High proportion of CD95+ and CD38+ in cultured CD8+ T cells predicts acute rejection and infection,respectively, in kidney recipients

The aim of this study was to find noninvasive T-cell markers able to predict rejection or infection risk after kidney transplantation.We prospectively examined T-lymphocyte subsets after cell culture stimulation (according to CD38, CD69, CD95, CD40L, and CD25 expression) in 79 first graft recipients from four centers, before and after transplantation. Patients were followed up for one year.Patients who rejected within month-1 (n = 10) showed high pre-transplantation and week-1 post-transplantation percentages of CD95⁺, in CD4⁺ and CD8⁺ T-cells (P < 0.001 for all comparisons). These biomarkers conferred independent risk for early rejection (HR:5.05, P = 0.061 and HR:75.31, P = 0.004; respectively). The cut-off values were able to accurately discriminate between rejectors and non-rejectors and Kaplan–Meier curves showed significantly different free-of-rejection time rates (P < 0.005). Patients who rejected after the month-1 (n = 4) had a higher percentage of post-transplantation CD69⁺ in CD8⁺ T-cells than non-rejectors (P = 0.002). Finally, patients with infection (n = 41) previously showed higher percentage of CD38⁺ in CD8⁺ T-cells at all post-transplantation times evaluated, being this increase more marked in viral infections. A cut-off of 59% CD38⁺ in CD8⁺ T-cells at week-1, week-2 and month-2 reached 100% sensitivity for the detection of subsequent viral infections.In conclusion, predictive biomarkers of rejection and infection risk after transplantation were detected that could be useful for the personalized care of kidney recipients.     

Increased levels of CD4+ and CD8+ T cells expressing CCR1 in patients developing allograft dysfunction; a cohort study

BackgroundLeukocyte infiltration into the graft has pivotal effects on kidney transplantation outcome. The present study sought to determine whether the expression of sequential chemokine receptors on CD4⁺ and CD8⁺ T cells in human renal allograft can predict clinical episodes.MethodsBlood samples from 52 consecutive renal transplant patients were evaluated at the time of transplantation and at three times (2, 90 and 180 days) after transplantation to analyze the expression of CCR1 and CXCR3 on CD4⁺ and CD8⁺ T cells by flowcytometry. A total of 30 biopsies, including protocol biopsy (n = 24) and cause biopsy (n = 6), were investigated according to the Banff criteria.ResultsThe mean percentage of CD4⁺ and CD8⁺ T cells expressing CCR1 was significantly increased in patients with allograft dysfunction (n = 25) (p = 0.006, p = 0.004). The mean fluorescence intensity of CXCR3 on CD4⁺ and CD8⁺ T cells were found to be significantly higher in graft dysfunction than that in well-functioning grafts. (p < 0.001, p = 0.007). Receiver Operating Characteristic (ROC) Curve Analysis showed that the calculated AUC was 0.86 at the third month for CD4⁺ CCR1⁺ and CD8⁺ CCR1⁺ (p < 0.001). Multiple logistic regression analysis showed that an increase in CD4⁺ expressing CXCR3 leads to a lower risk of graft dysfunction (OR = 0.37), while an increase in CD8⁺ expressing CCR1 results in a higher risk of graft dysfunction (OR = 3.66).ConclusionDuring renal transplantation, CD4⁺ and CD8⁺ T cells expressing CCR1 were increased in patients who developed graft dysfunction. These findings may prospectively predict allograft dysfunction, and help elucidate the underlying pathogenic mechanisms.     

A pilot study on the characteristics of circulating T follicular helper cells in liver transplant recipients

