Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4⁺ regulatory T cells play a critical role in tolerance induction in transplantation. CD8⁺ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8⁺ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8⁺Foxp3⁺ T cells can be induced and significantly expanded by stimulating naïve CD8⁺ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8⁺Foxp3⁺ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8⁺Foxp3⁺ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8⁺Foxp3⁺ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4⁺Foxp3⁺ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8⁺Foxp3⁺ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.     

Delayed Anti‐CD3 Therapy Results in Depletion of Alloreactive T Cells and the Dominance of Foxp3+CD4+ Graft Infiltrating Cells

The engineered Fc‐nonbinding (crystallizable fragment‐nonbinding) CD3 antibody has lower mitogenicity and a precise therapeutic window for disease remission in patients with type 1 diabetes. Before anti‐CD3 can be considered for use in transplantation, the most effective timing of treatment relative to transplantation needs to be elucidated. In this study anti‐CD3F(ab′)₂ fragments or saline were administered intravenously for 5 consecutive days (early: d1–3 or delayed: d3–7) to mice transplanted with a cardiac allograft (H2^b‐to‐H2^k; d0). Survival of allografts was prolonged in mice treated with the early protocol (MST = 48 days), but most were rejected by d100. In contrast, in mice treated with the delayed protocol allografts continued to survive long term. The delayed protocol significantly inhibited donor alloreactivity at d30 as compared to the early protocol. A marked increase in Foxp3⁺ T cells (50.3 ± 1.6%) infiltrating the allografts in mice treated with the delayed protocol was observed (p < 0.0001 vs. early (24.9 ± 2.1%)) at d10; a finding that was maintained in the accepted cardiac allografts at d100. We conclude that the timing of treatment with anti‐CD3 therapy is critical for inducing long‐term graft survival. Delaying administration effectively inhibits the alloreactivity and promotes the dominance of intragraft Foxp3⁺ T cells allowing long‐term graft acceptance.     

Baseline Donor‐Specific Antibody Levels and Outcomes in Positive Crossmatch Kidney Transplantation

Renal transplant candidates with donor‐specific alloantibody (DSA) have increased risk of antibody‐mediated allograft injury. The goal of this study was to correlate the risk of antibody‐mediated rejection (AMR), transplant glomerulopathy (TG) and graft survival with the baseline DSA level (prior to initiation of pretransplant conditioning). These analyses include 119 positive crossmatch (+XM) compared to 70 negative crossmatch (?XM) transplants performed between April 2000 and July 2007. Using a combination of cell‐based crossmatch tests, DSA level was stratified into very high +XM, high +XM, low +XM and ?XM groups. In +XM transplants, increasing DSA level was associated with increased risk for AMR (HR = 1.76 [1.51, 2.07], p = 0.0001) but not TG (p = 0.18). We found an increased risk for both early and late allograft loss associated with very high DSA (HR = 7.71 [2.95, 20.1], p = 0.0001). Although lower DSA recipients commonly developed AMR and TG, allograft survival was similar to that of ?XM patients (p = 0.31). We conclude that the baseline DSA level correlates with risk of early and late alloantibody‐mediated allograft injury. With current protocols, very high baseline DSA patients have high rates of AMR and poor long‐term allograft survival highlighting the need for improved therapy for these candidates.     

Intragraft Regulatory T Cells in Protocol Biopsies Retain Foxp3 Demethylation and Are Protective Biomarkers for Kidney Graft Outcome

Presence of subclinical rejection (SCR) with IF/TA in protocol biopsies of renal allografts has been shown to be an independent predictor factor of graft loss. Also, intragraft Foxp3+ T_reg cells in patients with SCR has been suggested to differentiate harmful from potentially protective infiltrates. Nonetheless, whether presence of Foxp3 T_reg cells in patients with SCR and IF/TA may potentially protect from a deleterious graft outcome has not yet been evaluated. This is a case‐control study in which 37 patients with the diagnosis of SCR and 68 control patients with no cellular infiltrates at 6‐month protocol biopsies matched for age and time of transplantation were evaluated. We first confirmed that numbers of intragraft Foxp3‐expressing T cells in patients with SCR positively correlates with Foxp3 demethylation at the T_reg‐specific demethylation region. Patients with SCR without Foxp3+ T_reg cells within graft infiltrates showed significantly worse 5‐year graft function evolution than patients with SCR and Foxp3+ T_reg cells and those without SCR. When presence of SCR and IF/TA were assessed together, presence of Foxp3+ T_reg could discriminate a subgroup of patients showing the same graft outcome as patients with a normal biopsy. Thus, presence of Foxp3+ T_reg cells in patients with SCR even with IF/TA is associated with a favorable long‐term allograft outcome.     

