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The functional role of IL-12 in rheumatoid arthritis is controversial. Moreover, whether IL-12 contributes to regulation of Ab-induced joint inflammation remains unclear. To address these issues, we explored the functional roles of IL-12 in Ab-induced arthritis using the K/BxN serum transfer model. IL-12p35(-/-) and IL-12Rbeta(2)(-/-) mice were resistant to the development of arthritis. Injection of K/BxN serum into IL-12p40-yellow fluorescence protein reporter (yet40) mice induced CD11b(+) cells, CD11c(+) cells, and Gr-1(+) granulocytes to produce IL-12p40 in the joints. The levels of IFN-gamma, IL-4, and IL-6 production were lower in joint tissues of IL-12p35(-/-) and IL-12Rbeta(2)(-/-) mice than in B6 mice, whereas levels of TGF-beta expression were higher. Administering IL-12p35(-/-) mice rIL-12 or IFN-gamma restored joint inflammation and suppressed TGF-beta production in joint tissues. Moreover, administering neutralizing anti-TGF-beta mAb enhanced joint inflammation. Among the immune cells that infiltrated joint tissues during Ab-induced arthritis, NKT cells expressed IL-12beta(2) receptors. Furthermore, the adoptive transfer of splenocytes from B6 or Gr-1(+) granulocyte-depleted mice restored joint inflammation in IL-12Rbeta(2)(-/-) mice as much as in B6 mice, whereas splenocytes from Jalpha18(-/-) mice did not. These findings indicate that signals via IL-12beta(2) receptors on NKT cells play a critical role in the development of Ab-induced arthritis. The IL-12p35/IFN-gamma axis promotes Ab-induced joint inflammation by activating NKT cells and suppressing TGF-beta, which may provide novel information for the development of new therapeutic strategies for the inhibition of rheumatoid arthritis.

Regulatory T cells (Tregs) play a pivotal role in the maintenance of homeostasis between immune response and immune tolerance. The transcription factor Foxp3 and the surface protein CD25 are the two key molecules characterizing Tregs. In autoimmune and various other chronic inflammatory diseases, the expression of Foxp3 is severely down-regulated. However, the molecular mechanism underlying the down-regulation of Foxp3 is not understood yet. Because the IL-12p40 homodimer (p40(2)) is markedly up-regulated in response to various inflammatory stimuli, the present study was undertaken to explore the role of p40(2) in the regulation of Foxp3 in naive mouse splenocytes. IL-12p40(2) dose-dependently inhibited the expression of Foxp3 and CD25, but not CD4. Interestingly, this inhibition was absent in splenocytes of IL-12Rbeta1(-/-), but not IL-12Rbeta2(-/-), mice. Moreover, suppression of Foxp3 in wild-type and IL-12Rbeta2(-/-) splenocytes was accompanied by production of NO. Consistently, l-N(6)-(1-iminoethyl)-lysine hydrochloride, an inhibitor of inducible NO synthase (iNOS), and PTIO, a scavenger of NO, restored the expression of Foxp3 and CD25 in p40(2)-stimulated splenocytes, and p40(2) was unable to down-regulate Foxp3 and CD25 in splenocytes from iNOS(-/-) mice. Furthermore, NO, but not p40(2), was able to inhibit Foxp3 in purified CD4(+)CD25(+) T cells in the absence of iNOS-expressing cells. Hence, our results clearly demonstrate that p40(2) induces NO production via IL-12Rbeta1 and that NO subsequently suppresses Tregs in naive mouse splenocytes. This study, therefore, delineates an unprecedented biological function of p40(2) in the regulation of Foxp3 via IL-12Rbeta1-mediated NO production.

Inflammation can have both positive and negative effects on development of CD8 T cell memory, but the relative contributions and cellular targets of the cytokines involved are unclear. Using CD8 T cells lacking receptors for IL-12, type I IFN, or both, we show that these cytokines act directly on CD8 T cells to support memory formation in response to vaccinia virus and Listeria monocytogenes infections. Development of memory to vaccinia is supported predominantly by IL-12, whereas both IL-12 and type I IFN contribute to memory formation in response to Listeria. In contrast to memory formation, the inability to respond to IL-12 or type I IFN had a relatively small impact on the level of primary expansion, with at most a 3-fold reduction in the case of responses to Listeria. We further show that programming for memory development by IL-12 is complete within 3 days of the initial naive CD8 T cell response to Ag. This programming does not result in formation of a population that expresses killer cell lectin-like receptor G1, and the majority of the resulting memory cells have a CD62L(high) phenotype characteristic of central memory cells. Consistent with this, the cells undergo strong expansion upon rechallenge and provide protective immunity. These data demonstrate that IL-12 and type I IFN play an essential early role in determining whether Ag encounter by naive CD8 T cells results in formation of a protective memory population.

We have previously shown that mice lacking the IL-12-specific receptor subunit beta2 (IL-12Rbeta2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rbeta2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rbeta2(-/-) mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rbeta2-deficient mice to autoimmune diseases. T cells from IL-12Rbeta2(-/-) mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) in the thymus and spleen of IL-12Rbeta2(-/-) mice were comparable to those of WT mice. However, IL-12Rbeta2(-/-) mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-beta, as shown by significantly lower numbers of CD25(+)CD4(+) T cells that expressed Foxp3. Functionally, CD25(+)CD4(+) Tregs derived from IL-12Rbeta2(-/-) mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rbeta2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rbeta2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway.

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rbeta2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rbeta2 on OX40-stimulated CD4 T cells was tightly regulated and peaked approximately 4-6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rbeta2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF), a cell surface C3/C5 convertase regulator, exerts control over T cell responses. Extending these results, we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1(-/-)) APCs produce significantly more IL-12, C5a, and IFN-gamma compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-gamma-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC, demonstrating a link between DAF, local complement activation, IL-12, and T cell-produced IFN-gamma. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-gamma-producing effector T cells. Overall, our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production, this in turn, contributes to directing T cell differentiation toward an IFN-gamma-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity.