Dendritic cells activated by lipopolysaccharide after dexamethasone treatment induce donor-specific allograft hyporesponsiveness

BACKGROUND: Immature dendritic cells (imDC) can prolong allograft survival in murine transplantation models. Recent data indicate that semi-mature or alternatively activated DC (aaDC) may be even more tolerogenic. METHODS: We compared the phenotype and regulatory capacity of: a) imDC, cultured in the presence of dexamethasone (DEX), b) mature DC (matDC), activated with LPS, and c) aaDC, activated with LPS after pretreatment with DEX. RESULTS: As compared to imDC, aaDCs displayed a slight upregulation of CD40 while expression levels of MHC-II and CD86 remained low. The production of proinflammatory cytokines, in particular IL-12, by aaDC was much lower than by matDC while both produced similar amounts of the regulatory cytokine IL-10 leading to an increased IL-10/IL-12 ratio for aaDC. After infusion of donor type aaDCs, responder cells isolated from the recipient mice showed donor-specific hyporesponsiveness to restimulation by matDC. Infusion of matDC was immunogenic, while imDC induced partial hyporesponsiveness. Importantly, pretreatment with donor type aaDC (but not imDC) resulted in prolonged survival of a completely MHC-mismatched heart allograft. CONCLUSIONS: Alternatively activated DC are more efficacious than the classical imDC in the regulation of the alloimmune response, which may be related to a distinct cytokine profile characterized by an increased IL-10/IL12 ratio.     

Clinical significance of in vitro donor-specific hyporesponsiveness in renal allograft recipients as demonstrated by the MLR

A longitudinal study was carried out on 19 recipients of cadaveric renal allografts, monitoring their anti-donor and anti-third party responses in the mixed lymphocyte reaction (MLR) at the time of transplantation and at 3, 6, and 12 months post-transplant. Two patterns of responses were identified: in the first (n=11), patients showed, or later developed, donor-specific hyporesponsivenes, and in the second (n=8), patients had persistent antidonor and anti-third party responses. After 1 year, the serum creatinine, number of episodes of acute rejection and biopsy findings were compared in both groups. In the first group, the mean serum creatinine was 136.4 mmol/l, the total number of acute rejection episodes was three and in nine of the ten available biopsies, there were minimal cellular infiltrates and normal appearance of the glomeruli, tubules and blood vessels. In the second group, the mean serum creatinine was 163 mmol/l, the total number of acute rejection episodes was 12 and in five of the seven biopsies available, evidence of ongoing rejection was obtained. The difference in mean serum creatinine was not statistically significant (P>0.05), but the difference in the numbers of acute rejection episodes was (P>0.05). It is concluded that in some renal allograft recipients, a state of donor-specific hyporesponsiveness develops, and this state may be associated with better graft out-come at 1 year. These data may be useful in selecting patients for reduced immunosuppressive therapy.     

Reversibility with interleukin-2 suggests that T cell anergy contributes to donor-specific hyporesponsiveness in renal transplant patients

Data from various rodent models have implicated a role for anergic T cells in the maintenance of self and transplantation tolerance. The relevance of donor-specific T cell anergy to clinical transplantation, however, has not been demonstrated. Previous studies have reported that recipients of solid organ transplant often have reduced frequencies of CD4(+) T cells with anti-donor direct pathway allospecificity after transplantation. The underlying mechanism(s) of this donor-specific hyporesponsiveness is unclear but likely to contribute to the diminished immunosuppressive requirement of transplant patients with time after transplantation. This study shows that ex vivo treatment of CD4(+) T cells from renal transplant recipients with IL-2 could specifically increase the anti-donor frequency in all the patients with evidence of donor-specific hyporesponsiveness. It also shows that the IL-2-induced recovery of anti-donor frequency is unlikely to result from nonspecific stimulation or selective clonal expansion of activated, allospecific CD4(+) T cells. Taken together, the data suggest that T cell anergy plays an important role in the direct pathway hyporesponsiveness that evolves in many human renal transplant recipients.     

