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.     

Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation

Bone marrow‐derived mesenchymal stromal cells (MSC) have emerged as useful cell population for immunomodulation therapy in transplantation. Moving this concept towards clinical application, however, should be critically assessed by a tailor‐made step‐wise approach. Here, we report results of the second step of the multistep MSC‐based clinical protocol in kidney transplantation. We examined in two living‐related kidney transplant recipients whether: (i) pre‐transplant (DAY‐1) infusion of autologous MSC protected from the development of acute graft dysfunction previously reported in patients given MSC post‐transplant, (ii) avoiding basiliximab in the induction regimen improved the MSC‐induced Treg expansion previously reported with therapy including this anti‐CD25‐antibody. In patient 3, MSC treatment was uneventful and graft function remained normal during 1 year follow‐up. In patient 4, acute cellular rejection occurred 2 weeks post‐transplant. Both patients had excellent graft function at the last observation. Circulating memory CD8⁺ T cells and donor‐specific CD8⁺ T‐cell cytolytic response were reduced in MSC‐treated patients, not in transplant controls not given MSC. CD4⁺FoxP3⁺Treg expansion was comparable in MSC‐treated patients with or without basiliximab induction. Thus, pre‐transplant MSC no longer negatively affect kidney graft at least to the point of impairing graft function, and maintained MSC‐immunomodulatory properties. Induction therapy without basiliximab does not offer any advantage on CD4⁺FoxP3⁺Treg expansion ( ClinicalTrials.gov number: NCT 00752479).     

Reduced CD4+ CD25++ CD45RA− Foxp3hi activated regulatory T cells and its association with acute rejection in patients with kidney transplantation

BackgroundIt was found that regulatory T cells (Tregs) importantly affect the maintenance of the kidney graft. However, Tregs are a heterogeneous population with less to more suppressive activity. The aim of this study was to determine the effects of different subsets of Tregs, as well as their ratio to effector T cells (Teff), on kidney transplantation outcomes.MethodsA total of 58 participants were enrolled in this study and divided into four groups: (i) first kidney transplant recipients (stable 1); (ii) second kidney transplant recipients (stable 2); (iii) transplant recipients with acute rejection (AR); and (iv) healthy control subjects. By using flow cytometer, the frequencies of CD4⁺ CD25⁺⁺ CD45RA⁻ Foxp3^hi activated Tregs (aTregs), CD4⁺ CD25⁺ CD45RA⁺ Foxp3^lo resting Tregs (rTregs), CD4⁺ CD25⁺ CD45RA⁻ Foxp3^lo non-suppressive T cells, CD4⁺ CD25⁺ Foxp3⁻ cells Teff, and total Tregs were analyzed in all subjects.ResultsThe frequency of aTregs (as well as the ratio of aTregs/Tregs) was significantly lower in the AR patients than the other three groups. In contrast to AR patients, stables 1 and 2 had a higher aTreg/Treg ratio than those in the control group. Although patients with AR had a significantly lower total Tregs than the other three groups, the balance of total Tregs and Teff was similar between patients with and without AR.ConclusionPatients with AR had poorer immunoregulatory properties than those with normal graft functioning, as well as those in the control group. These reduced immunoregulatory properties in patients with AR could lead to graft rejection.     

Antigen-Specific Suppression by Induced CD4+CD25high Regulatory T Cells in Kidney Recipients

The biology and function of induced CD4⁺CD25^high regulatory T (Treg) cells have not been clarified for their specificity to a foreign antigen. To test whether the regulatory functions of the induced CD4⁺CD25^high Treg cells after transplantation require antigen-specific triggering, we analyzed the capacity of induced CD4⁺CD25^high Treg cells to inhibit the proliferation of conventional CD4⁺CD25^? T cells in response to T-cell receptor stimulation using donor cells or HLA-mismatched third-party cells in vitro. CD4⁺CD25^high Treg cells did not proliferate in response to allogeneic stimulation and suppressed proliferation of the co-cultured autologous CD4⁺CD25^? populations in a dose-dependent manner. The proliferation of CD4⁺CD25^?T cells from the same donor in mixed lymphocyte reactions was significantly inhibited at a 1:8 ratio of conventional T cells:Treg cells: 14,404 ± 673 cpm without CD4⁺CD25^high Treg cells versus 10,781 ± 539 cpm with CD4⁺CD25^high Treg cells P = .01). At the same 1:8 ratio, the proliferation of CD4⁺CD25^? cells derived from major histocompatibility complex–mismatched patients was not significantly inhibited: 14,404 ± 673 cpm without CD4⁺CD25^high Treg cells versus 12,471 ± 709 cpm with CD4⁺CD25^high Treg cells (P = .06). Antigen specificity of the induced CD4⁺CD25^high Treg cells was demonstrated, after transplantation, supporting the use of antigen-specific Treg cells as a therapeutic strategy.     