Kidney Intragraft Donor‐Specific Antibodies as Determinant of Antibody‐Mediated Lesions and Poor Graft Outcome

Allograft pathology, antibody–tissue interaction as demonstrated by C4d deposition and serological evidence of donor‐specific antibodies (DSA) are the cardinal diagnostic features of antibody‐mediated lesions (AML) in kidney transplantation. However, discrepancy between histological and serological findings is common, and more reliable diagnostic tools are called for. Here, we asked whether the in situ detection of DSA could serve as marker for AML. To that end, we applied the anti‐HLA single antigen flow bead assay to eluates from 51 needle core graft biopsies performed for cause. Intragraft antibody profiles were correlated to serum DSA (sDSA), histological data and transplant outcome. The prevalence and the mean number of intragraft DSA (gDSA) were lower than that of sDSA (15/51 gDSA+ vs. 37/51 sDSA+ patients; 1.64 gDSA vs. 2.24 sDSA per patient). DSA were detected in all anti‐HLA antibody‐positive biopsies (15/15). The presence of gDSA was significantly associated with (1) microcirculation lesions taken individually (g, cg) and analyzed in functional clusters (ptc + g + cg > 0, cg + mm > 0), (2) C4d positivity and (3) a worse short‐term transplant outcome (p = 0.05). These associations were not found for patients presenting only sDSA. Taken together, these results indicate that gDSA is a severity marker of antibody‐mediated pathogenic process.     

Donor‐Specific HLA Antibodies in a Cohort Comparing Everolimus With Cyclosporine After Kidney Transplantation

Donor‐specific HLA antibodies (DSA) have a negative impact on kidney graft survival. Therefore, we analyzed the occurrence of DSA and antibody‐mediated rejection (AMR) in patients from two prospective randomized trials in our center. At 3–4.5 months posttransplant 127 patients were randomized to continue cyclosporine or converted to everolimus therapy. The presence of DSA was prospectively assessed using Luminex assays. AMR was defined according to the Banff 2009 classification. Antibody screening was available in 126 patients with a median follow‐up of 1059 days. Seven out of 65 (10.8%) patients on cyclosporine developed DSA after a median of 991 days. In comparison, 14/61 patients (23.0%) randomized to everolimus developed DSA after 551 days (log‐rank: p = 0.048). Eight patients on everolimus compared to two patients on cyclosporine developed AMR (log‐rank: p = 0.036). Four of 10 patients with AMR—all in the everolimus group—lost their graft. A multivariate regression model revealed everolimus, >3 mismatches and living donor as significant risk factors for DSA. Acute rejection within the first year, >3 mismatches, everolimus and living donor were independent risk factors for AMR. This single center analysis demonstrates for the first time that everolimus‐based immunosuppression is associated with an increased risk for the development of DSA and AMR.     

Significance of C4d Banff Scores in Early Protocol Biopsies of Kidney Transplant Recipients with Preformed Donor‐Specific Antibodies (DSA)

The significance of C4d‐Banff scores in protocol biopsies of kidney transplant recipients with preformed donor‐specific antibodies (DSA) has not been determined. We reviewed 157 protocol biopsies from 80 DSA+ patients obtained at 3 months and 1 year post‐transplant. The C4d Banff scores (1,2,3) were associated with significant increments of microcirculation inflammation (MI) at both 3 months and 1 year post‐transplant, worse transplant glomerulopathy and higher class II DSA‐MFI (p < 0.01). Minimal‐C4d had injury intermediate between negative and focal, while focal and diffuse‐C4d had the same degree of microvascular injury. A total of 54% of patients had variation of C4d score between 3 months and 1 year post‐transplant. Cumulative (3 month + 1 year) C4d scores correlated with long‐term renal function worsening (p = 0.006). However, C4d staining was not a sensitive indicator of parenchymal disease, 55% of C4d‐negative biopsies having evidence of concomitant MI. Multivariate analysis demonstrated that the presence of MI and class II DSA at 3 months were associated with a fourfold increased risk of progression to chronic antibody‐mediated rejection independently of C4d (p < 0.05). In conclusion, the substantial fluctuation of C4d status in the first year post‐transplant reflects a dynamic humoral process. However, C4d may not be a sufficiently sensitive indicator of activity, MI and DSA being more robust predictors of bad outcome.     

