Graft produced interleukin-6 functions as a danger signal and promotes rejection after transplantation

BACKGROUND: Interleukin (IL)-6 is a pleiotropic cytokine that functions in both the innate and adaptive immune responses. However, the role of IL-6 in allograft rejection remains poorly understood. METHODS: In this study, we demonstrate a critical role for graft-produced IL-6 in allograft rejection in a murine model of cardiac allograft transplantation. RESULTS: The results show that IL-6-deficient grafts transplanted into allogeneic wild-type recipients have significantly prolonged survival, approximately three times the survival time of wild-type controls. In contrast, allogeneic cardiac transplants into IL-6-deficient recipients do not have prolonged graft survival, indicating that donor graft cells are the relevant source of IL-6. Our investigation of potential mechanisms shows that graft-produced IL-6 promotes the activation of peripheral CD4 and CD8 T cells. Furthermore, we show that IL-6 deficiency prolongs graft survival only in the presence of CD25+ T cells that have a phenotype consistent with regulatory T cells. Interestingly, IL-6 production by the graft is triggered by antigen-independent innate immune mechanisms. CONCLUSIONS: Thus, our results suggest the paradigm that graft rejection versus tolerance is determined by a balance between the activation of effector T cells versus immune suppression by regulatory T cells, and that after transplantation, IL-6 functions as a systemic danger signal that overcomes constitutive immune suppression mediated by regulatory T cells and promotes the activation of effector T cells.     

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。     

Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice

INTRODUCTION: The purpose of the current study was to investigate the capacity of CD4+, CD8+, or non-T cells to independently initiate acute rejection of allogeneic hepatocytes using reconstituted SCID, CD4 or CD8 knockout (KO) recipient mice. METHODS: Allogeneic hepatocytes (FVB/N, H-2q) were transplanted into C57BL/6.SCID (H-2b), CD4 KO (H-2b), CD8 KO (H-2b), or beige/beige (H-2b) mice. SCID mice with functioning hepatocellular allografts subsequently received purified non-T cells (NTC), CD4+, or CD8+ splenocytes. Some mice were treated with anti-CD4, anti-CD8, and/or anti-nkl.1 mAb. Recipient mice were also assessed for donor-reactive delayed-type hypersensitivity (DTH) responses and donor-reactive alloantibody production. RESULTS: Median hepatocellular allograft survival time (MST) was 28 days in CD4+ reconstituted SCID mice and 14 days in CD8+ reconstituted SCID mice. SCID hosts reconstituted with NTC demonstrated indefinite hepatocellular allograft survival (>120 days). MST was 10 days in untreated beige/beige (NK cell deficient) mice. MST was 14 days in untreated, 35 days in anti-CD4 mAb treated, and 10 days in anti-nkl.1 mAb treated CD8 KO mice. MST was 10 days in untreated, 35 days in anti-CD8 mAb treated, and 7 days in anti-nk1.1 mAb treated CD4 KO mice. Donor-reactive DTH responses were not detected in reconstituted SCID mice, were minimal in CD4 KO mice, and were prominent in CD8 KO mice after rejection of allogeneic hepatocytes. Similarly, donor-reactive alloantibody, was not detected in CD4 KO hosts, but was readily detected in CD8 KO hosts. CONCLUSIONS: These studies show that both CD4+ and CD8+ T cells (but not host NTC) can independently initiate the rejection of allogeneic hepatocytes. While hepatocyte rejection by isolated CD4+ T cells is not surprising, rejection by CD8+ T cells (in the absence of CD4+ T cells) was unusual, and may explain the failure of "standard" immunosuppressive regimens to suppress acute rejection of allogeneic hepatocytes, as noted in prior studies. Furthermore, NK cells do not appear to be required for either CD4+ T cell or CD8+ T cell initiated hepatocyte rejection.     

Effect of Inflammation on Costimulation Blockade-Resistant Allograft Rejection

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8⁺ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8⁺ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8⁺ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8⁺ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8⁺ T cells to induce costimulation blockade-resistant allograft rejection.     