Circulating CD4⁺ CXCR5⁺ T follicular helper cells (cTfh) have been demonstrated to be involved in B cell-mediated systemic autoimmune diseases and alloreactive responses following kidney transplantation; however, whether cTfh cells are involved in alloreactive responses after liver transplantation (LT) remains unclear. Our present study aimed to investigate the characteristics of cTfh, as well as CXCR3⁺ CCR6⁻ Tfh1, CXCR3⁻ CCR6⁻ Tfh2, and CXCR3⁻ CCR6⁺ Tfh17 subsets in liver allograft recipients. A total of 30 liver transplant recipients were enrolled in this study. The frequencies of cTfh, Tfh1, Tfh2, and Tfh17 subsets, and interleukin (IL)-21-producing Tfh cells in the circulating blood were analyzed by flow cytometry. The capacity of cTfh cells to help B cells differentiate into plasmablasts was determined one day before and one month after LT. The results revealed that the frequency of cTfh cells remained unaltered before and after LT. However, the frequency of the cTfh subsets (e.g., Tfh1 and Tfh2 cells) and B cells were reduced one month after LT. Functionally, the capacity of Tfh cells to produce IL-21 was reduced one month after LT. In addition, cTfh cells exhibited the capacity to help B cells differentiate into plasmablasts in an IL-21-dependent manner in vitro, which was reduced after LT, despite the unaltered production of IgM and IgG by plasmablasts. Thus, our data suggest that cTfh cells may be involved in alloreactive responses following LT via helping B cells differentiate into plasmablasts and plasma cells.     

ASP0028 in combination with suboptimal-dose of tacrolimus in Cynomolgus monkey renal transplantation model

FTY720, a S1P-receptor modulator, has shown to be effective in several transplant and autoimmune disease models, via modulating lymphocyte homing into secondary lymphoid organs (SLOs), and thereby reducing these cells in peripheral blood. ASP0028, a newly developed S1P₁/S1P₅-selective agonist, presented comparable efficacy to FTY720 and wider safety margins than FTY720. In this study, we assessed the efficacy and safety of ASP0028 co-administered with suboptimal-dose of tacrolimus in the Cynomolgus monkey renal transplantation model. Seven animals in group-1 or group-2 received mono-tacrolimus 1.0 mg/kg once a day (QD), or ASP0028 0.6 mg/kg plus tacrolimus 1.0 mg/kg QD, respectively. Eight animals in group-3 received ASP0028 1.2 mg/kg plus tacrolimus 1.0 mg/kg QD. The allograft median survival time (MST) in group-2 and group-3 were significantly extended to 41 and 61.5 days, versus that of 28 days in group-1 (p = 0.036 and 0.001, respectively). ASP0028 administration remarkably reduced absolute numbers of peripheral lymphocytes, particularly subsets of CD4⁺/ or CD8⁺/naive and central memory cells, CD4⁺/Treg cells, and to a lesser extent on B cells, but not CD4⁺/ or CD8⁺/effector memory cells and NK cells. These data show ASP0028 combined with suboptimal-dose of tacrolimus effectively prolongs renal allograft survival in nonhuman primates (NHPs) with well tolerated safety, supporting its further investigation to optimize CNI-sparing regimens.     

Effects of B cell depletion on T cell allogeneic Immune responses: A strategy to reduce allogeneic sensitization

BackgroundB cell depletion has been employed to treat antibody-mediated organ transplantation rejection, although the effects on cellular immune responses have not been extensively investigated.MethodsA model of B cell depletion used SCID/beige mice reconstituted with BALB/c splenocytes either depleted of B cells (BD) or not (BN). BD and B/N mice received C57BL/6 skin grafts and were sacrificed after 6 weeks (BD-S6 and BN-S6).ResultsRecall proliferative responses of BD-S6 splenocytes to C57BL/6 were significantly reduced compared to BN-S6, and central memory T cells' proportions (CD4⁺CD44⁺CD62L⁺ or CD8⁺CD44⁺CD62L⁺) were significantly decreased in BD-S6 spleens. Recall IFN-γ production by BD-S6 splenocytes was significantly reduced compared to BN-S6 splenocytes (p = 0.0028). Survival times of C57BL/6 heart grafts were significantly longer in SCID/beige mice reconstituted with BD-S6 splenocytes (8.5 ± 1.1 days) than for SCID/beige reconstituted with BN-S6 splenocytes (6.0 ± 1.1 days; p = 0.0006). Under cyclosporine therapy, C57BL/6 heart survival was significantly longer for SCID/beige reconstituted with BD-S6 splenocytes (17.5 ± 6.4 days) than those reconstituted with BN-S6 splenocytes (6.2 ± 1.5 days; p < 0.0001).ConclusionB cell depletion during allogeneic sensitization decreased memory T cells and recalls IFN-γ production and reduced second-set allograft rejection.     

Utility of CD127 combined with FOXP3 for identification of operational tolerance after liver transplantation

Loss of cell surface expression of CD127 on CD4⁺ CD25⁺⁺ regulatory T-cells (Tregs) may be a useful marker to efficiently isolate Tregs. As FOXP3 was specifically used to identify Tregs, combining these two markers could give better identification for patient with operational tolerance (OT) after liver transplantation. To testify this mixed lymphocyte reaction (MLR), the function of circulating CD4⁺ CD25⁺⁺ CD127^dim cells (CD127^dim cells) was examined in immunosuppression (IS)-free pediatric recipients after liver transplantation (LTx) (group operational tolerance: OT) (Gr-tol n = 25) compared to recipients who could not stop IS due to clinically overt rejection (group intolerance) (Gr-intol n = 18), recipients who were weaning IS (Gr-weaning n = 11) and age-matched healthy volunteers (Gr-vol n = 11). In addition, the frequencies of CD127^dim cells vs CD4⁺ CD25⁺⁺ CD127^dimFOXP3⁺ (CD127^dimFOXP3⁺) cells were compared in these four groups by FACS analyses. Our results showed that The proliferation of CD4 cells to donor antigens was reduced compared to third-party antigens only in Gr-tol (P = 0.022) but not in other groups (P = NS). Depletion of CD127^dim cells resulted in a donor antigen-specific abrogation of this MLR hyporesponsiveness in Gr-tol (P < 0.001) but not other groups (P = NS). This implied that CD127 efficiently isolated donor antigen-specific Tregs. The frequencies of CD127^dim cells were significantly lower in Gr-intol (5.2% ± 1.9%) compared to those in Gr-tol (7.8% ± 1.8%) (P < 0.001) as were the frequencies of CD127^dim FOXP3⁺ cells (Gr-tol: 5.4% ± 1.7% vs Gr-intol: 2.9% ± 1.0%, P < 0.001). Of interest, there were fewer CD127^dimFOXP3⁺ cells in Gr-intol (2.9% ± 1%) than in Gr-weaning (5.1% ± 1.8%) (P = 0.002), but no difference in CD127^dim cells (Gr-intol: 5.2% ± 1.9% vs Gr-weaning: 6.7% ± 2.0%) (NS). Thus, combining FOXP3 with CD127 for phenotype analysis demonstrated an unequivocal difference between Gr-intol and Gr-weaning that was not detected by CD127 alone. In conclusion CD127 was a useful surface marker to isolate donor-antigen-specific-Tregs in OT after LTx. The additive effect of its combination with FOXP3 is important in phenotypical Treg analyses of OT patients.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. II. Interferon gamma (IFN-γ) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4⁺ T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4⁺ T cells.Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for > 100 days with no further immunosuppression. In previous studies, we found the CD4⁺ T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3 days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4⁺ T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4⁺ T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4⁺ T cells. Tolerant CD4⁺ CD25⁺ T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4⁺ CD25⁺ T cells to third-party Lewis. Tolerant CD4⁺ CD25⁺ T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4⁺ CD25⁺ T cells.     

Ibrutinib suppresses alloantibody responses in a mouse model of allosensitization

BackgroundIbrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses.Materials and methodsA mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions. Donor-specific antibody (DSA) levels were measured in a flow-cytometric antibody binding assay. Splenic T and B cell subsets and plasma cells were analyzed in flow cytometry.ResultsControl mice developed peak levels of DSA IgM at day 14 PTx while the ibrutinib treated mice had significantly lower levels of DSA IgM (p = 0.0047). Control mice developed HLA.A2-specific IgG antibodies at day 14 (230 ± 60 MFI) and reached peak levels at day 21 (426 ± 61 MFI). In contrast, mice in the treatment group had low levels of HLA.A2-specific IgG at day 14 (109 ± 59 MFI, p = 0.004) and day 21 (241 ± 86 MFI, p = 0.003). FACS analysis found a reduction of B220⁺ or CD19⁺ B cell population (p < 0.05). In addition, ibrutinib attenuated recall DSA IgG responses to re-sensitization (p < 0.05) and reduced CD38⁺ CD138⁺ plasma cells (p < 0.05) in the spleens.ConclusionsIbrutinib is effective in suppressing alloantibody responses through blocking BTK-mediated BCR signaling, leading to reduction of B cells and short-lived plasma cells in the spleens. Use of ibrutinib may provide benefits to HLA-sensitized transplant patients for alloantibody suppression.     

Low utility of serum 25–hydroxyvitamin D3 and 1, 25-dihydroxyvitamin D3 in predicting peripheral Treg and Th17 cell counts in ESRD and renal transplant patients

BackgroundVitamin D has shown an immune-modulatory effect in different studies. Vitamin D stimulates Tregs and inhibits Th17 cells. The immune-modulatory role of vitamin D in chronic kidney disease (CKD) and renal transplant patients is unclear. We measured whether different serum levels of vitamin D were associated with an increased or decreased presence of lymphocyte subsets including Treg and Th17 cells in end-stage renal disease (ESRD) and renal transplant recipients.MethodsEighty-seven renal transplant recipients and 53 end-stage renal disease (ESRD) patients were enrolled in this study. The absolute counts of CD4 + and CD8 + T, CD16 + CD56 + NK, CD19 + B, CD4 + CD25 + CD127- Foxp3 + (Tregs), Helios + Tregs, CD38 + Tregs, and CD4 + CD17 + (Th17) cells were analyzed in peripheral blood in both patient groups. In addition, serum 25 (OH) D₃, 1, 25 (OH)₂ D₃, IL-6, IL-17, IL-23, and TGF-β₁ were measured. The association between lymphocyte subset counts and 1, 25 (OH)₂ D₃ or 25 (OH) D₃ was studied, as was the association between serum IL-6, IL-17, IL-23, or TGF-β₁ and 1,25 (OH)₂ D₃ or 25 (OH) D₃.ResultsSerum 25 (OH) D₃ and 1,25 (OH)₂ D₃ levels were not independently associated with peripheral CD4 + T, CD19 + B, CD16 + CD56 + NK, Treg, or Th17 cell counts. In contrast to serum 25 (OH) D₃, serum1, 25 (OH)₂ D₃ was positively associated with CD8 + T cells counts in renal transplant recipients.ConclusionOur findings indicate low utility of serum 25 (OH) D₃ and 1, 25 (OH)₂ D₃ levels in predicting a change in lymphocyte subset counts in ESRD and renal transplant patients.     

Blockade of CD27/CD70 pathway to reduce the generation of memory T cells and markedly prolong the survival of heart allografts in presensitized mice

BackgroundAlloreactive memory T cells are a major obstacle to transplantation acceptance due to their capacity for accelerated rejection.MethodsC57BL/6 mice that had rejected BALB/c skin grafts 4 weeks earlier were used as recipients. The recipient mice were treated with anti-CD154/LFA-1 with or without anti-CD70 during the primary skin transplantation and anti-CD154/LFA-1 or not during the secondary transplantation of BALB/c heart. We evaluated the impact of combinations of antibody-mediated blockade on the generation of memory T cells and graft survival after fully MHC-mismatched transplantations.ResultsOne month after the primary skin transplantation, the proportions of CD4⁺ memory T cells/CD4⁺ T cells and CD8⁺memory T cells/CD8⁺ T cells in the anti-CD154/LFA-1 combination group were 47.32 ± 4.28% and 23.18 ± 2.77%, respectively. In the group that included anti-CD70 treatment, the proportions were reduced to 34.10 ± 2.71% and 12.19 ± 3.52% (P < 0.05 when comparing the proportion of memory T cells between the two groups). The addition of anti-CD70 to the treatment regimen prolonged the mean survival time following secondary heart transplantation from 10 days to more than 90 days (P < 0.001). Furthermore, allogenic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly decreased. Meanwhile, the proportion of regulatory T cells was increased to 9.46 ± 1.48% on day 100 post-transplantation (P < 0.05).ConclusionsThe addition of anti-CD70 to the anti-CD154/LFA-1 combination given during the primary transplantation reduced the generation of memory T cells. This therapy regimen provided a potential means to alleviate the accelerated rejection mediated by memory T cells during secondary heart transplantation and markedly prolong the survival of heart allografts.     

