Allospecific Memory B Cell Responses Are Dependent on Autophagy.

Long-lived, donor-reactive memory B cells (Bmems) can produce alloantibodies that mediate transplant injury. Autophagy, an intrinsic mechanism of cell organelle/component recycling, is required for Bmem survival in infectious and model antigen systems, but whether autophagy affects alloreactive Bmem is unknown. We studied mice with an inducible yellow fluorescent protein (YFP) reporter expressed under the activation-induced cytidine deaminase (AID) promoter active in B cells undergoing germinal center reactions. Up to 12 months after allogeneic sensitization, splenic YFP+ B cells were predominantly IgD- IgM- IgG+ and expressed CD73, CD80, and PD-L2, consistent with Bmems. Labeled cells contained significantly more cells with autophagosomes and more autophagosomes per cell than unlabeled, naïve B cells. To test for a functional link, we quantified alloantibody formation in mice with B cells conditionally deficient in the requisite autophagy gene ATG7. These experiments revealed absent B cell ATG7 (1) prevented B cell autophagy, (2) inhibited secondary alloantibody responses without altering primary alloantibody formation, and (3) diminished frequencies of alloreactive Bmems. Pharmacological autophagy inhibition with 3-methyladenine had similar effects on wild-type mice. Together with new documentation of increased autophagosomes within human Bmems, our data indicate that targeting autophagy has potential for eliminating donor-reactive Bmems in transplant recipients.

B Cell Receptor Genes Associated With Tolerance Identify a Cohort of Immunosuppressed Patients With Improved Renal Allograft Graft Function.

We previously reported that two B cell receptor genes, IGKV1D-13 and IGKV4-1, were associated with tolerance following kidney transplantation. To assess the potential utility of this "signature," we conducted a prospective, multicenter study to determine the frequency of patients predicted tolerant within a cohort of patients deemed to be candidates for immunosuppressive minimization. At any single time point, 25-30% of patients were predicted to be tolerant, while 13.7% consistently displayed the tolerance "signature" over the 2-year study. We also examined the relationship of the presence of the tolerance "signature" on drug use and graft function. Contrary to expectations, the frequency of predicted tolerance was increased in patients receiving tacrolimus and reduced in those receiving corticosteroids, mycophenolate mofetil, or Thymoglobulin as induction. Surprisingly, patients consistently predicted to be tolerant displayed a statistically and clinically significant improvement in estimated glomerular filtration rate that increased over time following transplantation. These findings indicate that the frequency of patients consistently predicted to be tolerant is sufficiently high to be clinically relevant and confirm recent findings by others that immunosuppressive agents impact putative biomarkers of tolerance. The association of a B cell-based "signature" with graft function suggests that B cells may contribute to the function/survival of transplanted kidneys.

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.

Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study.

Identification of biomarkers that assess posttransplant risk is needed to improve long-term outcomes following heart transplantation. The Clinical Trials in Organ Transplantation (CTOT)-05 protocol was an observational, multicenter, cohort study of 200 heart transplant recipients followed for the first posttransplant year. The primary endpoint was a composite of death, graft loss/retransplantation, biopsy-proven acute rejection (BPAR), and cardiac allograft vasculopathy (CAV) as defined by intravascular ultrasound (IVUS). We serially measured anti-HLA- and auto-antibodies, angiogenic proteins, peripheral blood allo-reactivity, and peripheral blood gene expression patterns. We correlated assay results and clinical characteristics with the composite endpoint and its components. The composite endpoint was associated with older donor allografts (p < 0.03) and with recipient anti-HLA antibody (p < 0.04). Recipient CMV-negativity (regardless of donor status) was associated with BPAR (p < 0.001), and increases in plasma vascular endothelial growth factor-C (OR 20; 95%CI:1.9-218) combined with decreases in endothelin-1 (OR 0.14; 95%CI:0.02-0.97) associated with CAV. The remaining biomarkers showed no relationships with the study endpoints. While suboptimal endpoint definitions and lower than anticipated event rates were identified as potential study limitations, the results of this multicenter study do not yet support routine use of the selected assays as noninvasive approaches to detect BPAR and/or CAV following heart transplantation.

What's hot, what's new in basic science: report from the American Transplant Congress 2015.