Role of Natural Killer Cell Subsets in Cardiac Allograft Rejection

To achieve donor-specific immune tolerance to allogeneic organ transplants, it is imperative to understand the cell types involved in acute allograft rejection. In wild-type mice, CD4(+) T cells are necessary and sufficient for acute rejection of cardiac allografts. However, when T-cell responses are suboptimal, such as in mice treated with costimulation-targeting agents or in CD28-deficient mice, and perhaps in transplanted patients taking immunosuppressive drugs, the participation of other lymphocytes such as CD8(+) T cells and NK1.1(+) cells becomes apparent. We found that host NK but not NKT cells were required for cardiac rejection. Ly49G2(+) NK cells suppressed rejection, whereas a subset of NK cells lacking inhibitory Ly49 receptors for donor MHC class I molecules was sufficient to promote rejection. Notably, rejection was independent of the activating receptors Ly49D and NKG2D. Finally, our experiments supported a mechanism by which NK cells promote expansion and effector function of alloreactive T cells. Thus, therapies aimed at specific subsets of NK cells may facilitate transplantation tolerance in settings of impaired T-cell function.     

Alloantigen-driven T cell death mediated by Fas ligand and tumor necrosis factor-alpha is not essential for the induction of allograft acceptance

BACKGROUND: Fas ligand (FasL)-Fas and tumor necrosis factor alpha (TNFalpha)-tumor necrosis factor receptor (TNFR) interactions regulate immune responses and contribute to self-tolerance by mediating antigen-driven T cell apoptosis. It is not known whether FasL and TNFalpha, expressed by the recipient's lymphoid or nonlymphoid cells, are essential for the apoptosis of alloreactive T lymphocytes and the induction of allograft acceptance. METHODS: We compared the survival of fully allogeneic vascularized cardiac allografts between wild-type (wt) and FasL-mutant (gld) recipient mice. In addition, we studied cardiac allograft survival in gld mice injected with TNFalpha-neutralizing antibody. Allograft acceptance (graft survival >100 days) was induced by treating the recipients with CTLA4Ig, a recombinant fusion protein that blocks B7-CD28 T cell costimulation. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T lymphocytes was analyzed in mice repeatedly stimulated with allogeneic splenocytes. RESULTS: We found that CTLA4Ig induces 100% long-term acceptance of cardiac allografts in wt and gld mice. Similarly, CTLA4Ig induced 100% allograft acceptance in gld recipients injected with TNFalpha-neutralizing antibody. In vivo alloantigen-driven apoptosis of mature CD4+ and CD8+ T cells was significantly reduced in gld mice and in wt mice treated with anti-TNFalpha antibody. However, neutralizing TNFalpha activity in gld mice failed to abrogate alloantigen-driven T cell apoptosis. CONCLUSIONS: These data indicate that: (1) FasL and TNFalpha expression are not obligatory for the induction of long-term allograft acceptance by CTLA4Ig and (2) FasL- and TNFalpha-independent death pathways contribute to alloantigen-driven T cell apoptosis.     

CD4+ T cells are critical for corneal, but not skin, allograft rejection

BACKGROUND: The relative contribution of CD4+ or CD8+ T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. METHODS: We compared the ability of CD4- knockout (KO), CD8- KO, and normal CD4+/CD8+ mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. RESULTS: Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8- KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 +/- 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 +/- 1 days), although most CD4- KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4- KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4- KO mice that accepted such grafts or that were not grafted. CONCLUSIONS: This study indicates that mice lacking CD4+ T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4+ T cells, the likely mechanism for rejection appears to involve the generation of CD8+ CTLs.     

Immunomodulatory properties of FK734, a humanized anti-CD28 monoclonal antibody with agonistic and antagonistic activities

