Complement Dependence of Murine Costimulatory Blockade-Resistant Cellular Cardiac Allograft Rejection.

Building on studies showing that ischemia-reperfusion-(I/R)-injury is complement dependent, we tested links among complement activation, transplantation-associated I/R injury, and murine cardiac allograft rejection. We transplanted BALB/c hearts subjected to 8-h cold ischemic storage (CIS) into cytotoxic T-lymphocyte associated protein 4 (CTLA4)Ig-treated wild-type (WT) or c3-/- B6 recipients. Whereas allografts subjected to 8-h CIS rejected in WT recipients with a median survival time (MST) of 37 days, identically treated hearts survived >60 days in c3-/- mice (p < 0.05, n = 4-6/group). Mechanistic studies showed recipient C3 deficiency prevented induction of intragraft and serum chemokines/cytokines and blunted the priming, expansion, and graft infiltration of interferon-γ-producing, donor-reactive T cells. MST of hearts subjected to 8-h CIS was >60 days in mannose binding lectin (mbl1-/- mbl2-/- ) recipients and 42 days in factor B (cfb-/- ) recipients (n = 4-6/group, p < 0.05, mbl1-/- mbl2-/- vs. cfb-/- ), implicating the MBL (not alternative) pathway. To pharmacologically target MBL-initiated complement activation, we transplanted BALB/c hearts subjected to 8-h CIS into CTLA4Ig-treated WT B6 recipients with or without C1 inhibitor (C1-INH). Remarkably, peritransplantation administration of C1-INH prolonged graft survival (MST >60 days, p < 0.05 vs. controls, n = 6) and prevented CI-induced increases in donor-reactive, IFNγ-producing spleen cells (p < 0.05). These new findings link donor I/R injury to T cell-mediated rejection through MBL-initiated, complement activation and support testing C1-INH administration to prevent CTLA4Ig-resistant rejection of deceased donor allografts in human transplant patients.

Anti-huCD20 antibody therapy for antibody-mediated rejection of renal allografts in a mouse model.

We have reported that B6.CCR5(-/-) mice reject renal allografts with high serum donor-specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody-mediated rejection (AMR). B6.huCD20/CCR5(-/-) mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti-huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long-term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti-huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti-huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.

Acute and chronic rejection: compartmentalization and kinetics of counterbalancing signals in cardiac transplants.

Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell-deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy.

Interferon Gamma ELISPOT Testing as a Risk-Stratifying Biomarker for Kidney Transplant Injury: Results From the CTOT-01 Multicenter Study.

Previous studies suggest that quantifying donor-reactive memory T cells prior to kidney transplantation by interferon gamma enzyme-linked immunosorbent spot assay (IFNγELISPOT) can assist in assessing risk of posttransplant allograft injury. Herein, we report an analysis of IFNγELISPOT results from the multicenter, Clinical Trials in Organ Transplantation-01 observational study of primary kidney transplant recipients treated with heterogeneous immunosuppression. Within the subset of 176 subjects with available IFNγELISPOT results, pretransplant IFNγELISPOT positivity surprisingly did not correlate with either the incidence of acute rejection (AR) or estimated glomerular filtration rate (eGFR) at 6- or 12-month. These unanticipated results prompted us to examine potential effect modifiers, including the use of T cell-depleting, rabbit anti-thymocyte globulin (ATG). Within the no-ATG subset, IFNγELISPOT(neg) subjects had higher 6- and 12-month eGFRs than IFNγELISPOT(pos) subjects, independent of biopsy-proven AR, peak PRA, human leukocyte antigen mismatches, African-American race, donor source, and recipient age or gender. In contrast, IFNγELISPOT status did not correlate with posttransplant eGFR in subjects given ATG. Our data confirm an association between pretransplant IFNγELISPOT positivity and lower posttransplant eGFR, but only in patients who do not receive ATG induction. Controlled studies are needed to test the hypothesis that ATG induction is preferentially beneficial to transplant candidates with high frequencies of donor-reactive memory T cells.

Endogenous memory CD8 T cells directly mediate cardiac allograft rejection.

