CCR5 Is Required for Regulation of Alloreactive T-Cell Responses to Single Class II MHC-Mismatched Murine Cardiac Grafts

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2^bm12 cardiac allografts is restricted by CD4⁺CD25⁺ regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2^bm12 cardiac allografts. In contrast to the long-term survival of B6.H-2^bm12 allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5^−/− recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-γ and IL-4 were markedly increased in spleens of B6.CCR5^−/− versus wild-type recipients. Wild-type and B6.CCR5^−/− mice had equivalent numbers of splenic FoxP3⁺ Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro . However, diminished numbers of FoxP3⁺ Tregs infiltrated B6.H-2^bm12 allografts in B6.CCR5^−/− recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4⁺CD25⁺ Tregs to CCR5^−/− recipients restored long-term survival of B6.H-2^bm12 cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.     

Genetic deletion of chemokine receptor CXCR3 or antibody blockade of its ligand IP-10 modulates posttransplantation graft-site lymphocytic infiltrates and prolongs functional graft survival in pancreatic islet allograft recipients

BACKGROUND: Interaction of chemokine receptor CXCR3 with its ligand IP-10 mediates effector cell trafficking to sites of allograft rejection in murine models of whole organ allotransplantation. We hypothesized that blocking the CXCR3/IP-10 interaction would impair posttransplantation leukocyte trafficking to and delay rejection of pancreatic islet allografts. METHODS: A/J strain murine islets were implanted to the kidney capsule of H-2 disparate, streptozotocin-induced diabetic wild type (WT), CXCR3 deficient (CXCR3(-/-)) or IP-10 antibody-treated WT (alphaIP-10) C57BL/6 recipients. Representative grafts from each group were harvested at day 7. Ribonuclease protection assay was used to determine gene expression for cell markers F4/80 (macrophages), CD8 (type I T cells), CD4 (type II T cells), and CD 19 (natural killer cells), and for chemokines IP-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES. Immunohistochemistry was used to confirm ribonuclease protection assay infiltrate data. Graft-site chemokine gene expression and cellular infiltrate were correlated with time to functional graft rejection. RESULTS: Untreated WT recipients demonstrated heavy graft-site cell infiltrates and increased graft-site gene expression for cell markers F4/80, CD8, CD4, and CD19, and for chemokines RANTES, IP-10, and MIP-1beta at day 7. In comparison with untreated WT, alphaIP-10-treated WT and CXCR3(-/-) recipients demonstrated the same degree of chemokine gene expression but less lymphocytic infiltrate. The mean length of allograft survival was 12.7 +/- 3.1 days in untreated WT versus 20.2 +/- 2.7 days (P <.05) for CXCR3(-/-)- and 19.7 +/- 2.3 days (P <.05) for alphaIP-10-treated WT recipients. CONCLUSIONS: CXCR3 gene deletion or alphaIP-10 antibody therapy modulates posttransplantation lymphocytic graft infiltration and statistically prolongs graft survival in murine islet allograft recipients.     

Role of CXCR3 and CCR5 in allograft rejection

Chemokines are known to participate in allograft rejection by mediating leukocyte trafficking. Despite redundancy in chemokine family, several chemokine-chemokine receptor interactions have proven critical in alloimmune responses. We sought to determine the effect of combined blockade of CXCR3 and CCR5, two critical chemokine receptors, in acute rejection. METHODS: Heterotopic heart transplantation was performed using BALB/c to B6/129 mice deficient in CCR5. Following transplantation these mice were treated with goat anti-CXCR3 serum every other day. In the control group, BALB/c hearts were transplanted in wild type B6/129 recipients and treated with goat serum alone. No immunosuppression was given to either group. Recipient mice were then assessed daily for allograft function by abdominal palpation, and graft survival was confirmed by laparotomy. RESULTS: The donor hearts in the control group were rejected at 6 +/- 1 days posttransplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival versus control; all allografts survived to 24 days. In addition, there was a decrease in graft infiltrating CD4 and CD8 lymphocytes in the experimental group at 24 days. CONCLUSION: Combined CXCR3 and CCR5 blockade is effective in prolonging allograft survival in a fully MHC mismatched murine model. Combined chemokine blockade holds promise in control of acute rejection in organ transplantation.     

