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.     

Attenuation of Donor‐Reactive T Cells Allows Effective Control of Allograft Rejection Using Regulatory T Cell Therapy

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long‐term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor‐specific transfusion followed by cyclophosphamide treatment deleted 70–80% donor‐reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 10⁶ Tregs with direct donor reactivity or 25 × 10⁶ polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen‐specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8⁺ T cells and concomitant increase in endogenous Tregs among graft‐infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor‐reactive T cells will be an important component of Treg‐based therapies in transplantation.     

T Regulatory Cells Play a Significant Role in Modulating MHC Class I Antibody‐Induced Obliterative Airway Disease

The molecular mechanisms leading to the development of chronic lung allograft dysfunction following de novo development of antibodies to mismatched donor MHC remain undefined. We demonstrated that intrabronchial administration of antibodies to MHC class I resulted in induction of both innate and adaptive cellular immune responses characterized by a predominance of Th17 specific to lung associated self‐antigens Kα1‐tubulin and Collagen‐V leading to the development of obliterative airway lesions (OAD), correlate of chronic rejection following human lung transplantation. To determine the role of regulatory T cells (Treg) in the pathogenesis of OAD, we administered anti‐MHC class I to mice, in which Treg were depleted by conditional ablation of FoxP3+cells. Under this condition, we observed a threefold increase in pulmonary cellular infiltration, luminal occlusion and fibrous deposition when compared anti‐MHC class I Ab administered mice maintaining FoxP3. OAD lesions were accompanied with enhanced accumulation of neutrophils along with self‐antigen‐specific Th17 and humoral responses. However, IL‐17‐blockade or adoptive transfer of Treg abrogated OAD. We conclude that Treg exerts a suppressive effect on anti‐MHC induced IL‐8‐mediated neutrophil infiltration and innate immune responses that leads to inhibition of Th17 immune responses to lung associated self‐antigens which is critical for development of OAD.     

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

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

Regulatory T Cells in AKI

Human AKI is manifested by inflammation, and an early feature in the pathogenesis is the accumulation of immune cells in the kidney. To understand the pathophysiology of AKI, results from animal models have shown a causal relation between the leukocyte activation and infiltration to the kidney after kidney ischemia-reperfusion. Blocking the activation or trafficking of proinflammatory leukocytes into the kidney preserves renal function and histologic integrity. In contrast, the anti-inflammatory lymphocytes called regulatory T cells have an intrinsic renal-protective function and may represent a novel therapeutic approach and/or target for pharmacological manipulation to ameliorate AKI. This review will highlight the recent insight gained into the role and mechanisms of regulatory T cells in AKI.AKI is a common problem in hospitalized patients, and it affects >5% of all inpatients,¹ with rates of 40% or more in those patients who are critically ill.² AKI significantly increases the risk of chronic renal disease, ESRD, and death, presenting a major burden to patients and the health care system.² Because of its high metabolic activity, caused, in part, by the handling and transport of filtered ions, amino acids, and other small molecules, the kidney is highly susceptible to acute injury from exposure to and accumulation of nephrotoxic substances or lack of sufficient perfusion. One common response of renal cells to injury is the production and/or release of proinflammatory proteins. Modulating the immune system’s response to the proinflammatory signals produced in the injured kidney can have a profound effect on the extent of renal dysfunction and tissue damage. Whereas a great deal is known about the cells and other mediators in the immune system that contribute to and extend renal damage after an insult,³ recent studies have begun to illuminate the role of regulatory T cells (Tregs), which suppress renal inflammation and preserve renal function.     

Low‐Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

Regulatory T cells (T_reg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote T_reg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human T_reg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low‐dose rapamycin and subtherapeutic T_reg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2^?/?Il2rg^?/? mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T‐cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4⁺ but not CD8⁺ T lymphocytes were sensitive to T_reg and rapamycin‐induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of T_reg‐based immunosuppressive protocols in clinical practice. By inhibiting TA, T_reg and rapamycin may prevent chronic transplant dysfunction and improve long‐term allograft survival.     

Generation and Large‐Scale Expansion of Human Inducible Regulatory T Cells That Suppress Graft‐Versus‐Host Disease

Adoptive transfer of thymus‐derived natural regulatory T cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft‐versus‐host disease (GVHD). TGFß induces Foxp3 expression and suppressive function in stimulated murine CD4+25‐ T cells, and these induced Treg (iTregs), like nTreg, suppress auto‐ and allo‐reactivity in vivo. However, while TGFß induces Foxp3 expression in stimulated human T cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFß‐dependent Foxp3 expression and induces a potent suppressor function in naive (CD4+ 25–45RA+) T cells. Rapa/TGFß iTregs are anergic, express CD25 at levels higher than expanded nTregs and few cells secrete IL‐2, IFNγ or IL‐17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFß induces suppressive function even in the presence of memory CD4+ T cells. A single apheresis unit of blood yields an average ～240 × 10⁹ (range ～70–560 × 10⁹) iTregs from CD4+25‐ T cells in ≤2 weeks of culture. Most importantly, Rapa/TGFß iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.     