Pre‐transplant immune state defined by serum markers and alloreactivity predicts acute rejection after living donor kidney transplantation

Acute rejection (AR) remains a major cause for long‐term kidney allograft failure. Reliable immunological parameters suitable to define the pre‐transplant immune state and hence the individual risk of graft rejection are highly desired to preferably adapt the immunosuppressive regimen in advance. Donor and third party alloreactivities were determined by mixed lymphocyte cultures. Soluble forms of CD25, CD30, and CD44 were detected in patients' serum by ELISA. Various lymphocyte subpopulations were measured using flow cytometry. All patients received triple immunosuppression (tacrolimus/mycophenolate mofetil/steroids) and were grouped according to biopsy results within the first year: rejection‐free (RF, n = 13), borderline (BL, n = 5), or acute rejection (AR, n = 7). Patients with AR showed the highest pre‐transplant alloreactivities and serum levels (sCD25/sCD30/sCD44) according to the pattern RF < BL < AR. Relying on serum analysis only, multivariate logistic regression (logit link function) yielded a prognostic score for prediction of rejection with 75.0% sensitivity and 69.2% specificity. Patients with rejection showed markedly higher pre‐transplant frequencies of CD4⁺/CD8⁺ T cells lacking CD28, but lower numbers of CD8⁺CD161^bright T cells and NK cells than RF individuals. Pre‐transplant immune state defined by alloreactivity, serum markers, and particular lymphocyte subsets seems to correlate with occurrence of graft rejection after kidney transplantation. A prognostic score based on pre‐transplant serum levels has shown great potential for prediction of rejection episodes and should be further evaluated.     

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.     

Permanent CNI Treatment for Prevention of Renal Allograft Rejection in Sensitized Hosts Can Be Replaced by Regulatory T Cells

Recent data suggest that donor‐specific memory T cells (T_mem) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg). We established a clinically more relevant, life‐supporting rat kidney transplant model using a high responder (DA to LEW) recipients that received donor‐specific CD4⁺/ 8⁺ GFP⁺ T_mem before transplantation to achieve similar pre‐transplant frequencies of donor‐specific T_mem as seen in many patients. T cell depletion alone induced long‐term graft survival in naïve recipients but could not prevent acute rejection in T_mem⁺ rats, like in patients. Only if T cell depletion was combined with permanent CNI‐treatment, the intragraft inflammation, and acute/chronic allograft rejection could be controlled long‐term. Remarkably, combining 10 days CNI treatment and adoptive transfer of Tregs (day 3) but not Treg alone also induced long‐term graft survival and an intragraft tolerance profile (e.g. high TOAG‐1) in T_mem⁺ rats. Our model allows evaluation of novel therapies under clinically relevant conditions.     

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. Interleukin-4 does not maintain alloantigen specific CD4+CD25+ Treg

IL-4 is thought to promote induction of transplantation tolerance and alloantigen-specific CD4⁺CD25⁺ T regulatory cells (Treg). This study examined the effect of IL-4 on the induction and maintenance of the CD4⁺ T regulatory cells (Treg) that mediate transplantation tolerance. Tolerance was induced in DA rats with PVG heterotopic cardiac allografts by a short course of cyclosporine. Naïve and tolerant lymphocytes, including the CD4⁺ and CD4⁺CD25⁺ T cell subsets, were assayed in mixed lymphocyte cultures with or without recombinant (r)IL-4 or other cytokines. The proliferation, cell surface and cytokine phenotype of these cells was examined, as was their capacity to adoptively transfer tolerance. rIL-4 enhanced the proliferation of naïve and tolerant lymphoid cells, including CD4⁺ and CD4⁺CD25⁺ T cells, but this was not alloantigen specific. Naïve or tolerant CD4⁺ T cells cultured with rIL-4 and donor PVG antigen effected rapid graft rejection, even though before culture tolerant CD4⁺ T cells transferred antigen-specific tolerance. These rIL-4 cultured CD4⁺ T cells had a phenotype consistent with activated CD4⁺CD25⁺FoxP3⁻ Th2 cells. While naïve natural CD4⁺CD25⁺ T cells (nTreg) cultured with alloantigen and rIL-4 had enhanced proliferation and capacity to suppress rejection in vivo, the culture of tolerant CD4⁺CD25⁺ T cells with alloantigen and rIL-4 could not sustain their proliferation against specific donor, nor their capacity to transfer tolerance to specific donor allograft. Thus, IL-4 promotes both regulatory and effector T cells early in the immune response, but once alloimmune tolerance is established, IL-4 promoted the activation of effector cells to mediate rejection and did not support alloantigen-specific Treg that could transfer specific tolerance.     

Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response

Singh AK, Seavey CN, Horvath KA, Mohiuddin MM. Ex‐vivo expanded baboon CD4⁺ CD25^Hi Treg cells suppress baboon anti‐pig T and B cell immune response. Xenotransplantation 2012; 19: 102–111. © 2012 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ FoxP3⁺ regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex‐vivo expansion of naturally occurring CD4⁺ CD25⁺ T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens. Methods: Naturally occurring baboon CD4⁺ CD25⁺ regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti‐CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL‐2. Treg cells were also enriched directly from CD4⁺ T cells cultured in the presence of rapamycin (0.1–10 nm ). Mixed lymphocyte culture and polyclonal B cell stimulation with ex‐vivo Treg cells were performed to assess the function of ex‐vivo expanded Treg cells. Results: The nTreg cells were expanded to more than 200‐fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2‐fold increase in enrichment of CD4⁺ CD25⁺ FoxP3⁺ Treg cells from CD4⁺ cells was observed with rapamycin compared to cultures without rapamycin. The ex‐vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti‐porcine xenogeneic T and B cell immune response in‐vitro efficiently (more than 90% suppression at 1 : 1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1 : 256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1 : 1 ratio, and their suppressive ability was reduced to ≤50% at 1 : 16 ratio. Furthermore, we have found that ex‐vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex‐vivo expanded Treg cells were added to the culture at a 1 : 1 ratio. The addition of CD4⁺ CD25^Neg cells however induced vigorous proliferation. Conclusion: Ex‐vivo expanded CD4⁺ CD25⁺ FoxP3⁺ Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex‐vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models.     

Virus‐Specific CD8+ T Cells Cross‐Reactive to Donor‐Alloantigen Are Transiently Present in the Circulation of Kidney Transplant Recipients Infected With CMV and/or EBV

T cells play a dual role in transplantation: They mediate transplant rejection and are crucial for virus control. Memory T cells generated in response to pathogens can cross‐react to alloantigen, a phenomenon called heterologous immunity. Virus‐specific CD8⁺ T cells cross‐reacting to donor‐alloantigen might affect alloimmune responses and hamper tolerance induction following transplantation. Here, we longitudinally studied these cross‐reactive cells in peripheral blood of 25 kidney transplant recipients with a cytomegalovirus and/or Epstein‐Barr virus infection. Cross‐reactive T cells were identified by flow cytometry as virus‐specific T cells that proliferate in response to donor cells in a mixed‐lymphocyte reaction. In 13 of 25 patients, we found cross‐reactivity to donor cells for at least 1 viral epitope before (n = 7) and/or after transplantation (n = 8). Cross‐reactive T cells were transiently present in the circulation, and their precursor frequency did not increase following transplantation or viral infection. Cross‐reactive T cells expressed interferon‐γ and CD107a in response to both alloantigen and viral peptide and resembled virus‐specific T cells in phenotype and function. Their presence was not associated with impaired renal function, proteinuria, or rejection. In conclusion, virus‐specific T cells that cross‐react to donor‐alloantigen are transiently detectable in the circulation of kidney transplant recipients.     

