Ana L. Mora

NF-kappa B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes

Ontogenetic, homeostatic, and functional deficiencies within immunoregulatory natural T (iNKT) lymphocytes underlie various inflammatory immune disorders including autoimmunity. Signaling events that control cell fate specification and molecular differentiation of iNKT cells are only partly understood. Here we demonstrate that these processes within iNKT cells require classical NF-κB signaling. Inhibition of NF-κB signaling blocks iNKT cell ontogeny at an immature stage and reveals an apparent, novel precursor in which negative selection occurs. Most importantly, this block occurs due to a lack of survival signals, as Bcl-xL overexpression rescues iNKT cell ontogeny. Maturation of immature iNKT cell precursors induces Bcl-2 expression, which is defective in the absence of NF-κB signaling. Bcl-xL overexpression also rescues this maturation-induced Bcl-2 expression. Thus, antiapoptotic signals relayed by NF-κB critically control cell fate specification and molecular differentiation of iNKT cells and, hence, reveal a novel role for such signals within the immune system.

T Cell-Intrinsic Requirement for NF-κB Induction in Postdifferentiation IFN-γ Production and Clonal Expansion in a Th1 Response

NF-κB/Rel transcription factors are linked to innate immune responses and APC activation. Whether and how the induction of NF-κB signaling in normal CD4+ T cells regulates effector function are not well-understood. The liberation of NF-κB dimers from inhibitors of κB (IκBs) constitutes a central checkpoint for physiologic regulation of most forms of NF-κB. To investigate the role of NF-κB induction in effector T cell responses, we targeted inhibition of the NF-κB/Rel pathway specifically to T cells. The Th1 response in vivo is dramatically weakened when T cells defective in their NF-κB induction (referred to as IκBα(ΔN) transgenic cells) are activated by a normal APC population. Analyses in vivo, and IL-12-supplemented T cell cultures in vitro, reveal that the mechanism underlying this T cell-intrinsic requirement for NF-κB involves activation of the IFN-γ gene in addition to clonal expansion efficiency. The role of NF-κB in IFN-γ gene expression includes a modest decrease in Stat4 activation, T box expressed in T cell levels, and differentiation efficiency along with a more prominent postdifferentiation step. Further, induced expression of Bcl-3, a trans-activating IκB-like protein, is decreased in T cells as a consequence of NF-κB inhibition. Together, these findings indicate that NF-κB induction in T cells regulates efficient clonal expansion, Th1 differentiation, and IFN-γ production by Th1 lymphocytes at a control point downstream from differentiation.

NF-κB/Rel Participation in the Lymphokine-Dependent Proliferation of T Lymphoid Cells

Proliferative responses of lymphoid cells to IL-2 and IL-4 depend on activation of the cells, but the mechanism(s) by which activation enhances cellular competence to respond to cytokines is not fully understood. The NF-κB/Rel family represents one signal transduction pathway induced during such activation. We show in this study that inhibition of NF-κB through the expression of an IκBα (inhibitory protein that dissociates from NF-κB) mutant refractory to signal-induced degradation (IκBα(ΔN)) interfered with the acquisition of competence to proliferate in response to IL-4 as well as IL-2. Thymocytes and T cells from IκBα(ΔN) transgenic mice expressed normal levels of IL-2R subunits. However, transgenic cells exhibited a dramatic defect in Stat5A activation treatment with IL-2, and a similar defect was observed for IL-4-induced Stat5. In contrast, T lymphoid cells with inhibition of NF-κB showed normal insulin receptor substrate-2 phosphorylation and only a modest decrease in Stat6 activation and insulin receptor substrate-1 phosphorylation after IL-4 stimulation. These results indicate that the NF-κB/Rel/IκBα system can regulate cytokine receptor capacitation through effects on the induction of downstream signaling by the Stat transcription factor family.

IL-4 signaling, gene transcription regulation, and the control of effector T cells

The central goal of our laboratory is to understand the regulation of lymphoid cells through molecular mechanisms of signal transduction and transcriptional control. A long-standing focus has been on changes that influence the effector function of mature lymphocytes. Work in the laboratory is oriented toward the identification of new regulatory mechanisms using cell lines and primary cells, and the validation of these in vitro findings in mouse models of immune responses and diseases. In this review, we summarize key insights into the regulation of T helper cell function during the phase of immunity where effector responses arise de novo. Particular interest has been centered on cytokine gene regulation as part of T cell differentiation into the Th1 and Th2 subsets. Information on IL-4 receptor signaling and the role of NF-κB transcription factors is reviewed. Our more recent work is designed to understand how regulation at the Th 1/2 effector stages is related to the control of memory T cell survival, immune recall responses, and the role of these responses in immune-mediated disease.

