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.

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.

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.

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-12rß2) 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-12rß2, 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.

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.

Studies on naïve CD4+CD25+T cells inhibition of naïve CD4+CD25- T cells in mixed lymphocyte cultures.

BACKGROUND: Naïve CD4+CD25+T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4+CD25+T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood. METHOD: The alloreactivity of naïve CD4+CD25+, CD4+CD25(-) and unfractionated CD4+T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (LewisxPVG)F1 cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4+CD25+T cells. RESULTS: Proliferation of CD4+CD25(-)T cells was significantly greater than unfractionated CD4+T cells to both allogeneic and syngeneic stimulator cells. CD4+CD25+T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3(-) cells. Admixing CD4+CD25+T cells with CD4+CD25(-)T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4+ T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-gamma, IL-10, TGF-beta and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4+CD25+ T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4+CD25(-)T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-gamma, IL-10, TGF-beta, IL-5 or iNOS did not prevent CD4+CD25+T cell's inhibition of CD4+CD25(-)T cell proliferation. Blocking IFN-gamma or iNOS enhanced CD4+CD25(-)T cell proliferation only in the absence of CD4+CD25+T cells. Depletion of CD4+CD25+T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators. CONCLUSIONS: Two independent mechanisms that control proliferation of CD4+CD25(-)T cells in MLC were identified that naive CD4+CD25+T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4+CD25(-)T cells producing IFN-gamma to induce iNOS.

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 (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta 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. (PVGxLewis)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-beta. 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-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.

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)-gamma 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-gamma, and IL-12p70 or monoclonal antibody that blocked IL-4, IL-5, and transforming growth factor-beta did not prevent acceptance of PVG skin grafts. CONCLUSIONS: These studies in a model of tolerance regulated by CD4CD25 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-beta and was not inactivated or overwhelmed by administration of Th1 cytokines, IL-2, IFN-gamma or IL-12p70.

IL-13 prolongs allograft survival: association with inhibition of macrophage cytokine activation.

Th2 cytokines, especially IL-4 and IL-10, may facilitate transplant tolerance induction but the role of IL-13, another Th2 cytokine, is not known. This study examined the effects of rat recombinant IL-13 (rIL-13) on alloimmune responses. In vitro effects of rIL-13 were compared in mixed lymphocyte cultures (MLC) on rat lymphocytes cultured with PVG stimulator cells. DA rats grafted with fully allogeneic PVG neonatal heart grafts were treated with 40,000 units of rIL-13 for 10 days and graft survival monitored by ECG. Cytokine mRNA expression in the graft and lymphoid tissues was studied by RT-PCR and alloantibody levels assayed. rIL-13 had no effect on MLC, unlike rIL-4 which enhanced proliferation and induced Th2 and inhibited Th1 cytokines in MLC. rIL-13 inhibited IL-12p35, IL-12p40 and TNF-alpha mRNA induction in dendritic cell cultures. Treatment with rIL-13 prolonged fully allogeneic PVG neonatal heart graft survival to 18-21 (13-27) days (median (range)); compared to 12 (9-15) days in untreated normal rejection (p<0.05) and 14 (10-24) days in sham treated controls (p<0.05). RT-PCR studies on graft tissue identified reduced mRNA expression for the dendritic cell/macrophage molecules iNOS, TNF-alpha and IL-12 compared to normal rejection. rIL-13 treatment did not increase Th2 cytokines as compared to normal rejection, or the Th2 dependent IgG1 alloantibody response, while IL-4 did. These studies demonstrated that rIL-13 can prolong allograft survival associated with inhibition of IL-12, TNF-alpha and iNOS mRNA induction, and suggest IL-13 could modify graft rejection by inhibition of dendritic cell and/or macrophage function.