BACKGROUND: We describe immunomodulatory effects of FK734, a humanized version of a mouse anti-human CD28 mAb (clone TN228), in vitro and in a chimeric human-mouse model of allograft rejection. METHODS: Cytokine production and proliferation were assessed in a mixed lymphocyte reaction containing FK734, human T cells, and endothelial cells or monocytes. FK734 was also administered to SCID mice engrafted with human skin and adoptively transferred with human peripheral blood mononuclear cells allogeneic to the skin graft. RESULTS: In vitro, FK734 enhanced secretion of interleukin-2 and interferon-gamma as well as proliferation of CD4+ and CD8+ T cells stimulated by allogeneic human leukocyte antigen (HLA)-DR+ human umbilical vein endothelial cells (which lack B7 molecules and FcgammaRs) or by blood monocytes (which express low levels of B7 molecules and FcgammaRs) compared with control mAb, but these effects were significantly smaller than those provided by mAb 28.2, a stimulatory mouse anti-human CD28 mAb, at comparable concentrations. However, FK734 generally inhibited cytokine secretion and T cell proliferation in cocultures with human umbilical vein endothelial cells transduced to express CD86. In vivo using SCID/beige mice bearing human skin with adoptively transferred peripheral blood mononuclear cells, administration of FK734 protected human endothelial cell-lined microvessels, significantly but incompletely reducing endothelial cell injury and T cell infiltration into the graft one or two weeks later. CONCLUSIONS: FK734 is a partial agonist of CD28 signaling that can reduce human T cell alloresponses in the presence of strong costimulation by B7 molecules in vitro and can reduce T cell-mediated skin allograft rejection in vivo.     

Aggressive skin allograft rejection in CD28-/- mice independent of the CD40/CD40L costimulatory pathway.

CD28-/- mice have been utilized to study the role of B7/CD28 and B7-CTLA4 interactions. There is evidence that CTLA4 ligation may be critical for tolerance induction. The aim of the current study is to further investigate rejection responses of CD28-/- mice and to define the role of B7-CTLA4 interactions in the absence of the CD40 and CD28 pathways. Balb/c skin allografts were transplanted onto C57BL/6 (B6) wild type or CD28-/- mice treated with anti-CD40L, CTLA4-Ig, or combination blockade. To investigate the cellular mechanism of rejection in CD28-/- recipients, mice were treated with anti-CD4 or anti-CD8 antibodies prior to treatment with costimulation blockade. The fluoroscein dye CFSE was utilized to study T cell expansion in vivo. Surprisingly, treatment of B6 CD28-/- mice with CTLA4-Ig alone (MST 12d), anti-CD40L alone (MST 13d), or combined blockade (MST 13d) had no effect on allograft survival compared to untreated B6 CD28 mice (MST 11d). CD28-/- recipients depleted of CD4+ cells and treated with CTLA4-Ig, anti-CD40L, or combination blockade also did not have prolonged survival compared with untreated mice (MST 10d). In contrast, CD28-/- recipients depleted of CD8+ cells had markedly prolonged allograft survival when treated with either anti-CD40L alone (MST 49d) or with combination blockade (MST 57d). Studies utilizing CFSE demonstrated that CD28-/- CD8+ T cells are not defective in in vivo proliferation responses compared with wild type CD8 cells. Thus, CD28-/- CD8+ T cells are responsible for aggressive rejection responses of CD28-/- mice independent of the CD40 pathway. In addition, CD40L blockade does not result in CD4+ T cell tolerance in CD28 recipients, despite an intact B7-CTLA4 pathway.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Mechanism of allorecognition and skin graft rejection in CD28 and CD40 ligand double-deficient mice

BACKGROUND: It has been shown that simultaneous blockade of CD28- and CD40-mediated costimulatory signals significantly prolongs allograft survival. Although these results led to an expectation of the establishment of specific immunotolerant therapy for organ transplantation, it became evident that these treatments rarely resulted in indefinite allograft survival. To uncover the mechanisms underlying these costimulation blockade-resistant allograft rejections, we studied the process of allogenic skin graft rejection in CD28 and CD40 ligand (L) double-deficient (double-knockout [dKO]) mice. METHODS: Skin grafts from BALB/c or BALB.B mice were transplanted to C57BL/6 background dKO mice. The frequency of CD4+ and CD8+ T cells responding to alloantigens presented by direct or indirect pathways were defined by the use of a cytostaining assay. RESULTS: BALB/c skin grafts were rapidly rejected by dKO mice. This CD28 and CD40L independent allograft rejection was inhibited by the depletion of CD8+ T cells. In vitro studies indicated that CD8+ T cells from BALB/c skin-grafted dKO mice responded to donor antigen presented only by the direct pathway. Unlike major histocompatibility complex (MHC)-mismatched donors, allogenic skin grafts from MHC-matched donors were accepted by dKO mice. CONCLUSION: In the absence of CD28 and CD40 costimulatory signals, CD8+ T cells recognize MHC antigens by the direct pathway, resulting in the rejection of skin grafts from MHC-mismatched donors. In contrast, MHC-matched and non-MHC-mismatched donor skin grafts indefinitely survive in dKO mice. These results indicated that donor-host MHC matching may still be critical to costimulation blockade therapy for organ transplantation.     

