Combined Anti‐CD154/CTLA4Ig Costimulation Blockade‐Based Therapy Induces Donor‐Specific Tolerance to Vascularized Osteomyocutaneous Allografts

Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor‐derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2^d) mice were transplanted into C57BL/6 (H2^b) recipients. Immunosuppression consisted of 1 mg anti‐CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0–7, then every other day for 3 weeks). Long‐term allograft survival, donor‐specific tolerance and donor–recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long‐term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor‐specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor‐specific tolerance was further assessed in vivo by secondary donor‐specific skin graft survival and third‐party graft rejection. A significant increase of Foxp3⁺ regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti‐CD154/CTLA4Ig costimulation blockade‐based therapy induces donor‐specific tolerance in a stringent murine alloOMC transplant model.     

The influence of CTLA‐4 single nucleotide polymorphisms on acute kidney allograft rejection in Turkish patients

Cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) is a cell surface protein, which down‐regulates the immune response at CTLA‐4/CD28/B7 pathway. We aimed to investigate the influence of the ?318C/T, +49A/G, ?1661A/G and CT60A/G, and CTLA‐4 gene polymorphisms on acute rejection of kidney allograft in Turkish patients. The study design was a case–control study that consists of three groups: Group 1 (n = 34) represented the kidney transplant (Ktx) recipients who experienced acute rejection, Group 2 (n = 47) was randomly assigned Ktx recipients without acute rejection, and Group 3 (n = 50) consisting of healthy volunteers to evaluate the normal genomic distribution. The polymerase chain reaction–restriction fragment length polymorphism technique was used to determine the polymorphisms. Genotype and allele frequencies among three groups denoted similar distributions for +49A/G, ?1661A/G, and CT60A/G. Conversely, ?318C/T genotype was three times more frequent in the acute rejection group than in the non‐rejection group (OR = 3.45; 95%CI = 1.18–10.1, p = 0.015) and two times more frequent than the healthy control group (OR = 2.45; 95% CI = 0.98 – 6.11, p = 0.047). Additionally, having a T allele at ?318 position was significantly associated with acute rejection (0.147 vs. 0.043, OR = 3.45; 95% CI = 1.13–10.56, p = 0.02). 318C/T gene polymorphism and T allelic variant were found to be associated with increased acute rejection risk in Turkish kidney allograft recipients.     

Rapamycin Interferes With Postdepletion Regulatory T Cell Homeostasis and Enhances DSA Formation Corrected by CTLA4‐Ig

Previously, we demonstrated that alemtuzumab induction with rapamycin as sole maintenance therapy is associated with an increased incidence of humoral rejection in human kidney transplant patients. To investigate the role of rapamycin in posttransplant humoral responses after T cell depletion, fully MHC mismatched hearts were transplanted into hCD52Tg mice, followed by alemtuzumab treatment with or without a short course of rapamycin. While untreated hCD52Tg recipients acutely rejected B6 hearts (n = 12), hCD52Tg recipients treated with alemtuzumab alone or in conjunction with rapamycin showed a lack of acute rejection (MST > 100). However, additional rapamycin showed a reduced beating quality over time and increased incidence of vasculopathy. Furthermore, rapamycin supplementation showed an increased serum donor‐specific antibodies (DSA) level compared to alemtuzumab alone at postoperation days 50 and 100. Surprisingly, additional rapamycin treatment significantly reduced CD4⁺CD25⁺FoxP3⁺ T reg cell numbers during treatment. On the contrary, ICOS⁺PD‐1⁺CD4 follicular helper T cells in the lymph nodes were significantly increased. Interestingly, CTLA4‐Ig supplementation in conjunction with rapamycin corrected rapamycin‐induced accelerated posttransplant humoral response by directly modulating Tfh cells but not Treg cells. This suggests that rapamycin after T cell depletion could affect Treg cells leading to an increase of Tfh cells and DSA production that can be reversed by CTLA4‐Ig.     

