CD4+CD25+ T regulatory cells in renal transplantation.

The immune response to an allograft activates lymphocytes with the capacity to cause rejection. Activation of CD4+CD25+Foxp3+T regulatory cells (Treg) can down-regulate allograft rejection and can induce immune tolerance to the allograft. Treg represent <10% of peripheral CD4+T cells and do not markedly increase in tolerant hosts. CD4+CD25+Foxp3+T cells include both resting and activated Treg that can be distinguished by several markers, many of which are also expressed by effector T cells. More detailed characterization of Treg to identify increased activated antigen-specific Treg may allow reduction of non-specific immunosuppression. Natural thymus derived resting Treg (tTreg) are CD4+CD25+Foxp3+T cells and only partially inhibit alloantigen presenting cell activation of effector cells. Cytokines produced by activated effector cells activate these tTreg to more potent alloantigen-activated Treg that may promote a state of operational tolerance. Activated Treg can be distinguished by several molecules they are induced to express, or whose expression they have suppressed. These include CD45RA/RO, cytokine receptors, chemokine receptors that alter pathways of migration and transcription factors, cytokines and suppression mediating molecules. As the total Treg population does not increase in operational tolerance, it is the activated Treg which may be the most informative to monitor. Here we review the methods used to monitor peripheral Treg, the effect of immunosuppressive regimens on Treg, and correlations with clinical outcomes such as graft survival and rejection. Experimental therapies involving ex vivo Treg expansion and administration in renal transplantation are not reviewed.

Transplant Tolerance, Not Only Clonal Deletion.

The quest to understand how allogeneic transplanted tissue is not rejected and how tolerance is induced led to fundamental concepts in immunology. First, we review the research that led to the Clonal Deletion theory in the late 1950s that has since dominated the field of immunology and transplantation. At that time many basic mechanisms of immune response were unknown, including the role of lymphocytes and T cells in rejection. These original observations are reassessed by considering T regulatory cells that are produced by thymus of neonates to prevent autoimmunity. Second, we review "operational tolerance" induced in adult rodents and larger animals such as pigs. This can occur spontaneously especially with liver allografts, but also can develop after short courses of a variety of rejection inhibiting therapies. Over time these animals develop alloantigen specific tolerance to the graft but retain the capacity to reject third-party grafts. These animals have a "split tolerance" as peripheral lymphocytes from these animals respond to donor alloantigen in graft versus host assays and in mixed lymphocyte cultures, indicating there is no clonal deletion. Investigation of this phenomenon excludes many mechanisms, including anti-donor antibody blocking rejection as well as anti-idiotypic responses mediated by antibody or T cells. This split tolerance is transferred to a second immune-depleted host by T cells that retain the capacity to effect rejection of third-party grafts by the same host. Third, we review research on alloantigen specific inhibitory T cells that led to the first identification of the CD4+CD25+T regulatory cell. The key role of T cell derived cytokines, other than IL-2, in promoting survival and expansion of antigen specific T regulatory cells that mediate transplant tolerance is reviewed. The precise methods for inducing and diagnosing operational tolerance remain to be defined, but antigen specific T regulatory cells are key mediators.

Interleukin-5 (IL-5) Therapy Prevents Allograft Rejection by Promoting CD4+CD25+ Ts2 Regulatory Cells That Are Antigen-Specific and Express IL-5 Receptor.

CD4+CD25+Foxp3+T cell population is heterogenous and contains three major sub-groups. First, thymus derived T regulatory cells (tTreg) that are naïve/resting. Second, activated/memory Treg that are produced by activation of tTreg by antigen and cytokines. Third, effector lineage CD4+CD25+T cells generated from CD4+CD25- T cells' activation by antigen to transiently express CD25 and Foxp3. We have shown that freshly isolated CD4+CD25+T cells are activated by specific alloantigen and IL-4, not IL-2, to Ts2 cells that express the IL-5 receptor alpha. Ts2 cells are more potent than naïve/resting tTreg in suppressing specific alloimmunity. Here, we showed rIL-5 promoted further activation of Ts2 cells to Th2-like Treg, that expressed foxp3, irf4, gata3 and il5. In vivo, we studied the effects of rIL-5 treatment on Lewis heart allograft survival in F344 rats. Host CD4+CD25+T cells were assessed by FACS, in mixed lymphocyte culture and by RT-PCR to examine mRNA of Ts2 or Th2-like Treg markers. rIL-5 treatment given 7 days after transplantation reduced the severity of rejection and all grafts survived ≥60d whereas sham treated rats fully rejected by day 31 (p<0.01). Treatment with anti-CD25 or anti-IL-4 monoclonal antibody abolished the benefits of treatment with rIL-5 and accelerated rejection. After 10d treatment with rIL-5, hosts' CD4+CD25+ cells expressed more Il5ra and responded to specific donor Lewis but not self. Enriched CD4+CD25+ cells from rIL-5 treated rats with allografts surviving >60 days proliferated to specific donor only when rIL-5 was present and did not proliferate to self or third party. These cells had more mRNA for molecules expressed by Th2-like Treg including Irf4, gata3 and Il5. These findings were consistent with IL-5 treatment preventing rejection by activation of Ts2 cells and Th2-like Treg.

