Gisen Kim

Recognition of bacterial glycosphingolipids by natural killer T cells

Natural killer T (NKT) cells constitute a highly conserved T lymphocyte subpopulation that has the potential to regulate many types of immune responses through the rapid secretion of cytokines. NKT cells recognize glycolipids presented by CD1d, a class I-like antigen-presenting molecule. They have an invariant T-cell antigen receptor (TCR) α-chain, but whether this invariant TCR recognizes microbial antigens is still controversial. Here we show that most mouse and human NKT cells recognize glycosphingolipids from Sphingomonas, Gram-negative bacteria that do not contain lipopolysaccharide. NKT cells are activated in vivo after exposure to these bacterial antigens or bacteria, and mice that lack NKT cells have a marked defect in the clearance of Sphingomonas from the liver. These data suggest that NKT cells are T lymphocytes that provide an innate-type immune response to certain microorganisms through recognition by their antigen receptor, and that they might be useful in providing protection from bacteria that cannot be detected by pattern recognition receptors such as Toll-like receptor 4.

Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis

Regulatory T cells (Treg cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b+ myeloid cells in recombination-activating gene 1–deficient (Rag1−/−) recipient mice was needed to prevent the colitis induced by transferred CD4+CD45RBhi T cells. In Il10−/−Rag1−/− mice, Treg cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor–deficient (Il10rb−/−) Treg cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the Treg cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on Treg cells to maintain Foxp3 expression.

Transcriptional reprogramming of mature CD4 + helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes

TCRαβ thymocytes differentiate into either CD8αβ+ cytotoxic T lymphocytes or CD4+ helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II–restricted CD4+ thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4+ T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4+ T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II–restricted CD4+ cytotoxic T lymphocytes.

Retinoic Acid Can Directly Promote TGF-β-Mediated Foxp3+ Treg Cell Conversion of Naive T Cells