Regulatory T cell expressed MyD88 is critical for prolongation of allograft survival

MyD88 signaling directly promotes T‐cell survival and is required for optimal T‐cell responses to pathogens. To examine the role of T‐cell‐intrinsic MyD88 signals in transplantation, we studied mice with targeted T‐cell‐specific MyD88 deletion. Contrary to expectations, we found that these mice were relatively resistant to prolongation of graft survival with anti‐CD154 plus rapamycin in a class II‐mismatched system. To specifically examine the role of MyD88 in Tregs, we created a Treg‐specific MyD88‐deficient mouse. Transplant studies in these animals replicated the findings observed with a global T‐cell MyD88 knockout. Surprisingly, given the role of MyD88 in conventional T‐cell survival, we found no defect in the survival of MyD88‐deficient Tregs in vitro or in the transplant recipients and also observed intact cell homing and expression of Treg effector molecules. MyD88‐deficient Tregs also fail to protect allogeneic bone marrow transplant recipients from chronic graft‐versus‐host disease, confirming the observations of defective regulation seen in a solid organ transplant system. Together, our data define MyD88 as having a divergent requirement for cell survival in non‐Tregs and Tregs, and a yet‐to‐be defined survival‐independent requirement for Treg function during the response to alloantigen.     

GITR Ligation Blocks Allograft Protection by Induced CD25+CD4+ Regulatory T Cells without Enhancing Effector T-Cell Function

The induction of operational tolerance prior to transplant could provide a solution to the complications of current immunosuppression in transplantation. In rodents, operational tolerance frequently correlates with the presence of CD25(+)CD4(+) regulatory T cells (Tregs) but their function is usually demonstrated by adoptive transfer into lymphopenic hosts leading some to question their relevance to normal immunocompetent recipients. The role of these cells in primary transplant recipients has been explored using anti-CD25 antibody but specific targeting of Treg is not possible since CD25 is also up-regulated on activated effector T cells. To overcome this limitation we targeted the Treg associated molecule GITR in tolerized primary transplant recipients. This reverses regulation resulting in acute allograft rejection. This is not due to co-stimulation of effector cells since rejection mediated by isolated populations of CD4(+)CD25(-) or CD8(+)CD25(-) T cells transferred into Rag(-/-) mice was not enhanced by anti-GITR antibody. Furthermore, GITR cross-linking does not provide co-stimulation for in vitro proliferation of the same CD4(+)CD25(-) or CD8(+)CD25(-) T-cell populations in response donor-strain APC. Thus, CD4(+)CD25(+)GITR(+) Treg play an essential role in early graft protection in primary transplant recipients following tolerance induction providing further support for protocols that might generate similar populations in clinical transplantation.     

Human FOXP3+ Regulatory T Cells in Transplantation

CD4+CD25+FOXP3+ suppressive regulatory T cells (Treg) represent a subset of immune regulatory cells. Based on experimental results, Treg have recently been considered as a potential treatment option in several diseases. Compared with murine Treg, human CD4+CD25+FOXP3+ cells are less well characterized and understood, so a thorough understanding of their biology is vital before clinical applications can be initiated. This review summarizes knowledge on generation, phenotypic characteristics and function of human Treg. The possible role of these cells in organ transplantation, as well as interactions between immunosuppression and Treg are also discussed.     

