Early barriers to neonatal porcine islet engraftment in a dual transplant model.

Porcine islet xenografts have the potential to provide an inexhaustible source of islets for ß cell replacement. Proof-of-concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood-mediated inflammatory reaction and a thorough understanding of early xeno-specific immune responses. A paucity of data exist comparing xeno-specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1 hour, 24 hours, and 7 days. Within the first 24 hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])-positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24 hours compared with 1 hour in both NPI (wild-type) and AIs. At 7 days, IgM and macrophages were highly specific for NPIs (α1,3-galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.

Belatacept-Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells.

Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28-CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept- or a tacrolimus-based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28+ CD8+ TEMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28+ CD8+ TEMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28- . These data suggest that CD28+ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28+ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation-independent rejection.

Lessons Learned: Early Termination of a Randomized Trial of Calcineurin Inhibitor and Corticosteroid Avoidance Using Belatacept.

The intent of this National Institutes of Health-sponsored study was to compare a belatacept-based immunosuppressive regimen with a maintenance regimen of tacrolimus and mycophenolate. Nineteen primary, Epstein-Barr virus-immune renal transplant recipients with a negative cross-match were randomized to one of three groups. All patient groups received perioperative steroids and maintenance mycophenolate mofetil. Patients in groups 1 and 2 were induced with alemtuzumab and maintained on tacrolimus or belatacept, respectively. Patients in group 3 were induced with basiliximab, received 3 mo of tacrolimus, and maintained on belatacept. There was one death with a functioning allograft due to endocarditis (group 1). There were three graft losses due to vascular thrombosis (all group 2) and one graft loss due to glomerular disease (group 1). Biopsy-proven acute cellular rejection was more frequent in the belatacept-treated groups, with 10 treated episodes in seven participants compared with one episode in group 1; however, estimated GFR was similar between groups at week 52. There were no episodes of posttransplant lymphoproliferative disorder or opportunistic infections in any group. Protocol enrollment was halted prematurely because of a high rate of serious adverse events. Such negative outcomes pose challenges to clinical investigators, who ultimately must weigh the risks and benefits in randomized trials.

Thrombalexin: Use of a Cytotopic Anticoagulant to Reduce Thrombotic Microangiopathy in a Highly Sensitized Model of Kidney Transplantation.

Early activation of coagulation is an important factor in the initiation of innate immunity, as characterized by thrombotic microangiopathy (TMA). In transplantation, systemic anticoagulation is difficult due to bleeding. A novel "cytotopic" agent, thrombalexin (TLN), combines a cell-membrane-bound (myristoyl tail) anti-thrombin (hirudin-like peptide [HLL]), which can be perfused directly to the donor organ or cells. Thromboelastography was used to measure time to clot formation (r-time) in both rhesus and human blood, comparing TLN versus HLL (without cytotopic tail) versus negative control. Both TLN- and HLL-treated rhesus or human whole blood result in significantly prolonged r-time compared to kaolin controls. Only TLN-treated human endothelial cells and neonatal porcine islets prolonged time to clot formation. Detection of membrane-bound TLN was confirmed by immunohistochemistry and fluorescence activated cell sorter. In vivo, perfusion of a nonhuman primate kidney TLN-supplemented preservation solution in a sensitized model of transplantation demonstrated no evidence of TLN systemically. Histologically, TLN was shown to be present up to 4 days after transplantation. There was no platelet deposition, and TMA severity, as well as microvascular injury scores (glomerulitis + peritubular capillaritis), were less in the TLN-treated animals. Despite promising evidence of localized efficacy, no survival benefit was demonstrated.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model.

Costimulation blockade (CoB) via belatacept is a lower-morbidity alternative to calcineurin inhibitor (CNI)-based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept and increased adhesion molecule expression. One such molecule is leukocyte function antigen (LFA)-1. LFA-1 exists in two forms: a commonly expressed, low-affinity form and a transient, high-affinity form, expressed only during activation. We have shown that antibodies reactive with LFA-1 regardless of its configuration are effective in eliminating memory T cells but at the cost of impaired protective immunity. Here we test two novel agents, leukotoxin A and AL-579, each of which targets the high-affinity form of LFA-1, to determine whether this more precise targeting prevents belatacept-resistant rejection. Despite evidence of ex vivo and in vivo ligand-specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity before efficacy, while AL-579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA-1 blockade may not be a suitable adjuvant agent for CoB-resistant rejection.