Multiple sclerosis patients have reduced resting and increased activated CD4+CD25+FOXP3+T regulatory cells.

Resting and activated subpopulations of CD4+CD25+CD127loT regulatory cells (Treg) and CD4+CD25+CD127+ effector T cells in MS patients and in healthy individuals were compared. Peripheral blood mononuclear cells isolated using Ficoll Hypaque were stained with monoclonal antibodies and analysed by flow cytometer. CD45RA and Foxp3 expression within CD4+ cells and in CD4+CD25+CD127loT cells identified Population I; CD45RA+Foxp3+, Population II; CD45RA-Foxp3hi and Population III; CD45RA-Foxp3+ cells. Effector CD4+CD127+ T cells were subdivided into Population IV; memory /effector CD45RA- CD25-Foxp3- and Population V; effector naïve CD45RA+CD25-Foxp3-CCR7+ and terminally differentiated RA+ (TEMRA) effector memory cells. Chemokine receptor staining identified CXCR3+Th1-like Treg, CCR6+Th17-like Treg and CCR7+ resting Treg. Resting Treg (Population I) were reduced in MS patients, both in untreated and treated MS compared to healthy donors. Activated/memory Treg (Population II) were significantly increased in MS patients compared to healthy donors. Activated effector CD4+ (Population IV) were increased and the naïve/ TEMRA CD4+ (Population V) were decreased in MS compared to HD. Expression of CCR7 was mainly in Population I, whereas expression of CCR6 and CXCR3 was greatest in Populations II and intermediate in Population III. In MS, CCR6+Treg were lower in Population III. This study found MS is associated with significant shifts in CD4+T cells subpopulations. MS patients had lower resting CD4+CD25+CD45RA+CCR7+ Treg than healthy donors while activated CD4+CD25hiCD45RA-Foxp3hiTreg were increased in MS patients even before treatment. Some MS patients had reduced CCR6+Th17-like Treg, which may contribute to the activity of MS.

Autoantigen specific IL-2 activated CD4+CD25+T regulatory cells inhibit induction of experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) induced by peripheral nerve myelin (PNM) is self-limiting and re-immunization with PNM does not re-activate disease. This study showed inhibition of EAN by CD4+CD25+T cells both from sensitized hosts or from naïve hosts after ex-vivo activation by PNM and rIL-2. Transfer of naïve CD4+CD25+T cells has no effect on EAN, nor did naïve CD4+CD25+T cells activated with rIL-2 and renal tubular antigen. Culture of naive CD4+CD25+Treg with rIL-2 and PNM induced mRNA for the IFN-gamma receptor. We showed naïve CD4+CD25+T cells activated by specific auto-antigen and rIL-2 produced more potent antigen-specific Treg that may have therapeutic potential.

Alloactivation of Naïve CD4+CD8-CD25+T Regulatory Cells: Expression of CD8α Identifies Potent Suppressor Cells That Can Promote Transplant Tolerance Induction.

Therapy with alloantigen-specific CD4+CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rß2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+CD25+Foxp3+Treg. After culture of naïve DA CD4+CD8-CD25+T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10-20% and CD8ß on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+CD8α+CD25+Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+CD8-CD25+T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+CD8-CD25+Foxp3+T cells was a marker of activated and potent Treg that included alloantigen-specific Treg.

Interleukin-5 Mediates Parasite-Induced Protection against Experimental Autoimmune Encephalomyelitis: Association with Induction of Antigen-Specific CD4+CD25+ T Regulatory Cells.