Evolution and Clinical Pathologic Correlations of De Novo Donor‐Specific HLA Antibody Post Kidney Transplant

The natural history for patients with de novo donor‐specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pretransplant DSA, with a mean follow‐up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA posttransplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years posttransplant. Independent predictors of dnDSA were HLA‐DRβ1 MM > 0 (OR 5.66, p < 0.006); and nonadherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p = 0.061). The median 10‐year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody‐mediated injury can occur and progress in patients with dnDSA in the absence of graft dysfunction and furthermore, nonadherence and cellular rejection contribute to dnDSA development and progression to graft loss.     

Pretransplant Donor‐Specific HLA Class‐I and ‐II Antibodies Are Associated With an Increased Risk for Kidney Graft Failure

Pretransplant risk assessment of graft failure is important for donor selection and choice of immunosuppressive treatment. We examined the relation between kidney graft failure and presence of IgG donor specific HLA antibodies (DSA) or C1q‐fixing DSA, detected by single antigen bead array (SAB) in pretransplant sera from 837 transplantations. IgG‐DSA were found in 290 (35%) sera, whereas only 30 (4%) sera had C1q‐fixing DSA. Patients with both class‐I plus ‐II DSA had a 10 yr graft survival of 30% versus 72% in patients without HLA antibodies (p < 0.001). No significant difference was observed in graft survival between patients with or without C1q‐fixing DSA. Direct comparison of both assays showed that high mean fluorescence intensity values on the pan‐IgG SAB assay are generally related to C1q‐fixation. We conclude that the presence of class‐I plus ‐II IgG DSA as detected by SAB in pretransplant sera of crossmatch negative kidney recipients is indicative for an increased risk for graft failure, whereas the clinical significance of C1q‐fixing IgG‐DSA could not be assessed due to their low prevalence.     

Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation

Short‐term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen‐related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end‐stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3⁺CD4⁺CD25⁺CD127^lo surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg‐specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.     

Intragraft CD11b+IDO+ Cells Mediate Cardiac Allograft Tolerance by ECDI‐Fixed Donor Splenocyte Infusions

We have previously shown that pre‐ and post‐transplant infusions of donor splenocytes treated with 1‐ethyl‐3‐(3’‐dimethylaminopropyl)‐carbodiimide (ECDI‐SPs) provide permanent donor‐specific protection of islet allografts. The efficacy of donor ECDI‐SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI‐SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b⁺IDO⁺ cells in the cardiac allograft, and that the presence of this population is dependent on Gr1⁺ cells. Consequently, depletion of Gr1⁺ cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI‐SPs infusions. In addition, T cells from ECDI‐SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI‐SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI‐SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

Role of IFNγ in Allograft Tolerance Mediated by CD4+CD25+ Regulatory T Cells by Induction of IDO in Endothelial Cells

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.     

Pretransplant Interferon‐γ Secretion by CMV‐Specific CD8+ T Cells Informs the Risk of CMV Replication After Transplantation

In this prospective study we analyzed pretransplant interferon‐γ secretion by cytomegalovirus (CMV)‐specific CD8+ T cells to assess its possible utility in determining the risk of CMV replication after solid organ transplantation. A total of 113 lung and kidney transplant patients were enrolled in the study but only 55 were evaluable. All CMV‐seronegative recipients were pretransplant “nonreactive” (IFNγ <0.2 IU/mL) (11/11), whereas 30/44 (68.2%) CMV‐seropositive (R+) recipients were “reactive” (IFNγ ≥0.2 IU/mL) and 14/44 (31.8%) were “nonreactive”. In the R(+) “nonreactive” group, 7/14 (50%) developed posttransplant CMV replication, whereas the virus replicated only in 4/30 (13.3%) of the R(+) “reactive” patients (p = 0.021). According to the best multivariate model, pretransplant “nonreactive” recipients receiving an organ from a CMV‐seropositive donor had a 10‐fold increased risk of CMV replication compared to pretransplant “reactive” recipients (adjusted OR 10.49, 95% CI 1.88–58.46). This model displayed good discrimination ability (AUC 0.80) and calibration (Hosmer–Lemeshow test, p = 0.92). Negative and positive predictive values were 83.7% and 75%, respectively. The accuracy of the model was 82%. Therefore, assessment of interferon‐γ secretion by cytomegalovirus (CMV)‐specific CD8+ T cells prior to transplantation is useful in informing the risk of posttransplant CMV replication in solid organ transplant patients.     

