Chronic antagonism of Mig inhibits cellular infiltration and promotes survival of class II MHC disparate skin allografts

BACKGROUND: The goal of the current study was to test the ability of monokine induced by IFN-gamma (Mig)-specific antibodies to inhibit long-term T cell infiltration into class II major histocompatibility complex (MHC) disparate skin allografts and to test cellular and molecular changes in the graft during the rejection observed following cessation of treatment. METHODS: C57BL/6 recipients of B6.H-2bm12 skin grafts were treated with normal rabbit serum (NRS) or rabbit Mig antiserum (Mig AS) every other day from day 7 until day 21 posttransplant and then weekly thereafter. Allografts were retrieved during the course of treatment and following cessation. Tissue sections were prepared and stained to compare infiltration by macrophages and CD4+ and CD8+ T cells and to assess collagen deposition in the grafts. RNA was prepared and tested by ribonuclease protection assay for intragraft levels of Mig, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T-cell expressed and secreted (RANTES). RESULTS: T cell and macrophage infiltration into allografts was inhibited and graft survival maintained as long as Mig-specific antibodies were given. Following cessation of treatment, T cells and macrophages infiltrated the allografts. In contrast to the histology of acute rejection observed in allografts from NRS-treated recipients, the resulting rejection of the allografts from Mig AS-treated recipients was accompanied by dense collagen deposition and high level expression of Mig and RANTES. CONCLUSIONS: Mig directs T cell infiltration into B6.H-2bm12 skin allografts on C57BL/6 recipients. Delayed T cell and macrophage infiltration and rejection of the grafts following cessation of Mig AS treatment results in rejection that is histologically and molecularly distinct from acute rejection.     

MHC-identical heart and hepatocyte allografts evoke opposite immune responses within the same host

BACKGROUND: Purified allogeneic hepatocytes are highly antigenic and elicit immune responses that are not easily controlled. However, it is not clear whether hepatocytes are not capable of protective immune mechanisms or whether they are not to protection by immune mechanisms that permit long-term survival of other allografts. The purpose of the current study was to determine whether donor-matched allogeneic hepatocytes are protected from rejection in mice that have been induced to accept heart allografts. METHODS: Transient treatment with anti-CD4 monoclonal antibody (mAb) or gallium nitrate (GN) was used to induce acceptance of heterotopic FVB/N (H-2(q)) heart allografts by C57BL/6 (H-2(b)) mice. Transgenic hA1AT-FVB/N hepatocytes were sequentially transplanted into C57BL/6 mice that had accepted FVB/N heart allografts more than 60 days (heart acceptor mice), CD8 depleted C57BL/6 heart acceptor mice, or B-cell knockout (BCKO, H-2(b)) heart acceptor mice. Hepatocyte survival was determined by the detection of secreted transgenic product hA1AT by enzyme-linked immunosorbent assay (ELISA). RESULTS: FVB/N hepatocytes were rejected by day 10-14 posttransplant, while FVB/N heart allografts continued to function in C57BL/6, BCKO, and CD8 depleted heart acceptor mice. When FVB/N hepatocytes and heart allografts were transplanted into C57BL/6 or BCKO mice under short-term cover of anti-CD4 mAb or GN, hepatocyte rejection occurred by day 10 posttransplant, while most heart allografts survived for more than 60 days. CONCLUSIONS: Hepatocyte rejection does not appear to interfere with the of mechanisms that permit heart allograft acceptance. However, immune responses to allogeneic hepatocytes are not to regulation by mechanisms induced in heart acceptor mice. The simultaneous rejection of FVB/N allogeneic hepatocytes and continued acceptance of FVB/N-matched heart allografts is independent of host CD8+ T cells and humoral immunity.     

The influence of MHC and non-MHC genes on the nature of murine cardiac allograft rejection. I. Kinetic analysis of mononuclear cell infiltrate and MHC-class I/class II expression in donor tissue