OBJECTIVE: To examine if the protective effect of parasite infection on experimental autoimmune encephalomyelitis (EAE) was due to interleukin (IL)-5, a cytokine produced by a type-2 response that induces eosinophilia. We hypothesize that, in parasite infections, IL-5 also promotes expansion of antigen-specific T regulatory cells that control autoimmunity. METHODS: Nippostrongylus brasiliensis larvae were used to infect Lewis rats prior to induction of EAE by myelin basic protein. Animals were sham treated, or given blocking monoclonal antibodies to interleukin 4 or 5 or to deplete CD25+ T cells. Reactivity of CD4+CD25+ T regulatory cells from these animals was examined. RESULTS: Parasite-infected hosts had reduced severity and length of EAE. The beneficial effect of parasitic infection was abolished with an anti-IL-5 or an anti-CD25 monoclonal antibody (mAb), but not anti-IL-4 mAb. Parasite-infected animals with EAE developed antigen-specific CD4+CD25+ T regulatory cells earlier than EAE controls and these expressed more Il5ra than controls. Treatment with IL-5 also reduced the severity of EAE and induced Il5ra expressing CD4+CD25+ T regulatory cells. INTERPRETATION: The results of this study suggested that IL-5 produced by the type-2 inflammatory response to parasite infection promoted induction of autoantigen-specific CD25+Il5ra+ T regulatory cells that reduced the severity of autoimmunity. Such a mechanism may explain the protective effect of parasite infection in patients with multiple sclerosis where eosinophilia is induced by IL-5, produced by the immune response to parasites.

Changes in Reactivity In Vitro of CD4+CD25+ and CD4+CD25- T Cell Subsets in Transplant Tolerance.

Transplant tolerance induced in adult animals is mediated by alloantigen-specific CD4+CD25+ T cells, yet in many models, proliferation of CD4+ T cells from hosts tolerant to specific-alloantigen in vitro is not impaired. To identify changes that may diagnose tolerance, changes in the patterns of proliferation of CD4+, CD4+CD25+, and CD4+CD25- T cells from DA rats tolerant to Piebald Virol Glaxo rat strain (PVG) cardiac allografts and from naïve DA rats were examined. Proliferation of CD4+ T cells from both naïve and tolerant hosts was similar to both PVG and Lewis stimulator cells. In mixed lymphocyte culture to PVG, proliferation of naïve CD4+CD25- T cells was greater than naïve CD4+ T cells. In contrast, proliferation of CD4+CD25- T cells from tolerant hosts to specific-donor PVG was not greater than CD4+ T cells, whereas their response to Lewis and self-DA was greater than CD4+ T cells. Paradoxically, CD4+CD25+ T cells from tolerant hosts did not proliferate to PVG, but did to Lewis, whereas naïve CD4+CD25+ T cells proliferate to both PVG and Lewis but not to self-DA. CD4+CD25+ T cells from tolerant, but not naïve hosts, expressed receptors for interferon (IFN)-γ and IL-5 and these cytokines promoted their proliferation to specific-alloantigen PVG but not to Lewis or self-DA. We identified several differences in the patterns of proliferation to specific-donor alloantigen between cells from tolerant and naïve hosts. Most relevant is that CD4+CD25+ T cells from tolerant hosts failed to proliferate or suppress to specific donor in the absence of either IFN-γ or IL-5. The proliferation to third-party and self of each cell population from tolerant and naïve hosts was similar and not affected by IFN-γ or IL-5. Our findings suggest CD4+CD25+ T cells that mediate transplant tolerance depend on IFN-γ or IL-5 from alloactivated Th1 and Th2 cells.

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 100days 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.

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 >100days 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 3days 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.

CD4+CD25+ T Regulatory Cells in Transplantation Tolerance: 25 Years On.

In the 1970s, the capacity of T cells to inhibit immunity and those from transplant tolerant hosts to transfer alloantigen-specific suppression to lymphopenic recipients was described. CD4 T suppressor cells that ex vivo reverted to effector cells were described in the 1980s. Their antigen-specific suppressor function could be preserved by stimulation by specific donor alloantigen and cytokines from activated lymphocytes. This led to the finding that alloantigen-specific T suppressor cells express IL-2 receptor (CD25) and that IL-2 in part promotes their survival.Whether these alloantigen-specific CD4CD25FOXP3 regulatory T (Treg) cells are progeny of thymic-derived CD4CD25FOXP3 Treg (tTreg) cells or are induced from peripheral effector CD4CD25FOXP3T cells (iTreg) cells is still debated.In vitro studies of antigen specific Treg has been difficult as they die in the absence of cytokines produced by immune activated cells. The antigen-specific CD4CD25 T cells that control rejection in tolerant hosts differ from the naive tTreg. Thymic-derived Treg cells are not antigen-specific, and very high ratios are required to suppress rejection. Thymic-derived Treg cells can be expanded ex vivo and are currently being tested in trials to control allograft rejection and graft versus host disease.Thymic-derived Treg cells with specific receptors for alloantigen are activated by either IL-2 or IL-4 but rapidly become dependent on other cytokines, respectively, IFN-γ or IL-12 if activated by IL-2, or IL-5 if activated by IL-4. The Th1 and Th2 pathways for activation of tTreg produce more potent antigen-specific Treg that only suppress specific donor rejection. After 25 years, much remains unknown about antigen-specific CD4CD25FOXP3 Treg-mediating transplant tolerance.