The Jak Inhibitor CP‐690,550 Preserves the Function of CD4+CD25brightFoxP3+ Regulatory T Cells and Inhibits Effector T Cells

The Jak inhibitor CP‐690,550 inhibits alloreactivity and is currently being investigated for prevention of allograft rejection after transplantation. In this study, we examined the effect of CP‐690,550 on IL‐2‐mediated Jak/STAT5 phosphorylation by CD4⁺CD25^brightFoxP3⁺CD127^?/low T cells (Treg) and CD4⁺CD25^neg effector T cells (Teff) in kidney transplant (KTx) patients. Phosphospecific flow cytometry was used to study the effect of CP‐690,550 on IL‐2‐induced intracellular STAT5‐phosphorylation. IL‐2‐induced phosphorylation of STAT5 (P‐STAT5) in both Treg and Teff, which was significantly higher for CD4⁺CD25^bright Treg (increased by 71%, mean) than for CD4⁺CD25^neg Teff (increased by 42%). In the presence of 100 ng/mL CP‐690,550, a clinically relevant exposure, IL‐2‐induced P‐STAT5 was partially inhibited in CD4⁺CD25^brightTreg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC₅₀ was 2–3 times higher for Treg (104 ng/mL) than for Teff (40 ng/mL, p = 0.02). In the presence of CP‐690,550, Treg exhibited additional suppressive activities on the alloactivated proliferation of Teff (56%, mean). In addition, CD4⁺CD25^bright Treg from KTx‐patients receiving CP‐690,550 vigorously suppressed the proliferation of Teff (87%, mean). Our findings show that CP‐690,550 effectively inhibits Teff function but preserves the suppressive activity of CD4⁺CD25^bright regulatory T cells.     

Acquisition of C3d‐Binding Activity by De Novo Donor‐Specific HLA Antibodies Correlates With Graft Loss in Nonsensitized Pediatric Kidney Recipients

Alloantibody‐mediated graft injury is a major cause of kidney dysfunction and loss. The complement‐binding ability of de novo donor‐specific antibodies (dnDSAs) has been suggested as a prognostic tool to stratify patients for clinical risk. In this study, we analyzed posttransplant kinetics of complement‐fixing dnDSAs and their role in antibody‐mediated rejection development and graft loss. A total of 114 pediatric nonsensitized recipients of first kidney allograft were periodically monitored for dnDSAs using flow bead assays, followed by C3d and C1q assay in case of positivity. Overall, 39 patients developed dnDSAs, which were C1q⁺ and C3d⁺ in 25 and nine patients, respectively. At follow‐up, progressive acquisition over time of dnDSA C1q and C3d binding ability, within the same antigenic specificity, was observed, paralleled by an increase in mean fluorescence intensity that correlated with clinical outcome. C3d‐fixing dnDSAs were better fit to stratify graft loss risk when the different dnDSA categories were evaluated in combined models because the 10‐year graft survival probability was lower in patients with C3d‐binding dnDSA than in those without dnDSAs or with C1q⁺/C3d^? or non‐complement‐binding dnDSAs (40% vs. 94%, 100%, and 100%, respectively). Based on the kinetics profile, we favor dnDSA removal or modulation at first confirmed positivity, with treatment intensification guided by dnDSA biological characteristics.     

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4⁺CD25⁺Foxp3⁺ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4⁺CD25⁺Foxp3⁺ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG^?/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4⁺CD25⁺Foxp3⁺ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4⁺CD25⁺Foxp3⁺ iTregs were further able to induce endogenous naïve T cells to convert to CD4⁺CD25⁺Foxp3⁺ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro‐generated donor‐specific CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.