The article by Hill et al. (2008), published in the November 14, 2008 issue of Immunity, describes a mechanism by which retinoic acid (RA) enhances TGF-β-induced Foxp3 expression. The authors propose that RA does not act directly on naive T cells during activation in culture but rather indirectly via negative regulation of an accompanying population of effector or memory CD4+ CD44hi cells. They reasoned that the increased generation of Foxp3+ cells in response to RA in culture, as described previously (Coombes et al., 2007; Elias et al., 2008; Mucida et al., 2007; Sun et al., 2007; Xiao et al., 2008), represented the lifting by RA of inhibition imparted by accompanying CD4+CD44hi T cells, rather than by direct or indirect effects of RA on the Foxp3 expression of the primed naive T cells themselves. In order to assess the effects of RA on naive T cells in the absence of accompanying CD4+CD44hi T cells, we sorted (CD4+CD25−CD44low CD62L+) GFP− T cells (more than 99.7% purity) from Foxp3-eGFP reporter mice (Figure S1A available online) by flow cytometry. After 4 days of stimulation with anti-CD3 and anti-CD28, we stained CD4 cells with 7AAD to exclude dead cells; additionally, forward and side scatter (area versus width) was used to exclude doublets, and we evaluated Foxp3 expression via GFP staining. Addition of RA enhanced Foxp3 induction more than 50% by use of 1 or 10 ng/ml doses of TGF-β (Figure S1B). Because the sorting efficiency is not 100%, it is possible that extremely low numbers of “accompanying” memory or effector cells could still influence these results. To exclude this possibility, we used FACS-sorted CD4+CD25−CD44lo CD62L+ T cells, isolated from B7-1 and B7-2 double-deficient mice (Cd80−/− Cd86−/−), which even before sorting already contain less than 5% of memory or effector CD44hi cells (data not shown). RA also greatly enhanced Foxp3 induced by TGF-β in CD4+CD25−CD44lo CD62L+ naive T cells isolated from Cd80−/− Cd86−/− mice (Figure S1C). Moreover, we showed previously that RA is able to counterbalance the inhibitory effects of costimulation on TGF-β-mediated Foxp3 induction, with either CD4+CD25− or CD4+Foxp3− T cells (Benson et al., 2007). To confirm these results, we used OTII TCR transgenic CD4+CD25−CD44lo CD62L+ cells sorted by flow cytometry and tested the effects of RA by using increasing doses of anti-CD28 stimulation. We found that RA markedly enhanced TGF-β-mediated Foxp3 induction on pure naive CD4+ T cells that were stimulated with anti-CD3 and various doses of anti-CD28 (Figure S1D). The enhanced Foxp3 expression mediated by RA is more pronounced on naive monoclonal OTII TCR transgenic T cells as compared to polyclonal T cells, consistent with a lesser frequency of “contaminating” memory T cells. Finally, because we showed previously that RA-mediated enhanced expression of Foxp3 is greatly reduced in the absence of IL-2 (Mucida et al., 2007), we investigated the effects of RA on naive T cells with various doses of exogenous IL-2. Although IL-2-deficient mice develop inflammatory disorders, Il2−/−Cd80−/−Cd86−/− mice are healthy and, more importantly, they do not contain T regulatory cells. At steady state, ~99% of all CD4+ T cells isolated from Il2−/−Cd80−/−Cd86−/− mice are naive (data not shown). The CD4+ T cells were further sorted by flow cytometry so that highly purified naive CD4+CD25−CD44lo CD62L+ cells (more than 99.9% purity) were obtained. The sorted naive CD4+ Il2−/−Cd80−/−Cd86−/− T cells were tested for TGF-β-induced Foxp3 expression in the presence of increasing doses of IL-2 and anti-CD3 and anti-CD28 coated beads, with or without RA. The data showed that 1 nM RA distinctly enhanced TGF-β (1 ng/ml)-mediated Foxp3 induction in pure naive CD4+ T cells at all doses of IL-2 examined (Figures S1E and S1F). Strikingly, although the expression of Foxp3 was much reduced, RA enhanced TGF-β-mediated Foxp3 induction not only in the absence of memory or effector T cells but also in the absence of IL-2. These data demonstrate that RA mediates enhanced TGF-β-induced Foxp3 expression upon activation of pure naive T cells in the absence of accompanying CD4+CD44hi T cells. In addition, we confirmed, as Hill et al. (2008) proposed, that RA also efficiently counteracts inhibitory effects of CD44hi T cells on Foxp3 induction (data not shown), which indicates that RA is able to enhance Foxp3 expression both, via effects directly on the primed naive T cells as well as indirectly via inhibitory effects on accompanying CD4+CD44hi T cells. There is no doubt that the new findings by Hill et al. (2008) add an important new pathway by which RA can enhance Foxp3 induction, which had been suggested previously (Elias et al., 2008; Mucida et al., 2007; Xiao et al., 2008).Nevertheless, published data, together with the data presented here, disagree with the central statement proposed by Hill et al. (2008) that the enhanced expression of TGF-β driven Foxp3 mediated by RA is an indirect effect that requires suppression of accompanying CD4+CD44hi T cells rather than via direct or indirect effects on the primed T cells themselves. Under physiological conditions, naive T cells may be exposed to cytokines and effector or memory cells, and hence it is likely that during priming of naive T cells, both mechanisms of RA-mediated enhanced TGF-β driven Foxp3 expression in the primed T cells will synergize in vivo. Therefore, elucidating and understanding both processes by which RA affects naive and already differentiated T cells is important and may lead to the identification of possible targets for therapeutic interventions to treat various inflammatory and autoimmune diseases.

Cutting Edge: CD4+CD25+ Regulatory T Cells Impaired for Intestinal Homing Can Prevent Colitis

Transfer of CD4+CD45RBhigh T cells into RAG−/− mice causes colitis, which can be prevented by CD4+CD25+ regulatory T cells (Treg). Colitis induction by CD4+CD45RBhigh T cells requires β7 integrin-dependant intestinal localization, but the importance of β7 integrins for Treg function is unknown. In this study, we show that β7−/− Treg were effective in preventing colitis. Treg expanded in vivo to the same extent as CD4+CD45RBhigh T cells after transfer and they did not inhibit CD4+CD45RBhigh T cell expansion in lymphoid tissues, although they prevented the accumulation of Th1 effector cells in the intestine. β7−/− Treg were significantly reduced in the large intestine, however, compared with wild-type Treg, and regulatory activity could not be recovered from the intestine of recipients of β7−/− Treg. These data demonstrate that Treg can prevent colitis by inhibiting the accumulation of tissue-seeking effector cells and that Treg accumulation in the intestine is dispensable for colitis suppression.