T Cells: Soldiers and Spies--The Surveillance and Control of Effector T Cells by Regulatory T Cells.

Traditionally, T cells were CD4+ helper or CD8+ cytotoxic T cells, and with antibodies, they were the soldiers of immunity. Now, many functionally distinct subsets of activated CD4+ and CD8+ T cells have been described, each with distinct cytokine and transcription factor expression. For CD4+ T cells, these include Th1 cells expressing the transcription factor T-bet and cytokines IL-2, IFN-γ, and TNF-ß; Th2 cells expressing GATA-3 and the cytokines IL-4, IL-5, and IL-13; and Th17 cells expressing RORγt and cytokines IL-17A, IL-17F, IL-21, and IL-22. The cytokines produced determine the immune inflammation that they mediate. T cells of the effector lineage can be naïve T cells, recently activated T cells, or memory T cells that can be distinguished by cell surface markers. T regulatory cells or spies were characterized as CD8+ T cells expressing I-J in the 1970s. In the 1980s, suppressor cells fell into disrepute when the gene for I-J was not present in the mouse MHC I region. At that time, a CD4+ T cell expressing CD25, the IL-2 receptor-α, was identified to transfer transplant tolerance. This was the same phenotype of activated CD4+ CD25+ T cells that mediated rejection. Thus, the cells that could induce tolerance and undermine rejection had similar badges and uniforms as the cells effecting rejection. Later, FOXP3, a transcription factor that confers suppressor function, was described and distinguishes T regulatory cells from effector T cells. Many subtypes of T regulatory cells can be characterized by different expressions of cytokines and receptors for cytokines or chemokines. In intense immune inflammation, T regulatory cells express cytokines characteristic of effector cells; for example, Th1-like T regulatory cells express T-bet, and IFN-γ-like Th1 cells and effector T cells can change sides by converting to T regulatory cells. Effector T cells and T regulatory cells use similar molecules to be activated and mediate their function, and thus, it can be very difficult to distinguish soldiers from spies.

Induction of antigen specific CD4(+)CD25(+)Foxp3(+)T regulatory cells from naïve natural thymic derived T regulatory cells.

CD4(+)CD25(+)FOXP3(+)T regulatory cells (Treg) play a major role in prevention of induction and control of immune responses, and contribute to induction of immune tolerance. Natural or thymic Treg (tTreg) have non-antigen specific suppressor action. Tolerance to a specific antigen is also mediated by CD4(+)CD25(+)FOXP3(+)Treg, but the source of these cells is disputed. Many suggest that they are derived from effector lineage CD4(+)CD25(-)FOXP3(-)T cells and are induced Treg (iTreg). Our work shows that tTreg with specific TCR for the antigen can be activated to more potent antigen specific Treg. We have demonstrated that initial activation of tTreg with antigen and IL-2 induces antigen specific Treg that express receptors for the late Th1 cytokines IFN-γ and IL-12. These antigen specific Treg suppress effector lineage T cells at much lower ratios than tTreg, and we call these Ts1 cells as they are activated by Th1 cytokines and express receptors for Th1 cytokines. Further activation of Ts1 cells with specific antigen and late Th1 cytokines such as IL-12 induces very potent Th1-like Treg, that express t-bet, the transcription factor for Th1 cells, as well as the Th1 cytokine IFN-γ. Similar Th1-like Treg can be induced in IL-2 activated tTreg, by IFN-γ or IL-27. tTreg activated by antigen in the presence of IL-4 induces antigen specific Treg that express the IL-5 receptor, and these Ts2 cells can be induced to Th2-like Treg by IL-5 and antigen. tTreg can be activated to antigen specific Tregs that induce tolerance and have therapeutic potential.

Acute renal failure and metformin-associated lactic acidosis following colonoscopy.

Two patients with type 2 DM developed acute kidney injury and lactic acidosis following colonoscopy despite withholding metformin. We recommend that DM patients on metformin also withhold ACEI, ARB until their dehydration is reversed after colonoscopy. This should reduce the risk of acute renal failure (ARF) and of lactic acidosis.

High-sensitivity troponin as a predictor of cardiac events and mortality in the stable dialysis population.

