Antigen Location Contributes to the Pathological Features of a Transplanted Heart Graft
2004; Elsevier BV; Volume: 164; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63227-0
ISSN1525-2191
AutoresYifa Chen, Yilmaz Demir, Anna Valujskikh, Peter S. Heeger,
Tópico(s)Immune Cell Function and Interaction
ResumoOrgan-specific injury after transplantation presents with a variety of clinical and pathological phenotypes, yet the factors influencing development of each outcome are poorly understood. Because primed T lymphocytes must re-encounter their antigen within the target organ to engage effector functions, we postulated that the cellular location of antigen within that organ could significantly impact the induced pathology. We challenged female Marilyn CD4 T-cell receptor transgenic mice, in which all T cells are specific for the male minor transplantation antigen, with male heart transplants expressing the relevant peptide: major histocompatibility complex on either graft parenchymal/vascular cells or alternatively, on graft-infiltrating mononuclear cells. The two different graft donors led to equivalent activation of recipient T cells as assessed by frequency, cell surface marker expression, cytokine production, and the ability to traffic to the graft. Nonetheless, if the target antigen was expressed on graft vascular and/or parenchymal cells, the outcome was acute graft destruction. In contrast, if the antigen was expressed only on graft-infiltrating mononuclear cells the same effector T-cell repertoire caused chronic rejection and vasculopathy. This unique result, that target antigen location can influence pathological outcome, has significant implications for understanding the pathogenesis of chronic allograft injury in humans. Organ-specific injury after transplantation presents with a variety of clinical and pathological phenotypes, yet the factors influencing development of each outcome are poorly understood. Because primed T lymphocytes must re-encounter their antigen within the target organ to engage effector functions, we postulated that the cellular location of antigen within that organ could significantly impact the induced pathology. We challenged female Marilyn CD4 T-cell receptor transgenic mice, in which all T cells are specific for the male minor transplantation antigen, with male heart transplants expressing the relevant peptide: major histocompatibility complex on either graft parenchymal/vascular cells or alternatively, on graft-infiltrating mononuclear cells. The two different graft donors led to equivalent activation of recipient T cells as assessed by frequency, cell surface marker expression, cytokine production, and the ability to traffic to the graft. Nonetheless, if the target antigen was expressed on graft vascular and/or parenchymal cells, the outcome was acute graft destruction. In contrast, if the antigen was expressed only on graft-infiltrating mononuclear cells the same effector T-cell repertoire caused chronic rejection and vasculopathy. This unique result, that target antigen location can influence pathological outcome, has significant implications for understanding the pathogenesis of chronic allograft injury in humans. 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3: 23-27Crossref PubMed Scopus (43) Google Scholar The complexity of such transplant-reactive immune repertoires has limited our ability to independently test whether antigen location in the target organ affects the downstream pathological consequences. In an effort to circumvent this limitation, we performed a series of experiments using a CD4 T cell receptor (TCR) transgenic mouse as transplant recipients such that we could control the specificity of the induced effector T cells. The results provide evidence that the pathological characteristics of the injured target organ can be specifically attributed to where the target antigen is expressed in the graft at the effector stage.Materials and MethodsAnimalsMale and female C57BL/6 (H-2b) and C3H (H-2k) mice, age 6 to 8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME). Male and female C57BL/10NA;-(Tg)TCR Marilyn-(KO) Rag2 N11, N2 mice (H-2b, Marilyn), age 6 to 8 weeks, were