An increase in the survival of murine H-2-mismatched cultured fetal pancreas allografts using depleting or nondepleting anti-CD4 monoclonal antibodies, and a further increase with the addition of cyclosporine

Depletion of CD4+ T lymphocytes with monoclonal antibodies (mAbs) has been shown to prolong allograft survival in mice. In this study, two rat anti-CD4 mAbs, H129.19 and GK1.5, were administered either alone or in combination with cyclosporine (CsA) to recipients of MHC-mismatched (H-2k to H-2d) cultured fetal pancreas allografts to determine their effect on graft survival. When compared with control mice, splenic CD4+ cells of GK1.5-treated mice were depleted by greater than 95%, but in H129.19-treated mice no depletion of CD4+ cells occurred. Instead, rat Ig was present on the surface of CD4+ cells in H129.19-treated mice. Anti-CD4 therapy with either H129.19 or GK1.5 prolonged fetal pancreas allograft survival to a similar extent, but did not lead to indefinite survival. Blockade of the CD4 antigen by the mAb H129.19 was as effective as the depletion of CD4+ cells by GK1.5 in prolonging allograft survival. Rejection of grafts by day 28 posttransplantation occurred in the absence of CD4+ cells, as determined by both flow cytometric examination of spleen cells and immunoperoxidase staining of the graft site. CsA alone did not prolong graft survival, but its addition to either H129.19 or GK1.5 mAb treatment significantly increased the survival rate of grafts at 28 days compared with mAb treatment alone. These results suggest that CD4+ cell depletion is not essential for effective anti-CD4 mAb therapy--and, further, that CsA may have a direct inhibitory effect on CD8+ cells during allograft rejection.     

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. III. Late allograft rejection

We have studied organ allograft survival in rhesus monkeys conditioned with myeloablative total-body irradiation and T cell-depleted autologous bone marrow transplantation then given a heterotopic MHC-mismatched cardiac allograft in the immediate postmyeloablative period. This model has enabled us to investigate the role of T cells in vascularized organ allograft rejection. We previously reported (1) that recipients of marrow depleted of T cells below a critical threshold (0.16% residual marrow T cells, or 0.14 x 10(5) infused T cells/kg) experienced a period of freedom from acute rejection associated with a profound nonspecific immune deficiency (determined by skin grafting). Resolution of the nonspecific immune deficiency was associated with late graft rejection. In the present report, we correlate the results of peripheral immune reconstitution studies and direct immunohistochemical analysis with allograft status in order to study T cell subsets involved in late rejection. We report that, in contrast with CD8+/CD28- T cells, CD16+ NK cells, and CD20+ B cells, late allograft rejection was associated with the return of peripheral CD4+ T cells and CD8+/CD28+ T cells, suggesting a critical role for one or both of these subsets in late allograft rejection in this model.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.     

