Modulation of human allogeneic and syngeneic pluripotent stem cells and immunological implications for transplantation.

Tissues derived from induced pluripotent stem cells (iPSCs) are a promising source of cells for building various regenerative medicine therapies; from simply transplanting cells to reseeding decellularized organs to reconstructing multicellular tissues. Although reprogramming strategies for producing iPSCs have improved, the clinical use of iPSCs is limited by the presence of unique human leukocyte antigen (HLA) genes, the main immunologic barrier to transplantation. In order to overcome the immunological hurdles associated with allogeneic tissues and organs, the generation of patient-histocompatible iPSCs (autologous or HLA-matched cells) provides an attractive platform for personalized medicine. However, concerns have been raised as to the fitness, safety and immunogenicity of iPSC derivatives because of variable differentiation potential of different lines and the identification of genetic and epigenetic aberrations that can occur during the reprogramming process. In addition, significant cost and regulatory barriers may deter commercialization of patient specific therapies in the short-term. Nonetheless, recent studies provide some evidence of immunological benefit for using autologous iPSCs. Yet, more studies are needed to evaluate the immunogenicity of various autologous and allogeneic human iPSC-derived cell types as well as test various methods to abrogate rejection. Here, we present perspectives of using allogeneic vs. autologous iPSCs for transplantation therapies and the advantages and disadvantages of each related to differentiation potential, immunogenicity, genetic stability and tumorigenicity. We also review the current literature on the immunogenicity of syngeneic iPSCs and discuss evidence that questions the feasibility of HLA-matched iPSC banks. Finally, we will discuss emerging methods of abrogating or reducing host immune responses to PSC derivatives.

Clinical Implications of Basic Science Discoveries: Microchimerism Finds a Major Role in Reproductive Success; but Does It Also Contribute to Transplant Success?

Conventional wisdom argues against inbreeding, to maintain hybrid vigor and increase MHC diversity in response to pathogens. A recent report from the laboratory of Sing-Sing Way uses a mouse model to test a hypothesis put forward by Ray D. Owen more than 60 years ago: that a certain amount of inbreeding is a good thing. Owen proposed that antigens not inherited from the mother (noninherited maternal antigens), when replicated on the mate of the daughter, could protect the latter's developing child from fetal wastage due to immune attack during her pregnancy. Kinder et al use elegant mouse breeding models and MHC class II peptide tetramers to show that Owen's hypothesis, based only on humoral (anti-Rh IgG) data and a small sample size, was indeed correct. The mediators of this cross-generational protection turn out to be a special kind of Foxp3+ T regulatory cell, the development of which requires the persistence of maternal microchimerism into adulthood. The implications of this discovery for the role of microchimerism in tolerance to transplants are discussed.

Longitudinal studies of a B cell-derived signature of tolerance in renal transplant recipients.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

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.

Tumor necrosis factor-alpha and tumor growth factor-beta1 genotype: partial association with intragraft gene expression in two cases of long-term peripheral tolerance to a kidney transplant.

Genomic DNA was obtained from peripheral blood samples of patients JB and DS each of whom received a kidney transplant at 16 years of age from a serologically HLA-DR matched and HLA-class I -mismatched donor. Both patients discontinued immunosuppression after 1-2 years and retained good renal function for an additional 5 years or more. DNA was analyzed for genetic polymorphisms in the tumor necrosis factor-alpha (TNFalpha) and tumor growth factor-beta1 (TGFbeta1) loci. Biopsy samples obtained during stable function (DS, JB) and during rejection (JB) were analyzed by RT/PCR for cytokine gene expression. Both patients had a high responder genotype for TGFbeta1. DS had a low responder TNFalpha genotype, while JB and his donor were both genotypically TNFalpha intermediate responders. DS had a high TGFbeta1: TNFalpha mRNA ratio in two biopsies obtained during tolerance, while JB, who eventually lost his graft, had more TNFalpha than TGFbeta1 mRNA. The results suggest a possible role for cytokine immunogenetics in the stability of peripheral tolerance.

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 dendritic cell persistence in organ allograft recipients in the absence of immunosuppression.