CD57(+) CD4 T Cells Underlie Belatacept-Resistant Allograft Rejection.

Belatacept is a B7-specific fusion protein used to prevent allograft rejection by blocking T cell costimulation. Generally efficacious, it fails to prevent acute rejection in a sizable minority of patients. In experimental models, memory T cells mediate costimulation blockade-resistant rejection (CoBRR), but this remains undefined in humans. To explore relationships between individual patients' immune cell phenotypes and CoBRR, we studied patients receiving belatacept or conventional calcineurin inhibitor-based immunosuppression. We identified a population of CD57(+) PD1(-) CD4 T cells present prior to transplantation that correlated with CoBRR. Contrary to data recognizing CD57 as a marker of senescence on CD8 T cells, we discovered a nonsenescent, cytolytic phenotype associated with CD57 on CD4 T cells. Moreover, CD57(+) CD4 T cells expressed high levels of adhesion molecules implicated in experimental CoBRR, were CD28(-) , expressed a transcriptional phenotype broadly defining allograft rejection and were shown to be present in rejecting human kidney allografts. These data implicate CD57(+) CD4 T cells in clinical CoBRR. If prospectively validated, this characteristic could identify patients at higher risk for acute rejection on belatacept-based therapy.

Antibody-Mediated Rejection in Sensitized Nonhuman Primates: Modeling Human Biology.

We have established a model of sensitization in nonhuman primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, and donor-specific antibody (DSA) response was monitored by flow cross-match. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate, and methylprednisolone. Three animals received basiliximab induction; compared with nonsensitized animals, they showed a shorter mean survival time (4.7 ± 3.1 vs. 187 ± 88 days). Six animals were treated with T cell depletion (anti-CD4/CD8 mAbs), which prolonged survival (mean survival time 21.6 ± 19.0 days). All presensitized animals showed antibody-mediated rejection (AMR). In two of three basiliximab-injected animals, cellular rejection (ACR) was prominent. After T cell depletion, three of six monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three monkeys survived 27-44 days, with mixed AMR and ACR. Most T cell-depleted animals experienced a rebound of DSA that correlated with deteriorating kidney function. We also found an increase in proliferating memory B cells (CD20(+) CD27(+) IgD(-) Ki67(+) ), lymph node follicular helper T cells (ICOS(+) PD-1(hi) CXCR5(+) CD4(+) ), and germinal center (GC) response. Depletion controlled cell-mediated rejection in sensitized nonhuman primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies.

Studies Introducing Costimulation Blockade for Vascularized Composite Allografts in Nonhuman Primates.

Vascularized composite allografts (VCAs) are technically feasible. Similar to other organ transplants, VCAs are hampered by the toxicity and incomplete efficacy associated with conventional immunosuppression. Complications attributable to calcineurin inhibitors remain prevalent in the clinical cases reported to date, and these loom particularly large given the nonlifesaving nature of VCAs. Additionally, acute rejection remains almost ubiquitous, albeit controllable with current agents. Costimulation blockade offers the potential to provide prophylaxis from rejection without the adverse consequences of calcineurin-based regimens. In this study, we used a nonhuman-primate model of VCA in conjunction with immunosuppressive regimens containing combinations of B7-specific costimulation blockade with and without adhesion blockade with LFA3-Ig to determine what adjunctive role these agents could play in VCA transplantation when combined with more conventional agents. Compared to tacrolimus, the addition of belatacept improved rejection free allograft survival. The combination with LFA3-Ig reduced CD2(hi) memory T cells, however did not provide additional protection against allograft rejection and hindered protective immunity. Histology paralleled clinical histopathology and Banff grading. These data provide the basis for the study of costimulation blockade in VCA in a relevant preclinical model.

Dual islet transplantation modeling of the instant blood-mediated inflammatory reaction.