BACKGROUND: High-sensitivity cardiac troponin T (hs-cTnT) is a biomarker used in diagnosing myocardial injury. The clinical utility and the variation of this biomarker over time remain unclear in hemodialysis (HD) and peritoneal dialysis (PD) patients. We sought to determine whether hs-cTnT concentrations were predictive of myocardial infarction (MI) and death and to examine hs-cTnT variability over a 1-year period. METHODS: A total of 393 nonacute HD and PD patients (70% HD and 30% PD) were followed in a prospective observational study for new MI and death. RESULTS: Median hs-cTnT was 57 ng/L (interquartile range, 36-101 ng/L) with no observed difference between HD and PD patients (P = 0.11). Incremental increases in mortality (P = 0.024) and MI (P = 0.001) were observed with increasing hs-cTnT quartiles. MI incidence increased significantly across quartiles in both HD and PD patients (P = 0.012 and P = 0.025, respectively), whereas mortality increased only in HD patients (P = 0.015). For every increase of 25 ng/L in hs-cTnT, the unadjusted hazard ratio (HR) was 1.10 for mortality in the whole group (95% CI, 1.04-1.16, P = 0.001) and 1.16 for MI (95% CI, 1.08-1.23, P < 0.001). Adjusted HR for mortality was 1.07 (95% CI, 1.01-1.15, P = 0.04) and 1.14 for MI (95% CI, 1.06-1.22, P < 0.001). Changes in hs-cTnT from baseline concentrations after 1 year were minimal (55 ng/L vs 53 ng/L, P = 0.22) even in patients who had an MI (P = 0.53). CONCLUSIONS: hs-cTnT appears to have a useful role in predicting MI and death in the dialysis population. Over a 1-year period concentrations remained stable even in patients who sustained a new cardiac event.

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.

Do Natural T Regulatory Cells become Activated to Antigen Specific T Regulatory Cells in Transplantation and in Autoimmunity?

Antigen specific T regulatory cells (Treg) are often CD4(+)CD25(+)FoxP3(+) T cells, with a phenotype similar to natural Treg (nTreg). It is assumed that nTreg cannot develop into an antigen specific Treg as repeated culture with IL-2 and a specific antigen does not increase the capacity or potency of nTreg to promote immune tolerance or suppress in vitro. This has led to an assumption that antigen specific Treg mainly develop from CD4(+)CD25(-)FoxP3(-) T cells, by activation with antigen and TGF-ß in the absence of inflammatory cytokines such as IL-6 and IL-1ß. Our studies on antigen specific CD4(+)CD25(+) T cells from animals with tolerance to an allograft, identified that the antigen specific and Treg are dividing, and need continuous stimulation with specific antigen T cell derived cytokines. We identified that a variety of cytokines, especially IL-5 and IFN-γ but not IL-2 or IL-4 promoted survival of antigen specific CD4(+)CD25(+)FoxP3(+) Treg. To examine if nTreg could be activated to antigen specific Treg, we activated nTreg in culture with either IL-2 or IL-4. Within 3 days, antigen specific Treg are activated and there is induction of new cytokine receptors on these cells. Specifically nTreg activated by IL-2 and antigen express the interferon-γ receptor (IFNGR) and IL-12p70 (IL-12Rß2) receptor but not the IL-5 receptor (IL-5Rα). These cells were responsive to IFN-γ or IL-12p70. nTreg activated by IL-4 and alloantigen express IL-5Rα not IFNGR or IL-12p70Rß2 and become responsive to IL-5. These early activated antigen specific Treg, were respectively named Ts1 and Ts2 cells, as they depend on Th1 or Th2 responses. Further culture of Ts1 cells with IL-12p70 induced Th1-like Treg, expressing IFN-γ, and T-bet as well as FoxP3. Our studies suggest that activation of nTreg with Th1 or Th2 responses induced separate lineages of antigen specific Treg, that are dependent on late Th1 and Th2 cytokines, not the early cytokines IL-2 and IL-4.

IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity.

Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.

Distinct regulatory CD4+T cell subsets; differences between naïve and antigen specific T regulatory cells.

Effector T cells have functional subpopulations with distinct cytokine, cytokine receptor, chemokine receptor and transcription factors. We review how activation of antigen specific Treg induces expression of cytokines, cytokine receptors and chemokine receptors depending upon the effector lineage they are activated by. Activated Treg express receptors that are directly related to the effector T cell lineage. Other classes of Treg are induced in the periphery from effector lineage CD4(+)CD25(-)FOXP3(-)CD127(high)T cells, either by IL-10 or TGF-ß or by association with activated CD4(+)CD25(+)FOXP3(+)Treg. Thus Treg are produced and adapt to the specific immune inflammatory environment they are activated within. Activated Treg produce different molecules to mediate suppression, which are tailored to the immune response they are activated by and control.