obtained as a generous gift from Polly Matzinger (National Institutes of Health, Bethesda, MD) and Olivier Lantz (INSERM, Paris, France). All animals were maintained and bred in the pathogen-free animal facility at the Cleveland Clinic Foundation.Flow CytometryFluorescein isothiocyanate-conjugated anti-mouse CD44, CD62L, biotin-conjugated anti-mouse CD44, streptavidin-phycoerythrin, and streptavidin-PerCP were purchased from BD PharMingen (San Diego, CA). Spleen cells from the heart graft recipients or naïve mice were labeled and incubated on ice for 30 minutes with appropriate antibody followed by three washes in phosphate-buffered saline (PBS) 0.1% bovine serum albumin. When biotin-conjugated antibodies were used, cells were additionally incubated with streptavidin-phycoerythrin or streptavidin-PerCP followed by three more washes in PBS. The labeled cells were analyzed on a Becton-Dickinson FACScan using CellQuest software (Becton Dickinson, Mountain View, CA). Events (n = 10,000 to 100,000) were acquired and analyzed for each experiment.PeptidesHYDbyp (NAGFNSNRANSSRSS) and chicken ovalbumin 323-339 (OVA323–339, KISQAVHAAHAEINEAG) were synthesized by Research Genetics, Huntsville, AL) at >90% purity.Placement and Evaluation of Heart and Skin GraftsFull thickness skin grafts were placed as performed by our laboratory,20Valujskikh A Pantenburg B Heeger PS Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.Am J Transplant. 2002; 2: 501-509Crossref PubMed Scopus (198) Google Scholar, 31Matesic D Lehmann PV Heeger PS High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.Transplantation. 1998; 65: 906-914Crossref PubMed Scopus (82) Google Scholar, 35Benichou G Valujskikh A Heeger PS Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice.J Immunol. 1999; 162: 352-358PubMed Google Scholar bandages were removed on day 7 and the grafts were inspected daily. Rejection was defined as >90% necrosis. Vascularized heterotopic cardiac allografts were placed in the abdomen as described20Valujskikh A Pantenburg B Heeger PS Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.Am J Transplant. 2002; 2: 501-509Crossref PubMed Scopus (198) Google Scholar, 38Valujskikh A Lantz O Celli S Matzinger P Heeger PS Cross-primed CD8(+) T cells mediate graft rejection via a distinct effector pathway.Nat Immunol. 2002; 3: 844-851Crossref PubMed Scopus (167) Google Scholar and palpated daily for evidence of a heartbeat. Rejection was defined as a loss of palpable heartbeat. All grafts were harvested at the time of rejection or at predetermined time points after transplant. Graft survival was compared using Kaplan-Meier analysis.Histology and ImmunohistochemistryFormalin-fixed paraffin sections of graft tissues were stained with hematoxylin and eosin (H&E) and for elastin as described.20Valujskikh A Pantenburg B Heeger PS Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.Am J Transplant. 2002; 2: 501-509Crossref PubMed Scopus (198) Google Scholar, 38Valujskikh A Lantz O Celli S Matzinger P Heeger PS Cross-primed CD8(+) T cells mediate graft rejection via a distinct effector pathway.Nat Immunol. 2002; 3: 844-851Crossref PubMed Scopus (167) Google Scholar For single-color immunohistochemistry, frozen sections of cardiac tissue were acetone-fixed, hydrated in PBS for 10 minutes, blocked with the avidin-biotin blocking system (DAKO, Carpinteria, CA), washed with PBS three times, treated with 3% H2O2 (Sigma, St. Louis, MO) for 8 to 10 minutes, washed three times in PBS, and incubated for 60 minutes at room temperature with biotinylated anti-CD4 (1:50 dilution in PBS-1% bovine serum albumin; BD Pharmingen). After three additional PBS washes, the sections were incubated with peroxidase-conjugated streptavidin (stock concentration, DAKO) and developed using the diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA), counterstained, dehydrated with ethanol, and mounted.For two-color immunohistochemistry, frozen sections of cardiac tissue were acetone-fixed, hydrated in PBS for 10 minutes, blocked with the biotin blocking system (DAKO), washed with PBS three times, and incubated for 60 minutes at room temperature with biotinylated anti-H-2/I-Ab (1:50 dilution