Differences in levels of environmentally induced memory T cells that cross-react with donor MHC molecules are postulated to account for the efficacy of allograft tolerance-inducing strategies in rodents versus their failure in nonhuman primates and human transplant patients. Strategies to study the impact of donor-reactive memory T cells on allografts in rodents have relied on the pretransplant induction of memory T cells cross-reactive with donor allogeneic MHC molecules through recipient viral infection, priming directly with donor antigen or adoptive transfer of donor antigen primed memory T cells. Each approach accelerates allograft rejection and confers resistance to tolerance induction, but also biases the T cell repertoire to strong donor reactivity. The ability of endogenous memory T cells within unprimed mice to directly reject an allograft is unknown. Here, we show a direct association between increased duration of cold ischemic allograft storage and numbers and enhanced functions of early graft infiltrating endogenous CD8 memory T cells. These T cells directly mediate rejection of allografts subjected to prolonged ischemia and this rejection is resistant to costimulatory blockade. These findings recapitulate the clinically significant impact of endogenous memory T cells with donor reactivity in a mouse transplant model in the absence of prior recipient priming.

Graft-derived CCL2 increases graft injury during antibody-mediated rejection of cardiac allografts.

The pathogenic role of macrophages in antibody-mediated rejection (AMR) remains unclear. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic factor for monocytes and macrophages. The current studies used a murine model of AMR to investigate the role of graft-derived CCL2 in AMR and how macrophages may participate in antibody-mediated allograft injury. B6.CCR5−/−/CD8−/− recipients rejected MHC-mismatched WT A/J allografts with high donor-reactive antibody titers and diffuse C4d deposition in the large vessels and myocardial capillaries, features consistent with AMR. In contrast, A/J.CCL2−/− allografts induced low donor-reactive antibody titers and C4d deposition at Day 7 posttransplant. Decreased donor-reactive CD4 T cells producing interferon gamma were induced in response to A/J.CCL2−/− versus WT allografts. Consequently, A/J.CCL2−/− allograft survival was modestly but significantly longer than A/J allografts. Macrophages purified from WT allografts expressed high levels of IL-1ß and IL-12p40 and this expression and the numbers of classically activated macrophages were markedly reduced in CCL2-deficient allografts on Day 7. The results indicate that allograft-derived CCL2 plays an important role in directing classically activated macrophages into allografts during AMR and that macrophages are important contributors to the inflammatory environment mediating graft tissue injury in this pathology, suggesting CCL2 as a therapeutic target for AMR.

IL-15 induces alloreactive CD28(-) memory CD8 T cell proliferation and CTLA4-Ig resistant memory CD8 T cell activation.

The presence of CD28(-) memory CD8 T cells in the peripheral blood of renal transplant patients is a risk factor for graft rejection and resistance to CTLA-4Ig induction therapy. In vitro analyses have indicated poor alloantigen-induced CD28(-) memory CD8 T cell proliferation, raising questions about mechanisms mediating their clonal expansion in kidney grafts to mediate injury. Candidate proliferative cytokines were tested for synergy with alloantigen in stimulating CD28(-) memory CD8 T cell proliferation. Addition of IL-15, but not IL-2 or IL-7, to co-cultures of CD28(-) or CD28(+) memory CD8 T cells and allogeneic B cells rescued proliferation of the CD28(-) and enhanced CD28(+) memory T cell proliferation. Proliferating CD28(-) memory CD8 T cells produced high amounts of interferon gamma and tumor necrosis factor alpha and expressed higher levels of the cytolytic marker CD107a than CD28(+) memory CD8 T cells. CTLA-4Ig inhibited alloantigen-induced proliferation of CD28(+) memory CD8 T cell proliferation but had no effect on alloantigen plus IL-15-induced proliferation of either CD28(-) or CD28(+) memory CD8 T cells. These results indicate the ability of IL-15, a cytokine produced by renal epithelial during inflammation, to provoke CD28(-) memory CD8 T cell proliferation and to confer memory CD8 T cell resistance to CTLA-4Ig-mediated costimulation blockade.

Key role for CD4 T cells during mixed antibody-mediated rejection of renal allografts.