Efficacy and Safety of Everolimus and Mycophenolic Acid With Early Tacrolimus Withdrawal After Liver Transplantation: A Multicenter Randomized Trial

SIMCER was a 6‐mo, multicenter, open‐label trial. Selected de novo liver transplant recipients were randomized (week 4) to everolimus with low‐exposure tacrolimus discontinued by month 4 (n = 93) or to tacrolimus‐based therapy (n = 95), both with basiliximab induction and enteric‐coated mycophenolate sodium with or without steroids. The primary end point, change in estimated GFR (eGFR; MDRD formula) from randomization to week 24 after transplant, was superior with everolimus (mean eGFR change +1.1 vs. ?13.3 mL/min per 1.73 m² for everolimus vs. tacrolimus, respectively; difference 14.3 [95% confidence interval 7.3–21.3]; p < 0.001). Mean eGFR at week 24 was 95.8 versus 76.0 mL/min per 1.73 m² for everolimus versus tacrolimus (p < 0.001). Treatment failure (treated biopsy‐proven acute rejection [BPAR; rejection activity index score >3], graft loss, or death) from randomization to week 24 was similar (everolimus 10.0%, tacrolimus 4.3%; p = 0.134). BPAR was more frequent between randomization and month 6 with everolimus (10.0% vs. 2.2%; p = 0.026); the rate of treated BPAR was 8.9% versus 2.2% (p = 0.055). Sixteen everolimus‐treated patients (17.8%) and three tacrolimus‐treated patients (3.2%) discontinued the study drug because of adverse events. In conclusion, early introduction of everolimus at an adequate exposure level with gradual calcineurin inhibitor (CNI) withdrawal after liver transplantation, supported by induction therapy and mycophenolic acid, is associated with a significant renal benefit versus CNI‐based immunosuppression but more frequent BPAR.     

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade‐induced murine allograft survival

Costimulatory blockade‐induced murine cardiac allograft survival requires intragraft accumulation of CD11b⁺Ly6C^loLy6G^? regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60 days in anti‐CD154 (MR1)‐treated or cytotoxic T‐lymphocyte associated protein 4 (CTLA4)‐Ig–treated wild‐type (WT) recipients, they were rejected at ~30 days in MR1‐treated or CTLA4‐Ig–treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1^fl/flxLysM‐Cre). This accelerated rejection was associated with ~2‐fold more donor‐reactive T cells and ~40% less expansion of donor‐reactive Tregs. Analysis of graft‐infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6C^lo monocytes in C5ar1^fl/flxLysM‐Cre recipients. Expression profiling of intragraft Ly6C^lo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1^fl/fl and C5ar1^fl/flxLysM‐Cre myeloid cells into MR1‐treated allograft recipients resulted in less accumulation of C5ar1^?/? cells within the allografts, and in vitro assays confirmed that Ly6C^hi myeloid cells migrate to C5a/C5aR1‐initiated signals. Together, our results newly link myeloid cell–expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.     

Renal Transplantation With Final Allocation Based on the Virtual Crossmatch

Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However, the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor–recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long‐term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor‐specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within 1 year (AR <1 year) for FCXM+ (n = 54) and FCXM? (n = 454) recipients. Median follow‐up is 7.1 years. FCXM+ recipients were significantly different from FCXM? recipients for the following risk factors: living donor (24% vs. 39%, p = 0.03), duration of dialysis (31.0 months vs. 13.5 months, p = 0.008), retransplants (17% vs. 7.3%, p = 0.04), % sensitized (63% vs. 19%, p = 0.001), and PRA >80% (20% vs. 4.8%, p = 0.001). Despite these differences, 5‐year actual graft survival rates are 87% and 84%, respectively. AR <1 year occurred in 13% FCXM+ and 12% FCXM? recipients. Crossmatch status was not associated with graft outcomes in any univariate or multivariate model. Renal transplantation can be performed successfully, using SPI as the definitive test for donor–recipient compatibility.     