Donor-derived leukocytes are known to persist in the peripheral blood of organ allograft recipients after withdrawal of all immunosuppressive drug therapy and can exert a donor-specific veto effect. Antigen-presenting cells (APC), in particular dendritic cells (DC), have been proposed as a candidate for this veto leukocyte. Myeloid DC were derived from the peripheral blood of two ion-compliant organ transplant recipients: D. S., a heart transplant recipient, and J. M., a liver transplant recipient. Donor-specific signal was enriched in the cultured DC fraction relative to whole blood for both patients. The clinical outcome in each patient was different: D. S. lost his heart allograft due to biopsy-proven acute and chronic rejection 2.5 years after discontinuing anti-rejection medication; J. M. continues to maintain adequate liver function. The results have important implications for the planned withdrawal of immunosuppression in tolerance protocols as DC may play a role either in the maintenance of tolerance or immune activation.

Peptide-conformed beta2m-free class I heavy chains are intermediates in generation of soluble HLA by the membrane-bound metalloproteinase.

Molecular mechanisms of soluble HLA-release by a membrane-bound metalloproteinase (MPase) are not defined. We have investigated the possibility that certain beta2-microglobulin (beta2m)-free heavy chains (HC) retain peptide-induced conformations before and after the cleavage by using mutant HLA-A2.242K HC with reduced affinity for beta2m. We show that dissociation of HC/beta2m complexes on the surface of C1R lymphoblastoid cells generates both conformed and non-conformed beta2m-free HC recognized by conformation-dependent antibodies. Conformed HC, having bound the HLA-A2-specific peptide HTLV-1 tax 11-19, can retain their proper conformations after dissociation of beta2m. Further, conformed and non-conformed surface beta2m-free HC are cleaved by the MPase, and some released HC preserve their conformations. Exogenous beta2m binds only to conformed HC, and protects them from cleavage as effectively as the MPase inhibitor BB-2116. We propose that soluble HLA-release requires generation of peptide-conformed beta2m-free HC intermediates on the cell surface, which are then cleaved by the MPase and in solution may reassociate with beta2m. Given the role of soluble HLA in the indirect allorecognition, the activity of this MPase may be important in transplant rejection.

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.

Chimerism after organ transplantation: is there any clinical significance?

Historically, attempts to clinically correlate microchimerism and cytotoxic T lymphocyte (CTL) dysfunction with allograft tolerance (via a "veto effect") have not been highly successful. We have re-examined this question by studying the case of JB, a maternal-renal allograft recipient who did not develop symptoms of acute or chronic rejection after ceasing immunotherapy of his own volition. A biopsy showed immunologic activity in the allograft that did not progress to full-scale rejection. A polymerase chain reaction (PCR) assay showed traces of donor-derived DNA in JB's peripheral blood leukocytes (PBL), which also were shown to display a "split tolerance" toward donor stimulators (MLC+/CTL-) in vitro. Subsequent experiments in our laboratory conclusively demonstrated that JB's suppressed CTL function was donor-specific and that small numbers of donor-derived cells in his PBL could cause the CTL suppression similar to the "veto effect" observed by other researchers. This confirmed the clinical link between microchimerism, CTL dysfunction, and allograft tolerance in a single patient. We propose a testable model for this unusual type of allograft tolerance. The model is based on donor-derived predendritic cell lineages that survive in the host and that exert a powerful "veto effect" by interfering with the normal development and function of donor-specific CD8+ T cells, perhaps even leading to their apoptosis. Additional experiments to test the details of our hypothetical model are under way.

Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient.