Association of Higher CD4+CD25highCD127low,FoxP3+, and IL‐2+ T Cell Frequencies Early After Lung Transplantation With Less Chronic Lung Allograft Dysfunction at Two Years

Regulatory T cells (Treg) can regulate alloantigens and may counteract chronic lung allograft dysfunction (CLAD) in lung transplantation. We analyzed Treg in peripheral blood prospectively and correlated percentages of subpopulations with the incidence of CLAD at 2 years. Among lung‐transplanted patients between January 2009 and July 2011, only patients with sufficient Treg measurements were included into the study. Tregs were measured immediately before lung transplantation, at 3 weeks and 3, 6, 12, and 24 months after transplantation and were defined as CD4⁺CD25^high T cells and further analyzed for CTLA4, CD127, FoxP3, and IL‐2 expressions. Between January 2009 and July 2011, 264 patients were transplanted at our institution. Among the 138 (52%) patients included into the study, 31 (22%) developed CLAD within 2 years after transplantation. As soon as 3 weeks after lung transplantation, a statistically significant positive association was detected between Treg frequencies and later absence of CLAD. At the multivariate analysis, increasing frequencies of CD4⁺CD25^highCD127^low, CD4⁺CD25^highFoxP3⁺ and CD4⁺CD25^highIL‐2⁺ T cells at 3 weeks after lung transplantation emerged as protective factors against development of CLAD at 2 years. In conclusion, higher frequencies of specific Treg subpopulations early after lung transplantation are protective against CLAD development.     

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.     

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.     

Isolated CD39 Expression on CD4+ T Cells Denotes both Regulatory and Memory Populations

Foxp3⁺ regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4⁺CD39⁺ T‐cell pool contains two roughly equal size Foxp3⁺ and Foxp3^? populations. While Foxp3⁺CD39⁺ cells are CD73^bright and are the bone fide Tregs, Foxp3^?CD39⁺ cells do not have suppressive activity and are CD44⁺CD62L^?CD25^?CD73^dim/?, exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP‐induced apoptosis. Compared with Foxp3^?CD39^? naïve T cells, Foxp3^?CD39⁺ cells freshly isolated from non‐immunized mice express at rest significantly higher levels of mRNA for T‐helper lineage‐specific cytokines IFN‐γ (Th1), IL‐4/IL‐10 (Th2), IL‐17A/F (Th17), as well as pro‐inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3^?CD39⁺ cells inhibits TGF‐β induction of Foxp3 in Foxp3^?CD39^? cells. Furthermore, when transferred in vivo, Foxp3^?CD39⁺ cells rejected MHC‐mismatched skin allografts in a much faster tempo than Foxp3^?CD39^? cells. Thus, besides Tregs, CD39 is also expressed on pre‐existing memory T cells of Th1‐, Th2‐ and Th17‐types with heightened alloreactivity.     

T regulatory cells in xenotransplantation

Abstract: The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long‐term graft survival. In this regard, the induction of donor‐specific tolerance through isolation and expansion of xenoantigen‐specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4⁺CD25⁺Foxp3⁺ T cells, CD8⁺CD28^? Treg, double negative CD4^?CD8^? T cells, and natural killer Treg. Although only studied in vitro so far, human CD4⁺CD25⁺Foxp3⁺ Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8⁺CD28^? Treg and double negative CD4^?CD8^? Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4⁺Vα14⁺ Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4⁺CD25⁺Foxp3⁺ Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.     

Cytomegalovirus-specific regulatory and effector T cells share TCR clonality--possible relation to repetitive CMV infections

Cytomegalovirus (CMV) infections have a major impact on morbidity and mortality of transplant patients. Among the complex antiviral T‐cell response, CMV‐IE‐1 antigen‐specific CD8+ cells are crucial for preventing CMV disease but do not protect from recurring/lasting CMV reactivation. Recently, we confirmed that adoptive transfer of autologous IE‐1/pp65‐specific T‐cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV‐induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection. In fact, kidney transplant patients with recurring CMV infections expressed enhanced suppression on CMV response. Analysis of in vitro expanded CD4+ epitope‐specific cells revealed that CMV‐specific CD4+CD25^high Treg cells functionally suppress CD25^low effector T cells (Teff) upon epitope‐specific reactivation. Their phenotype is similar to iTreg – CD39^high/Helios‐/IL‐2^low/IFNγ^high/IL‐10±/TGFß‐LAP±/FOXP3+ and methylated foxp3 locus. Remarkably, in vitro expanded CD4+CD25^high iTreg share the same dominant TCR‐Vβ‐CDR3 clones with functionally distinct CD4+CD25^low Teff. Moreover, the same clones were present in freshly isolated CD4+CD25^high and CD4+CD25^low T cells suggesting their in vivo generation. These findings directly demonstrate that Teff and iTreg can differentiate from one “mother” clone with specificity to the same viral epitope and indicate that peripheral iTreg generation is related to frequent antigen appearance.     

