Exhaustive differentiation of alloreactive CD8+ T cells: critical for determination of graft acceptance or rejection

BACKGROUND: The precise role that CD8+ T cells play in the rejection and acceptance of different types of allograft is unclear and has been shown to vary between donor-recipient combinations. METHODS: The response of adoptively transferred CD8+ T cells reactive to the donor alloantigen H2Kb was examined after transplantation of H2Kb liver, kidney, and heart grafts in mice. RESULTS: After transfer of 6 x 10(6) alloreactive CD8+ T cells to T-cell depleted syngeneic mice spontaneous long-term acceptance of liver grafts was observed, whereas kidney and heart grafts were acutely rejected. Within 5 days of liver transplantation, we found that the entire H2Kb-reactive T-cell pool was stimulated to proliferate and differentiate into memory or effector cells that were detectable within lymphoid tissues as well as the liver graft itself. However, despite the generation of effector or memory T cells, liver allografts were accepted, which correlated with the exhaustion or deletion of such cells. In contrast, although activation and proliferation of H2Kb-reactive CD8+ T cells was observed after transplantation of heart or kidney grafts, unactivated, H2Kb-reactive CD8+ T cells were still present in the spleen even long term. Interestingly, differences in the effector function of liver and kidney graft infiltrating donor-reactive CD8+ T cells were not detected after adoptive transfer into immunodeficient mice, despite a reduction in Th1-type cytokines within liver grafts. CONCLUSIONS: The rapid and extensive initial activation and differentiation of donor-reactive CD8+ T cells that occurs after liver transplantation leads to clonal exhaustion or deletion of the alloreactive CD8+ T-cell repertoire resulting in spontaneous tolerance induction.     

Lymphocyte depletion and risk of acute rejection in renal transplant recipients at increased risk for delayed graft function

Delayed graft function (DGF) is a risk factor for acute rejection (AR) in renal transplant recipients, and KDIGO guidelines suggest use of lymphocyte‐depletion induction when DGF is anticipated. We analyzed the United Network for Organ Sharing/Organ Procurement and Transplantation Network (UNOS/OPTN) database to assess the impact of induction immunosuppression on the risk of AR in deceased kidney recipients based on pretransplant risk of DGF using a validated model. Recipients were categorized into 4 groups based upon the induction immunosuppression: (1) Rabbit anti‐thymocyte globulin (rATG); (2) Alemtuzumab (C1H); (3) IL2‐receptor antagonists (IL2‐RA; basiliximab or daclizumab), and (4) No antibody induction. The primary endpoint for analysis was a composite endpoint of treated AR or graft failure by 1‐year posttransplantation. Compared to no antibody induction, rATG and C1H had consistently lower adjusted odds of the composite endpoint across all risk strata for DGF risk, whereas IL2‐Ra was associated with increased adjusted odds of the composite endpoint with increasing DGF risk. When the induction agents were compared, rATG and C1H were associated with decreasing adjusted odds for the composite endpoint with increasing risk of DGF, especially at the higher risk spectrum of DGF. Consideration must be given to use of lymphocyte‐depletion induction when the anticipated risk of DGF is increased.     