CD103分子介导CD8+T淋巴细胞对同种胰岛移植物的损伤

目的 检验CD103分子是否介导了CD8+T淋巴细胞对同种移植胰岛的免疫损伤.方法 用流式细胞仪检测野生型C57BL/6小鼠外周血CD8+T淋巴细胞表达CD103的情况.以Balb/c小鼠为供者,C57BL/6小鼠为受者,制作同种胰岛移植模型.受者分为3组:M290-SAP组小鼠注射CD103免疫毒素M290-SAP;M290组小鼠注射抗CD103单克隆抗体M290;另以仅接受胰岛移植、不注射任何药物的小鼠为未处理组.检测移植胰岛CD3、CD8、CD44和CD103阳性细胞的表达,检测肠系膜淋巴结中CD3、CD8和CD103阳性细胞的表达.移植物功能丧失或观察期结束时获取移植胰岛,行HE染色和免疫组织化学染色.结果 野生型C57BL/6小鼠外周血的CD8+T淋巴细胞中有44.06%表达CD103.未处理组移植胰岛浸润的细胞成分中有29%的CD8+T淋巴细胞表达CD103.M290-SAP组小鼠淋巴细胞不仅丧失了CD103的表达,而且CD8+T淋巴细胞的绝对数量也减少,该组小鼠血糖稳定时间超过100 d(未处理组为13 d,P＜0.05),移植胰岛组织学形态良好.结论 CD8+T淋巴细胞免疫损伤同种移植胰岛必须表达CD103,CD103有可能成为胰岛移植抗排斥反应治疗的新靶点.

Abstract：

Objective To test whether the CD103 molecule mediates CD8+ T lymphocytes on allogeneic islet graft immune injury. Methods By using flow cytometry, the expression of CD103 in peripheral CD8+ T lymphocytes in wild-type C57BL/6 mice was detected. Allogenic islet transplantation models were made using Balb/c donor mice and C57BL/6 recipient mice. Recipients were divided into 3 groups: M290-SAP-treated mice were injected with CD103 immunotoxin M290-SAP; M290-treated mice were injected with CD103 monoclonal antibody M290; untreated mice were only transplanted islet without any drug treatment. CD3, CD8, CD44 and CD103 positive cells were counted in islet allograft infiltrative lymphocytes. CD3, CD8, and CD103 positive cells were measured in the mesenteric lymph node. The islet allografts were removed and subjected to HE staining and immunohistochemical staining at the time of graft loss or the end of the observation period. Results 44. 06% peripheral CD8+ T cells expressed CD103 in wild-type C57BL/6 mice. 29 % CD8+ T cells expressed CD103 in the infiltrative lyrnphocytes of islet allografts in the untreated mice. In M290-SAP-treated mice, the lymphocytes had no CD103 expression and the absolute number of CD8+ lymphocytes was decreased as well The blood glucose was maintained stable for more than 100 days (13 days in untreated group, P＜0.05) in the M290-SAP-treated mice. Moreover, the transplanted islets retained intact. Conclusion CD103 expression is required for destruction of pancreatic islet allograft by CD8+ T cells. CD103 might provide a novel target for therapeutic intervention in islet allograft rejection.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.

Abstract：

Objective To investigate the expression of CXCR6 in allograft rejection and effect of CXCL16/CXCR6 interaction on allograft survival Methods Intra-abdominal heterotopic heart transplantation was performed using wild type (WT) Balb/c mice (H-2d) (allogeneic) as donors or WT C57BL/6 mice (B6, H-2b) (syngeneic) as donors, and using WT B6 mice as recipients. The intragraft expression of CXCR6 and expression of CXCR6 in CD8+ T cells of the spleens from syngeneic and allogeneic recipients were examined. The allogeneic recipients were further divided into the experimental group (n = 5) and control group (n = 6) randomly. The experiment group and control group were injected with anti-CXCL16 mAb or control mAb respectively until rejection occurred. The cardiac allograft survival in experimental group and control group was evaluated. Results Rejected allografts showed higher expression of CXCR6 than syngeneic cardiac grafts. More importantly,expression of CXCR6 in CD8+ T cells was also up-regulated by allograft rejection. However, injection of anti-CXCL16 mAb could not inhibit cytotoxic activity of CD8+ T cells. Moreover, experimental group could not prolong the cardiac graft survival time as compared with control group. Conclusion Expression of CXCR6 in CD8+ T cells is up-regulated in allograft rejection.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.     

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.     

Selective CD28 Blockade Attenuates Acute and Chronic Rejection of Murine Cardiac Allografts in a CTLA‐4‐Dependent Manner

Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single‐chain Fv‐based reagent (α28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA‐4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154‐blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA‐4 during α28scFv‐based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)‐1 and 2,3‐indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA‐4 is thus a promising approach to inhibit pathogenic alloimmunity.