Functional analysis of CD4+ CD25bright T cells in kidney transplant patients: improving suppression of donor-directed responses after transplantation

Abstract: Background: The role of CD4⁺ CD25^bright regulatory T cells (Treg) in controlling alloreactivity is established, but little is known whether antigen‐specific Treg are induced in fully immunosuppressed kidney transplant patients. Methods: The frequency and function of CD25^bright T cells of nine stable kidney transplant patients before and 0.5–2 yr after transplantation were measured. Patients received triple therapy consisting of cyclosporine, mycophenolate mofetil and prednisone. To investigate the influence of transplantation and immunosuppression on Treg function, we compared their suppressive capacities pre‐ and post‐transplantation using mixed lymphocyte reactions and kept the CD25^?/dim effector T‐cell (Teff) population constant. Results: After transplantation, the percentage of CD4⁺ CD25^bright T cells significantly decreased from 8.5% pre‐transplant to 6.9% post‐transplant (median, p = 0.05). However, the lower percentage of post‐transplant CD4⁺ CD25^bright T cells was not associated with reduced, but rather improved suppressor function of these cells. The proliferative response of pre‐transplant Teff to donor‐antigens was more profoundly suppressed by post‐transplant Treg than by pre‐transplant Treg (pre‐transplant 18% vs. post‐transplant 55% median, p = 0.03) and was comparable against third party antigens at a CD25^bright:CD25^?/dim ratio of 1:20. Conclusions: In immunosuppressed kidney transplant patients, the donor‐directed suppressive capacity of CD4⁺ CD25^bright regulatory T cells improved, which may contribute to the development of donor‐specific hyporesponsiveness against the graft.     

Ex Vivo Expansion of Human Tregs by Rabbit ATG Is Dependent on Intact STAT3‐Signaling in CD4+ T Cells and Requires the Presence of Monocytes

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG‐mediated Treg expansion. CD4⁺ T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell–cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially “reprograms” CD4⁺ T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4⁺ cells. These reprogrammed CD4⁺ T cells subsequently secrete GM‐CSF and IL‐10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14⁺CD11c⁺ dendritic cells characterized by enhanced IL‐10 and decreased IL‐12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM‐CSF and Bcl‐2, but not TGF‐β, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4⁺ T cells in a STAT3‐dependent but TGF‐β‐independent manner, leading to the generation of monocyte‐derived dendritic cells with a tolerogenic cytokine profile.     

Expression of CD39 by Human Peripheral Blood CD4+CD25+ T Cells Denotes a Regulatory Memory Phenotype

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4⁺Foxp3⁺ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood‐derived human CD4⁺CD25⁺CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4⁺CD39⁺ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4⁺ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL‐17. These latter cell populations are increased, with a concomitant decrease in the CD4⁺CD25⁺CD39⁺ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell‐populations to allow tracking of these in health and disease, as in renal allograft rejection.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

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.     

Characterization of Human CD8+TCR− Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγnull Mouse Model

CD8⁺/TCR^? facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8⁺/TCR^?/CD56 negative (CD56^neg); the remainder are CD8⁺/TCR^?/CD56 bright (CD56^bright). The CD56^neg FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL‐2 receptor γ‐chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56^neg FC subpopulation promotes rapid reconstitution of donor HSPCs without graft‐versus‐host disease (GVHD); recipients of CD56^bright FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56^neg or CD56^bright FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56^bright FCs with HSPCs upregulates cathelicidin and β‐defensin 2, factors that prime responsiveness of HSPCs to stromal cell–derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell‐based therapy for enhancement of engraftment and prevention of GVHD.

     

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.     

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.     

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.     