Induction of donor-specific hyporesponsiveness and prolongation of cardiac allograft survival by jejunal administration of donor splenocytes

BACKGROUND: Donor-specific immunosuppression is important in transplantation surgery. We examined the immunosuppressive effects of donor splenocytes administered postoperatively into the jejunum and the effect of such treatment on the survival of heterotopic vascularized cardiac allograft in rats. METHODS: Lewis (LEW, RT-1l) recipient rats were treated with 5x10(7) Brown Norway (BN, RT-1n) donor splenocytes for 5 days orally, intrajejunally, or subcutaneously. The immune responses of LEW treated with either donor BN or irrelevant Wistar King A (WKA, RT-1k) were examined by mixed lymphocyte reaction (MLR) and delayed type hypersensitivity (DTH). The effect of postoperative enteral treatment for 6 days with suboptimal dose of cyclosporine (CsA) on heterotopic cardiac allotransplantation was investigated. We measured the production of cytokines (interleukin [IL]-2, IL-4, IL-10, and interferon-gamma [IFN-gamma]) in the supernatant of MLR by ELISA. The effect of intravenous dose of GdCls to block Kupffer cell function was also investigated before the administration of splenocytes. RESULTS: MLR and DTH responses were strongly inhibited in a BN-restricted manner after jejunal or oral feeding of donor BN splenocytes but not by subcutaneous injection or injections by any routs of WKA splenocytes. The effect was more prominent in jejunal than oral feeding. Immunosuppression was associated with a significant inhibition of IL-2 and IFN-gamma production and increased concentrations of IL-4 and IL-10 in MLR supernatants. Immunosuppression was abrogated by pretreatment with GdCl3. Postoperative intrajejunal feeding of donor splenocytes with CsA significantly prolonged cardiac allograft survival time (18.7+/-7.3 vs. 9.9+/-1.7 days for control animals). CONCLUSION: Jejunal administration of splenocytes produces donor-specific immunosuppression and prolongs cardiac allograft survival. Our results suggest the involvement of T helper (Th) 2 cytokines and Kupffer cells in the induction of immune hyporesponsiveness, and indicate that this method represents a unique approach for induction of donor-specific immunosuppression.     

A strategy to achieve donor-specific hyporesponsiveness in cadaver renal allograft recipients by donor haematopoietic stem cell transplantation into the thymus and periphery.

INTRODUCTION: We designed a prospective, randomized clinical trial to evaluate the immune response to thymic and peripheral infusions of donor haematopoietic stem cells (HSCs) to create tolerance in recipients of cadaver renal allografts. METHOD: We divided 24 patients into two equal groups. For group A, 350 ml of unfractionated bone marrow (BM) was aspirated from the anterior iliac crests of donor cadavers. A 2 ml aliquot of concentrated marrow was infused into the thymus of the subject and 100 ml into the BM before surgery; the remaining 250 ml was infused peripherally post-transplantation. The mean nucleated cell count inoculated into the thymus was 3.3 x 10(4) cells/cm(3) and into the periphery 8.6 x 10(7) cells/kg body weight. Group B (controls) underwent renal transplantation directly. Recipients were lymphocytotoxicity cross-match negative in both groups. Group A received low dose prednisolone and cyclosporin; controls also received azathioprine. RESULTS: Over a mean follow-up of 703 days for both groups, group A had significantly better graft function with minimum acute rejection episodes or cytomegalovirus (CMV) infections, a mean serum creatinine (SCr) of 1.23 mg/dl and no graft or patient loss. Group B, with a mean SCr of 2.19 mg/dl had three patients with single acute rejection episodes, two of whom died following uncontrolled rejection-associated infections. The third patient maintained an SCr of 2.5 mg%. Actuarial graft survival was 87.5% in controls at the end of 2 years compared with group A with 100% graft survival at the end of 2 years. CONCLUSION: This novel approach of introducing unfractionated HSCs into the thymus and periphery to create tolerance is safe and efficacious and gives significantly better graft function, minimum acute rejection and no CMV disease with monotherapy.     

Donor-specific cytotoxic T lymphocyte hyporesponsiveness following renal transplantation in patients pretreated with donor-specific transfusions

Our purpose was to investigate the mechanism of the continuing beneficial effect of donor-specific transfusions in the cyclosporine era. We describe the development of donor-specific cytotoxic T lymphocyte hyporesponsiveness in peripheral blood lymphocytes obtained up to 2 years posttransplant in patients preconditioned with 3 DST plus azathioprine. In a group of 12 such patients, hyporesponsiveness developed gradually, becoming detectable in some patients as early as 1 month posttransplant and becoming statistically significant for the entire group at 9-12 months posttransplant. A complete specificity for donor alloantigens was seen in the hyporesponsiveness of some patients; in others, partial suppression of the response to a third party HLA-mismatched control was also seen. Although slight suppression of the mixed lymphocyte culture response was seen in some patients, overall there were no statistically significant differences in MLC responses to control or donor stimulators at any time point posttransplant as compared with pretransplant, pre-DST. The mechanism of donor-specific CTL hyporesponsiveness 2 years posttransplant was explored in one patient (HLA A1, 2, B 57, 60; DR 3, 6) who had received a 2-HLA haplotype-mismatched kidney transplant from her husband (HLA A2,--; B5, 8; DR4,--) following DST plus AZA pretreatment. Bulk culture CTL analysis showed specific nonresponsiveness to donor stimulators; however in the presence of exogenous recombinant IL-2, the antidonor response was restored to the level of pretransplant PBL. Limiting dilution analysis using recombinant IL-2 revealed equivalent precursor frequency of antidonor CTL in pre- and posttransplant PBL. These data suggest that the hyporesponsive PBL contained donor-specific CTL precursors but were deficient in helper function necessary for CTL maturation.     