水溶性一氧化碳释放分子抑制供肾树突状细胞活化减轻移植后排斥反应

目的 研究水溶性一氧化碳释放分子3 (CORM-3)减轻小鼠肾移植后排斥反应的作用及机制.方法 以C57BL/6J小鼠为供鼠,Balb/c小鼠为受鼠,采用随机数字表法将供、受鼠分为2组,制备小鼠肾移植模型.CORM-3组受鼠接受经CORM-3预处理的供肾,iCORM组受鼠接受经无CO活性的iCORM预处理的供肾.术后检测各组受鼠的血清肌酐(SCr)水平,观察各组移植肾组织的病理改变,记录各组移植肾存活时间.以C.FVB-Tg (Itgax-DTR/GFP) 57Lan/J小鼠为供鼠,Balb/c小鼠为受鼠,肾移植后24 h通过流式细胞仪检测移植肾内供鼠rDC的活化情况.结果 术后iCORM组和CORM-3组间移植肾中位存活时间分别为40.5和70 d(P＜0.05)；术后两组受鼠的SCr水平均进行性升高,但CORM-3组受鼠的SCr水平始终保持在较低的水平(P＜0.05或P＜0.01)；与正常小鼠肾组织相比,术后第4周iCORM组受鼠的移植肾间质出现弥漫性单个核细胞浸润,中度肾小管炎症,以及部分肾小球硬化；CORM-3组移植肾组织单个核细胞浸润较iCORM组明显减轻,肾小球与肾小管形态基本正常.移植后24 h,iCORM组和CORM-3组移植肾内供鼠rDC表面均高表达CD80和CD86,表明rDC处于活化状态,而CORM-3组rDC表面CD80和CD86的表达较iCORM组明显减少(P＜0.05).结论 CORM-3可显著减轻同种肾移植小鼠的排斥反应,改善移植肾功能,其机制可能是CORM-3抑制了移植后供鼠rDC的活化.     

Acute rejection in kidney grafts with delayed onset of graft function

Forty-five kidney transplant recipients with delayed onset of diuresis due to acute tubular necrosis (ATN) were examined with duplex ultrasonography (DU). Resistive index (RI) was measured on the 4th post-transplant day. Eleven grafts (24%) developed acute rejection. Mean RI prior to rejection of the 4th postoperative day in these grafts was 0.97 and in the 34 grafts which did not develop rejection mean RI was 0.82. There were 2/26 rejections (8%) in the group of grafts with an initial RI below 0.9 and 9/19 rejections (47%) in the group of grafts with RI of 0.9 or above on the 4th post-transplant day. Six months postoperatively there were 2/26 nonfunctioning grafts in the group with lower initial RI values (<0.9) and 6/19 nonfunctioning grafts in the group with higher indices (0.9). In nonfunctioning grafts a high initial RI (0.9) indicates that these grafts will be prone to developing actute rejection.     

乌司他丁对脂多糖介导的小鼠肾脏固有树突状细胞成熟的抑制作用

目的 研究乌司他丁对脂多糖介导的小鼠肾脏固有树突状细胞(rDC)成熟的影响.方法 取C57BL/6J小鼠肾脏,制备单细胞悬液,使用免疫磁珠法分选CD11c+的rDC,流式细胞仪鉴定rDC纯度.脂多糖刺激24 h,促使rDC成熟,给予不同浓度(50、100和200 U/L)乌司他丁处理,酶联免疫吸附试验检测上清液中白细胞介素12(IL-12)的分泌水平.100 U/L的乌司他丁处理后,流式细胞仪检测rDC表面主要组织相容性复合物(MHC)Ⅱ类分子及CD80和CD86分子,以观察乌司他丁对rDC表型及功能的影响.检测趋化因子受体7(CCR7)的表达,计数受巨噬细胞炎症蛋白3β(MIP-3β)趋化进入细胞培养小室下层的CD11c+细胞的数目,分析乌司他丁对rDC趋化能力的影响.结果脂多糖刺激促使rDC成熟,高表达MHCⅡ、CD80、CD86分子,并分泌大量IL-12,而乌司他丁能够抑制脂多糖介导的rDC表型和功能成熟.经乌司他丁处理,CCR7分子的表达由69.2％下降至37％,并且受趋化的CD11c+细胞数目下降为脂多糖处理者的45.3％.结论 乌司他丁能够抑制脂多糖介导的小鼠rDC的成熟和趋化能力上调.     

Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function

Delayed graft function (DGF) often occurs in kidney transplants from deceased donors. We wanted to provide studies giving more accurate non-invasive tests for acute rejection (AR). Using real-time PCR, we examined the expression of cytolytic molecules such as perforin, granzyme B, and fas-ligand along with serpin proteinase inhibitor-9. We also measured the expression of FOXP3, a characteristic gene of T-regulatory cells known to be involved in AR. These studies were conducted on peripheral blood monocytes, urinary cells, and 48 surveillance kidney biopsies taken from a total of 35 patients with DGF. Of these patients, 20 had a histopathological diagnosis of AR, whereas other 28 had characteristics of acute tubular necrosis (ATN). Expression of cytolytic and apoptotic-associated genes in the biopsy tissue, peripheral blood leukocytes, and urinary cells was significantly higher in patients with AR than that in patients with ATN. Diagnostic parameters associated with FOXP3 gene expression were most accurate in peripheral blood leukocytes and urine cells with sensitivity, specificity, positive and negative predictive values, and accuracy between 94 and 100%. Our study shows that quantification of selected genes in peripheral blood leukocytes and urinary cells from renal transplant patients with DGF may provide a useful and accurate non-invasive diagnosis of AR.     

Does delayed kidney graft function increase the risk of chronic rejection?

Abstract  The impact of delayed graft function (DGF) on later renal graft loss due to chronic rejection was studied in a single center using uniform protocol for organ procure merit and post transplant patient care. DGF function was observed in 34 % of 829 consecutive first cadaveric renal transplants in adults and in 47 % of 169 retransplantations (P < 0.05). There were no significant differences in graft survival between groups with early graft function (EGF) and DGF, either in first transplantations or retransplantations. The half-life in EGF and DGF groups of first transplants was 12.3 years and 10.5 years, respectively, and of retransplantants was 8.0 years and 6.5 years, respectively. DGF was divided in three subgroups according to the day of onset. If graft function started during the first or second week after transplantation there were no significant differences in long-term graft survival rates compared with EGF. Only in re-transplants, if graft function started later than 2 weeks postoperatively, were long-term graft survival rates significantly lower when compared with EGF and the difference persisted if other causes of graft loss except chronic rejection were censored.     

脾移植前去除成熟树突状细胞预防移植排斥反应的体外研究

目的 建立治疗性单抗溶液持续灌注大鼠脾脏模型,应用抗大鼠树突状细胞单克隆抗体（ＷＺＤ）降低大鼠移植脾的免疫原性。方法 以ＷＺＤ溶液持续灌注移植脾,筛选最佳方案并观察灌注后混合淋巴细胞培养（ｍｉｘｄｅ ｌｙｍｐｈｏｃｙｔｅ ｃｕｌｔｕｒｅ,ＭＬＣ）淋巴细胞增殖情况及脾细胞的超微结构,以及灌注后移植脾细胞的免疫组化情况。结果 ＷＺＤ灌注组ＭＬＣ淋巴细胞增殖能力明显降低,与对照组及应ＣｓＡ组相比差异有显     

肾脏固有树突状细胞在肾缺血-再灌注期间的变化

目的  探讨在肾脏缺血-再灌注损伤(IRI)期间肾脏固有树突状细胞(rDC)的变化。方法  采用C57BL/6J小鼠建立双侧肾脏热缺血模型,再灌注24 、48 h后取肾组织制备单细胞悬液,流式细胞仪分析CD45⁺细胞和CD11c⁺rDC的比例变化;采用绿色荧光蛋白和白喉毒素受体标记(CD11c⁺GDTR)的小鼠肾组织制作单细胞悬液,流式细胞仪分析CD11c⁺rDC的比例及表型;采用CD11c⁺GDTR小鼠建立双侧肾脏热缺血模型,再灌注24 h后取肾组织制备单细胞悬液,MACS磁珠富集CD45⁺细胞,经流式细胞仪分析rDC表面共刺激分子表达情况。结果  再灌注24 h后C57BL/6J小鼠肾脏内CD45⁺细胞比例明显增加,48 h后其比例进一步升高,再灌注24 h后CD11c⁺rDC数量同样持续升高,但其占CD45⁺细胞的比例出现明显下调,48 h后恢复并较Sham组轻度升高;CD11c⁺GDTR小鼠正常肾脏CD45⁺细胞比例低于1%,其中约40%为CD11c⁺的肾脏rDC,主要呈CD11b^intF4/80^-MHC Ⅱ⁺;再灌注24 h后CD11c⁺F4/80^-亚群rDC表面共刺激分子CD40、CD80、CD86均显著升高。结论  热IRI后rDC比例、数量及其表面共刺激分子表达均增加,提示热IRI后肾脏rDC浸润增多且表型成熟。     