Research reports presented at the American Transplant Congress 2015 provided an array of basic science findings of relevance to the transplant community. Among key themes is the concept that ischemia-reperfusion injury and early posttransplantation inflammation is linked to adaptive alloimmunity and transplant injury. Molecular and cellular mechanisms contributing to these interactions were highlighted. The relevance of understanding how blocking costimulation, including CD40/CD154 interactions, affects various aspects of the alloimmune response was enhanced by the description of preclinical studies demonstrating efficacy of a unique, blocking anti-CD40 monoclonal antibody that could potentially be used in humans. The identification of mechanisms underlying interactions among T cell subsets and B cells, including follicular helper T cells, regulatory T cells, effector B cells, and regulatory B cells, provides multiple previously unrecognized targets for future therapeutic interventions. Additional reports of interest include novel insights into effects of the gut microbiome on graft survival and the ability to differentiate insulin-secreting, islet-like cells from induced pluripotent stem cells. Overall, the reported basic science findings from American Transplant Congress 2015 add to the fundamental understanding of innate and adaptive alloimmunity and provide novel and testable hypotheses that have the potential to be translated into improved clinical care of transplant patients.

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.

Strategies to inhibit alloantibody production in alloprimed murine recipients of hematopoietic stem cell grafts.

Alloantibody, not primed T cells, is the major barrier to bone marrow (BM) engraftment in allosensitized mice. We have shown that a single intravenous injection of donor splenocytes, to mimic a blood transfusion, results in high, sustained levels of serum alloantibody sufficient to eliminate donor BM within 3 h, resulting in uniform mortality in lethally irradiated allogeneic recipients. Current studies focused preventing and treating allopriming. Blockade of B cell survival signals with mTACI-Ig pre- and postpriming was ineffective, as was the B cell but not plasma cell depleting anti-CD20 mAb. Germinal center formation inhibition by lymphotoxin-beta receptor-Ig (LßR-Ig) diminished allosensitization, although conditional Prmd1 (Blimp-1) deletion in CD19+ cells was highly effective. By combining anti-CD20 mAb to reduce B cells and LTßR-Ig to diminish the frequency of B cells that could form germinal centers pre- and postpriming, allosensitization was precluded, permitting long-term survival in T- and NK-depleted, irradiated allogeneic recipients, whereas combined therapy postpriming alone was ineffective. As evidence of the critical role of B cells, the proteosomal inhibitor, bortezomib, given unencapsulated or encapsulated, proved ineffective in influencing allosensitization. These data extend our understanding of allopriming and provide a potential therapy for patients at risk for allosensitization and BM graft rejection.

Costimulatory blockade-induced allograft survival requires Beclin1.

Autophagy is required for T cell homeostasis and activation-induced T cell expansion. Whether autophagy participates in tolerance induction to foreign antigens, including allografts, is unknown. We tested the role of an essential autophagy protein, Beclin1, in heart transplant survival in mice. We observed that long-term allograft survival induced by donor-specific transfusion plus anti-CD154 mAb required homozygous lymphocyte expression of Beclin1. Following adoptive transfer into allogeneic recipients, autophagy-deficient, Beclin1 heterozygous effector T cells (Teffs) exhibited enhanced proliferation with diminished cell death and increased production of interferon gamma. Whereas the induction and function of regulatory T cells (Tregs) in Beclin1 heterozygous mice were normal, Teffs from these mice were resistant to Treg-mediated suppression. Our findings identify a requisite role for Beclin1 in facilitating Teff death during tolerance induction.

Immune cell-derived C3a and C5a costimulate human T cell alloimmunity.

Emerging evidence indicates that complement provides costimulatory signals for murine T cells but whether complement impacts human T cells remains unclear. We observed production of complement activation products C3a and C5a during in vitro cultures of human T cells responding to allogeneic dendritic cells (DC). Both partners expressed the receptors for C3a (C3aR) and C5a (C5aR) and C3aR- and C5aR-antagonists inhibited T cell proliferation. Recombinant C3a/C5a promoted CD4(+) T cell expansion, bypassed the inhibitory effects of CTLA4-Ig, and induced AKT phosphorylation, the latter biochemically linking C3aR/C5aR to known T cell signaling pathways. Lowering DC C3a/C5a production by siRNA knockdown of DC C3 reduced T cell alloresponses. Conversely downregulating DC expression of the complement regulatory protein decay-accelerating factor increased immune cell C3a/C5a and augmented T cell proliferation, identifying antigen presenting cells as the dominant complement source. Pharmacological C5aR blockade reduced graft versus host disease (GVHD) scores, prolonged survival, and inhibited T cell responses in NOD scid γc(null) mouse recipients of human peripheral blood mononuclear cells, verifying that the mechanisms apply in vivo. Together our findings unequivocally document that immune cell-derived complement impacts human T cell immunity and provide the foundation for future studies targeting C3aR/C5aR as treatments of GVHD and organ transplant rejection in humans.