We utilized mouse models to elucidate the immunologic mechanisms of functional graft loss during mixed antibody-mediated rejection of renal allografts (mixed AMR), in which humoral and cellular responses to the graft occur concomitantly. Although the majority of T cells in the graft at the time of rejection were CD8 T cells with only a minor population of CD4 T cells, depletion of CD4 but not CD8 cells prevented acute graft loss during mixed AMR. CD4 depletion eliminated antidonor alloantibodies and conferred protection from destruction of renal allografts. ELISPOT revealed that CD4 T effectors responded to donor alloantigens by both the direct and indirect pathways of allorecognition. In transfer studies, CD4 T effectors primed to donor alloantigens were highly effective at promoting acute graft dysfunction, and exhibited the attributes of effector T cells. Laser capture microdissection and confirmatory immunostaining studies revealed that CD4 T cells infiltrating the graft produced effector molecules with graft destructive potential. Bioluminescent imaging confirmed that CD4 T effectors traffic to the graft site in immune replete hosts. These data document that host CD4 T cells can promote acute dysfunction of renal allografts by directly mediating graft injury in addition to facilitating antidonor alloantibody responses.

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.

Transient lymphopenia breaks costimulatory blockade-based peripheral tolerance and initiates cardiac allograft rejection.

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long-term graft acceptance. After vascularized MHC-mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti-CD4 plus anti-CD8 mAb. Long-term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44(high) effector/memory T cells in lymphatic organs and strong recovery of donor-reactive T cell responses, indicating lymphopenia-induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4(+) Foxp(3+)) cell and B cell numbers as well as donor-specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor-reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.

Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury.

Noninvasive biomarkers are needed to assess immune risk and ultimately guide therapeutic decision-making following kidney transplantation. A requisite step toward these goals is validation of markers that diagnose and/or predict relevant transplant endpoints. The Clinical Trials in Organ Transplantation-01 protocol is a multicenter observational study of biomarkers in 280 adult and pediatric first kidney transplant recipients. We compared and validated urinary mRNAs and proteins as biomarkers to diagnose biopsy-proven acute rejection (AR) and stratify patients into groups based on risk for developing AR or progressive renal dysfunction. Among markers tested for diagnosing AR, urinary CXCL9 mRNA (odds ratio [OR] 2.77, positive predictive value [PPV] 61.5%, negative predictive value [NPV] 83%) and CXCL9 protein (OR 3.40, PPV 67.6%, NPV 92%) were the most robust. Low urinary CXCL9 protein in 6-month posttransplant urines obtained from stable allograft recipients classified individuals least likely to develop future AR or a decrement in estimated glomerular filtration rate between 6 and 24 months (92.5-99.3% NPV). Our results support using urinary CXCL9 for clinical decision-making following kidney transplantation. In the context of acute dysfunction, low values can rule out infectious/immunological causes of injury. Absent urinary CXCL9 at 6 months posttransplant defines a subgroup at low risk for incipient immune injury.

Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling.

Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold-changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.

Antibody-mediated rejection of single class I MHC-disparate cardiac allografts.

Murine CCR5(-/-) recipients produce high titers of antibody to complete MHC-mismatched heart and renal allografts. To study mechanisms of class I MHC antibody-mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild-type C57BL/6 (H-2(b)) and B6.CCR5(-/-) recipients. Donor-specific antibody titers in CCR5(-/-) recipients were 30-fold higher than in wild-type recipients. B6.K(d) allografts survived longer than 60 days in wild-type recipients whereas CCR5(-/-) recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils and macrophages, and C4d deposition in the graft capillaries. B6.K(d) allografts were rejected by CD8(-/-)/CCR5(-/-), but not µMT(-/-)/CCR5(-/-), recipients indicating the need for antibody but not CD8 T cells. Grafts recovered at day 10 from CCR5(-/-) and CD8(-/-)/CCR5(-/-) recipients and from RAG-1(-/-) allograft recipients injected with anti-K(d) antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of antidonor class I MHC antibody can mediate allograft rejection, that donor-reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity.

The spleen is the major source of antidonor antibody-secreting cells in murine heart allograft recipients.

Antibody-mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor-specific alloantibody (DSA)-producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody-secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC-mismatched heart allografts were transplanted into three groups of recipients: nonsensitized wild type, alloantigen-sensitized wild-type and CCR5(-/-) mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of antidonor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody-secreting cells and the serum titers of antidonor IgG alloantibody were equivalent in sensitized and nonsensitized recipients 6 weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor-reactive ASCs than bone marrow at days 7-21 after transplantation. Furthermore, individual spleen ASCs produced more antidonor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor-reactive alloAb early after transplantation in both sensitized and nonsensitized recipients.

LFA-1 antagonism inhibits early infiltration of endogenous memory CD8 T cells into cardiac allografts and donor-reactive T cell priming.