Islet xenotransplantation is a potential treatment for diabetes without the limitations of tissue availability. Although successful experimentally, early islet loss remains substantial and attributed to an instant blood-mediated inflammatory reaction (IBMIR). This syndrome of islet destruction has been incompletely defined and characterization in pig-to-primate models has been hampered by logistical and statistical limitations of large animal studies. To further investigate IBMIR, we developed a novel in vivo dual islet transplant model to precisely characterize IBMIR as proof-of-concept that this model can serve to properly control experiments comparing modified xenoislet preparations. WT and α1,3-galactosyltransferase knockout (GTKO) neonatal porcine islets were studied in nonimmunosuppressed rhesus macaques. Inert polyethylene microspheres served as a control for the effects of portal embolization. Digital analysis of immunohistochemistry targeting IBMIR mediators was performed at 1 and 24 h after intraportal islet infusion. Early findings observed in transplanted islets include complement and antibody deposition, and infiltration by neutrophils, macrophages and platelets. Insulin, complement, antibody, neutrophils, macrophages and platelets were similar between GTKO and WT islets, with increasing macrophage infiltration at 24 h in both phenotypes. This model provides an objective and internally controlled study of distinct islet preparations and documents the temporal histology of IBMIR.

A pilot trial targeting the ICOS-ICOS-L pathway in nonhuman primate kidney transplantation.

Costimulation blockade with the B7-CD28 pathway-specific agent belatacept is now used in clinical kidney transplantation, but its efficacy remains imperfect. Numerous alternate costimulatory pathways have been proposed as targets to synergize with belatacept, one of which being the inducible costimulator (ICOS)-ICOS ligand (ICOS-L) pathway. Combined ICOS-ICOS-L and CD28-B7 blockade has been shown to prevent rejection in mice, but has not been studied in primates. We therefore tested a novel ICOS-Ig human Fc-fusion protein in a nonhuman primate (NHP) kidney transplant model alone and in combination with belatacept. ICOS-Ig did not prolong rejection-free survival as a monotherapy or in combination with belatacept. In ICOS-Ig alone treated animals, most graft-infiltrating CD4(+) and CD8(+) T cells expressed ICOS, and ICOS(+) T cells were present in peripheral blood to a lesser degree. Adding belatacept reduced the proportion of graft-infiltrating ICOS(+) T cells and virtually eliminated their presence in peripheral blood. Graft-infiltrating T cells in belatacept-resistant rejection were primarily CD8(+) CD28(-) , but importantly, very few CD8(+) CD28(-) T cells expressed ICOS. We conclude that ICOS-Ig, alone or combined with belatacept, does not prolong renal allograft survival in NHPs. This may relate to selective loss of ICOS with CD28 loss.

Neutralizing BAFF/APRIL with atacicept prevents early DSA formation and AMR development in T cell depletion induced nonhuman primate AMR model.

Depletional strategies directed toward achieving tolerance induction in organ transplantation have been associated with an increased incidence and risk of antibody-mediated rejection (AMR) and graft injury. Our clinical data suggest correlation of increased serum B cell activating factor/survival factor (BAFF) with increased risk of antibody-mediated rejection in alemtuzumab treated patients. In the present study, we tested the ability of BAFF blockade (TACI-Ig) in a nonhuman primate AMR model to prevent alloantibody production and prolong allograft survival. Three animals received the AMR inducing regimen (CD3-IT/alefacept/tacrolimus) with TACI-Ig (atacicept), compared to five control animals treated with the AMR inducing regimen only. TACI-Ig treatment lead to decreased levels of DSA in treated animals at 2 and 4 weeks posttransplantation (p < 0.05). In addition, peripheral B cell numbers were significantly lower at 6 weeks posttransplantation. However, it provided only a marginal increase in graft survival (59 ± 22 vs. 102 ± 47 days; p = 0.11). Histological analysis revealed a substantial reduction in findings typically associated with humoral rejection with atacicept treatment. More T cell rejection findings were observed with increased graft T cell infiltration in atacicept treatment, likely secondary to the graft prolongation. We show that BAFF/APRIL blockade using concomitant TACI-Ig treatment reduced the humoral portion of rejection in our depletion-induced preclinical AMR model.

Banff fibrosis study: multicenter visual assessment and computerized analysis of interstitial fibrosis in kidney biopsies.

Increasing interstitial fibrosis (IF) in native and kidney transplant biopsies is associated with functional decline and serves as a clinical trial end point. A Banff 2009 Conference survey revealed a range in IF assessment practices. Observers from multiple centers were asked to assess 30 renal biopsies with a range of IF and quantitate IF using two approaches on trichrome, Periodic acid-Schiff (PAS) and computer-assisted quantification of collagen III immunohistochemistry (C-IHC) slides, as well as assessing percent of cortical tubular atrophy% (TA%) and Banff total cortical inflammation score (ti-score). C-IHC using whole slide scans was performed. C-IHC assessment showed a higher correlation with organ function (r = -0.48) than did visual assessments (r = -0.32--0.42); computerized and visual C-IHC assessment also correlated (r = 0.64-0.66). Visual assessment of trichrome and C-IHC showed better correlations with organ function and C-IHC, than PAS, TA% and ti-score. However, visual assessment of IF, independent of approach, was variable among observers, and differences in correlations with organ function were not statistically significant among C-IHC image analysis and visual assessment methods. C-IHC image analysis correlated among three centers (r > 0.90, p < 0.0001, between all centers). Given the difficulty of visual IF assessment standardization, C-IHC image could potentially accomplish standardized IF assessment in multicenter settings.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection.