Disengaging the IL‐2 Receptor with Daclizumab Enhances IL‐7–Mediated Proliferation of CD4+ and CD8+ T Cells

Allograft rejection is mainly driven by the production of IL‐2, which expands T cells by linking the IL‐2 receptor (IL‐2R) composed of three subunits: CD25, CD122 and CD132. Daclizumab, widely used in immunosuppression, is a humanized anti‐CD25 antibody that disrupts IL‐2 signaling by binding to CD25 and preventing the assembly of the high‐affinity IL‐2R. Here we show that Daclizumab, while blocking the T‐cell response to IL‐2, increases CD4⁺ and CD8⁺ T‐cell proliferative response to the homeostatic cytokine IL‐7. The IL‐7R shares CD132 with the IL‐2R and blocking of CD25 by Daclizumab results in the enhanced formation of the IL‐7R that in turn allows IL‐7 to bind more efficiently on the cell surface. The consequently increased IL‐7R signaling boosts intracellular phosphorylated STAT5 and T‐cell proliferation. In addition, treatment with Daclizumab delays the internalization of CD127 upon IL‐7 treatment, retaining T‐cell sensitivity to IL‐7 for a prolonged time. This effect of Daclizumab highlights the redundancy of the cytokine system, which may influence T‐cell proliferation in transplanted patients, and provides information to improve future immunosuppressive strategies.     

Absolute of CD4(+)CD25(+)FOXP3(+) regulatory T-cell count rather than its ratio in peripheral blood is related to long-term survival of renal allografts

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) may reflect the immune status of kidney transplant (KTx) recipients. Since individual KTx recipients show different lymphocyte counts, we hypothesized that the Treg absolute count rather than its peripheral ratio was more related to long-term survival. We enrolled 42 patients with more than 5-year KTx survival: 32 patients with stable graft function and 10 suffering chronic rejection (CR group). The stable group was divided into four subgroups according to graft survival time: subgroups A (5-6 years); B (6-7 years); C (7-8 years); and D (>8 years). Healthy volunteers were enrolled as controls. We compared the peripheral ratio and absolute count of CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25⁺Foxp3⁺ Tregs. Treg peripheral ratio was not significantly different among the three groups. However, the Treg absolute count was higher among the stable than the CR group (P < .01). Meanwhile, both Treg ratios and absolute counts were altered with renal graft survival. Treg absolute count in subgroups B and C were not only higher than that in subgroup A (P < .05), but also was significantly higher than that in the CR control group or (P < .05). Interestingly, both the Treg ratio and absolute count in subgroup D were lower than those in subgroups B and C (P < .05). Treg quantitation, which alters with graft survival time, may contribute to long-term acceptance of renal allografts. Compared to Treg peripheral ratio, the absolute count may be a superior index to estimate KTx recipient immune status.     

Low‐Dose IL‐2 for In Vivo Expansion of CD4+ and CD8+ Regulatory T Cells in Nonhuman Primates

IL‐2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high‐dose IL‐2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL‐2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low‐dose IL‐2 (1 million IU/m² BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low‐dose IL‐2 therapy significantly expanded peripheral blood CD4⁺ and CD8⁺ Tregs in vivo with limited expansion of non‐Treg cells. These expanded Tregs are mainly CD45RA^? Foxp3 ^high activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.     

Impact of Human Mutant TGFβ1/Fc Protein on Memory and Regulatory T Cell Homeostasis Following Lymphodepletion in Nonhuman Primates

Transforming growth factor β1 (TGFβ1) plays a key role in T cell homeostasis and peripheral tolerance. We evaluated the influence of a novel human mutant TGFβ1/Fc (human IgG4 Fc) fusion protein on memory CD4⁺ and CD8⁺ T cell (Tmem) responses in vitro and their recovery following antithymocyte globulin (ATG)–mediated lymphodepletion in monkeys. TGFβ1/Fc induced Smad2/3 protein phosphorylation in rhesus and human peripheral blood mononuclear cells and augmented the suppressive effect of rapamycin on rhesus Tmem proliferation after either alloactivation or anti‐CD3/CD28 stimulation. In combination with IL‐2, the incidence of CD4⁺CD25^hiFoxp3^hi regulatory T cells (Treg) and Treg:Th17 ratios were increased. In lymphodepleted monkeys, whole blood trough levels of infused TGFβ1/Fc were maintained between 2 and 7 μg/mL for 35 days. Following ATG administration, total T cell numbers were reduced markedly. In those given TGFβ1/Fc infusion, CD8⁺ T cell recovery to predepletion levels was delayed compared to controls. Additionally, numbers of CD4⁺CD25^hiCD127^lo Treg increased at 4–6 weeks after depletion but subsequently declined to predepletion levels by 12 weeks. In all monkeys, CD4⁺CD25^hiFoxp3^hi Treg/CD4⁺IL‐17⁺ cell ratios were reduced, particularly after stopping TGFβ1/Fc infusion. Thus, human TGFβ1/Fc infusion may delay Tmem recovery following lymphodepletion in nonhuman primates. Combined (low‐dose) IL‐2 infusion may be required to improve the Treg:Th17 ratio following lymphodepletion.     