Complement Independent Antibody‐Mediated Endarteritis and Transplant Arteriopathy in Mice

Complement fixation, as evidenced by C4d in the microvasculature, is a widely accepted criterion of antibody‐mediated rejection. Complement fixation has been shown to be essential in acute antibody‐mediated rejection, but its role in chronic rejection has not been addressed. Previous studies showed that passive transfer of complement fixing monoclonal IgG2a anti‐H‐2K^k into B6.RAG1^?/? KO recipients of B10.BR hearts led to progressive chronic transplant arteriopathy (CTA) over 14–28 days, accompanied by C4d deposition. The present studies were designed to test whether complement was required for these lesions. We report that a noncomplement fixing donor‐specific alloantibody (DSA, monoclonal IgG1 anti‐H‐2K^k) injected into B6.RAG1^‐/‐ KO recipients of B10.BR hearts also promotes CTA, without C4d deposition. Furthermore, a passive transfer of DSA (monoclonal IgG2a anti‐H‐2K^k) initiated endarteritis followed by CTA in B6.RAG1^?/‐ mice genetically deficient in the third component of complement (RAG1^?/?C3^?/?). These studies indicate that antibody to class I MHC antigens can trigger chronic arterial lesions in vivo without complement participation, in contrast to acute antibody‐mediated rejection. This pathway may be relevant to C4d‐negative chronic rejection sometimes observed in patients with DSA, and argues that lack of C4d deposition does not exclude antibody‐mediated chronic rejection.     

Peritransplant VLA‐4 blockade inhibits endogenous memory CD8 T cell infiltration into high‐risk cardiac allografts and CTLA‐4Ig resistant rejection

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC‐mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen‐4 (VLA‐4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti‐VLA‐4 mAb given to C57BL6 (H‐2^b) recipients of AJ (H‐2^a) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra‐allograft IFN‐γ‐induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA‐4Ig treated recipients. Anti‐VLA‐4 mAb also inhibited priming of donor‐specific T cells producing IFN‐γ until at least day 7 posttransplant. Peritransplant anti‐VLA plus anti‐CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti‐VLA‐4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.     

Macrophage subpopulations and their impact on chronic allograft rejection versus graft acceptance in a mouse heart transplant model

Macrophages infiltrating the allografts are heterogeneous, consisting of proinflammatory (M1 cells) as well as antiinflammatory and fibrogenic phenotypes (M2 cells); they affect transplantation outcomes via diverse mechanisms. Here we found that macrophage polarization into M1 and M2 subsets was critically dependent on tumor necrosis factor receptor–associated factor 6 (TRAF6) and mammalian target of rapamycin (mTOR), respectively. In a heart transplant model we showed that macrophage‐specific deletion of TRAF6 (LysM^CreTraf6 ^fl/fl) or mTOR (LysM^CreMtor^fl/fl) did not affect acute allograft rejection. However, treatment of LysM^CreMtor^fl/fl recipients with CTLA4‐Ig induced long‐term allograft survival (>100 days) without histological signs of chronic rejection, whereas the similarly treated LysM^CreTraf6 ^fl/fl recipients developed severe transplant vasculopathy (chronic rejection). The presentation of chronic rejection in CTLA4‐Ig–treated LysM^CreTraf6 ^fl/fl mice was similar to that of CTLA4‐Ig–treated wild‐type B6 recipients. Mechanistically, we found that the graft‐infiltrating macrophages in LysM^CreMtor^fl/fl recipients expressed high levels of PD‐L1, and that PD‐L1 blockade readily induced rejection of otherwise survival grafts in the LysM^CreMtor^fl/fl recipients. Our findings demonstrate that targeting mTOR‐dependent M2 cells is critical for preventing chronic allograft rejection, and that graft survival under such conditions is dependent on the PD‐1/PD‐L1 coinhibitory pathway.     

Deletion of the activating NK cell receptor NKG2D accelerates rejection of cardiac allografts

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co‐stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC‐disparate BALB/c‐derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1^?/?). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46⁺ NK cells in Klrk1^?/? recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4⁺, but a lesser infiltration of CD8⁺ T cell frequencies. Contrary to published observations, co‐stimulation blockade with CTLA4‐Ig resulted in a significant acceleration of cardiac rejection by Klrk1^?/? recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1^?/? recipients were characterized by significantly higher levels of interferon‐γ mRNA, and both CD4⁺ and CD8⁺ T cells displayed a greater capacity for degranulation and interferon‐γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.     

Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen

BACKGROUND: The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. METHODS: Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). RESULTS: ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). CONCLUSIONS: Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.     

Dynamics of anti‐human leukocyte antigen antibodies after renal transplantation and their impact on graft outcome

The purpose of this study was to sequentially monitor anti‐HLA antibodies and correlate the results with antibody‐mediated rejection (AMR), graft survival (GS), and graft function (GF). We collected sera from 111 kidney transplant recipients on transplant days 0, 7, 14, 30, 60, 90, 180, and 360 and analyzed PRA levels by ELISA. DSAs were analyzed by single‐antigen beads in rejecting kidneys. At pre‐transplant, 79.3% of the patients were non‐sensitized (PRA = 0%) and 20.7% were sensitized (PRA > 1%). After transplant, patients were grouped by PRA profile: no anti‐HLA antibodies pre‐ or post‐transplant (group HLApre?/post?; n = 80); de novo anti‐HLA antibodies post‐transplant (group HLApre?/post+; n = 8); sensitized pre‐transplant/increased PRA post‐transplant (group HLApre+/post↑; n = 9); and sensitized pre‐transplant/decreased PRA post‐transplant (group HLApre+/post↓; n = 14). De novo anti‐HLA antibodies were detected at 7–180 d. In sensitized patients, PRA levels changed within the first 30 d post‐transplant. Incidence of AMR was higher in HLApre?/post+ and HLApre+/post↑ than in HLApre?/post?, and HLApre+/post↓ (p < 0.001) groups. One‐yr death‐censored GS was 36% in group HLApre+/post↑, compared with 98%, 88% and 100% in groups HLApre?/post?, HLApre?/post+, and HLApre+/post↓, respectively (p < 0.001). Excluding first‐year graft losses, GF and GS were similar among the groups. In conclusion, post‐transplant antibody monitoring can identify recipients at higher risk of AMR.     

Glutamate Receptor Interacting Protein 1 Regulates CD4+ CTLA‐4 Expression and Transplant Rejection

PDZ domains are common 80‐ to 90‐amino‐acid regions named after the first three proteins discovered to share these domains: postsynaptic density 95, discs large, and zonula occludens. PDZ domain‐containing proteins typically interact with the C‐terminus of membrane receptors. Glutamate receptor interacting protein 1 (GRIP1), a seven–PDZ domain protein scaffold, regulates glutamate receptor surface expression and trafficking in neurons. We have found that human and mouse T cells also express GRIP1. T cell–specific GRIP1^?/? mice >11 weeks old had prolonged cardiac allograft survival. Compared with wild‐type T cells, in vitro stimulated GRIP1^?/? T cells had decreased expression of activation markers and increased apoptotic surface marker expression. Surface expression of the strong T cell inhibitory molecule cytotoxic T lymphocyte antigen‐4 (CTLA‐4) was increased on GRIP1^?/? T cells from mice >11 weeks old. CTLA‐4 increases with T cell stimulation and its surface expression on GRIP1^?/? T cells remained high after stimulation was removed, indicating a possible internalization defect in GRIP1‐deficient T cells. CTLA‐4–blocking antibody treatment following heart transplantation led to complete rejection in T cell GRIP1^?/? mice, indicating that increased CTLA‐4 surface expression contributed to the extended graft survival. Our data indicate that GRIP1 regulates T cell activation by regulating CTLA‐4 surface expression.     

Co‐localized immune protection using dexamethasone‐eluting micelles in a murine islet allograft model

The broad application of ß cell transplantation for type 1 diabetes is hindered by the requisite of lifelong systemic immunosuppression. This study examines the utility of localized islet graft drug delivery to subvert the inflammatory and adaptive immune responses. Herein, we have developed and characterized dexamethasone (Dex) eluting Food and Drug Administration‐approved micro‐Poly(lactic‐co‐glycolic acid) micelles and examined their efficacy in a fully major histocompatibility complex‐mismatch murine islet allograft model. A clinically relevant dose of 46.6 ± 2.8 μg Dex per graft was confirmed when 2 mg of micelles was implemented. Dex‐micelles + CTLA‐4‐Ig (n = 10) resulted in prolonged allograft function with 80% of the recipients demonstrating insulin independence for 60 days posttransplant compared to 40% in empty micelles + CTLA‐4‐Ig recipients (n = 10, P = .06). Recipients of this combination therapy (n = 8) demonstrated superior glucose tolerance profiles, compared to empty micelles + CTLA‐4‐Ig recipients (n = 4, P < .05), and significantly reduced localized intragraft proinflammatory cytokine expression. Histologically, increased insulin positive and FOXP3⁺ T cells were observed in Dex‐micelles + CTLA‐4‐Ig grafts compared to empty micelles + CTLA‐4‐Ig grafts (P < .01 and P < .05, respectively). Localized drug delivery via micelles elution has the potential to alter the inflammatory environment, enhances allograft survival, and may be an important adjuvant approach to improve clinical islet transplantation outcomes.     

In Vivo Function of Immune Inhibitory Molecule B7‐H4 in Alloimmune Responses

B7 ligands deliver both costimulatory and coinhibitory signals to the CD28 family of receptors on T lymphocytes, the balance between which determines the ultimate immune response. Although B7‐H4, a recently discovered member of the B7 family, is known to negatively regulate T cell immunity in autoimmunity and cancer, its role in solid organ allograft rejection and tolerance has not been established. Targeting the B7‐H4 molecule by a blocking antibody or use of B7‐H4^?/? mice as recipients of fully MHC‐mismatched cardiac allografts did not affect graft survival. However, B7‐H4 blockade resulted in accelerated allograft rejection in CD28‐deficient recipients. B7‐1/B7‐2‐double‐deficient recipients are truly independent of CD28/CTLA‐4:B7 signals and usually accept MHC‐mismatched heart allografts. Blockade of B7‐H4 in these mice also precipitated rejection, demonstrating regulatory function of this molecule independent of an intact CD28/CTLA‐4:B7 costimulatory pathway. Accelerated allograft rejection was always accompanied by increased frequencies of alloreactive IFN‐γ‐, IL‐4‐ and Granzyme B‐producing splenocytes. Finally, intact recipient, but not donor, B7‐H4 is essential for prolongation of allograft survival by blocking CD28/CTLA4:B7 pathway using CTLA4‐Ig. These data are the first to provide evidence of the regulatory effects of B7‐H4 in alloimmune responses in a murine model of solid organ transplantation.     

Selective CD28 Blockade Results in Superior Inhibition of Donor‐Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4‐Ig

Donor‐specific antibodies (DSAs) are a barrier to improved long‐term outcomes after kidney transplantation. Costimulation blockade with CTLA4‐Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4⁺ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8⁺ T cells relative to CTLA4‐Ig, but the impact of CD28‐specific blockade on CD4⁺ Tfh cells is unknown. Thus, we identified and characterized donor‐reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti‐CD28 domain antibody (dAb) on the donor‐specific Tfh cell–mediated immune response. We observed that the anti‐CD28 dAb led to superior inhibition of donor‐reactive CXCR5⁺PD‐1^high Tfh cells, CD95⁺GL7⁺ germinal center B cells and DSA formation compared with CTLA4‐Ig. Interestingly, donor‐reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti‐CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.     

Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen‐Pulsed Autologous Regulatory Dendritic Cells

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte‐derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg‐pulsed autologous DCreg (1.4–3.6 × 10⁶/kg) were administered intravenously, 1 day before MHC‐mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag‐pulsed DCreg (n = 5) was associated with evidence of modulated host CD4⁺ and CD8⁺ T cell responses to donor Ag and attenuation of systemic IL‐17 production. Circulating anti‐donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag‐pulsed DCreg rejected its graft in association with progressively elevated anti‐donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag‐pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.     