De novo donor-specific antibody (DSA) after organ transplantation promotes antibody-mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti-CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti-CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM(+) B cells, bela- and 2C10R4-added groups maintained a predominant population of IgM(+) B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4(+) CD28(+) CD95(+)) as well as PD-1(hi) CD4(+) T cells were decreased in both bela-added and 2C10R4-added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC-follicular helper T (Tfh) cells, and IL-21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.

Inhibition of αvß6 promotes acute renal allograft rejection in nonhuman primates.

The integrin αvß6 activates latent transforming growth factor-ß (TGF-ß) within the kidney and may be a target for the prevention of chronic allograft fibrosis after kidney transplantation. However, TGF-ß also has known immunosuppressive properties that are exploited by calcineurin inhibitors (CNIs); thus, the net benefit of αvß6 inhibition remains undetermined. To assess the acute impact of interference with αvß6 on acute rejection, we tested a humanized αvß6-specific monoclonal antibody (STX-100) in a randomized, double-blinded, placebo-controlled nonhuman primate renal transplantation study to evaluate whether αvß6 blockade alters the risk of acute rejection during CNI-based immunosuppression. Rhesus monkeys underwent renal allotransplantation under standard CNI-based maintenance immunosuppression; 10 biopsy-confirmed rejection-free animals were randomized to receive weekly STX-100 or placebo. Animals treated with STX-100 experienced significantly decreased rejection-free survival compared to placebo animals (p = 0.049). Immunohistochemical analysis confirmed αvß6 ligand presence, and αvß6 staining intensity was lower in STX-100-treated animals (p = 0.055), indicating an apparent blockade effect of STX-100. LAP, LTBP-1 and TGF-ß were all decreased in animals that rejected on STX-100 compared to those that rejected on standard immunosuppression alone, suggesting a relevant effect of αvß6 blockade on local TGF-ß. These data caution against the use of αvß6 blockade to achieve TGF-ß inhibition in kidney transplantation.

Belatacept and sirolimus prolong nonhuman primate renal allograft survival without a requirement for memory T cell depletion.

Belatacept is an inhibitor of CD28/B7 costimulation that is clinically indicated as a calcineurin inhibitor (CNI) alternative in combination with mycophenolate mofetil and steroids after renal transplantation. We sought to develop a clinically translatable, nonlymphocyte depleting, belatacept-based regimen that could obviate the need for both CNIs and steroids. Thus, based on murine data showing synergy between costimulation blockade and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched renal allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on costimulation blockade-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Belatacept and sirolimus therapy successfully prevented rejection in all animals. Tolerance was not induced, as animals rejected after withdrawal of therapy. The regimen did not deplete T cells. Alefecept did not add a survival benefit to the optimized belatacept and sirolimus regimen, despite causing an intended depletion of memory T cells, and caused a marked reduction in regulatory T cells. Furthermore, alefacept-treated animals had a significantly increased incidence of CMV reactivation, suggesting that this combination overly compromised protective immunity. These data support belatacept and sirolimus as a clinically translatable, nondepleting, CNI-free, steroid-sparing immunomodulatory regimen that promotes sustained rejection-free allograft survival after renal transplantation.

Patterns of de novo allo B cells and antibody formation in chronic cardiac allograft rejection after alemtuzumab treatment.

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody-mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti-human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody-mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody-mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long-term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor-specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection.

Evidence for kidney rejection after combined bone marrow and renal transplantation despite ongoing whole-blood chimerism in rhesus macaques.

Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism-induced tolerance are not clearly elucidated. To address this, we used an MHC-defined primate model to determine the impact of impermanent, T cell-poor, mixed-chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant ("BMT/renal") and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40-directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole-blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28-/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28-negative Tem and costimulation blockade-resistant rejection. These results suggest that in some settings, transient T cell-poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.