A crucial role for HVEM and BTLA in preventing intestinal inflammation

The interaction between the tumor necrosis factor (TNF) family member LIGHT and the TNF family receptor herpes virus entry mediator (HVEM) co-stimulates T cells and promotes inflammation. However, HVEM also triggers inhibitory signals by acting as a ligand that binds to B and T lymphocyte attenuator (BTLA), an immunoglobulin super family member. The contribution of HVEM interacting with these two binding partners in inflammatory processes remains unknown. In this study, we investigated the role of HVEM in the development of colitis induced by the transfer of CD4+CD45RBhigh T cells into recombination activating gene (Rag)−/− mice. Although the absence of HVEM on the donor T cells led to a slight decrease in pathogenesis, surprisingly, the absence of HVEM in the Rag−/− recipients led to the opposite effect, a dramatic acceleration of intestinal inflammation. Furthermore, the critical role of HVEM in preventing colitis acceleration mainly involved HVEM expression by radioresistant cells in the Rag−/− recipients interacting with BTLA. Our experiments emphasize the antiinflammatory role of HVEM and the importance of HVEM expression by innate immune cells in preventing runaway inflammation in the intestine.

TSC1 regulates the balance between effector and regulatory T cells

Mammalian target of rapamycin (mTOR) plays a crucial role in the control of T cell fate determination; however, the precise regulatory mechanism of the mTOR pathway is not fully understood. We found that T cell-specific deletion of the gene encoding tuberous sclerosis 1 (TSC1), an upstream negative regulator of mTOR, resulted in augmented Th1 and Th17 differentiation and led to severe intestinal inflammation in a colitis model. Conditional Tsc1 deletion in Tregs impaired their suppressive activity and expression of the Treg marker Foxp3 and resulted in increased IL-17 production under inflammatory conditions. A fate-mapping study revealed that Tsc1-null Tregs that lost Foxp3 expression gained a stronger effector-like phenotype compared with Tsc1-/- Foxp3+ Tregs. Elevated IL-17 production in Tsc1-/- Treg cells was reversed by in vivo knockdown of the mTOR target S6K1. Moreover, IL-17 production was enhanced by Treg-specific double deletion of Tsc1 and Foxo3a. Collectively, these studies suggest that TSC1 acts as an important checkpoint for maintaining immune homeostasis by regulating cell fate determination.

HVEM signalling at mucosal barriers provides host defence against pathogenic bacteria

The herpes virus entry mediator (HVEM), a member of the tumour-necrosis factor receptor family, has diverse functions, augmenting or inhibiting the immune response. HVEM was recently reported as a colitis risk locus in patients, and in a mouse model of colitis we demonstrated an anti-inflammatory role for HVEM, but its mechanism of action in the mucosal immune system was unknown. Here we report an important role for epithelial HVEM in innate mucosal defence against pathogenic bacteria. HVEM enhances immune responses by NF-κB-inducing kinase-dependent Stat3 activation, which promotes the epithelial expression of genes important for immunity. During intestinal Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli infection, Hvem−/− mice showed decreased Stat3 activation, impaired responses in the colon, higher bacterial burdens and increased mortality. We identified the immunoglobulin superfamily molecule CD160 (refs 7 and 8), expressed predominantly by innate-like intraepithelial lymphocytes, as the ligand engaging epithelial HVEM for host protection. Likewise, in pulmonary Streptococcus pneumoniae infection, HVEM is also required for host defence. Our results pinpoint HVEM as an important orchestrator of mucosal immunity, integrating signals from innate lymphocytes to induce optimal epithelial Stat3 activation, which indicates that targeting HVEM with agonists could improve host defence.

Protein kinase C-η controls CTLA-4–mediated regulatory T cell function

Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η–deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4–PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η–deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4–PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

A Novel Role for IL-27 in Mediating the Survival of Activated Mouse CD4 T Lymphocytes

IL-27, an IL-12 family cytokine, has pleiotropic functions in the differentiation and expansion of CD4+ T cell subsets. In this study, we discovered a novel function of IL-27. CD4+CD45RBhigh T cells from mice deficient for the α-chain of IL-27 receptor failed to induce colitis in Rag−/− recipients, because of an inability of activated donor cells to survive. Interestingly, IL-27 was indispensable for the prevention of colitis by regulatory T cells, also because of a defect in long-term cell survival. IL-27 affected the survival of activated T lymphocytes, rather than promoting cell proliferation, by inhibiting Fas-mediated activation-induced T cell death, acting through the STAT3 signaling pathway. The addition of IL-27 during activation resulted in an increased cell number, which was correlated with decreased activation of both caspases 3 and 8. This prosurvival effect was attributed to downregulation of FasL and to the induction of the antiapoptotic protein cFLIP. Although activation induced cell death is an important mechanism for the maintenance of immunological homeostasis, protection of lymphocytes from excessive cell death is essential for effective immunity. Our data indicate that IL-27 has a crucial role in the inhibition of activation-induced cell death, thereby permitting Ag-driven T cell expansion.