Soluble donor-like MHC class I proteins induce CD4+CD25+CD8−FoxP3+ cells with potential to ameliorate graft chronic injury

Conventional immunosuppressive therapies failed to prevent allograft chronic rejection. New approaches to modulate recipient immune response are needed. Donor-like MHC class I soluble proteins demonstrated therapeutic potential to suppress chronic rejection. The present study was designed to clarify the ability of MHC class I soluble proteins to induce T regulatory cells with true regulatory potential in a fully allogeneic rat cardiac transplant model. Donor-like MHC class I proteins upregulate small population of splenic CD8^? negative CD4⁺CD25⁺FoxP3⁺ positive cells. CD4⁺ splenocytes after MHC therapy suppress lymphocyte proliferation against donor antigens in vitro. ACI recipients of WF hearts treated with CD4⁺ cells, induced with donor-like MHC class I proteins (CD4-MHC), demonstrated stable survival of the transplanted organ (MST > 120 days; n = 17). Histology revealed that grafts of recipients treated with CD4-MHC had 23.6% vessels affected 100 days postgrafting. On the contrary, hearts obtained from long-term surviving hosts treated with CD4⁺ cells induced with high-dose CsA (CD4-CsA) had 50–70% of affected vessels. CD4-MHC class I treated transplants were mostly CD3^? negative, had low level of mast and FoxP3⁺ cell infiltration compared to CD4-CsA treated hearts. Intragraft CD4⁺ cells were close to mast cells in morphology. The same graft tissues had similar number of CD4⁺ positive cells and mast cells suggesting existence of CD4⁺ positive mast cells. On the other hand, a negligible number of FoxP3⁺ positive cells in the grafts after CD4-MHC treatment supports the idea of CD4⁺ positive FoxP3⁺ negative mast cells population. We demonstrate that donor-like MHC class I protein therapy induces population of CD4⁺CD25⁺CD8^?FoxP3⁺ cells with potential to ameliorate development of transplant vascular disease and evoke CD4⁺ positive FoxP3 negative mast cells in the secondary hosts.     

Pre-transplant infusion of donor-derived dendritic cells maintained at the immature stage by sinomenine increases splenic Foxp3+ Tregs in recipient rats after renal allotransplantation

ObjectiveThe immunosuppressive mechanism of sinomenine in organ allotransplantation was investigated, especially its effect of blocking dendritic cell (DC) maturation, which might influence the frequency of regulatory T cells (Tregs).MethodsBone marrow cells from male donor Wistar rats were induced to differentiate into DCs in vitro in the presence or absence of sinomenine, and characterized by flow cytometry. These two groups of DCs were respectively injected into male recipient Sprague-Dawley rats via the tail vein, at both high and low doses. Sprague-Dawley rats receiving saline injection were used as controls. Seven days later, renal transplantation was performed from donor Wistar rats to the recipient Sprague-Dawley rats. Seven days after transplantation, spleens were collected from the recipients. The proportions of Tregs and Foxp3⁺ Tregs to CD4⁺ T cells were determined using flow cytometry.ResultsWith sinomenine treatment, the frequency of mature DCs was reduced, as indicated by lower expression of the surface markers CD80, CD86, and RT1B. In recipient Sprague-Dawley rats that received sinomenine-treated DCs before renal allotransplantation, the proportions of splenic Tregs and Foxp3⁺ Tregs were significantly higher than in control recipients receiving saline or DCs without sinomenine treatment (all p < 0.05). A high dose of sinomenine-treated DCs (10⁶ cells) had a more obvious effect in increasing Tregs than the low dose (10⁵ cells) (p < 0.05).ConclusionPre-transplant infusion of donor-derived sinomenine-induced maturation arrested DCs could result in the increase of Foxp3⁺ Tregs in the spleens of recipients after renal allotransplantation.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4⁺ T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4⁺ CD25⁺ FOXP3⁺ Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg.This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4⁺, especially CD4⁺ CD25⁺ T cells. CD4⁺ T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100 days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4⁺ T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4⁺ CD25⁺ T cells' response to rIL-5 and expression of IL-5Rα was also assessed.rIL-5 was sufficient to promote transplant tolerance mediating CD4⁺ T cells' survival in culture with specific-donor alloantigen. Tolerant CD4⁺ T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4⁺ T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4⁺ CD25⁺ T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4⁺ CD25⁺ T cells expressed IL-5Rα.This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4⁺ CD25⁺ T cells that mediate transplant tolerance.     

Influence of immune activation on the risk of allograft rejection in human immunodeficiency virus-infected kidney transplant recipients

BackgroundHIV infection is associated with high rates of acute rejection following kidney transplantation. The underlying mechanisms for such predisposition are incompletely understood. Pathological immune activation is a hallmark of chronic HIV infection that persists despite effective antiretroviral therapy. We hypothesized that the baseline levels of T cell activation in HIV⁺ candidates would correlate with their risk of acute rejection following kidney transplantation.MethodsSingle-center retrospective cohort analysis of HIV⁺ adult kidney transplants performed between October 2006 and September 2013. The frequency of CD3⁺ HLA-DR⁺ cells measured by flow cytometry served as a surrogate marker of immune activation. Patients were categorized into tertiles of activation, and the rates of biopsy-proven acute rejection were compared across groups.Results(1) Compared to matched HIV⁻ controls, the baseline number of CD3⁺ HLA-DR⁺ cells was higher in HIV⁺ kidney transplant candidates. (2) Abnormally high levels of activation did not decrease with transplant-associated immunosuppression. (3) Patients categorized within the lower and middle CD3⁺ HLA-DR⁺ tertiles had higher probability of rejection during the first 3 years post-transplant compared to those in the higher activation tertile (36.9% vs. 0%; log-rank P = 0.04).ConclusionsPathological immune activation in HIV⁺ transplant candidates does not explain their increased susceptibility to allograft rejection. Paradoxically, those with the highest levels of immune activation seem to be less prone to rejection.     

IFNy + and IFNy − Treg subsets with stable and unstable Foxp3 expression in kidney transplant recipients with good long-term graft function

BackgroundTreg are a heterogenous cell population. In the present study we attempted to identify Treg subsets that might contribute to stable and good long-term graft function.MethodLymphocyte and Treg subsets were studied in 136 kidney transplant recipients with good long-term graft function and in 52 healthy control individuals using eight-color-fluorescence flow cytometry. Foxp3 TSDR methylation status was investigated in enriched IFNy + and IFNy − Treg preparations using high resolution melt analysis.ResultsCompared with healthy controls, patients showed strong associations of IFNy secreting Helios + and Helios − Treg with Treg that co-expressed perforin and/or CTLA4 (CD152; p < 0.01). Moreover they showed associations of IFNy − Helios + Treg with Treg that produced TGFβ and/or perforin and of IFNy − Helios − Treg with TGFβ production (all p < 0.01). Only in patients, but not in healthy controls, were IFNy − Helios + and Helios − Treg associated with higher CD45 +, CD3 +, (CD4 +), CD19 + lymphocyte counts (p < 0.001). In addition IFNy − Helios + Treg were associated with CD16 + 56 + lymphocytes (p < 0.001). Enriched IFNy − Treg from female but not male patients showed an association of Foxp3 methylation with higher total Treg and higher Helios + IFNy −, CXCR3 + Lselectin + (CD183 + CD62L +), CXCR3 − Lselectin + and CD28 + HLADR + Treg subsets (p < 0.01). Enriched IFNy + Treg from male patients showed an association of demethylated Foxp3 with total Treg and IL10 − TFGβ + Treg counts, and in enriched IFNy − Treg an association of methylated Foxp3 with APO1/FasR + FasL + (CD95 + CD178 +) Treg (p < 0.01).ConclusionsKidney recipients with good long-term graft function possess IFNy + and IFNy − Treg with stable and unstable Foxp3 expression in the blood. They co-express CD28, HLADR, CTLA4, CXCR3, Lselectin, TGFβ, perforin and FasL and might contribute to the establishment and maintenance of good long-term graft function.     