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.

Eosinophils are not required for the induction and maintenance of an alloantibody response.

Eosinophil-produced cytokines have been shown to participate in the maintenance of antigen-specific plasma cells (PC) in bone marrow (BM), suggesting that eosinophils are required in the development and/or maintenance of alloantibody responses posttransplant. To test this hypothesis, we sensitized eosinophil-deficient ΔdblGATA1 mice and wild-type (WT) control mice with allogeneic splenocytes or with allogeneic heart grafts and compared the kinetics and titers of serum donor-specific antibodies (DSA), as well as BM and spleen CD130 + B220 low PC populations between groups. Spleen cells from naïve ΔdblGATA1 BALB/c mice contained higher percentages of PC than WT without detectable differences in BM PCs. After sensitization with allogeneic splenocytes, BALB/c ΔdblGATA1 mice contained fewer BM PCs but more splenic PCs compared to controls. These differences were associated with modestly lower titers of serum DSA 4 and 12 weeks after sensitization but secondary immunizations induced similar increases in both groups. Moreover, the kinetics and strength of DSA did not differ in WT and ΔdblGATA1 BALB/c mice transplanted with B6 cardiac allografts, nor did they differ in transplanted ΔdblGATA1 and WT mice on a B6 background. Therefore, eosinophils are not required for alloantibody formation or maintenance in mice and are thus unlikely to be effective targets for antibody desensitization.

Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R(2) ), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

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.

Standardization and cross validation of alloreactive IFNγ ELISPOT assays within the clinical trials in organ transplantation consortium.

Emerging evidence indicates memory donor-reactive T cells are detrimental to transplant outcome and that quantifying the frequency of IFNγ-producing, donor-reactive PBMCs by ELISPOT has potential utility as an immune monitoring tool. Nonetheless, differences in assay performance among laboratories limit the ability to compare results. In an effort to standardize assays, we prepared a panel of common cellular reagent standards, developed and cross validated a standard operating procedure (SOP) for alloreactive IFNγ ELISPOT assays in several research laboratories supported by the NIH-funded Clinical Trials in Organ Transplantation (CTOT) Consortium. We demonstrate that strict adherence to the SOP and centralized data analysis results in high reproducibility with a coefficient of variance (CV) of ≈ 30%. This standardization of IFNγ ELISPOT assay will facilitate interpretation of data from multicenter transplantation research studies and provide the foundation for developing clinical laboratory testing strategies to guide therapeutic decision-making in transplant patients.

Immunosuppressive effects of the traditional Chinese herb Qu Mai on human alloreactive T cells.

Current therapies for transplant rejection are suboptimally effective. In an effort to discover novel immunosuppressants we used cytokine ELISPOT and ELISAs to screen extracts from 53 traditional Chinese herbs for their ability to suppress human alloreactive T cells. We identified a dichloromethane-soluble fraction (Qu Mai fraction AD [QMAD]) of Qu Mai (Dianthus superbus) as a candidate. High-performance liquid chromatography (HPLC) analysis of QMAD revealed three dominant peaks, each with a MW ~600 Daltons and distinct from cyclosporine and rapamycin. When we added QMAD to human mixed lymphocyte cultures, we observed dose-dependent inhibition of proliferation and IFNγ production, by naïve and memory alloreactive T cells, and observed an increased frequency of Foxp3(+) CD4(+) T cells. To address whether QMAD induces regulatory T cells we added QMAD to anti-CD3/CD28-stimulated naïve CD4 T cells and observed a dose-dependent upregulation of Foxp3 associated with new suppressive capacity. Mechanistically, QMAD did not induce T cell IL-10 or TGFß but blocked T cell AKT phosphorylation, a key signaling nexus required for T cell proliferation and expansion, that simultaneously prevents Foxp3 transcription. Our findings provide novel insight into the antiinflammatory effects of one traditional Chinese herb, and support the need for continued isolation, characterization and testing of QMAD-derived components as immune suppressants for transplant rejection.

Anti-complement component C5 mAb synergizes with CTLA4Ig to inhibit alloreactive T cells and prolong cardiac allograft survival in mice.