Prolongation of corneal xenotransplant survival by T-cell vaccination-induced T-regulatory cells

Abstract: Background: Corneal xenotransplantation is an alternative approach for overcoming shortage of allograft in clinics. However, the mechanism of acute corneal xenograft rejection and the method of prolonging xenograft survival have not been well defined. Methods: In this study, we used an orthotopic corneal guinea pig‐to‐rat xenotransplantation model to study the effects of CD4 and CD8 T cells, T‐cell vaccination (TCV) and TCV‐induced T‐regulatory (Treg) cells on xenograft survival. Results: The acute rejection of xenografts occurred in untreated rats as early as 6 days post‐transplantation, while TCV significantly prolonged xenograft survival from 6–12 to 21–27 days. The lymph node cells of the TCV‐treated rats exhibited significant response to the anti‐guinea pig T cells and the responding cell populations contained two Treg cell subsets, CD4⁺ CD25^? and CD8⁺ CD28^? T cells, both of which lack expression of Foxp3. Adoptive transfer of CD8⁺ CD28^? T cells resulted in profound inhibition of corneal xenograft rejection, while transfer of CD4⁺ CD25^? T cells alone exhibited no significant inhibition. However, transfer of the CD4⁺ CD25^? and CD8⁺ CD28^? T‐cell mixture remarkably enhanced the in vivo protective activity against xenograft rejection. Conclusions: These data suggest that TCV induces the activation of specific Treg cell subsets, CD4⁺ CD25^? and CD8⁺ CD28^? T cells, which may act cooperatively to mediate prolongation of corneal xenograft survival. Therefore, TCV can be used as immunotherapy for suppression of acute xenograft rejection.     

Evaluation of Alloreactivity in Kidney Transplant Recipients Treated with Antithymocyte Globulin Versus IL‐2 Receptor Blocker

Induction therapy is used in kidney transplantation to inhibit the activation of donor‐reactive T cells which are detrimental to transplant outcomes. The choice of induction therapy is decided based on perceived immunological risk rather than by direct measurement of donor T‐cell reactivity. We hypothesized that immune cellular alloreactivity pretransplantation can be quantified and that blocking versus depleting therapies have differential effects on the level of donor and third‐party cellular alloreactivity. We studied 31 kidney transplant recipients treated with either antithymocyte globulin (ATG) or an IL‐2 receptor blocker. We tested pre‐ and posttransplant peripheral blood cells by flow cytometry to characterize T‐cell populations and by IFN‐γ ELISPOT assays to assess the level of cellular alloreactivity. CD8⁺ T cells were more resistant to depletion by ATG than CD4⁺ T cells. Posttransplantation, frequencies of donor‐reactive T cells were markedly decreased in the ATG‐treated group but not in the IL‐2 receptor blocker group, whereas the frequencies of third‐party alloreactivity remained nearly equivalent. In conclusion, when ATG is used, marked and prolonged donor hyporesponsiveness with minimal effects on nondonor responses is observed. In contrast, induction with the IL‐2 receptor blocker is less effective at diminishing donor T‐cell reactivity.     

Transplant Tolerance to Pancreatic Islets Is Initiated in the Graft and Sustained in the Spleen

The immune system is comprised of several CD4⁺ T regulatory (Treg) cell types, of which two, the Foxp3⁺ Treg and T regulatory type 1 (Tr1) cells, have frequently been associated with transplant tolerance. However, whether and how these two Treg‐cell types synergize to promote allograft tolerance remains unknown. We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3⁺ Treg and Tr1 cells. Here, we show that Foxp3⁺ Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non‐antigen (Ag) specific manner. Whereas CD4⁺CD25^? T cells, which contain Tr1 cells, act from the spleen and are key to the maintenance of long‐term tolerance. Importantly, the role of Foxp3⁺ Treg and Tr1 cells is not redundant once they are simultaneously expanded/induced in the same host. Moreover, our data show that long‐term tolerance induced by Foxp3⁺ Treg‐cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen.

     

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.