Prolongation of cardiac and islet allograft survival by a blocking hamster anti-mouse CXCR3 monoclonal antibody

BACKGROUND: Acute allograft rejection requires a multifaceted immune response involving trafficking of immune cells into the transplant and expression of effector cell functions leading to graft destruction. The chemokine receptor CXCR3 and its ligands, CXCL9, CXCL10 and CXCL11, constitute an important pathway for effector cell recruitment posttransplant. However, analysis of CXCR3 expression and function has been hampered by a general lack of availability of a neutralizing anti-CXCR3 monoclonal antibody (mAb) for use in experimental models. METHODS: We report the generation, characterization, and use of CXCR3-173, a new hamster mAb specific for mouse CXCR3 that recognizes CXCR3 on cells from wild-type but not CXCR3-/- mice. RESULTS: Using CXCR3-173 mAb, we demonstrate CXCR3 expression on primary memory phenotype CD4+ and CD8+ T cells, naturally occurring CD4+CD25+ Foxp3+ regulatory T cells, natural killer T cells, and approximately 25% of NK cells. CXCR3-173 blocked chemotaxis in vitro in response to CXCL10 or CXCL11 but not CXCL9. When injected into mice, this mAb significantly prolonged both cardiac and islet allograft survival. When combined with a subtherapeutic regimen of rapamycin, CXCR3-173 mAb induced long-term (>100 day) survival of cardiac and islet allografts. The in vivo effects of CXCR3-173 mAb were not associated with effector lymphocyte depletion. CONCLUSION: These data highlight the utility of CXCR3-173 mAb in developing immunotherapeutic approaches to inhibit transplant rejection and potentially other immune-mediated diseases in murine models.     

Homeostatic Repopulation by CD28−CD8+ T Cells in Alemtuzumab-Depleted Kidney Transplant Recipients Treated With Reduced Immunosuppression

Alemtuzumab (CAMPATH-1H) is a depleting agent introduced recently in transplantation and often used with reduced maintenance immunosuppression. In the current study we investigated the immune response of 13 kidney allograft recipients treated with alemtuzumab followed by weaned immunosuppression with reduced dose of mycophenolate mofetil (MMF) and tacrolimus. Tacrolimus was switched to sirolimus at 6 months and MMF withdrawn at 12 months after transplantation.
We found that after alemtuzumab induction the recovery of CD8⁺ T cells was much faster than that of CD4⁺ T cells. It was complete 6 months posttransplant while CD4⁺ T cells did not fully recover even 15 months posttransplant. Repopulating CD8⁺ T cells were mainly of immunosenescent CD28⁻CD8⁺ phenotype. In a series of in vitro experiments we showed that CD28⁻CD8⁺ T cells might suppress proliferation of CD4⁺ T cells. There were three successfully treated acute rejections during the study (first at +70 day, two others +12 months) that occurred in patients with the lowest level of CD28⁻CD8⁺ T cells.
We hypothesize that expanded CD28⁻CD8⁺ T cells might compete for 'immune space' with CD4⁺ T cells suppressing their proliferation and therefore delaying CD4⁺ T-cells recovery. This delay might be associated with the clinical outcome as CD4⁺ T cells, notably CD4⁺ T effector memory cells, were shown to be associated with 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.     

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.     

Prolonged rat allograft survival induced by temporary elimination of α/β T cells with monoclonal antibody

Abstract We tested the ability of lewis (LEW; RT-1¹) recipients to reject DA (RT-1^av1) cardiac allografts following the selective elimination of α/β T cells with the mouse monoclonal antibody R73. One group of adult rats (6 weeks old) received 1000 μ R73 i.p. on days 2 and 1 before transplantation, and 100 pg R73 every third day after transplantation up to day 18. Prolonged cardiac graft survival was noted (30, 30, 32, 51, 62, 108, > 500, > 500, > 500 days). Untreated controls ( n = 10) rejected their grafts within 7 ± 1 days. R73 therapy induced a dramatic decrease in α/β T cells from 69% before treatment to 5% within the first 5 days, followed by an increase to 64% by day 8. The T cell increase was paralleled by the appearance of anti-mouse antibody. A second group of adult rats (10 weeks old) received the same treatment. These "older" recipients rejected their grafts within 20 ± 5 days. Chronic R73 therapy from birth until the day of transplantation (100 μ R73 i.p. twice a week) resulted in graft survival of 37 ± 9 days in eight animals. Two rats had a graft survival of more than 200 days. When chronic R73 therapy was continued to day 70 after transplantation, DA hearts were accepted well in all animals for more than 100 days. α/β T cells were virtually absent throughout the whole time of treatment. Antibodies against R73 were not detected. We concluded that selective elimination of a/B T cells has a strong effect on allograft survival.     