in PBS-1% bovine serum albumin; BD Pharmingen). After three additional PBS washes, the sections were incubated with peroxidase-conjugated streptavidin (stock concentration, DAKO) and developed using the Novared substrate kit (Vector Laboratories). Sections were then washed three times in PBS, and incubated for 90 minutes at room temperature with fluorescein isothiocyanate-conjugated anti-CD4 antibody (BD Pharmingen). After three washes with PBS, slides were incubated with rabbit F(ab′) alkaline-phosphatase-conjugated anti-fluorescein isothiocyanate (DAKO). After three final washes in PBS the slides were developed using the Vector Blue alkaline-phosphatase substrate kit (Vector Laboratories). Sections were dehydrated with ethanol and mounted for analysis.ELISPOT AssaysAssays were performed as outlined previously in detail.20Valujskikh A Pantenburg B Heeger PS Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.Am J Transplant. 2002; 2: 501-509Crossref PubMed Scopus (198) Google Scholar, 31Matesic D Lehmann PV Heeger PS High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.Transplantation. 1998; 65: 906-914Crossref PubMed Scopus (82) Google Scholar, 35Benichou G Valujskikh A Heeger PS Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice.J Immunol. 1999; 162: 352-358PubMed Google Scholar, 38Valujskikh A Lantz O Celli S Matzinger P Heeger PS Cross-primed CD8(+) T cells mediate graft rejection via a distinct effector pathway.Nat Immunol. 2002; 3: 844-851Crossref PubMed Scopus (167) Google Scholar Briefly, MultiScreen ELISPOT plates (Millipore, Bedford, MA) were coated overnight with the capture antibodies (BD Pharmingen) in sterile PBS, blocked with sterile 1% bovine serum albumin in PBS, and washed three times with sterile PBS. Spleen cells (0.2 to 1 × 106 per well) were plated in HL-1 medium (BioWhittaker, Walkersville, MD) with or without mitomycin C-treated stimulator cells (400,000 per well) and/or soluble antigens (HY2pb and OVAp at 0.1 to 10 μmol/L) and then incubated at 37°C, 5% CO2 for 24 hours. After washing with PBS followed by PBS 0.025% Tween (PBST), detection antibodies (BD PharMingen) were added overnight. After washing with PBST, alkaline phosphatase-conjugated anti-biotin antibody (Vector Laboratories) diluted 1:1000 in PBST was added for 2 hours at room temperature. The plates were developed as previously described.20Valujskikh A Pantenburg B Heeger PS Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.Am J Transplant. 2002; 2: 501-509Crossref PubMed Scopus (198) Google Scholar, 31Matesic D Lehmann PV Heeger PS High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.Transplantation. 1998; 65: 906-914Crossref PubMed Scopus (82) Google Scholar, 35Benichou G Valujskikh A Heeger PS Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice.J Immunol. 1999; 162: 352-358PubMed Google Scholar, 38Valujskikh A Lantz O Celli S Matzinger P Heeger PS Cross-primed CD8(+) T cells mediate graft rejection via a distinct effector pathway.Nat Immunol. 2002; 3: 844-851Crossref PubMed Scopus (167) Google Scholar The resulting spots were counted on an ImmunoSpot Series 1 Analyzer (Cellular Technology, Cleveland, OH).20Valujskikh A Pantenburg B Heeger PS Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.Am J Transplant. 2002; 2: 501-509Crossref PubMed Scopus (198) Google Scholar, 31Matesic D Lehmann PV Heeger PS High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.Transplantation. 1998; 65: 906-914Crossref PubMed Scopus (82) Google Scholar, 35Benichou G Valujskikh A Heeger PS Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice.J Immunol. 1999; 162: 352-358PubMed Google Scholar, 38Valujskikh A Lantz O Celli S Matzinger P Heeger PS Cross-primed CD8(+) T cells mediate graft rejection via a distinct effector pathway.Nat Immunol. 