CD25+ regulatory T cells in the suppression of autoimmunity and graft rejection, and we therefore examined whether CD154 blockade is effective because of its blockade of inflammatory T-cell activation or because of a direct impact on the regulatory T cells. METHODS: RAG(-/-) mice were adoptively transfused with CD4+ T cells or a subset of the population (CD4+CD25+ or CD4+CD25- T cells) alone or in combination with donor-specific transfusion and anti-CD154 and given an allo-skin transplant. The longevity of the transplant was determined over time. CD154(-/-)CD4+ T cells were used to assess the importance of CD154 in graft rejection and acceptance. RESULTS: CD154 blockade (or loss of CD154) on CD4+CD25+ regulatory T cells enhanced their immunosuppressive activities and was a contributing factor to anti-CD154-induced immune suppression in vivo. In a model of allograft tolerance, suppression was elicited by antigen and anti-CD154 or antigen alone if the CD4+CD25+ regulatory T cells were deficient in CD154 expression. CONCLUSIONS: Neutralizing the function of CD154 on regulatory T cells upon antigen exposure induces heightened levels of suppressive activities and is likely a contributing factor to the long-lived therapeutic effects of anti-CD154 treatment.     

Regulatory function of human CD4+ cytolytic T lymphocytes.

Allograft rejection is mediated by both CD4+ and CD8+ T cells. The lytic function of the classic CD8+ cytolytic T lymphocytes (CTL) occurs through recognition of allogeneic major histocompatibility complex (MHC) class I on the surface of the graft. CD4+ CTL recognize MHC class II through a direct recognition pathway or an indirect pathway where MHC peptides are presented in the context of self MHC class II. Lytic CD4+ cells may destroy graft tissue or, we hypothesize, the indirect CD4+ T cell may down regulate CD8+ CTL by recognition of donor MHC peptides presented by self MHC class II expressed on CD8+ T cells. To define the role of CD4+ CTL in allograft outcome we used a CD4+ CTL that is MHC class II restricted, recognizing human leucocyte antigen (HLA)-A1 and HLA-B8 peptides in the context of HLA-DR4. This line (MDSxA1/B8) will lyse DR4+ B lymphoblastoid cells (LCL) pulsed with HLA-A1/B8 peptides (amino acids 60-84 of the alpha1 domain of the MHC class I molecule). These T cells will also lyse peptide-pulsed antigen-specific T cell clones, both CD4+ and CD8+, that express HLA-DR4. These clones must process and present the MHC class I peptides for recognition and lysis to occur. These results suggest a possible mechanism to explain allograft tolerance. Lytic CD4+ T cells, that recognize donor HLA peptides through an indirect antigen presentation pathway, down-regulate donor-specific CTL through peptide-specific lysis resulting in graft tolerance.     

Mechanism of portal venous tolerant long-term MHC Class I L(d)-specific skin graft survival in transgenic 2CF1 mice

BACKGROUND: Administration of alloantigen via the portal vein (PV) in non-transgenic animals has been shown to promote immunologic tolerance and enhance transplant allograft survival. The underlying mechanisms remain unclear. In 2C x dm2 F1 (2CF1) transgenic mice, the monoclonal antibody, 1B2, identifies specific 2C TCR transgenic CD8+ T cells that are cytotoxic against Class I MHC L(d). In these mice, the specific response by these cells to L(d+) skin grafts after PV administration of L(d+) antigen was determined. MATERIALS AND METHODS: Saline (control) or allogeneic C57BL/6 x BALB/c F1 (CB6F1) spleen cells (25 x 10(6)), which differ from 2CF1 only at L(d), were injected PV into 2CF1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these 2CF1 mice. Skin graft rejection was defined as >50% loss of the graft. Parallel experiments were performed in non-transgenic littermates [B6F1 (C57BL/6 x dm2)]. FACS analysis of 2CF1 peripheral blood for 1B2+, CD4+, and CD8+ T cells was performed 2 days before PV injection (9 days prior to skin grafting), 5 days after PV injection (2 days prior to skin grafting), and 7, 14, 21, 28, and 60 days after skin grafting. FACS analysis of nai;ve, saline control, and CB6F1 PV-treated 2CF1 thymocytes was also performed. Responsiveness of saline (control)-treated and PV-treated 2CF1 splenocytes was measured by in vitro cytotoxic T lymphocyte (CTL). RESULTS: All CB6F1 skin grafts were rejected in <14 days by PV saline controls. However, a single PV injection of donor L(d+) CB6F1 cells was sufficient to induce indefinite CB6F1 (L(d+)) skin allograft survival in 100% of non-transgenic B6F1 and transgenic 2CF1 (anti-L(d)) TCR transgenic recipients. FACS analysis of 1B2+ T cells demonstrated that PV injection of donor antigen followed by a CB6F1 skin graft led to a 70% decrease in peripheral donor-reactive 1B2+ CD8+ T cells by day 7, while central thymocytes were unchanged. CTL of 2CF1 splenocytes following PV CB6F1 demonstrated that they were hyporesponsive to L(d) compared to saline-treated 2CF1 splenocytes. Despite recovery of peripheral CD8+ T cells to near normal levels by 60 days post-transplantation, skin graft survival persisted indefinitely. CONCLUSIONS: Administration of specific PV antigen results in exquisite long-term L(d+) skin allograft acceptance. This tolerance induction is related to a significant peripheral deletion of donor-reactive 1B2+ CD8+ transgenic T cells and anergy of the residual T cells.