While normal cardiac tissue expresses low levels of MHC-class I, undetectable levels of MHC-class II antigens, and no mononuclear cell infiltrates, posttransplantation allogeneic donor cardiac tissue demonstrates dramatic increases of MHC-class I/class II expression coincident with the infiltration of the tissue with mononuclear cells. Results of this study demonstrate that the kinetics of MHC-class I/II antigen expression and the phenotype of mononuclear cell infiltrate are influenced, to a great degree, by the genetic H-2, intra-H-2 and non-H-2 incompatibility between donor and recipient strains of mice. Increases of MHC-class I precede class II expression in cells from donor cardiac tissue from completely allogeneic BALB/c, H-2-disparate B10.D2, B10.BR, and K, I-A and I-E-disparate B10.T (6R) strains of mice implanted in B10 recipients. In contrast, increase in the level of MHC-class II precedes MHC-class I increases in donor cardiac tissue from H-2-identical but non-H-2-incompatible A.By and the I-E + H-2D end-different B10.A(5R) donor tissue. The completely allogeneic, H-2-disparate or K, I-A, I-E-disparate donor cardiac tissue induced the infiltration of predominantly CD8+ T cells, whereas the non H-2 and I-E + H-2D end-different donor cardiac tissue induced the infiltration of predominantly CD4+ T cells. Finally, whereas bm1 donor cardiac tissue is rejected by B6 recipients by day 32, the (bm1 x bm12)F1 allografts are rejected by day 20, and both express MHC-class I antigens followed by MHC-class II antigens, and contain predominantly CD8+ T cells. In contrast, bm12 allografts are not rejected by B6 recipients, express chronic low levels of both MHC-class I and II antigens, and contain predominantly CD4+ T cells. Of interest is our preliminary finding that bm12 allografts placed in one ear of B6 recipients appear to modify the kinetics of MHC antigen expression and the predominant phenotype of mononuclear cell infiltrates in bm1 allografts placed in the opposite ear. Cumulatively, these data suggest that the type of genetic disparity between cardiac donor and recipient greatly influences the quantitative and qualitative host responses.     

T-cell mediated induction of allogeneic endothelial cell chemokine expression

BACKGROUND: The goal of the current study was to test the ability of T cells to stimulate allogeneic endothelial cells to express chemokines, particularly the T-cell recruiting factors monokine induced by interferon-gamma (Mig) and inducible protein (IP)-10. METHODS: Lymph node cells from C57BL/6 (H-2b) recipients of C3H (H-2k) skin grafts or from na?ve mice were added to monolayers of C3H-derived endothelial cell line 2F-2B. After 5 or 24 hr, the lymph node cells were removed, and RNA was prepared from the endothelial cells and tested by ribonuclease protection assay or Northern blot hybridization for endothelial cell expression of chemokines. RESULTS: Alloantigen-primed T cells induced endothelial cell expression of regulated on activation normal T-cell expressed and secreted (RANTES), IP-10, Mig, monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and macrophage inflammatory protein-1beta within 5 hr of coculture. In vitro chemotaxis assays demonstrated the production of T-cell chemoattractants by the endothelial cells. With the exception of low levels of monocyte chemotactic protein-1 and RANTES, culture with na?ve C57BL/6 lymph node T cells did not induce endothelial cell chemokine expression. Alloantigen-primed CD4 T cells induced endothelial expression of IP-10 and RANTES but none of the other chemokines tested, whereas primed CD8 T cells induced all of the chemokines tested. Expression of IP-10 and Mig was not induced when alloantigen-primed T cells from interferon-gamma deficient recipients of C3H skin grafts were cultured with the endothelial cells. This expression was blocked by addition of intercellular adhesion molecule-1 or lymphocyte function-associated antigen-1 specific antibodies to the cultures. CONCLUSIONS These results demonstrate the ability of alloantigen-primed CD8 T cells to quickly and directly stimulate endothelial cells to express and produce chemokines, including those recruiting T cells.     

Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts

BACKGROUND: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. METHODS: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. RESULTS: Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. CONCLUSIONS: Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.     

Surveillance of acute rejection in baboon renal transplantation by elevation of interferon-gamma inducible protein-10 and monokine induced by interferon-gamma in urine

BACKGROUND: CXCR3 binding chemokines play a key role in recruitment of inflammatory cells into an organ transplant. This study addresses the question of whether urinary excretion of these chemokines correlates with acute rejection in a baboon kidney transplantation model. METHODS: Seven outbred baboons underwent renal allotransplantation from major histocompatibility complex (MHC)-mismatched donors. The treatment of baboons consisted of anti-CD4 monoclonal antibody (mAb), anti-CD8 mAb, rapamycin, and mycophenolate mofetil (MMF). Urinary levels of interferon-gamma inducible protein-10 (IP-10) and monokine induced by interferon-gamma (Mig) were determined by ELISA. Renal biopsies were examined by immunohistochemical staining for CXCR3 and Mig. RESULTS: Urinary levels of IP-10 and Mig increased significantly in all of the five baboons at the time of acute rejection of renal transplant. The IP-10 and Mig levels did not rise in two nonrejecting baboons. In two baboons, urinary levels of IP-10 and Mig rose before the elevation of the serum creatinine. In renal biopsies, expression of Mig was detected in glomeruli, tubules, and infiltrating cells, and the expression was significantly elevated in biopsies with acute rejection (P<0.01). CXCR3 was constitutively expressed in tubular cells in biopsies derived from both normal grafts and grafts with acute rejection. Whereas the infiltrating cells were increased in the biopsies with acute rejection, the expression of CXCR3 was also significantly higher (P<0.01) in these infiltrating cells compared with those in the normal controls. CONCLUSIONS: This study shows an important correlation between urinary excretion of IP-10 and Mig and acute rejection in baboon kidney transplantation.     

Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray

BACKGOUND: Interferon (IFN)-gamma and the IFN-gamma-dependent pathway are prominent in vascularized allograft during acute rejection. However, IFN-gamma deficient (IFN-gamma-/-) mice can rapidly reject cardiac allografts. To bring the alternative pathway during allograft rejection into more precise focus, we investigated the gene expression profile in murine cardiac allografts in IFN-gamma-/- mice by means of DNA microarray. MATERIAL AND METHOD: We screened for gene expression changes in murine cardiac allografts of BALB/c H-2d into both wild-type C57BL/6 H-2b (n=3) and IFN-gamma-/- C57BL/6 H-2b(IFN-gamma-/-, n=4) using Affymetrix oligonucleotide arrays to monitor more than 11,000 genes and expressed sequence tag (ESTs). The heart was heterotopically transplanted. Transplanted hearts were harvested on day 5. As a control, isografts (C57BL/6 to C57BL/6) were also harvested on day 5. RESULTS: On day 5, 64 of the 84 genes induced in the allografts in wild-type mice were not up-regulated in IFN-gamma-/- mice. We identified a group of 54 genes that were up-regulated in allografts in IFN-gamma-/- mice. Several chemokine genes, including monocyte chemoattractant protein=1 and macrophage inflammatory protein, were induced in the allografts in both wild-type and IFN-gamma-/- mice. Interestingly, a group of genes, including C10-like chemokine and platelet factor 4, were specifically induced in the IFN-gamma-/- mice. CONCLUSION: DNA microarray analysis reveals a unique pattern of mRNA expression in allografts in IFN-gamma-/- mice as well as a group of genes induced in cardiac allografts in both wild-type and IFN-gamma-/- mice, including monocyte chemoattractant protein-1 and monocyte chemoattractant protein-1.     

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.     

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

Endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts within 12–24 h posttransplant in mice and are activated to proliferate and produce IFN‐γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti‐LFA‐1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG‐treated recipients on day 7 posttransplant, allografts from anti‐LFA‐1 mAb‐treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor‐reactive CD8 T cells producing IFN‐γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti‐LFA‐1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG‐3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.     

CD4+ T-cell-dependent immune damage of liver parenchymal cells is mediated by alloantibody

BACKGROUND: Allogeneic hepatocytes initiate both CD4- and CD8-dependent rejection responses. The current studies address the hypothesis that acute damage of allogeneic liver parenchymal cells by the CD4-dependent pathway is alloantibody-mediated and examines immune conditions which promote activation of this pathway. METHODS: The role of alloantibody in CD4-dependent hepatocyte rejection was evaluated by assessing hepatocyte (FVB/N, H-2q) survival in CD8-depleted B-cell knockout (KO) (H-2b) recipients and by monitoring hepatocyte survival in C57BL/6.SCID (H-2b) recipients transfused with donor-reactive alloantibody. The development of donor-reactive alloantibody in C57BL/6 (H-2b), CD8-depleted C57BL/6, CD8 KO (H-2b), IFN-gamma KO (H-2b), perforin KO (H-2b), and FasL mutant gld/gld (H-2b) hepatocyte recipients was assessed. RESULTS: Hepatocyte rejection in B-cell KO mice was significantly delayed by CD8+ T-cell depletion (median survival time [MST], 35 days) when compared to untreated (MST, 8 days) and CD4-depleted (MST, 10 days) recipient mice. Transfusion of donor-reactive alloantibody into SCID recipients with functional hepatocellular allografts was sufficient to precipitate rejection in a dose-dependent fashion. Donor-reactive alloantibody was minimal in the serum of C57BL/6 hepatocyte recipients, but was produced in significant quantities in hepatocyte recipients genetically deficient in or depleted of CD8+ T cells and in recipients with impaired cytotoxic effector mechanisms. In addition, recipients with defects in Th1 immunity, such as IFN-gamma KO recipients, also produced readily detectable alloantibody. CONCLUSIONS: Collectively, these data support the hypothesis that acute immune damage of allogeneic hepatocytes by the CD4-dependent pathway is mediated by alloantibody and that this pathway is favored when Th1- or cell-mediated cytotoxic effector immune mechanisms are impaired.     

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.     

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.     

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.     

