Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses.

IL17-dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)-dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1ß, tumor necrosis factor α and CD14(+) cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL-1ß responses, we investigated its role in Th17-, Th1/17- and Th1-mediated proinflammatory responses. Transfer of antigen-pulsed peripheral blood mononucleated cells (PBMCs) from Col V-reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT-specific swelling responses. P2X7R inhibitors blocked IL-1ß induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V-induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3(neg) CCR4(+)/6(+) CD4(+) [Th17] versus CXCR3(+)CCR4/6(neg) CD4(+) [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17-mediated alloimmunity, in a heart transplant patient without affecting anti-viral Epstein-Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.

Early and limited use of tacrolimus to avoid rejection in an alemtuzumab and sirolimus regimen for kidney transplantation: clinical results and immune monitoring.

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27-39 months of follow-up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed-type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3-4 ng/mL. One patient showed clinical signs of rejection at month 9 post-transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor-specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid-free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1-year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Monitoring the patient off immunosuppression. Conceptual framework for a proposed tolerance assay study in liver transplant recipients.

The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.

Clonotype analysis of human alloreactive T cells: a novel approach to studying peripheral tolerance in a transplant recipient.

The recognition of allo-MHC and associated peptides on the surface of graft-derived APC by host T cells (direct pathway allorecognition) plays an important role in acute rejection after organ transplantation. However, the status of the direct pathway T cells in stable long term transplants remains unclear. To detect alloreactive T cell clones in PBL and the allograft during the transplant tolerance, we utilized RT-PCR instead of functional assays, which tend to underestimate their in vivo frequencies. We established alloreactive CD4+ and CD8+ T cell clones from peripheral blood sampled during the stable tolerance phase of a patient whose graft maintained good function for 9 years, 7 without immunosuppression. We analyzed the sequence of TCR Vbeta and Valpha genes and made clonotype-specific probes that allowed us to detect each clone in peripheral blood or biopsy specimens obtained during a 1-year period before and after the rapid onset of chronic rejection. We found an unexpectedly high level of donor HLA-specific T cell clonotype mRNA in peripheral blood during the late tolerance phase. Strong signals for two CD4+ clonotypes were detected in association with focal T cell infiltrates in the biopsy. Chronic rejection was associated with a reduction in direct pathway T cell clonotype mRNA in peripheral blood and the graft. Our data are inconsistent with the hypothesis that direct pathway T cells are involved only in early acute rejection events and suggest the possibility that some such T cells may contribute to the maintenance of peripheral tolerance to an allograft.

Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis.

We studied late graft rejection in a patient who had received a kidney transplant 9-10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DR beta 1 locus (donor: DR beta 1*1104; recipient: DR beta 1*1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient's creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient's peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the development of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DR beta 1*1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DR beta 1*1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DR beta 1*1104 and DR beta 1*1101 but not with the recipient DR beta 1*1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this "indirect" alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DR beta 1*1104 subtype.

Donor-derived human leukocyte antigen class I proteins in the serum of heart transplant recipients.

BACKGROUND: Human leukocyte antigen class I proteins are expressed on most cell types in all organ allografts but are constitutively secreted only by certain organs, for example, the liver. We hypothesized that detectable levels of donor-derived human leukocyte antigen proteins would be released from transplanted cardiac allografts only when the allograft was immunologically stimulated, that is, during rejection and perhaps during viral infection. If so, then the release of donor human leukocyte antigen might be a noninvasive monitor of these events. METHODS: We used an enzyme-linked immunosorbent assay to detect donor-derived human leukocyte antigen-A2 in the serum of 21 human leukocyte antigen-A2 negative recipients of human leukocyte antigen-A2-positive heart transplants. The level of donor human leukocyte antigen-A2 during the first 100 days after transplantation was correlated with the clinical status of the patient. RESULTS: We found little or no donor human leukocyte antigen in the serum of heart transplant recipients whose postoperative clinical course was unremarkable for infection or rejection. We did find donor-derived human leukocyte antigen in the serum of heart transplant recipients transiently in the week immediately after transplantation, continuously from patients in whom chronic rejection was developing, during cytomegalovirus infection, and during some, but not all, acute rejection episodes as determined by endomyocardial biopsy. CONCLUSIONS: These findings are consistent with the hypothesis that the donor human leukocyte antigen serum level reflects vascular diseases, rather than myocardial disease in the transplanted heart. Therefore, the serum level of donor human leukocyte antigen cannot be used as a monitor of cellular infiltration and myocyte damage as currently assessed by endomyocardial biopsy but may be an early indicator of the development of vascular disease such as chronic rejection.