Faculty Bibliography

The transcription factor Foxp3 has an indispensable role in establishing stable transcriptional and functional programs of regulatory T cells (T(reg) cells). Loss of Foxp3 expression in mature T(reg) cells results in a failure of suppressor function, yet the molecular mechanisms that ensure steady, heritable Foxp3 expression in the T(reg) cell lineage remain unknown. Using T(reg) cell-specific gene targeting, we found that complexes of the transcription factors Runx and CBFbeta were required for maintenance of Foxp3 mRNA and protein expression in T(reg) cells. Consequently, mice lacking CBFbetab exclusively in the T(reg) cell lineage had a moderate lymphoproliferative syndrome. Thus, Runx-CBFbeta complexes maintain stable high expression of Foxp3 and serve as an essential determinant of T(reg) cell lineage stability

The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease

MicroRNAs (miRNAs) are implicated in the differentiation and function of many cell types. We provide genetic and in vivo evidence that the two RNaseIII enzymes, Drosha and Dicer, do indeed function in the same pathway. These have previously been shown to mediate the stepwise maturation of miRNAs (Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim. 2003. Nature. 425:415-419), and genetic ablation of either within the T cell compartment, or specifically within Foxp3(+) regulatory T (T reg) cells, results in identical phenotypes. We found that miRNA biogenesis is indispensable for the function of T reg cells. Specific deletion of either Drosha or Dicer phenocopies mice lacking a functional Foxp3 gene or Foxp3(+) cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease, but later in life. Thus, miRNA-dependent regulation is critical for preventing spontaneous inflammation and autoimmunity.

Immunoglobulin A (IgA) is generated in the gut by both T cell-dependent and T cell-independent processes. The sites and the mechanisms for T cell-independent IgA synthesis remain elusive. Here we show that isolated lymphoid follicles (ILFs) were sites where induction of activation-induced cytidine deaminase (AID) and IgA class switching of B cells took place in the absence of T cells. We also show that formation of ILFs was regulated by interactions between lymphoid tissue-inducer cells expressing the nuclear receptor ROR gamma t (ROR gamma t(+)LTi cells) and stromal cells (SCs). Activation of SCs by ROR gamma t(+)LTi cells through lymphotoxin (LT)-beta receptor (LT beta R) and simultaneously by bacteria through TLRs induced recruitment of dendritic cells (DCs) and B cells and formation of ILFs. These findings provide insight into the crosstalk between bacteria, ROR gamma t(+)LTi cells, SCs, DCs, and B cells required for ILF formation and establish a critical role of ILFs in T cell-independent IgA synthesis in gut

Natural Killer (NK) cells are innate immune cells that mediate resistance against viruses and tumors. They express multiple activating receptors that couple to immunoreceptor tyrosine-based activation motif (ITAM) containing signaling chains for downstream cell activation. Ligation of activating NK cell receptors induces NK cell cytotoxicity and cytokine release. How these distinct events are selectively controlled is not well defined. Here we report the identification of a specific signaling pathway that operates downstream of the ITAM coupled NK cell receptors NK1.1, Ly49D, Ly49H and NKG2D. Using primary NK cells from Bcl10(-/-), Malt1(-/-), Carma1(-/-), and Card9(-/-) mice we demonstrate a key role for Bcl10 signalosomes in the activation of canonical NF-kappaB signaling as well as JNK and p38 MAPK upon NK cell triggering. Bcl10 directly cooperates with Malt1 and depends on Carma1 (Card11) but not on Card9 for NK cell activation. These Bcl10-dependent cascades selectively control cytokine and chemokine production but do not affect NK cell differentiation or killing. Thus, we identify a molecular basis for the segregation of NK cell receptor induced signals for cytokine release and target cell killing and extend the previously recognized roles for CARD-protein/Bcl10/Malt1 complexes in ITAM receptor signaling in innate and adaptive immune cells