输注供者抗原特异性调节性T淋巴细胞延长小鼠胰岛移植物的存活时间

目的 研究体外扩增获得的供者抗原特异性调节性T淋巴细胞(Treg细胞)对小鼠同种胰岛移植物存活时间的影响.方法 建立从Balb/c小鼠到C57小鼠的胰岛移植模型,通过观察移植后小鼠血糖情况来判断移植胰岛的存活时间.受鼠制成糖尿病模型后分为3组.对照组:不给予任何处理,只观察血糖变化;单纯胰岛移植组:移植胰岛,但不输注Treg细胞;实验组:术前1 d静脉给予1×106个供者特异性Treg细胞,然后行胰岛移植.结果胰岛移植后,对照组小鼠血糖持续高于16.7 mmol/L;单纯胰岛移植组小鼠血糖于术后1～2 d全部降至正常,到7～11 d时陆续开始升高,并维持在术前水平,存活时间为(8.50±1.6)d;实验组小鼠血糖于术后2 d内降至正常,之后维持在较低水平,至第21天开始有小鼠血糖升高超过16.7 mmol/L,存活时间为(26.2±3.9)d,明显长于单纯胰岛移植组(P＜0.05).结论 输注供者抗原特异性Treg细胞可以明显延长移植胰岛的存活时间,在胰岛移植耐受中有积极的作用.

Abstract：

Objective To investigate the effects of donor-specific regulatory T cells (Treg) transfusion on islet allograft survival. Methods Allogeneic fresh islets from Balb/c mice were transplanted to streptozotocin-induced diabetic C57 mice. The survival of islet allografts was observed. The experiment was divided into 3 groups: control group, nothing had been done to the recipients; simple islet transplantation group, the recipients received the islet transplantation only; experimental group, the recipients were given 1 ×106 Treg, then received islet transplantation. Results Blood glucose (BG) was above 16. 7 mmol/L after islet transplantation in control group; In simple islet transplantation group,BG level returned to normal level 1 to 2 days after transplantation, and hyperglycemia appeared 7 to 11 days after transplantation and maintained as the same as that before transplantation; In experimental group, BG level returned to normal level 2 days after transplantation and maintained at a low level,and at the 21st day after transplantation BG level was over 16. 7mmol/L in some recipients. Islet allograft survival in experimental group was significantly prolonged as compared with simple islet transplantation group. Conclusion Donor-specific Treg transfusion could prolong the islet allograft survival,and maybe have positive effect on tolerance induction of islet transplantation.     

The induction of immunologic hyporesponsiveness by preoperative donor-specific transfusions and cyclosporine in human cadaveric transplants. A preliminary trial.

A prospective randomized preliminary trial was performed in patients undergoing cadaveric renal transplantation to determine the potential benefits, disadvantages, and logistic problems associated with the administration of donor-specific transfusions and cyclosporine initiated 24 hr before transplantation. Ten patients received DST followed by continuous intravenous CsA approximately 24 hr before cadaveric renal transplantation from the same donor. Twelve patients receiving sequential therapy with Minnesota antilymphoblast globulin, azathioprine, and steroids with subsequent conversion to CsA served as controls. Patient demographics and the donor characteristics were evenly matched in the two groups. While the study group had longer cold ischemia time and more evidence of renal dysfunction within the first two weeks, subsequent renal function was identical in the groups and there were fewer episodes of severe rejection requiring treatment with OKT3 within the first six months in the DST group (5 vs. 0, P less than 0.05), which also had less reactivity in mixed lymphocyte cultures against preserved donor-specific lymphocytes than did the control group (stimulation index 9.0 +/- 3.0 vs. 25.3 +/- 6.0, respectively, P less than 0.05). The need for dialysis, incidence of infections and other complications, and subsequent immunosuppressive therapy were not different in the two groups. It is concluded that DSTs and intravenous CsA initiated 24 hr prior to transplantation are capable of inducing reduced immunologic responsiveness against the specific donor. Patients treated with this therapy should receive organs from "ideal" donors without risk factors and cold ischemia time should not exceed 30 hr. Further clinical studies of this approach are warranted.     