While activation of serum complement mediates antibody-initiated vascular allograft injury, increasing evidence indicates that complement also functions as a modulator of alloreactive T cells. We tested whether blockade of complement activation at the C5 convertase step affects T cell-mediated cardiac allograft rejection in mice. The anti-C5 mAb BB5.1, which prevents the formation of C5a and C5b, synergized with subtherapeutic doses of CTLA4Ig to significantly prolong the survival of C57BL/6 heart grafts that were transplanted into naive BALB/c recipients. Anti-C5 mAb treatment limited the induction of donor-specific IFNγ-producing T cell alloimmunity without inducing Th2 or Th17 immunity in vivo and inhibited primed T cells from responding to donor antigens in secondary mixed lymphocyte responses. Additional administration of anti-C5 mAb to the donor prior to graft recovery further prolonged graft survival and concomitantly reduced both the in vivo trafficking of primed T cells into the transplanted allograft and decreased expression of T cell chemoattractant chemokines within the graft. Together these results support the novel concept that C5 blockade can inhibit T cell-mediated allograft rejection through multiple mechanisms, and suggest that C5 blockade may constitute a viable strategy to prevent and/or treat T cell-mediated allograft rejection in humans.

Immune reconstitution following rabbit antithymocyte globulin.

Depletional induction therapies are routinely used to prevent acute rejection and improve transplant outcome. The effects of depleting agents on T-cell subsets and subsequent T-cell reconstitution are incompletely defined. We used flow cytometry to examine the effects of rabbit antithymocyte globulin (rATG) on the peripheral T-cell repertoire of pediatric and adult renal transplant recipients. We found that while rATG effectively depleted CD45RA+CD27+ naïve and CD45RO+CD27+ central memory CD4+ T cells, it had little effect on CD45RO+CD27- CD4+ effector memory or CD45RA+CD31-, CD45RO+CD27+ and CD45RO+CD27- CD8+ T cell subsets. When we performed a kinetic analysis of CD31+ recent thymic emigrants and CD45RA+/RO+ T cells, we found evidence for both thymopoiesis and homeostatic proliferation contributing to immune reconstitution. We additionally examined the impact of rATG on peripheral CD4+Foxp3+ T cells. We found that in adults, administration of rATG-induced peripheral expansion and new thymic emigration of T cells with a Treg phenotype, while CD4+Foxp3+ T cells of thymic origin predominated in children, providing the first evidence that rATG induces Treg in vivo. Collectively our data indicate that rATG alters the balance of regulatory to memory effector T cells posttransplant, providing an explanation for how it positively impacts transplant outcome.

25-OH-vitamin D deficiency and cellular alloimmunity as measured by panel of reactive T cell testing in dialysis patients.

Primed antidonor alloreactive T cells are detrimental to transplant outcome, but factors that impact the strength of this immune response prior to transplantation are unknown. We tested peripheral blood mononuclear cells from dialysis patients, against panels of allogeneic, primary B-cell lines in a newly standardized IFNγ ELISPOT panel of reactive T cell (PRT) assay. Results were correlated with known alloantibody-sensitizing events and other clinical parameters. As 25-OH-vitamin D deficiency is associated with enhanced cellular immunity, is common in dialysis patients and is correctable, we assessed the relationship between serum 25-OH-vitamin D and the PRT. Using independent test and validation cohorts we found that low serum levels of 25-OH-vitamin D (<26 ng/mL) correlated with high-PRT values (in the upper 50th percentile, OR 0.02, p = 0.01) independent of age, sex, race, previous transplant, transfusion, pregnancy, time on dialysis, panel of reactive antibody, iPTH, and treatment with 1,25-OH-vitamin D. The data provide a potential mechanism for the possible relationship between vitamin D deficiency and poor posttransplant outcome, and support studies to test the impact of 25-OH-vitamin D repletion on alloimmunity and allograft injury in kidney transplant candidates.

Evolving paradigms that determine the fate of an allograft.

Despite the many advances in both immunological knowledge and the practical application of clinical immunosuppression, the holy grail of indefinite graft survival with immune tolerance in clinical solid organ transplantation remains a distant dream. The tremendous progress made in understanding the molecular and cellular basis of allograft rejection has not been translated into durable modalities that have advanced clinical care and outcomes. Indeed, currently used drugs and treatment protocols, largely directed at inhibiting alloreactive T cells, have not optimally improved allograft survival or function. A shift in emphasis, focusing on under appreciated immune pathways must now be considered to make further improvement. We highlight three areas of recent interest, complement, NK cells and lymphatics, which reinforce the concept that the transplant community must direct attention on how the immune system as a whole responds to a transplant. The current challenge is to integrate molecular, cellular and anatomic concepts to achieve the equivalent of a unified field theory of the immune response to organ transplants.