Catherine M. Robinson

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.

CD4 + CD25 + T cells alloactivated ex vivo by IL-2 or IL-4 become potent alloantigen-specific inhibitors of rejection with different phenotypes, suggesting separate pathways of activation by Th1 and Th2 responses

CD4(+)CD25(+)Foxp3(+) T cells are regulatory/suppressor cells (Tregs) that include non-antigen (Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naive CD4(+)CD25(+) Treg develop into specific Tregs is unknown. Here, we generated adaptive Tregs by culture of naive CD4(+)CD25(+)Foxp3(+) T cells with allo-Ag and either interleukin-2 (IL-2) or IL-4. Within days, IL-2 enhanced interferon-gamma receptor (Ifngammar) and Il-5 mRNA and IL-4 induced a reciprocal profile with de novo IL-5Ralpha and increased IFN-gamma mRNA expression. Both IL-2- and IL-4-alloactivated CD4(+)CD25(+) Tregs within 3 to 4 days of culture had enhanced capacity to induce tolerance to specific donor but not to third-party cardiac allografts. These hosts became tolerant as allografts functioned more than 250 days, with a physiologic ratio of less than 10% CD4(+)CD25(+)Foxp3(+) T cells in the CD4(+) population. CD4(+)CD25(+) T cells from tolerant hosts given IL-2-cultured cells had increased Il-5 and Ifngammar mRNA. Those from hosts given IL-4-cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third-party allo-Ag. Thus, IL-2 and IL-4 activated allo-Ag-specific Tregs with distinct phenotypes that were retained in vivo. These findings suggested that T-helper 1 (Th1) and Th2 responses activate 2 pathways of adaptive Ag-specific Tregs that mediate tolerance. We propose they be known as T-suppressor 1 (Ts1) and Ts2 cells.

Interleukin-12p70 prolongs allograft survival by induction of interferon gamma and nitric oxide production

Background. Interleukin (IL)-12p70, a heterodimeric cytokine has been considered central to induction of Th1 responses with the assistance of IL-18 and IL-27. It was predicted IL-12p70 treatment would promote allograft rejection. In these studies, IL-12p70 delayed rejection. Methods. We compared Piebald Virol Glaxo (PVG) neonatal heart graft survival in fully allogeneic Dark Agoutti (DA) rats treated with IL-12p70 alone or in combination with other cytokines. The mechanism by which IL-12p70 induced delayed rejection was examined by reverse transcription polymerase chain reaction of cytokine mRNA and studying the role of interferon (IFN)-γ and inducible nitric oxide synthase (iNOS) that were induced by IL-12. Results. IL-12p70 treatment significantly delayed PVG neonatal heart graft rejection compared to normal rejection control and other control groups treated with supernatant from Chinese hamster ovary (CHO)-K1 cells transfected with IL-12p35, IL-12p40, or no cytokine gene. IL-12p70 had no effect on alloantibody response. IFN-γ and iNOS mRNA expression was increased in heart graft and regional lymph node compared to normal rejection and other treatment groups, consistent with Th1 response induction. IL-12p35 mRNA expression decreased in IL-12p70 treated rats but there was no difference in IL-12p40, Th2, or Tr1 cytokine mRNA expression. Coadministration of an iNOS inhibitor, L-NIL, or a monoclonal antibody (mAb) that blocks IFN-γ, inhibited IL-12p70’s ability to prolong allograft survival; as did co-treatment with IL-4 but not IL-13. Conclusions. IL-12p70 treatment may inhibit rejection by hyperinduction of Th1 responses, especially production of IFN-γ and nitric oxide. These effects may be by enhancing regulatory T-cell responses or by the activation of iNOS in macrophages to produce excessive nitric oxide that in turn inhibits alloimmune responses.