CXCL9 Antagonism Further Extends Prolonged Cardiac Allograft Survival in CCL19/CCL21-Deficient Mice

CCL19/MIP-3beta and CCL21/SLC are essential for chemotactic recruitment of mature dendritic cells (DC) to T-cell areas of secondary lymphoid tissue. Paucity of lymph node T-cells (plt/plt) mice lack CCL21-serine (ser) and CCL19 expression. We tested plt/plt and wild type (wt) BALB/c (H2d) mice as recipients of heart or skin allografts from C57BL/10J (H2b) donors. Donor DC trafficking to secondary lymphoid tissue was markedly reduced in plt heart but not skin allograft recipients. Heart, but not skin grafts survived significantly longer in plt recipients. Accordingly, T cells from plt heart transplant recipients demonstrated poor anti-donor responses in ex vivo MLR, compared to wt heart or wt and plt skin recipients. Moreover, donor-reactive T cells from plt heart recipients exhibited Th2-skewing in comparison to T cells from wt heart or skin graft recipients. Anti-CXCL9/Mig was administered for 2 weeks post-transplant to determine whether impairment of activated T-cell migration could further prolong cardiac allograft survival in plt recipients. CXCL9-antagonism extended graft survival significantly only in plt mice, likely due, in part, to retention of alloactivated T cells in secondary lymphoid tissue/reduction of graft-infiltrating T cells. Thus, targeting DC and activated T-cell migration concomitantly has additive effects in prolonging heart graft survival with potential for therapeutic application.     

Acute Humoral Rejection of Renal Allografts in CCR5–/– Recipients

Increasing detection of acute humoral rejection (AHR) of renal allografts has generated the need for appropriate animal models to investigate underlying mechanisms. Murine recipients lacking the chemokine receptor CCR5 reject cardiac allografts with marked C3d deposition in the parenchymal capillaries and high serum donor-reactive antibody titers, features consistent with AHR. The rejection of MHC-mismatched renal allografts from A/J (H-2^a) donors by B6.CCR5^–/– (H-2^b) recipients was investigated . A/J renal allografts survived longer than 100 days in wild-type C57BL/6 recipients with normal blood creatinine levels (28 ± 7 μmol/L). All CCR5^–/– recipients rejected renal allografts within 21 days posttransplant (mean 13.3 ± 4 days) with elevated creatinine (90 ± 31 μmol/L). The rejected allografts had neutrophil and macrophage margination and diffuse C3d deposition in peritubular capillaries, interstitial hemorrhage and edema, and glomerular fibrin deposition. Circulating donor-reactive antibody titers were 40-fold higher in B6.CCR5^–/– versus wild-type recipients. Depletion of recipient CD8 T cells did not circumvent rejection of the renal allografts by CCR5-deficient recipients. In contrast, μMT^–/–/CCR5^–/– recipients, incapable of producing antibody, did not reject most renal allografts. Collectively, these results indicate the rapid rejection of renal allografts in CCR5^–/– recipients with many histopathologic features observed during AHR of human renal allografts.     

Blockade of indirect recognition mediated by CD4+ T cells leads to prolonged cardiac xenograft survival

Abstract:  The T-cell response to xenografts is induced by direct and indirect recognition of xenoantigens. Although the importance of indirect recognition is well established in vitro, the contribution of this pathway to xenograft rejection in vivo remains to be fully elucidated. We herein investigated the direct contribution of indirect recognition to cardiac xenograft rejection in the rat-to-mouse (PVG.R8-to-C57BL/10) concordant model. Rat xenoantigens invoked a vigorous proliferative response in mouse T cells harvested from naïve or graft recipients at rejection. Indirect recognition predominated the response, as antibodies against mouse class II I-A^b, CD80, or CD86 molecules significantly (45 to 60%) blocked the proliferative response. Importantly, the blockade of indirect recognition in vivo by treating the graft recipients with a monoclonal antibody (mAb) against class II I-A^b molecule on days 0, 1, and 3 post-transplantation resulted in significant ( P  < 0.009) prolongation of cardiac xenograft survival (Mean Survival Time (MST) >94 ± 55 days vs. 7 ± 0.8 days for controls). In contrast, treatment of recipients with a mAb against mouse class I H-2K^b/D^b molecules did not significantly affect graft rejection (MST = 8 ± 1 days). These results demonstrate that indirect recognition mediated by CD4⁺ T cells plays a critical role in the rejection of cardiac grafts in the rat-to-mouse xenogeneic model.     