Partial donor-specific tolerance to delayed skin grafts after rejection of hematopoietic cell graft

BACKGROUND: Donor-specific tolerance (DST) is induced after allogeneic hematopoietic cell transplantation (HCT) and is a potential strategy for prolonging survival of solid organ grafts. DST may persist in recipients with transient mixed hematopoietic chimerism (MC) when solid organ transplantation and HCT are done concomitantly. METHODS: In a canine model of allogeneic HCT after nonmyeloablative conditioning, DST to skin grafts was evaluated in dog leukocyte antigen (DLA)-identical recipients with stable MC (n=11), or after rejection of the hematopoietic cell (HC) graft (n=19). RESULTS: There was significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in four recipients with MC developed an inflammatory reaction without skin graft loss. This may represent partial DST. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged compared to normal recipients even when skin grafting was delayed until after rejection of the HC graft (P=0.002). An inflammatory reaction developed in all nine of the surviving HC donor-derived skin grafts in this group, but there was no graft loss at last follow-up (median, 30 [range, 9-84] weeks). An increased time to rejection of the hematopoietic graft was associated with prolonged survival of the subsequent skin graft (P=0.02). CONCLUSION: In a model of stable MC, DST to skin grafts may be complete or partial. Partial DST can persist after HC graft rejection even if solid organ transplantation is delayed. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.     

Intrathymic immune modulation prevents acute rejection but not the development of graft arteriosclerosis (chronic rejection)

BACKGROUND: We showed previously that our intrathymic immune modulation protocol induces virtually permanent graft survival of simultaneously transplanted cardiac allografts in MHC-incompatible rat strain combinations. It is, however, unknown whether this procedure prevents the development of graft arterial disease (GAD). METHODS: Male AO recipient rats were intrathymically inoculated with 2.5x10(7) PVG splenocytes immediately followed by heterotopic transplantation of a PVG cardiac allograft (day 0). Immunosuppression consisted of 1 ml of antilymphocyte serum i.p. (day 0) and cyclosporine i.m. (15 mg/kg body weight) on days 1, 2, and 3 posttransplantation. Histological analysis, mixed lymphocyte reactions, and intragraft cytokine mRNA expression were performed at several time points after engraftment. RESULTS: Histological analysis revealed that GAD was already present 14 days after transplantation. At 200 days, virtually all vessels were affected and over 80% of the vessels showed severe intimal lesions. Infiltrate analysis displayed massive parenchymatous infiltrates (CD8+ cells and ED1+ macrophages) 2 weeks after transplantation. At later time points, infiltrates became epicardial and/or blood vessel associated and mainly consisted of CD4+, CD8+, and B cells. Mixed lymphocyte reactions showed nonspecifically decreased responses at 60 days but complete restoration of these responses at later time points (120 to 280 days). Intragraft cytokine mRNA expression showed decreased interleukin-2/interferon-gamma and sustained interleukin-10 expression 2 weeks after transplantation. Transforming growth factor-beta mRNA expression was increased >200 days after transplantation. CONCLUSIONS: Intrathymic immune modulation does not abolish alloreactivity, and despite induction of long-lasting graft survival, this procedure does not prevent and may even facilitate the development of GAD.     

The relative influence of delayed graft function and acute rejection on renal transplant survival

Three hundred and eight cadaveric renal transplants were analysed to establish the effects of acute rejection in the first 90 days and delayed graft function (DGF) on graft outcome. There were 120 patients (39%) with no DGF and no rejection (group 1), 101 patients (33%) with rejection but no DGF (group 2), 41 patients (13%) with DGF but no rejection (group 3) and 46 patients (15%) with both rejection and DGF (group 4). The actuarial 4-year graft survival rates for groups 1,2,3 and 40.4%, respectively. The acute rejection rate was 101/221 (46%) in patients with initial graft function compared with 46/87 (53%) for those with DGF (²=1.02, P=0.31). Cox stepwise logistic regression analysis demonstrated that DGF was a more powerful predictive factor for poor graft survival (P=0.001) than acute rejection occurring in the first 90 days post-transplant (P=0.034). Further efforts at improving graft outcome should concentrate on reducing the incidence of DGF.     