Interleukin-12 (IL-12p70) Promotes Induction of Highly Potent Th1-Like CD4(+)CD25(+) T Regulatory Cells That Inhibit Allograft Rejection in Unmodified Recipients

In rat models, CD4+CD25+ T regulatory cells (Treg) play a key role in the induction and maintenance of antigen-specific transplant tolerance, especially in DA rats with PVG cardiac allografts (1, 2). We have previously described generation of alloantigen-specific Treg (Ts1), by culture of naïve natural CD4+CD25+ Treg (nTreg) with specific alloantigen and IL-2 for 4 days. These cells express mRNA for IFN-γ receptor (ifngr) and suppress donor but not third party cardiac allograft rejection mediated by alloreactive CD4+ T cells at ratios of <1:10. Here, we show that Ts1 also expressed the IL-12p70 specific receptor (il-12rβ2) and that rIL-12p70 can induce their proliferation. Ts1 cells re-cultured with rIL-12p70 alone or rIL-12p70 and recombinant interleukin-2 (rIL-2), suppressed proliferation of CD4+ T cells in mixed lymphocyte culture at <1:1024, whereas Ts1 cells re-cultured with rIL-2 and alloantigen only suppressed at 1:32–64. The rIL-12p70 alloactivated Ts1 cells markedly delayed PVG, but not third party Lewis, cardiac allograft rejection in normal DA recipients. Ts1 cells re-cultured for 4 days with rIL-12p70 alone, but not those re-cultured with rIL-12p70 and rIL-2, expressed more il-12rβ2, t-bet, and ifn-γ, and continued to express the markers of Ts1 cells, foxp3, ifngr, and il-5 indicating Th1-like Treg were induced. Ts1 cells re-cultured with rIL-2 and alloantigen remained of the Ts1 phenotype and did not suppress cardiac graft rejection in normal DA rats. We induced highly suppressive Th1-like Treg from naïve nTreg in 7 days by culture with alloantigen, first with rIL-2 then with rIL-12p70. These Th1-like Treg delayed specific donor allograft rejection demonstrating therapeutic potential.

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-β in the absence of inflammatory cytokines such as IL-6 and IL-1β. 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-12Rβ2) 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-12p70Rβ2 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.

Transplant tolerance associated with a Th1 response and not broken by IL-4, IL-5, and TGF-beta blockade or Th1 cytokine administration.

Background. Specific transplant tolerance is mediated by CD4+ T cells that die unless supported by T-cell derived cytokines and donor antigen. This study examined the role of Th1 and Th2 cytokines in the maintenance of tolerance. Methods. Tolerance to fully allogeneic PVG cardiac allografts in DA rats was induced by short-term anti-CD3 monoclonal antibody therapy. Responses of tolerant cells to donor and third party antigen were assessed in vivo by examination of the infiltrate in the heart and application of skin grafts, and in vitro in mixed lymphocyte culture. Cell subsets were stained, induction of cytokine mRNA assayed by reverse-transcriptase polymerase chain reaction and the role of cytokines determined by treating with blocking monoclonal antibody to cytokines or cytokine administration. Results. Tolerated grafts had a T cell and macrophage infiltrate with increased mRNA for Th1 cytokines, interleukin (IL)-2, and interferon (IFN)-&ggr; but not Th2 cytokines. Peripheral lymphocytes proliferated in mixed lymphocyte culture and expressed Th1 cytokine mRNA. Tolerant hosts accepted PVG and rejected Lewis skin allografts and the lymph nodes draining both these grafts had similar induction of Th1 and Th2 cytokine mRNA. Treatment of tolerant rats with Th1 cytokines IL-2, IFN-&ggr;, and IL-12p70 or monoclonal antibody that blocked IL-4, IL-5, and transforming growth factor-&bgr; did not prevent acceptance of PVG skin grafts. Conclusions. These studies in a model of tolerance regulated by CD4+CD25+ T cells demonstrated there was no defect in Th1 responses. Tolerance was due to regulation that was not solely dependent on IL-4, IL-5, or transforming growth factor-&bgr; and was not inactivated or overwhelmed by administration of Th1 cytokines, IL-2, IFN-&ggr; or IL-12p70.

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens.

Background. The mechanisms by which CD4+T cells, especially CD4+CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. Methods. Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+CD25+ and CD4+CD25−T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVG×Lewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-&bgr; in the transfer of tolerance was assessed. Results. CD4+T cells transferred specific tolerance and suppressed naïve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVG×Lewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or naïve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-&bgr;. CD4+CD25−T cells from either naïve or tolerant hosts effected rejection. In contrast neither tolerant nor naïve CD4+CD25+T cells restored rejection. Conclusions. Specific tolerance transfer required CD4+ containing CD4+CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-&bgr; were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.

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.

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.

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.