Induction of T Regulatory Cells Attenuates Idiopathic Nephrotic Syndrome

Buffalo/Mna rats spontaneously develop FSGS and nephrotic syndrome as a result of an immune disorder. Similar to some humans with FSGS, the disease recurs after renal transplantation, suggesting the involvement of a circulating factor. Here, we tested the effect of several immunosuppressive treatments on these rats. Although corticosteroids, cyclosporin A, and anti–T cell receptor treatment reduced proteinuria, only the deoxyspergualin derivative LF15-0195 led to a rapid and complete normalization of proteinuria. Furthermore, this compound led to the regression of renal lesions during both the initial disease and posttransplantation recurrence. The frequency of splenic and peripheral CD4⁺CD25⁺FoxP3⁺ T lymphocytes significantly increased with remission. Moreover, the transfer of purified LF15-0195–induced CD4⁺CD25⁺ T cells to irradiated Buff/Mna rats significantly reduced their proteinuria compared with the transfer of untreated control cells, suggesting that LF15-0195 induces regulatory T cells that are able to induce regression of rat nephropathy. These data suggest that idiopathic nephrotic syndrome/FSGS disease can be regulated by cellular transfer, but how this regulation leads to the reorganization of the podocyte cytoskeleton remains to be determined.Idiopathic nephrotic syndrome (INS) with primary FSGS lesions is a disease of unknown cause that is defined by selective proteinuria, hypoalbuminemia, and nonspecific lesions with a glomerular sclerosis. Although treatments such as corticosteroids, cyclosporin A (CsA), and cyclophosphamide remain useful, at least 20% of affected patients ultimately require hemodialysis and/or kidney transplantation for end-stage renal failure. Moreover, despite treatment, the initial disease immediately relapses in 30 to 50% of transplant patients¹ and leads to the loss of the graft. This immediate (and iterative) recurrence, together with the beneficial effect of plasmatic exchanges² or immunoadsorptions,^3,4 strongly supports the presence of a circulating factor.The majority of animal models of FSGS present secondary forms^5–7 and do not supply a causal model with a permeability factor.⁸ The Buffalo/Mna rat strain develops a spontaneous glomerulonephritis (with albuminuria, edema, and lipidic disorders) at 3 mo of age, and FSGS lesions appear at 4 to 6 mo of age.^9,10 In addition to a genetic background predisposing to proteinuria¹¹ (a growing concept also noted in humans), we recently highlighted the involvement of an extrarenal circulating factor. We demonstrated both the recurrence of the initial disease after transplantation of normal rat kidneys into Buff/Mna recipients and remission when nephrotic Buff/Mna kidneys were transplanted into normal rats.¹² We also highlighted the involvement of activated macrophages and Th2 lymphocytes in this disease,¹³ as reported in the human disease. All of these findings suggest that this rat model may be relevant for studying the challenge of INS recurrence after transplantation in the clinic.In this study, we used the Buff/Mna strain to test the antiproteinuric effect of various immunoregulatory compounds. Only a deoxyspergualin (DSG) derivative, LF15-0195,¹⁴ specifically induced a rapid and complete remission of the initial kidney disease as well as its posttransplantation recurrence. This effect was manifested as a resolution of both proteinuria and histologic lesions. These findings may provide novel insights into the development of innovative, clinically applicable therapeutics for the treatment of INS.     

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.     

Regulatory T Cells in the Control of Transplantation Tolerance and Autoimmunity

A role for immunoregulatory T cells in the maintenance of self-tolerance and in transplantation tolerance has long been suggested, but the identification of such cells has not been achieved until recently. With the characterisation of spontaneously occurring CD4+CD25+ and NK1.1+ T subpopulations of T cells as regulatory cells in rodents and in humans, together with several in vitro generated regulatory T-cell populations, it seems possible that 'customised' regulatory cells possessing antidonor specificity may become therapeutic tools in clinical transplantation tolerance.     

Immunosuppressive Dendritic and Regulatory T Cells are Upregulated in Melanoma Patients

Background Immunologic therapies for melanoma rarely succeed, suggesting a persistent counter-regulatory immune modulation. Regulatory T cells (T_regs) and plasmacytoid subpopulations of dendritic cells (pDCs) inhibit the immune response. We hypothesize that melanoma upregulates T_regs and subpopulations of immunosuppressive dendritic cells (DCs). Methods Peripheral blood mononuclear cells (PBMCs) were obtained from healthy controls, stage I and stage IV melanoma patients. T_regs were identified as CD4+ and CD25^hi. Dendritic cells were identified using a DC cocktail of antibodies including CD11c+ myeloid dendritic cells (mDCs) and CD123+ pDCs. Serum transforming growth factor-β (TGF-β), interleukin-4 (IL-4) and interleukin-10 (IL-10) levels were determined by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (ANOVA). Results Stage IV melanoma patients had a doubling of regulatory T cells compared to both normal subjects and stage I melanoma patients. There was a significantly higher number of DCs in all melanoma patients compared to normal subjects. Stage I melanoma patients had a significantly higher number of pDCs than normal subjects, and all melanoma patients had a higher concentration of mDCs than controls. Serum IL-4 and IL-10 were not detectable but serum TGF-β levels were significantly higher in stage I and stage IV melanoma patients compared to normal controls. Conclusion Advanced melanoma is associated with increased numbers of circulating dendritic cells and regulatory T cells. These data suggest that melanoma induces immunosuppressive DCs and regulatory T cells in the systemic circulation.     