A patient was found to be functionally tolerant of a maternal kidney allograft as evidenced by good graft function 5 years after cessation of all immunosuppressive drug therapy. Despite normal in vitro proliferative and IL-2 responses, patient anti-donor 1 degree MLR cultures yielded little donor-specific CTL activity in either bulk or limiting dilution analysis (LDA) cultures. Using polymerase chain reaction, the patient's PBL and skin were found to contain donor-derived Bw6+ cells. Removal of Bw6+ donor cells from the patient PBL with mAb and immunomagnetic beads before stimulation with donor PBL on day 0 failed to restore donor-specific CTL in either bulk 1 degree MLR or LDA cultures. Restimulation of 1 degree cultures with donor stimulator cells plus exogenous IL-2, however, completely restored anti-donor HLA class I-specific CTL, indicating class I-specific CTL precursors were not clonally deleted. Fresh patient PBL, as well as donor cell-enriched fractions, when added at the initiation of 3 degrees MLR cultures, inhibited the generation of anti-donor CTL, whereas donor cell-depleted fractions did not. The inhibition was cell dose-dependent, was specific for the anti-donor response, and was radioresistant (1200 rad). Thus, the clinical tolerance observed in patients with microchimerism may be due to the presence of veto cells within the circulating donor cell pool.

Donor-specific cytotoxic T lymphocyte hyporesponsiveness following renal transplantation in patients pretreated with donor-specific transfusions.

Our purpose was to investigate the mechanism of the continuing beneficial effect of donor-specific transfusions in the cyclosporine era. We describe the development of donor-specific cytotoxic T lymphocyte hyporesponsiveness in peripheral blood lymphocytes obtained up to 2 years posttransplant in patients preconditioned with 3 DST plus azathioprine. In a group of 12 such patients, hyporesponsiveness developed gradually, becoming detectable in some patients as early as 1 month posttransplant and becoming statistically significant for the entire group at 9-12 months posttransplant. A complete specificity for donor alloantigens was seen in the hyporesponsiveness of some patients; in others, partial suppression of the response to a third party HLA-mismatched control was also seen. Although slight suppression of the mixed lymphocyte culture response was seen in some patients, overall there were no statistically significant differences in MLC responses to control or donor stimulators at any time point posttransplant as compared with pretransplant, pre-DST. The mechanism of donor-specific CTL hyporesponsiveness 2 years posttransplant was explored in one patient (HLA A1, 2, B 57, 60; DR 3, 6) who had received a 2-HLA haplotype-mismatched kidney transplant from her husband (HLA A2,--; B5, 8; DR4,--) following DST plus AZA pretreatment. Bulk culture CTL analysis showed specific nonresponsiveness to donor stimulators; however in the presence of exogenous recombinant IL-2, the antidonor response was restored to the level of pretransplant PBL. Limiting dilution analysis using recombinant IL-2 revealed equivalent precursor frequency of antidonor CTL in pre- and posttransplant PBL. These data suggest that the hyporesponsive PBL contained donor-specific CTL precursors but were deficient in helper function necessary for CTL maturation.

Adsorption of cytotoxic anti-HLA antibodies with HLA class I immunosorbant beads.

In an effort to generate HLA immunosorbants to specifically remove anti-HLA antibodies from sera of highly sensitized patients, we purified HLA proteins, covalently coupled them onto Sepharose, and adsorbed antisera from five patients with narrowly reactive cytotoxic anti-HLA antibodies and from one patient with broadly reactive antibodies. We found that an HLA-A2 immunosorbant depleted anti-HLA-A2 cytotoxic antibodies, but did not deplete anti-HLA-B7 or anti-HLA-B44 cytotoxic antibodies from the narrowly reactive patient sera. Patient S.C. developed high PRA (81%) with strong cytotoxicity against HLA-A1 and -A2 following rejection of an HLA-A1, -B57 mismatched kidney. We adsorbed his sera with five HLA immunosorbants including HLA-A2 and HLA-A1,28. We found that the HLA-A2 immunosorbant depleted antibodies to HLA-A2+ and HLA-B57+ cells but not to HLA-A1+ cells, while the HLA-A1,A28 immunosorbant depleted antibodies to both HLA-A1+ cells and to the HLA-A28 cross-reactive HLA-A2+ cells. Adsorption was specific for HLA-A alleles to which the patient was sensitized, since neither HLA-B-C immunosorbants (containing HLA-B7, -B8, -B13, -B27, or -B37 plus HLA-C gene products) nor the control immunosorbants (bovine serum albumin or diphtheria toxoid) depleted serum S.C. of cytotoxic anti-HLA antibodies. Our results indicate that HLA immunosorbants are stable to sequential cycles of adsorption and elution, and thus may be of future therapeutic value in treatment of sensitized patients.