Giang T. Tran

TREATMENT WITH INTERLEUKIN-4 PROLONGS ALLOGENEIC NEONATAL HEART GRAFT SURVIVAL BY INDUCING T HELPER 2 RESPONSES

BACKGROUND The T helper (Th) 2 cytokine interleukin (IL)-4 has been implicated as a major regulatory cytokine for the induction of transplant tolerance, but few studies have examined the capacity of IL-4 to induce tolerance. The effect of IL-4 therapy alone or with low doses of anti-CD4 monoclonal antibody (mAb) therapy on survival of fully allogeneic PVG neonatal heart graft in adult DA rats was examined. METHODS Rat recombinant (r) IL-4 was given at 30 microg (10(4) U)/kg daily intraperitoneally for 10 days and MRC OX35 (anti-CD4, nondepleting) or MRC OX81 (anti-IL-4) was given intraperitoneally on days 0, 3, 7, and 10. Semiquantitative reverse transcriptase-polymerase chain reaction was used to assay mRNA for cytokine in the graft, regional node and spleen and fluorescence-activated cell sorting was used to assay alloantibody Ig isotypes. RESULTS Grafts in rIL-4-treated rats survived a median period of 39 days (range, 28-52 days), significantly longer than in both untreated and nontransfected Chinese hamster ovary-K1 supernatant-treated controls (median, 14 days; range, 10-16 days, P=0.009). rIL-4 treatment with a suboptimal dose of anti-CD4 mAb prolonged median survival to 70 days (range, 63-80 days), which was longer than rIL-4 treatment alone or anti-CD4 mAb alone (median, 36 days; range, 30-55 days; P<0.0045). Combining MRC OX81 with MRC OX35 therapy led to earlier rejection at a median period of 26 days (range, 20-28 days); MRC OX81 alone had no effect on graft survival. Alloantibody titers, especially IgG1, were higher in rIL-4-treated animals and lower in anti-CD4 mAb-treated animals than in animals with normal rejection (P<0.05). IL-4 mRNA was increased in regional lymph nodes and spleen of the rIL-4-treated groups compared with all other groups, but there were no differences for IL-2, interferon-gamma, or IL-10. CONCLUSIONS rIL-4 therapy markedly prolonged neonatal cardiac allograft survival, and, with anti-CD4 therapy, it further prolonged survival. It induced IL-4 mRNA in lymphoid tissues and enhanced alloantibody production, especially IgG1, which demonstrated enhanced Th2 responses, but did not affect Th1 cytokines.

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-β 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.

Attenuation of Experimental Allergic Encephalomyelitis in Complement Component 6-Deficient Rats Is Associated with Reduced Complement C9 Deposition, P-Selectin Expression, and Cellular Infiltrate in Spinal Cords

The role of Ab deposition and complement activation, especially the membrane attack complex (MAC), in the mediation of injury in experimental allergic encephalomyelitis (EAE) is not resolved. The course of active EAE in normal PVG rats was compared with that in PVG rats deficient in the C6 component of complement (PVG/C6−) that are unable to form MAC. Following immunization with myelin basic protein, PVG/C6− rats developed significantly milder EAE than PVG/C rats. The anti-myelin basic protein response was similar in both strains, as was deposition of C3 in spinal cord. C9 was detected in PVG/C rats but not in PVG/C6−, consistent with their lack of C6 and inability to form MAC. In PVG/C6− rats, the T cell and macrophage infiltrate in the spinal cord was also significantly less than in normal PVG/C rats. There was also reduced expression of P-selectin on endothelial cells, which may have contributed to the reduced cellular infiltrate by limiting migration from the circulation. Assay of cytokine mRNA by RT-PCR in the spinal cords showed no differences in the profile of Th1 or Th2 cytokines between PVG/C and PVG/C6− rats. PVG/C rats also had a greater increase in peripheral blood white blood cell, neutrophil, and basophil counts than was observed in the PVG/C6−. These findings suggest that the MAC may have a role in the pathogenesis of EAE, not only by Ig-activated MAC injury but also via induction of P-selectin on vascular endothelium to promote infiltration of T cells and macrophages into the spinal cord.

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.