CCR6 Recruits Regulatory T Cells and Th17 Cells to the Kidney in Glomerulonephritis

T cells recruited to the kidney contribute to tissue damage in crescentic and proliferative glomerulonephritides. Chemokines and their receptors regulate T cell trafficking, but the expression profile and functional importance of chemokine receptors for renal CD4⁺ T cell subsets are incompletely understood. In this study, we observed that renal FoxP3⁺CD4⁺ regulatory T cells (Tregs) and IL-17–producing CD4⁺ T (Th17) cells express the chemokine receptor CCR6, whereas IFNγ-producing Th1 cells are CCR6⁻. Induction of experimental glomerulonephritis (nephrotoxic nephritis) in mice resulted in upregulation of the only CCR6 ligand, CCL20, followed by T cell recruitment, renal tissue injury, albuminuria, and loss of renal function. CCR6 deficiency aggravated renal injury and increased mortality (from uremia) among nephritic mice. Compared with wild-type (WT) mice, CCR6 deficiency reduced infiltration of Tregs and Th17 cells but did not affect recruitment of Th1 cells in the setting of glomerulonephritis. Adoptive transfer of WT but not CCR6-deficient Tregs attenuated morphologic and functional renal injury in nephritic mice. Furthermore, reconstitution with WT Tregs protected CCR6^−/− mice from aggravated nephritis. Taken together, these data suggest that CCR6 mediates renal recruitment of both Tregs and Th17 cells and that the reduction of anti-inflammatory Tregs in the presence of a fully functional Th1 response aggravates experimental glomerulonephritis.The currently defined four subsets of CD4⁺ T cells—namely Th1, Th2, Th17, and regulatory T cells (Tregs)—are central players in adaptive immunity.¹ Inappropriate or unbalanced T cell responses underlie several distinct types of autoimmune diseases, some of which target the kidney. In particular, infiltrating effector CD4⁺ T cells of the Th1 type are supposed to initiate and perpetuate glomerular and tubulointerstitial tissue damage in crescentic and proliferative forms of glomerulonephritis either directly by cytotoxic functions or cytokine secretion or indirectly by providing help for induction of autoantibodies and cytokines or immune complexes or by activating macrophages.² Another IL-17–producing CD4⁺ effector T cell subset, termed Th17, has been identified.³ Ongoing studies demonstrate that Th17 cells are involved in driving autoimmune processes previously thought to be exclusively Th1-mediated, such as rheumatoid arthritis,⁴ multiple sclerosis,⁵ and crescentic glomerulonephritis.^6,7 In contrast, the Th2 cell–mediated immune response seems to be of importance in nonproliferative forms of glomerulonephritis, such as minimal-change and membranous nephropathy.² In rodents and humans, a unique subset of CD4⁺CD25⁺FoxP3⁺ Tregs has been shown to control peripheral tolerance. An anti-inflammatory role of Tregs has also been suggested in human and experimental glomerulonephritis^8,9; however, the molecular basis of immunosuppression and the trafficking properties of Tregs are still unknown.Before T cells can exert their effects on renal damage or repair, they have to reach the site of injury. In recent years, it has become clear that a group of small proteins called chemokines serve as key regulators of directional T cell trafficking under inflammatory conditions.¹⁰ Target cell specificity is achieved by differential expression of corresponding chemokine receptors on the surface of leukocyte subsets. The different CD4⁺ T cell populations in humans and mice display distinct patterns of chemokine receptor expression. Th1-polarized cells preferentially express CXCR3, CCR5, and CXCR6, whereas Th2 cells express higher amounts of CCR3, CCR4, and CCR8.¹⁰ Although CCR6 and CXCR3 have been detected on Th17 cells,^11–13 the chemokine receptor expression profile of this subset has yet to be defined, particularly with respect to functional importance. Tregs with potential anti-inflammatory properties express a wider repertoire of chemokine receptors, many of which they share with proinflammatory T cell subsets, such as CCR4, CCR5, CCR6, CCR8, CXCR3, and CXCR6.^10,13 It remains unclear, however, which of these chemokine receptors (or which receptor combination) is crucial for guiding Tregs to the site of inflammation. In general, the precise molecular basis for the chemokine/chemokine receptor-mediated trafficking of CD4⁺ T cell subsets in glomerulonephritis is not defined.Several features of CCR6 and its only highly specific ligand CCL20 argue for a critical role of this chemokine–chemokine receptor pair in this context. CCR6 is expressed in human and mouse dendritic cell subpopulations,^14–16 B cells,^17,18 and T cell subsets.^13,19 Recent data suggest that, in particular, autoreactive Th17 cells, which contribute to renal tissue injury in nephritis, are highly CCR6⁺.^11,12 This study was designed to examine the potential role of CCR6 in glomerulonephritis. We therefore induced the T cell–dependent model of nephrotoxic nephritis (NTN) in wild-type (WT) and CCR6^−/− mice to address two major issues: (1) What is the expression pattern of CCR6 on infiltrating renal CD4⁺ T cell subsets? (2) Does CCR6 deficiency influence the clinical course of experimental glomerulonephritis, and, if so, what are the mechanisms?