Suppressor cell induction in donor-specific transfused mouse heart recipients

Administration of donor-specific blood presenting major or minor foreign histocompatibility antigens to the mouse recipient improved heart allograft survival when a single transfusion of 0.25 ml was given to the recipient prior to transplantation. Multiple transfusions did not prolong allograft survival, which suggested a presensitization effect. In addition, when the single transfusion volume was reduced to 0.025 ml, no significant effect in prolongation of graft survival was observed. Thus the amount of blood transfused seemed to be a critical factor in achieving the transfusion effect. Splenic suppressor cells after transfusion and transplantation (as determined by adoptive transfer) were present during the stable maintenance phase of graft survival in transfused recipients with long-term surviving heart allografts. Also, an intact spleen was required to achieve improved allograft survival in mice transfused with donor-specific blood. Thus a mechanism for the favorable effect of blood transfusion may be the generation of splenic suppressor cells in response to transfusion followed by transplantation.     

Application of recipient-derived dendritic cells to induce donor-specific T-cell hyporesponsiveness

Administration of donor-derived immature dendritic cells (DC) treated with NF-kappaB oligodeoxyribonucleotides (ODN) prevents allograft rejection. We attempted to explore the use of recipient-derived DC pulsed with donor antigens, in which the donor antigens were presented to host T cells via an indirect pathway (cross-priming). Expression of CD40, CD80, and CD86 on DC was significantly inhibited by treatment with NF-kappaB ODN, whereas MHC class I and II were minimally affected. Normal C3H DC pulsed with B10 antigens stimulated proliferative responses and donor-specific CTL activity in C3H T cells, both of which were, however, markedly inhibited when DC were treated with NF-kappaB ODN. This manipulation was associated with reduced IFN-gamma and increased IL-10 production in the supernate, suggesting a Th2 bias. More frequent apoptotic T cells were observed in cultures with NF-kappaB ODN DC. In contrast to administration of normal DC pulsed with donor antigens that accelerated rejection of B10 cardiac allografts (median survival time [MST] 7 days versus 10 days in no-DC treatment control, P < .05), a single injection of 2 x 10(6) NF-kappaB ODN DC significantly prolonged allograft survival (MST 50 days, P < .05 compared with no-DC treatment control). The anti-donor CTL activity in infiltrating T cells isolated from cardiac grafts in recipients that received NF-kappaB ODN DC was significantly suppressed. These data indicate that vaccination with immature DC, propagated from recipient BM is an attractive approach to induce T-cell hyporesponsiveness.     

Selective loss of functional antidonor cytolytic T cell precursors following donor-specific blood transfusions in long-term renal allograft recipients.

Pretransplant administration of donor-specific blood transfusions prolongs the survival of HLA-disparate renal allografts to a level comparable to that of HLA-identical transplants. To test the hypothesis that DST recipients develop immunologic tolerance of their donors, we examined antidonor T cell immunity in a group of DST recipients with long-term well-functioning allografts. Limiting dilution analysis studies indicated that 5/14 long-term DST recipients exhibit a complete absence of detectable antidonor cytolytic T lymphocyte precursors (CTLp). In contrast, 4/4 long-term non-DST recipients who received conventional or cyclosporine immunosuppression exhibited significant levels of antidonor CTLp. Nonresponsive DST recipients retained the capacity to mount antidonor responses in mixed lymphocyte reactions and IL-2 production assays, and exhibited normal levels of CTLp directed to third-party antigens. Addition of exogenous IL-2 to cell-mediated lympholysis cultures did not restore antidonor CTL responses. In summary, our data suggest that the DST conditioning regimen induces a complete yet selective loss of functional antidonor CTLp in some long-term renal transplant recipients.     