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti-LFA-1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti-LFA-1 mAb given to C57BL/6 6 (H-2(b)) recipients of A/J (H-2(a)) heart grafts on days -1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN-γ-induced genes. Donor-specific T cells producing IFN-γ were at low/undetectable numbers in spleens of anti-LFA-1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti-LFA-1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti-LFA-1 mAb inhibits early donor-reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.

Evaluation of alloreactivity in kidney transplant recipients treated with antithymocyte globulin versus IL-2 receptor blocker.

Induction therapy is used in kidney transplantation to inhibit the activation of donor-reactive T cells which are detrimental to transplant outcomes. The choice of induction therapy is decided based on perceived immunological risk rather than by direct measurement of donor T-cell reactivity. We hypothesized that immune cellular alloreactivity pretransplantation can be quantified and that blocking versus depleting therapies have differential effects on the level of donor and third-party cellular alloreactivity. We studied 31 kidney transplant recipients treated with either antithymocyte globulin (ATG) or an IL-2 receptor blocker. We tested pre- and posttransplant peripheral blood cells by flow cytometry to characterize T-cell populations and by IFN-γ ELISPOT assays to assess the level of cellular alloreactivity. CD8(+) T cells were more resistant to depletion by ATG than CD4(+) T cells. Posttransplantation, frequencies of donor-reactive T cells were markedly decreased in the ATG-treated group but not in the IL-2 receptor blocker group, whereas the frequencies of third-party alloreactivity remained nearly equivalent. In conclusion, when ATG is used, marked and prolonged donor hyporesponsiveness with minimal effects on nondonor responses is observed. In contrast, induction with the IL-2 receptor blocker is less effective at diminishing donor T-cell reactivity.

T cell populations primed by hapten sensitization in contact sensitivity are distinguished by polarized patterns of cytokine production: interferon gamma-producing (Tc1) effector CD8+ T cells and interleukin (Il) 4/Il-10-producing (Th2) negative regulatory CD4+ T cells.

Contact hypersensitivity (CHS) is a T cell-mediated response to hapten sensitization of the epidermis. The roles of CD4+ and CD8+ T cells in CHS have remained unclear, however, as studies to define either subset as the T cells mediating CHS have provided conflicting results. The goal of this study was to correlate the in vivo function of CD4+ and CD8+ T cells in CHS with the cytokines produced by each T cell population. Antibody-mediated depletion of CD4+ T cells before sensitization of BALB/c mice with 2,4-dinitrofluorobenzene (DNFB) or oxazolone (Ox) resulted in increased and prolonged CHS responses, indicating CD4+ T cells as negative regulators of the response. Depletion of CD8+ T cells resulted in low or abrogated responses, indicating CD8+ T cells as the effector cells in CHS. Sensitization with DNFB or Ox induced lymph node cell populations of CD8+ T cells producing interferon (IFN)-gamma and no interleukin (Il) 4 or Il-10, and CD4+ T cells producing Il-4 and Il-10 and no or little detectable IFN-gamma. The polarized patterns of cytokine production were stimulated by culture of hapten-primed lymph node cells either on anti-T cell receptor antibody-coated wells or with semipurified Langerhans cells isolated from hapten-sensitized mice. Stimulation of cytokine production during culture of hapten-primed CD4+ or CD8+ T cells with Langerhans cells was hapten specific and restricted to class II or class I major histocompatibility complex, respectively. The induction of the CD4+ and CD8+ T cells producing the polarized patterns of cytokines was not restricted to BALB/c mice, as cells from Ox sensitized C57B1/6 and B10.D2 mice produced the same patterns. Collectively, these results expose the induction of two polarized and functionally opposing populations of T cells by hapten sensitization to induce CHS: IFN-gamma-producing effector CD8+ T cells and Il-4/Il-10-producing CD4+ T cells that negatively regulate the response.

Recent progress and new perspectives in studying T cell responses to allografts.

Studies in the past decade advanced our understanding of the development, execution and regulation of T-cell-mediated allograft rejection. This review outlines recent progress and focuses on three major areas of investigation that are likely to guide the development of graft-prolonging therapies in the future. The discussed topics include the contribution of recently discovered molecules to the activation and functions of alloreactive T cells, the emerging problem of alloreactive memory T cells and recently gained insights into the old question of transplantation tolerance.