Induction of Passive Heymann Nephritis in Complement Component 6-Deficient PVG Rats

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6−) and normal PVG rats (PVG/c). PVG/c and PVG/C6− rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6− rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6− glomeruli, indicating C5b-9/MAC had not formed in PVG/C6− rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

IL-4 Therapy Prevents the Development of Proteinuria in Active Heymann Nephritis by Inhibition of Tc1 Cells

The role of IL-4, a key Th2 cytokine, in promoting or inhibiting active Heymann nephritis (HN) was examined. HN is induced by immunization with Fx1A in CFA, and proteinuria in HN is associated with subepithelial IgG and C3 deposition and infiltration of CD8+ T-cytotoxic 1 (Tc1) cells and macrophages into glomeruli, as well as induction of Abs to Crry. Treatment with rIL-4 from the time of Fx1A/CFA immunization stimulated an earlier IgG1 response to Fx1A, induced anti-Crry Abs, and up-regulated IL-4 mRNA in lymphoid tissue, but did not alter proteinuria. Treatment with MRCOx-81, an IL-4-blocking mAb, resulted in greater proteinuria, which suggests endogenous IL-4 regulated the autoimmune response. Delay of rIL-4 treatment until 4 wk post-Fx1A/CFA immunization and just before the onset of proteinuria prevented the development of proteinuria and reduced Tc1 cell infiltrate in glomeruli. Delayed treatment with IL-4 had no effect on titer or isotype of Abs to Fx1A or on Ig, C3, and C9 accumulation in glomeruli. Treatment with rIL-13, a cytokine that alters macrophage function such as rIL-4, but has no direct effect on T or B cell function, reduced glomerular macrophage infiltrate, but did not prevent proteinuria or CD8+ T cell infiltrate. Anti-Crry Abs were paradoxically only induced with rIL-4 therapy, not in HN controls with proteinuria. It was concluded that the rIL-4 effect was probably by inhibition of Tc1 cells, which normally mediate the glomerular injury that results in proteinuria.

Mycophenolate mofetil treatment accelerates recovery from experimental allergic encephalomyelitis.

Mycophenolate mofetil (MM) acts through its metabolite mycophenolic acid to inhibit inosine monophosphate dehydrogenase (IMPDH), an enzyme essential for purine synthesis in lymphocytes. Oral treatment with MM from the day of immunization for 2 weeks significantly delayed both the development of active experimental allergic encephalomyelitis (EAE) in Lewis rats and reduced the antibody response to myelin basic protein (MBP). MM did not deplete T and B cells, nor did it prevent induction of Th1 or Th2 cytokine in the regional nodes. Treatment of EAE with MM at the onset of clinical symptoms resulted in more rapid recovery from EAE than in control or cyclosporin A (CsA)-treated. MM-treated rats had less infiltration of T cells, B cells, macrophages and dendritic cells into brainstems than either the control or CsA-treated. MM-treated brainstems also had lower level of mRNA for Thl (IL-2, IL-12Rbeta2, IFN-gamma), Th2 (IL-4, IL-10) cytokines and TNF-alpha and TGF-beta compared to that in CsA and controls groups. This study shows MM was superior to CsA in the treatment of EAE and acted by reducing the inflammatory infiltrate, not by suppression of Ig response or by promotion of regulatory cells such as Th2 or Th3.

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.

Alloantigen specific T regulatory cells in transplant tolerance.

CD4(+)CD25(+)Foxp3(+)T cells are regulatory/suppressor cells (Treg) that include non-antigen(Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naïve CD4(+)CD25(+)Treg develop into specific Tregs is unknown. We have studied DA rats tolerant to fully allogeneic PVG cardiac grafts that survived with out immunosuppression for over 100 days and identified the cellular basis of alloantigen specific tolerance. Key observations from our studies will be reviewed including how CD4(+)CD25(+)Tregs were first identified and the cytokine dependence of CD4(+)T cells that transfer alloantigen specific transplant tolerance which died in culture unless stimulated with both cytokine rich ConA supernatant and specific donor alloantigen. Both the tolerant CD4(+)CD25(+) and CD4(+)CD25(-) T cell populations are required to transfer tolerance, yet alone the CD4(+)CD25(-) T cell effect rejection. Tolerance transfer occurs with a low ratio of CD4(+)CD25(+)T cells (<1:10), whereas to induce tolerance with naive CD4(+)CD25(+)T cells requires both a ratio of >1:1 and is not alloantigen specific. Recent findings on how naïve CD4(+)CD25(+)T cells developed into two separated pathways of alloantigen specific Tregs, by culturing them with alloAg with either IL-2 or IL-4 and donor alloantigen are described. IL-2 enhances IFN-gammaR and IL-5 mRNA while 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 3-4 days of culture can induce alloantigen specific tolerance at ratios of 1:10. Long term, CD4(+)CD25(+)T cells from tolerant hosts given IL-2 cultured cells have increased IL-5 and IFN-gammaR mRNA; whereas hosts given IL-4 cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third party alloAg. These findings suggest that Th1 and Th2 responses activate two pathways of alloantigen specific Tregs that can mediate transplant tolerance but are dependent upon cytokines produced by ongoing Th1 and/or Th2 immune responses.