Selective depletion of cross-presenting dendritic cells enhances islet allograft survival

MHC class I presentation of peptides derived from exogenous antigens (not synthesized within the antigen-presenting cell) is called cross-presentation and is mediated by selective subsets of dendritic cells (DC). A proportion of both donor and host DC may cross-present. Although there has been many studies that have investigated the role of donor versus host DC (i.e., direct vs. indirect pathway), what role cross-presenting DC play in allograft rejection has not been determined. We recently identified an agent, cytochrome c (cytc), that selectively depletes cross-presenting DC in vivo. By administering cytc we were able to study the impact of cross-presenting DC on rejection of islets grafted into fully mismatched mice. We found that cytc protected about half of the islet allografts from rejection. Our results indicate that cross-presenting DC can make potent contributions to the immune response to islet allografts, and contend that agents such as cytc that selectively target DC heralds a novel method of immunosuppression.     

Suppression of delayed xenograft rejection by specific depletion of elicited antibodies of the IgM isotype.

BACKGROUND: Hamster hearts transplanted into untreated rats undergo delayed xenograft rejection (DXR). This acute inflammatory response is associated with the deposition of anti-graft antibodies of the immunoglobulin (Ig)M isotype in the vasculature. We have previously shown that these antibodies are generated in a T cell-independent manner. In this study, we tested whether the generation of anti-graft IgM antibodies is involved in the pathogenesis of DXR. In addition, we tested whether the suppression of this antibody response would overcome DXR. METHODS: Hamster hearts were transplanted into rats treated with an anti-mu monoclonal antibodies (mAb) to deplete circulating IgM or with an isotype-matched control mAb recognizing the dinitrophenyl epitope. T cell immunosuppression was achieved with cyclosporin A (CsA). RESULTS: Depletion of circulating IgM by anti-mu mAb inhibited DXR, whereas the control mAb had no effect on DXR. In anti-mu-treated rats, xenografts were rejected 5-7 days after transplantation through a T cell-dependent mechanism associated with the generation of antibodies of the IgG isotype. Combination of anti-mu with CsA suppressed the anti-graft IgM and IgG response and resulted in long-term xenograft survival (> 50 days). Xenograft long term survival occurred despite the return of anti-graft IgM antibodies to the circulation, a phenomenon referred to as accommodation. CONCLUSION: This study demonstrates that the pathogenesis of DXR can be initiated by anti-graft antibodies of the IgM isotype, which are generated in a T-cell independent manner. In addition, we show that under T cell immunosuppression, specific depletion of this IgM response by anti-mu mAb administration results in xenograft long-term survival and accommodation.     

Indium-labeled presensitized T cells for diagnosis of graft rejection.

The aim of this experiment was to test a safe, noninvasive method for necessary, accurate diagnosis of early allograft rejection. Heart-lung allograft was performed heterotopically using Brown Norway (BN) rats as the donor and Lewis (LEW) rats as the recipient. T cell suspensions were prepared from lymphnodes of specifically sensitized LEW rats that had acutely rejected full-thickness BN skin graft. Cell count was adjusted 50 x 10(6) cells/ml. The suspension was incubated in vitro with 111I oxide (1 m Ci-ml). An aliquot of labeled cell suspension containing 40 x 10(6) cells and a total radioactivity of 200 mCi was administered intravenously to each animal 3 and 6 days after heart-lung transplant. The traffic of T cells was followed in vivo and in isolated organs under large field view gamma camera. The gamma camera revealed radioactivity on the graft starting Postoperative Day 5 when the heart was actively beating; no radioactivity was revealed at the site of the isografted organs. The histology showed mild to moderate cellular infiltration parallel to the grade of radioimaging intensity. The injection of indium-labeled presensitized T cells was able to detect the rejection process in an early phase when there are no clinical symptoms of rejection and/or the rejection cascade can be reversed. These results suggest that a similar method can be used in human organ transplantation for early diagnosis of rejection.