Absence of CD4CD25 regulatory T cell expansion in renal transplanted patients treated in vivo with Belatacept mediated CD28–CD80/86 blockade

AimsBelatacept is a new recombinant molecule (CTLA4-Ig) that interferes with the second activation signal of T lymphocytes. CTLA4-Ig induced T cell allograft tolerance in rodents but not in primates. We examined the changes in peripheral lymphocyte subsets, including regulatory T cells, in renal transplant patients treated with Belatacept.MethodsA cross-sectional immunological study was carried out 6 months after transplantation in 28 patients enrolled in the Belatacept phase II study. Eighteen patients received Belatacept, mycophenolate mofetil and steroids (Belatacept group), while the control group of 10 patients received cyclosporine, mycophenolate mofetil and steroids (CsA group). Lymphocyte subsets were examined by flow cytometry. Foxp3 mRNA expression was measured by quantitative PCR.ResultsThe number of T lymphocytes and the percentage of CD3+ T cells were similar in both groups. However, the percentage of CD3+ CD4+ T cells was lower in the Belatacept group than in the control CsA group (B = 42.5% ± 13.7 vs CsA = 52.9% ± 9, p < 0.005), and the percentage of CD3+ CD8+ cells was higher in the Belatacept group than in the control (B = 32.9% ± 6.7 vs CsA = 19.5% ± 8.2, p < 0.0002). The percentage of CD19+ cells was similar in both groups. Among CD56+cells, only the percentage of CD16+ cells was significantly higher in the Belatacept group than in the control (B = 82% ± 12 vs CsA = 59.7% ± 25, p = 0.01). Among CD4 and CD8 T cells the percentage of activated lymphocytes expressing CTLA4, HLA-DR or CD40L was similar in both groups. The percentage of CD4+CD25+ T cells was higher in the CsA group. The percentage of regulatory CD4+CD25+ cells with bright CD25 staining was similar in both groups (B = 3.6 ± 2.3% vs CsA = 4.7 ± 1.9%, ns) as was the expression of FoxP3.ConclusionOur results indicated that Belatacept did not induce regulatory T cell expansion in vivo. We suggest that Belatacept treatment should be maintained after transplantation to allow graft acceptance.     

Ex vivo detection of CD8 T cells specific for H-Y minor histocompatibility antigens in allogeneic hematopoietic stem cell transplant recipients

Immunologic disparities between minor histocompatibility antigen (mHAg) genes on Y (H-Y) and X (H-X) chromosomes contribute to graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects observed in male recipients of a female donor (FtoM) hematopoietic stem cell transplant (HCT). Using in silico prediction tools, a panel of HLA-A0201 restricted H-Y peptides was synthesized. Expression of CD137 was monitored on CD8⁺ T cells after brief stimulation with the H-Y peptides in FtoM HLA-A0201 HCT recipients (N = 29), and control groups (HLA-A0201 MtoM [N = 18], non-HLA-A0201 FtoM [N = 14], and HLA-A0201 female volunteers [N = 25]). Specific H-Y responses were significantly greater in HLA-A0201 FtoM than controls. CD8⁺ T-cell responses to novel H-Y epitopes were shared among multiple patients, showing marked CD45RA⁺CD27 cytolytic effector profiles. These data represent a proof of concept for our in silico/ex vivo CD8⁺ T-cell based approach of prediction and validation of H-Y mHAgs in HCT recipients, which may facilitate prospective studies to identify targets/biomarkers of GVHD/GVL.     

NKT cells are important mediators of hepatic ischemia-reperfusion injury

IntroductionIRI results from the interruption then reinstatement of an organ's blood supply, and this poses a significant problem in liver transplantation and resectional surgery.In this paper, we explore the role T cells play in the pathogenesis of this injury.Materials & methodsWe used an in vivo murine model of warm partial hepatic IRI, genetically-modified mice, in vivo antibody depletion, adoptive cell transfer and flow cytometry to determine which lymphocyte subsets contribute to pathology. Injury was assessed by measuring serum alanine aminotransfersase (ALT) and by histological examination of liver tissue sections.ResultsThe absence of T cells (CD3εKO) is associated with significant protection from injury (p = 0.010). Through a strategy of antibody depletion it appears that NKT cells (p = 0.0025), rather than conventional T (CD4 + or CD8 +) (p = 0.11) cells that are the key mediators of injury.DiscussionOur results indicate that tissue-resident NKT cells, but not other lymphocyte populations are responsible for the injury in hepatic IRI. Targeting the activation of NKT cells and/or their effector apparatus would be a novel approach in protecting the liver during transplantation and resection surgery; this may allow us to expand our current criteria for surgery.