BACKGROUND: The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRalphabeta lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRalphabeta prematurely at the double negative stage and abnormal TCRalphabeta populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice. METHODOLOGY AND PRINCIPAL FINDINGS: To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRalphabeta diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORgammat-positive CD4(+)8(+) (double positive, DP) stage to accumulate either as CD4(-)8(-) (double negative, DN) or as CD8alpha(+) T cells in lymph nodes or gut epithelium. Likewise, DN TCRalphabeta cells in lymphoid tissue of female mice were not derived from DP thymocytes. CONCLUSION: The results further support the hypothesis that the premature expression of the TCRalphabeta can divert DN thymocytes into gamma delta lineage cells

T helper cells that produce IL-17 (T(H)17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, T(H)17 cells are thought to protect the host from infection, whereas regulatory T (T(reg)) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-beta (TGF-beta), but depends on distinct transcription factors: RORgammat (encoded by Rorc(gammat)) for T(H)17 cells and Foxp3 for T(reg) cells. How TGF-beta regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-beta orchestrates T(H)17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-beta synergizes with interleukin (IL)-6 and IL-21 (refs 9-11) to promote IL-23 receptor (Il23r) expression, favouring T(H)17 cell differentiation. High concentrations of TGF-beta repress IL23r expression and favour Foxp3+ T(reg) cells. RORgammat and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-beta and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-beta-induced Foxp3 inhibits RORgammat function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORgammat alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORgammat, thereby promoting T(H)17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either T(H)17 or T(reg) cells depends on the cytokine-regulated balance of RORgammat and Foxp3

Apobec proteins are a family of cellular cytidine deaminases, among which several members have been shown to have potent antiviral properties. This antiviral activity is associated with the ability to cause hypermutation of retroviral cDNA. However, recent research has indicated that Apobec proteins are also able to inhibit retroviruses by other mechanisms that are independent of their deaminase activity. We have compared the antiviral activities of human and murine Apobec3 (A3) proteins, and we have found that, consistent with previous reports, human immunodeficiency virus (HIV) is able to resist human A3G but is sensitive to murine A3, whereas murine leukemia virus (MLV) is relatively resistant to murine A3 (mA3) but sensitive to human A3G. In contrast to previous studies, we observed that mA3 is packaged efficiently into MLV particles. The C-terminal cytidine deaminase domain (CDD2) is required for packaging of mA3 into MLV particles, and packaging did not depend on the MLV viral RNA. However, mA3 packed into MLV particles failed to cause hypermutation of viral DNA, indicating that its deaminase activity is blocked or inhibited. hA3G also caused significantly less hypermutation of MLV than of HIV DNA. Both mA3 and the splice variant mA3Delta5 exhibited some residual antiviral activity against MLV and caused a reduction in the ability of MLV particles to generate reverse transcription products. These results suggest that MLV has evolved specific mechanisms to block the ability of Apobec proteins to mediate deaminase-dependent hypermutation

The mechanism underlying the transient accumulation of CD4 at the immunological synapse (IS) and its significance for T cell activation are not understood. To investigate these issues, we mutated a serine phosphorylation site (S408) in the cytoplasmic tail of murine CD4. Preventing phosphorylation of S408 did not block CD4 recruitment to the IS; rather, it blocked the ability of CD4 to leave the IS. Surprisingly, enhanced and prolonged CD4 accumulation at the supramolecular activation cluster in the contact area had no functional consequence for T cell activation, cytokine production, or proliferation. Protein kinase C theta (PKCtheta)-deficient T cells also displayed enhanced and prolonged accumulation of wild-type CD4 at the IS, indicating that theta is the critical PKC isoform involved in CD4 movement. These findings suggest a model wherein recruitment of CD4 to the IS allows its phosphorylation by PKCtheta and subsequent removal from the IS. Thus, an important role for PKCtheta in T cell activation involves its recruitment to the IS, where it phosphorylates specific substrates that help to maintain the dynamism of protein turnover at the IS