2002; 3: 844-851Crossref PubMed Scopus (167) Google ScholarResultsExperimental RationaleWe sought to design a model system in which CD4+ T cells of defined specificity could be permitted to respond either directly to donor antigens expressed on donor graft parenchymal/vascular cells or alternatively, to donor antigens that were processed and presented only by recipient APCs infiltrating the donor organ. To this end, we studied the male H-Y transplant antigen using mouse models of skin and heart graft rejection. Female C57BL/6 (B6) mice reject syngeneic male skin through T-cell-dependent mechanisms and the peptide specificities of the responding CD4+ and CD8+ anti-H-Y T cells are known.34Simpson E Scott D Chandler P The male-specific histocompatibility antigen, H-Y: a history of transplantation, immune response genes, sex determination and expression cloning.Annu Rev Immunol. 1997; 15: 39-61Crossref PubMed Scopus (133) Google Scholar, 39Silvers WK Billingham RE Sanford BH The H-Y transplantation antigen: a Y-linked or sex-influenced factor?.Nature. 1968; 220: 401-403Crossref PubMed Scopus (33) Google Scholar, 40Silvers WK Collins NH The behavior of H-Y-incompatible neonatal skin grafts in rats.Transplantation. 1979; 28: 57-59Crossref PubMed Scopus (6) Google Scholar, 41Wang W Meadows LR den Haan JM Sherman NE Chen Y Blokland E Shabanowitz J Agulnik AI Hendrickson RC Bishop CE Hunt DF Goulmy E Engelhard VH Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein.Science. 1995; 269: 1588-1590Crossref PubMed Scopus (332) Google Scholar The predominant MHC class II-binding male peptide is I-Ab-restricted, derives from the Dby gene, and is heretofore called HYDbyp.34Simpson E Scott D Chandler P The male-specific histocompatibility antigen, H-Y: a history of transplantation, immune response genes, sex determination and expression cloning.Annu Rev Immunol. 1997; 15: 39-61Crossref PubMed Scopus (133) Google Scholar, 42Braun MY Grandjean I Feunou P Duban L Kiss R Goldman M Lantz O Acute rejection in the absence of cognate recognition of allograft by T cells.J Immunol. 2001; 166: 4879-4883PubMed Google Scholar We used female Marilyn (Mar) TCR transgenic mice, which are on a B6 RAG−/− background and only contain CD4+ T cells that are specific for I-Ab + HYDbyp complexes,42Braun MY Grandjean I Feunou P Duban L Kiss R Goldman M Lantz O Acute rejection in the absence of cognate recognition of allograft by T cells.J Immunol. 2001; 166: 4879-4883PubMed Google Scholar as recipients for our studies. Female Mar T cells do not cross-react with any antigen expressed on male or female allogeneic C3H (H-2k) tissues.42Braun MY Grandjean I Feunou P Duban L Kiss R Goldman M Lantz O Acute rejection in the absence of cognate recognition of allograft by T cells.J Immunol. 2001; 166: 4879-4883PubMed Google ScholarFemale Mar Mice Acutely Reject Syngeneic but Not Allogeneic Male Heart GraftsIn the first set of experiments we determined the functional capacity of the Mar T cells by challenging Mar females with cardiac allografts. We reasoned that transplantation of male B6 heart grafts would present donor male antigen on donor heart-derived male I-Ab+ APCs, permitting direct priming of recipient female Mar T cells (Figure 1). In this strain combination, the donor-derived male antigen could also theoretically be processed and presented by recipient APCs (I-Ab+ female APCs) so as to also activate recipient Mar T cells through the indirect pathway (Figure 1). In contrast, when female Mar mice are transplanted with male C3H heart grafts, the male antigen would be expressed on endogenous graft cells in the context of I-Ak or I-Ek and thus could not directly interact with Mar T cells. Nonetheless, the allogeneic C3H hearts should still prime the recipient T cells through the indirect pathway (Figure 1) because the donor male antigen can be processed and presented by recipient female I-Ab+ B6 APCs. Thus, by altering the donor strain, we can use this experimental design to permit T cells of single specificity to become activated through either the direct plus indirect pathways (male B6 grafts) or through the indirect pathway alone (male C3H grafts), and determine how the recognition pathway affects the subsequent induced effector T-cell frequency and function, and ultimately, graft outcome.Female Mar mice acutely rejected male B6 heart grafts, as all grafts ceased beating by day 35 (Figure 2). In contrast, Mar females rejected neither male nor female allogeneic C3H heart grafts—graft heartbeats were palpable fo
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