CD4 T Cells but Not Th17 Cells Are Required for Mouse Lung Transplant Obliterative Bronchiolitis

Lung transplant survival is limited by obliterative bronchiolitis (OB), but the mechanisms of OB development are unknown. Previous studies in a mouse model of orthotopic lung transplantation suggested a requirement for IL‐17. We have used this orthotopic mouse model to investigate the source of IL‐17A and the requirement for T cells producing IL‐17A. The major sources of IL‐17A were CD4⁺ T cells and γδ T cells. Depletion of CD4⁺ T cells led to a significantly decreased frequency and number of IL‐17A⁺ lymphocytes and was sufficient to prevent acute rejection and OB. However, mice with STAT3‐deficient T cells, which are unable to differentiate into Th17 cells, rejected lung allografts and developed OB similar to control mice. The frequency of IL‐17A⁺ cells was not decreased in mice with STAT3‐deficient T cells due mainly to the presence of IL‐17A⁺ γδ T cells. Deficiency of γδ T cells also did not affect the development of airway fibrosis. Our data suggest that CD4⁺ T cells are required for OB development and expansion of IL‐17A responses in the lung, while Th17 and γδ T cells are not absolutely required and may compensate for each other.     

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.     

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 CD4+CD25+ T cells induced by locally expressed ICOS‐Ig: the role of Foxp3, Perforin,Granzyme B and IL‐10 ‐ an experimental study

We have previously reported that ICOS‐Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4⁺CD25⁺Foxp3⁺ T cells and specific xenograft prolongation. In the present study we isolated and purified CD4⁺CD25⁺ T cells from ICOS‐Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4⁺CD25⁺ T cells isolated from xenografts secreting ICOS‐Ig were analysed by flow cytometry and gene expression by real‐time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre‐existing Foxp3⁺ Treg, IL‐10, perforin and granzyme B. CD4⁺CD25⁺Foxp3⁺ T cells isolated from xenografts secreting ICOS‐Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.     

The Effect of Costimulatory and Interleukin 2 Receptor Blockade on Regulatory T Cells in Renal Transplantation

Regulatory T cells (Treg) are critical regulators of immune tolerance. Both IL‐2 and CD28‐CD80/CD86 signaling are critical for CD4⁺CD25⁺FOXP3⁺ Treg survival in mice. Yet, both belatacept (a second‐generation CTLA‐4Ig) and basiliximab (an anti‐CD25 monoclonal antibody) are among the arsenal of current immunotherapies being used in kidney transplant patients. In this study, we explored the direct effect of basiliximab and belatacept on the Tregs in peripheral blood both in the short term and long term and in kidney biopsies of patients with acute rejection. We report that the combined belatacept/basiliximab therapy has no long‐term effect on circulating Tregs when compared to a calcineurin inhibitor (CNI)‐treated group. Moreover, belatacept‐treated patients had a significantly greater number of FOXP3⁺ T cells in graft biopsies during acute rejection as compared to CNI‐treated patients. Finally, it appears that the basiliximab caused a transient loss of both FOXP3⁺ and FOXP3^? CD25⁺ T cells in the circulation in both treatment groups raising important questions about the use of this therapy in tolerance promoting therapeutic protocols.     

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

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

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.