Type I Interferons Are Not Critical for Skin Allograft Rejection or the Generation of Donor-Specific CD8+ Memory T Cells

Type I interferons (IFN-I) link innate to adaptive immunity in microbial infection, autoimmune disease and tumor immunity. It is not known whether IFN-I have an equally central role in alloimmunity. Here we tested this possibility by studying skin allograft survival and donor-specific CD8⁺ T-cell responses in mice that lack the IFN-I receptor (IFN-IR^−/−). We found that IFN-IR^−/− mice reject fully allogeneic wild-type skin grafts at the same rate as wild-type recipients. Similarly, allograft rejection was not delayed if IFN-IR^−/− male skin was transplanted to syngeneic IFN-IR^−/− female mice. Quantitation of the male (H-Y)-specific CD8⁺ T-cell response in these mice revealed normal generation of donor-specific CD8⁺ effector T cells but fourfold reduction in CD8⁺ memory T cells. Memory CD8⁺ T cells generated in the absence of IFN-IR had normal phenotype and recall function, assessed by ex vivo cytokine production and the ability of IFN-IR^−/− mice to mount second set rejection. Finally, these memory T cells were maintained at a constant number despite their inability to respond to IFN-1. Our findings indicate that IFN-I cytokines are not critical for acute allograft rejection or for the expansion and differentiation of donor-specific CD8⁺ T cells into long-lived, functional memory T cells.     

Primed CD8+ T-Cell Responses to Allogeneic Endothelial Cells Are Controlled by Local Complement Activation

CD8 T cells primed by transplantation recognize allogeneic class I MHC molecules expressed on graft vascular endothelium and contribute to allograft injury. We previously showed that immune cell-derived complement activation fragments are integral to T cell activation/expansion. Herein we tested the impact of local complement production/activation on T cell/endothelial cell (EC) interactions. We found that proinflammatory cytokines upregulated alternative pathway complement production by ECs, yielding C5a. We further found that ECs deficient in the cell surface C3/C5 convertase regulator decay accelerating factor (DAF, CD55) induced greater CD8 T-cell proliferation and more IFNγ⁺ and perforin⁺ effector cells than wild-type (WT) ECs. Allogeneic C3^−/− EC induced little or no CD8 responses. Abrogation of responses following C5a receptor (C5aR) blockade, or augmentation following addition of recombinant C5a demonstrated that the effects were mediated through T-cell-expressed-C5aR interactions. Analyses of in vivo CD8 cell responses to transplanted heart grafts deficient in EC DAF showed similar augmentation. The findings reveal that EC-derived complement triggers secondary CD8 T-cell differentiation and expansion and argue that targeting complement and/or C5aR could limit T-cell-mediated graft injury.     

Role of CD4+ T cells in the rat to mouse cardiac xenotransplantation

Abstract T cell subsets involved in rejection of xenografts were analyzed using a rat to mouse cardiac xenotransplant model. Proliferating response and interleulin-2 (IL-2) production in recipients' spleen cells were almost completely abrogated by elimination of L3T4⁺ T cells, but not by elimination of Lyt2.1⁺ T cells. Cytotoxic T lymphocyte (CTL) activities were mediated by both L3T4⁺ and Lyt2.1⁺ T cells with the help of IL-2-producing L3T4⁺ T cells. Administration of anti-L3T4 monoclonal antibody (mAb) into recipient mice resulted in a significant prolongation of graft survival (mean graft survival was 29.2 days). Moreover, anti-L3T4 mAb treatment plus thymectomy led to indefinite graft survival. Anti-rat endothelial cell (EC) antibody production in the grafted mice was remarkably suppressed by anti-L3T4 mAb treatment. In contrast, Lyt2.1 mAb treatment did not prolong the graft survival and did not suppress anti-EC antibody production. These results indicated the absolute requirement of L3T4⁺ T cells in the rejection of rat to mouse cardiac xenografts.     

Strategies to Induce Marked Prolongation of Secondary Skin Allograft Survival in Alloantigen-Primed Mice

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4⁺CD44^hiOX40⁺ and CD8⁺CD44^hiCD122⁺ T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122⁺ T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122⁺CD8⁺ T cells and/or the OX40/OX40L costimulatory pathway.