CD4+ T cells responsive through the indirect pathway can mediate skin graft rejection in the absence of interferon-gamma

BACKGROUND: T cells responding through the indirect pathway can induce allograft rejection, but mechanisms of rejection are not known. Interferon-y (IFN-gamma) may be an important mediator of rejection under these circumstances. METHODS: We transferred CD4+ T cells from IFN-gamma-deficient (IFN-gamma-/-) mice into SCID recipients of MHC II-deficient (MHC II-/-) skin grafts. Under these conditions, rejection can only occur via the indirect pathway and cannot be mediated by T-cell production of IFN-gamma. RESULTS: IFN-gamma-/- CD4+ T cells rejected MHC II -/- skin grafts. Flow cytometry revealed only CD4+ T cells in the recipients. Cytokine enzyme-linked immunosorbent spot assays confirmed only indirect recognition with an associated expansion of an alloreactive population of IL-2-, IL-4-, and IL-5-secreting T cells. CONCLUSION: CD4+ T cells recognizing alloantigens via the indirect pathway can mediate skin graft rejection in the absence of IFN-gamma.     

Early increased chemokine expression and production in murine allogeneic skin grafts is mediated by natural killer cells

BACKGROUND: Increased expression of chemokine mRNA is observed in allogeneic but not syngeneic skin grafts 3-4 days after transplantation. The recipient cells mediating this early inflammatory response in allografts remain unidentified. METHODS: Isogeneic and allogeneic skin grafts were transplanted to euthymic and athymic nude mice. mRNA expression and protein production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and the murine homolog of Gro(alpha), i.e. KC, from graft homogenates retrieved 3-4 days posttransplantation was tested by Northern blot hybridization and ELISA. To deplete NK cells, recipients were treated with antiasialo GM1 (ASGM1) antisera or with anti-NK1.1 mAb before transplantation. RESULTS: Expression of KC, MIP-1alpha, and MIP-1beta mRNA was equivalent in C57BL/6 allogeneic skin grafts and BALB/c isografts at day 2 posttransplant. At day 3 posttransplant, chemokine mRNA levels decreased in isografts but were maintained at high levels in the allografts. Increased early chemokine mRNA was also observed in C57BL/6, but not BALB/c++ grafts on BALB/c athymi(nu/nu) recipients. Treatment of allograft recipients with ASGM1 or with anti-NK1.1 antibody eliminated NK cells from the spleen and allograft infiltrating cell populations and decreased early chemokine mRNA levels in allografts 60-70%. Analyses of allograft homogenates indicated increased levels of KC, MIP-1alpha, and MIP-1beta protein at day 4 posttransplant that were decreased in recipients depleted of NK cells. Early chemokine mRNA levels were equivalent in isogeneic and semiallogeneic F1 grafts. CONCLUSIONS: Early chemokine mRNA expression and protein production in allogeneic skin grafts is amplified by recipient natural killer (NK) cells. These results indicate a novel function for infiltrating NK cells in mediating early increased intra-allograft chemokine production and inflammation during the initiation of acute rejection.     

Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection.

INTRODUCTION: This is the first in a series of reports that characterizes immune responses evoked by allogeneic hepatocytes using a functional model of hepatocyte transplantation in mice. METHODS: "Donor" hepatocytes expressing the transgene human alpha-1-antitrypsin (hA1AT-FVB/N, H2q) were transplanted into C57BL/6 (H2b) or MHC II knockout (H2b) hosts treated with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). Hepatocyte rejection was determined as a loss of circulating ELISA-detectable transgene product (hA1AT). In addition, some C57BL/6 mice underwent transplantation with FVB/N heterotopic cardiac allografts and were treated with anti-CD4 mAb. Cardiac allograft rejection was determined by palpation. Graft recipients were tested for donor-reactive alloantibodies and donor-reactive delayed-type hypersensitivity (DTH) responses. RESULTS: The median survival time (MST) of allogeneic hepatocytes in normal C57BL/6 mice was 10 days (no treatment), 10 days (anti-CD4 mAb), 14 days (anti-CD8 mAb), and 35 days (anti-CD4 and anti-CD8 mAbs). The MST of hepatocytes in B6 MHC class II knockout mice was 10 days (no treatment) and 21 days (anti-CD8 mAb). The MST of cardiac allografts was 11 days (no treatment) and >100 days (anti-CD4 mAb). Donor-reactive DTH responses were readily detected in both untreated and mAb-treated recipients. Donor-reactive alloantibody was barely detectable in untreated hosts. CONCLUSIONS: These studies demonstrate that allogeneic hepatocytes are highly immunogenic and stimulate strong cell-mediated immune responses by both CD4+ and CD8+ T cells, even when treated with agents that can cause acceptance of cardiac allografts. Indeed, CD4+ or CD8+ T cells seem to independently cause hepatocellular allograft rejection. Allogeneic hepatocytes evoked strong donor-reactive DTH responses but were poor stimuli for donor-reactive antibody production. This is an unusual pattern of immune reactivity in allograft recipients.     

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.