Th17 Responses to Collagen Type V, kα1-Tubulin, and Vimentin Are Present Early in Human Development and Persist Throughout Life.

T helper 17 (Th17)-dependent autoimmune responses can develop after heart or lung transplantation and are associated with fibro-obliterative forms of chronic rejection; however, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we investigated whether removal of regulatory T cells or blockade of function reveals a similar autoantigen bias. We found that Th17 cells specific for collagen type V (Col V), kα1-tubulin, and vimentin were present in healthy adult peripheral blood mononuclear cells, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1[V]), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. Although the latter responded well to α1(V) fragments and peptides in an HLA-DR-restricted fashion, Th17 cells from healthy persons responded in an HLA-DR-restricted fashion to fragments but not to peptides. Col V, kα1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, preexisting Th17 response that is MHC class II restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis.

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.

Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Pretransplant immune-regulation predicts allograft tolerance.

CD4⁺ Tregs specific for noninherited maternal antigens (NIMA(d) ) are detectable in some but not all B6 × BDF1 backcross, H-2(b) homozygous offspring, and their presence is strongly correlated with extent of maternal (BDF1) microchimerism. We hypothesized that the level of pretransplant donor antigen-specific Tregs could predict allograft tolerance. To test this idea, mice were screened for bystander suppression in a DTH assay, followed 1 week later by DBA/2 heterotopic heart transplantation. NIMA(d) -exposed, H-2(b) offspring that failed to suppress DTH uniformly rejected heart allografts (12/12) by d15. In contrast, 5/6 NIMA(d) -exposed DTH 'regulators' accepted their allografts >100 days. The defect in 'nonregulator" offspring could be corrected by transfer of CD4⁺CD25⁺, but not CD4⁺ CD25(neg) or CD8⁺ T cells from transplant acceptor mice. In conclusion, donor-specific T reg screening of F1 backcross offspring correctly predicted which recipients would accept a heart allograft. If translated to the clinic, similar pretransplant Treg screening could greatly enhance the effectiveness of tolerance as a clinical strategy in transplantation between family members.

Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

Obliterative bronchiolitis (OB) is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H-2(b) mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3-A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL-17A, but not IL-17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL-10 transcripts and protein were increased only in non-OB mice. Neutralizing IL-17 prevented OB, down regulated acute rejection, and upregulated systemic IL-10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL-17A or augmenting IL-10 could be therapeutic interventions to prevent OB.

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.

Tolerance induction or sensitization in mice exposed to noninherited maternal antigens (NIMA).

Developmental exposure to noninherited maternal antigens (NIMA) exerts a tolerizing or sensitizing influence on clinical transplantation in humans and experimental animals. The aim of this study was to determine if strain and gender differences influence the NIMA effect. Six different mouse strain backcross matings of F(1) females with homozygous males ('NIMA backcross') and corresponding control breedings of F1 males with homozygous females were performed. H-2 homozygous offspring underwent heterotopic heart transplantation from fully allogeneic donors expressing noninherited H-2 antigens. A NIMA tolerizing effect on heart allograft outcome was found in three of six breeding models. In all three cases, the tolerizing antigens were from an H-2(d+) strain. The tolerogenic effect was greatest in male as compared with female recipients. Offspring from the three breeding models in which no tolerance was seen, appeared to be sensitized based on poorer graft survival, or enhanced T- or B-cell responses to the noninherited H-2(b or k) antigens. Significantly higher percentages of maternal antigen(+) cells were found in the peripheral blood of tolerant versus nontolerant strains of backcross mice prior to transplant. Our findings imply that transplants are predisposed to tolerance or rejection due to recipient developmental history and immunogenetic background.

Human allograft acceptance is associated with immune regulation.

The ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse "trans vivo" delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen-linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-beta or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.

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.

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.

A rapid assay for measuring both colony size and cytolytic activity of limiting dilution microcultures.

A combined 51Cr-release/MTT dye method is described for accurately measuring cytolytic activity and colony size in the same set of culture microwells. The method was applied to the study of cell-mediated lympholysis (CML) in limiting dilution analysis (LDA) cultures of human PBL from a renal transplant recipient and a healthy control. The results showed that the combined CML/MTT method could detect differences in lytic activity per cell in LDA cultures, and thus is a useful adjunct to standard precursor frequency analysis.

Expression of cell-defined H-Y antigen on mouse epidermal cells.

Cytotoxic T lymphocytes (CTL)5 of female origin that readily lysed syngeneic male lymphoid cells in specific, dose-dependent, H-2 restricted fashion had little or no activity against syngeneic male epidermal cells (EC) in short-term or long-term chromium-release assays. Moreover, although male EC were quite capable of priming syngeneic female lymphocytes in vivo for the accelerated rejection of male-specific skin grafts and for the subsequent generation of H-Y-specific CTL by exposure of primed female spleen cells (SC) to irradiated, syngeneic male SC in vitro, male EC themselves were incapable of stimulating the development of H-Y CTL when cocultured with primed female SC. Tests of EC from reciprocal male-female radiation chimeras revealed that keratinocytes, not marrow-derived EC (Langerhans cells), were responsible for the priming ability of EC in vivo. Moreover, H-Y antigen was serologically defined on EC that failed to express H-Y-specific CTL target-cell determinants. Alternative explanations of these findings are discussed, including the possibility that the inability of H-2-restricted T cells to lyse male EC results from the lack of association of H-Y antigen and H-2 restricting elements on the EC membrane.

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.

Improved renal allograft survival following donor-specific transfusions. III. Kinetics of mixed lymphocyte culture responses before and after transplantation.

The essence of the clonal deletion model of the donor-specific transfusion (DST) effect is synergy between DST-priming and post-transplant immunosuppression. Using a sensitive kinetics assay of the mixed lymphocyte culture (MLC) response to donor and third-party stimulators, we compared the responses of controls (non-transfused healthy individuals) with those of patients who either had no rejection episodes in the first week posttransplant (group 1) or who had DST-type (greater than 3d onset) rejection episodes (group 2). We found that group 2 patients had normal or above-normal MLC responses after DST plus azathioprine (AZA) pretransplant treatment, but had a reduced/delayed posttransplant anti-donor MLC following reversal of early rejections (P = .05 compared with controls). Group 1 patients had a nonspecifically reduced MLC after DST + AZA treatment (P less than .02 compared with controls), while posttransplant MLC responses showed a return to normal (pretransfusion) levels. These data suggest a synergy of DST with immunosuppressive drug that induced MLC hyporesponsiveness, but only in patients who received anti-lymphoblast globulin or a sustained high dose immunosuppression in the early posttransplant period.

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.