Adipose‐derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long‐term graft tolerance in vascularized composite allotransplantation

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation‐free conditioning protocol that combines anti‐lymphocyte serum (ALS), tacrolimus, and cytotoxic T‐lymphocyte‐associated protein 4 immunoglobulin (CTLA4‐Ig) with adipose‐derived stromal cells (ASCs) to allow VCA survival without long‐term systemic immunosuppression. Full‐mismatched rat hind‐limb‐transplant recipients received tacrolimus (0.5 mg/kg) for 14 days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC‐group received CTLA4‐Ig (10 mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1 × 10⁶ iv, POD 2, 4, 7, 15, 28); the ASC‐cyclophosphamide (CYP)‐group received CTLA4‐Ig, ASC plus cyclophosphamide (50 mg/kg ip, POD 3); the ASC‐ALS‐group received CTLA4‐Ig, ASCs plus ALS (500 µL ip, POD 1, 5). Banff grade III or 120 days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection‐free VCA survival was 28 days in CTRL (n = 7), 34 in ASC (n = 6), and 27.5 in ASC‐CYP (n = 4). In contrast, ASC‐ALS achieved significantly longer, rejection‐free VCA survival in 6/7 animals (86%), with persistent mixed donor‐cell chimerism, and elevated systemic and allograft skin T_regs, with no signs of acute cellular rejection. Taken together, a regimen comprised of short‐course tacrolimus, repeated CTLA4‐Ig and ASC administration, combined with ALS, promotes long‐term VCA survival without chronic immunosuppression.     

CMV‐infected kidney grafts drive the expansion of blood‐borne CMV‐specific T cells restricted by shared class I HLA molecules via presentation on donor cells

We aimed to determine the role of cytomegalovirus (CMV)‐infected donor cells in the development of a CMV‐specific immune response in kidney transplant recipients. We assessed the CMV pp65‐specific immune response by using interferon‐? ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R? 31, D+R + 44, D?R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN‐?‐producing anti‐CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV‐specific CD8⁺ T cells after transplantation, we compared the number of HLA‐A2–restricted CMV‐specific CD8⁺ T cells in primo‐infected recipients who received an HLA‐A2 or non–HLA‐A2 graft. The median of anti‐CMV pp65 T cells restricted by HLA‐A2 was very low for patients who received a non–HLA‐A2 graft vs an HLA‐A2 graft (300 [0‐14638] vs. 17972 [222‐85594] anti‐CMV pp65 CD8⁺ T cells/million CD8⁺ T cells, P = .001). This adds new evidence that CMV‐infected kidney donor cells present CMV peptides and drive an inflation of memory CMV‐specific CD8⁺ T cells, likely because of frequent CMV replications within the graft.     

Long-term outcomes of dual kidney transplantation—a single center experience

Abstract:  The shortage of kidney donors has led to broadening of the acceptance criteria for deceased donor organs beyond the traditional use of young donors. We determined long-term post-transplant outcomes in recipients of dual expanded criteria donor kidneys (dECD, n = 44) and compared them to recipients of standard criteria donor kidneys (SCD, n = 194) and single expanded criteria donor kidneys (sECD, n = 62). We retrospectively reviewed these 300 deceased donor kidney transplants without primary non-function (PNF) or death in the first two wk, at our center from 1996 to 2003. The three groups were similar in baseline characteristics. Kidney allograft survival and patient survival (nine yr) were similar in the three respective donor groups, SCD, sECD and dECD (60% vs. 59% vs. 64% and 82% vs. 73% vs. 73%). Acute rejection in the first three months was 23.2%, 16.1%, and 22.7% in SCD, sECD and dECD, respectively (p = 0.49) and delayed graft function was 25.2%, 31.9% and 17.1% in the three groups, respectively (p = 0.28). When PNF and death within the first two wk was included, there was no significant difference in graft survival between the three groups. In our population, recipients of dECD transplants have acceptable patient and graft survival with kidneys that would have usually been discarded.