Successful hepatitis B vaccination in liver transplant recipients with donor-specific hyporesponsiveness

Currently, patients are prescribed lifelong treatment with hepatitis B immunoglobulin (HBIg) after liver transplantation (LT) for hepatitis B virus (HBV)-related diseases in order to prevent reinfection with HBV. Active immunization with an HBV vaccine would be a preferable alternative; however, the immunosuppressive environment in LT recipients is believed to elicit a poor response to vaccination. Minimizing the exposure of the HBV-infected LT recipients to immunosuppressants would be beneficial in inducing adaptive immunity against HBV by vaccination. In this study, in addition to efforts to minimize immunosuppression, prophylaxis with HBV vaccination combined with continuous HBIg administration was performed in 17 LT recipients who had undergone transplantation attributable to HBV-related diseases. During the observation period, the overall response rate to HBV vaccination was 64.7%. The immune status of the recipients was evaluated by a mixed lymphocyte reaction assay in response to allostimulation. Patients showing a donor-specific hyporesponse with a well-maintained response to the third-party stimulus always achieved a sustained immune response to the vaccine, whereas patients showing a hyporesponse to both the donor and the third-party stimulus were unable to do so. Thus, inducing an anti-donor-specific immunosuppressive status by minimizing immunosuppression should enable post-transplant HBV vaccination to be a promising prophylactic strategy.     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受.方法将114只日本白色家兔和Dutch家兔分为对照组和实验组,日本白色家兔作为供体,Dutch家兔作为受体.对照组,在不使用免疫抑制剂的情况下,将12只日本白色家兔与12只Dutch家兔行相同面积的背部全厚皮肤互换移植,观察其成活时间.实验组,将45只日本白色家兔和45只Dutch家兔行全厚皮肤移植的同时行骨髓移植,然后将作为受体的Dutch家兔分为A,B,C,D四组,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植,观察移植皮肤的成活时间.结果对照组,供体与受体移植皮肤的平均成活时间分别为(12.0±1.7)天和(10.3±1.3)天.实验组,A,B,C,D四组移植皮肤的平均成活时间分别为(61.0± 7.2)、(80.7± 10.4)、(78.8± 12.7)、(88.0± 6.0)天.结论通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验,旨在为临床应用提供了理论基础及可靠依据,为同种组织重建提供一个新方法.     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的　证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受。方法　将 114只日本白色家兔和Dutch家兔分为对照组和实验组 ,日本白色家兔作为供体 ,Dutch家兔作为受体。对照组 ,在不使用免疫抑制剂的情况下 ,将 12只日本白色家兔与 12只Dutch家兔行相同面积的背部全厚皮肤互换移植 ,观察其成活时间。实验组 ,将 4 5只日本白色家兔和 4 5只Dutch家兔行全厚皮肤移植的同时行骨髓移植 ,然后将作为受体的Dutch家兔分为A ,B ,C ,D四组 ,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植 ,观察移植皮肤的成活时间。结果　对照组 ,供体与受体移植皮肤的平均成活时间分别为 (12 .0± 1.7)天和 (10 .3± 1.3)天。实验组 ,A ,B ,C ,D四组移植皮肤的平均成活时间分别为 (6 1.0± 7.2 )、(80 .7± 10 .4 )、(78.8± 12 .7)、(88.0± 6 .0 )天。结论　通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验 ,旨在为临床应用提供了理论基础及可靠依据 ,为同种组织重建提供一个新方法     

The roles of T cell subpopulations in allograft rejection

In principle, cell-mediated allograft rejection can be brought about by cytotoxic T cells or by a DTH-like reaction. The first part of this article reviews the relative importance of these two mechanisms in graft rejection and concludes that the bulk of evidence points to cytotoxic T cells as being of major importance. In the discussion an operational definition of DTH is adopted--namely a mechanism that mediates bystander killing and depends on the existence of a radiation-sensitive effector mechanism. The rejection of skin and organ allografts in mammals is T cell dependent and the demonstration that such rejection can occur in heavily-irradiated T cell reconstituted animals and in the absence of demonstrable bystander effects leads to the conclusion drawn. This is not to imply that concomitant DTH reactions may not augment the rejection process nor to deny the possibility that in special circumstances DTH reactions may play an essential part. In the second part of this article, the roles in graft rejection of CD4+ and CD8+ cytotoxic T cells are considered. The classical collaborative interaction between class II-restricted CD4+ cells that play an inductive role, and class I restricted CD8+ cells that differentiate into cytotoxic effector cells, seems to be unnecessary in some allograft responses. Thus, not only can CD4+ T cells differentiate into class II-restricted cytotoxic T cells but CD8+ T cells, at least in some class I MHC-incompatible strain combinations, can develop into mature effector cells without an inductive signal from CD4+ T cells. This autonomy seems to be limited to MHC incompatibilities so that CD8+ cells alone are unable to mediate the rejection of minor-H incompatible grafts. For these latter types of grafts, CD4+ T cells can bring about graft rejection if multiple minor-H determinants are present, but for weak minor-H responses the classical collaborative scheme is necessary.