Inefficient ZAP-70 Phosphorylation and Decreased Thymic Selection In Vivo Result from Inhibition of NF-κB/Rel

Signaling from the TCR regulates T lymphoid survival, deletion by apoptosis, and selective clonal expansion. One set of signaling pathways activated during thymic selection leads to degradation of a cytosolic retention protein, the inhibitor of κB (IκB)α, followed by nuclear translocation of the NF-κB/Rel family of transcription factors. It has been found previously that NF-κB proteins mediate a pathway signaling the survival of mature T cells and protection of thymocytes against TNF-induced apoptosis. In contrast, we show in this study that a transgenic inhibitor of NF-κB/Rel signaling interferes with the negative selection of immature thymocytes by endogenous MHC ligands in vivo. Positive selection of the H-Y TCR also was diminished. This attenuation of thymic selection efficiency was associated with decreased ZAP-70 phosphorylation and TCR signaling of CD69 induction. These findings demonstrate that the NF-κB transcriptional pathway plays an important role in normal processes of clonal deletion and they indicate that the NF-κB/IκB axis can regulate the efficiency of TCR signaling.

Sequence Motifs in IL-4Rα Mediating Cell-Cycle Progression of Primary Lymphocytes

IL-4 signaling through the IL-4Rα chain regulates the development and proliferation of the Th2 lineage of effector CD4+ T cells. Analyses of the IL-4R in factor-dependent cell lines led to the development of two apparently conflicting models of the primary structural determinants of IL-4R-mediated proliferative signaling. In one model, proliferation was dependent on the first conserved tyrosine in the cytoplasmic tail (Y1), while in the second, proliferation was independent of cytoplasmic tyrosines. We found that in activated primary T cells, mutation of only the Y1 residue resulted in a modest decrease in IL-4-induced S phase entry, a further decrease in cell-cycle completion, and a complete failure of IL-4 to induce p70S6 kinase phosphorylation. Consistent with a role for the PI3K/mammalian target of rapamycin pathway in mediating cytokine acceleration of G2/M transit, pretreatment of activated T cells with rapamycin resulted in only a modest decrease in IL-4-induced S phase entry, but a total block of cell-cycle completion. Strikingly, IL-4Rα chains that lacked all cytoplasmic tyrosines were competent to signal for STAT5 phosphorylation, mediated efficient S phase entry, and promoted cell-cycle progression. The ability of tyrosine-deficient IL-4Rs to mediate proliferative signaling and STAT phosphorylation was absolutely dependent on the presence of an intact ID-1 region. These findings show that IL-4Rα lacking cytoplasmic tyrosine residues is competent to induce ID-1-dependent proliferation, and indicate that IL-4 can promote G2/M progression via activation of the mammalian target of rapamycin pathway initiated at the Y1 residue.

Lineage‐specific differences among CD8+ T cells in their dependence of NF‐κB/Rel signaling

Whereas most CD8+ T cells in lymph nodes and spleen express the CD8α β heterodimer and depend absolutely on thymic competence for their development, a substantial population of T cells expressing CD8α α matures extrathymically. Although the existence of these CD8 sublineages is well established, relatively little is known about differences that might exist among CD8 cells in their requirement for particular transcriptional pathways during the development and maintenance of normal populations. Transgenic mice whose T lineage expresses an IκBα mutant exhibited decreased NF‐κB signaling and a diminution in mature CD8 T cells. We now have determined that although TCR‐dependent CD69 induction by CD8α α and CD8α β T cells was unaffected by inhibition of NF‐κB, TCRα β CD8α β T cells were preferentially reduced compared to their TCRα β CD8α α or TCRγ δ counterparts. This finding was most prominent in spleen, but was also apparent in Peyers patches of transgenic mice. In addition, diminished antiviral cytotoxic responses of CD8α β intraepithelial lymphocytes were observed after enteric reovirus infection. Taken together, these results indicate that NF‐κB signaling is more important for the thymus‐dependent TCRα β CD8α β population than for other CD8 lineages, and thus regulates the number, function, and normal balance of CD8 subsets in the periphery.

An IL-4Rα Allelic Variant, I50, Acts as a Gain-of-Function Variant Relative to V50 for Stat6, But Not Th2 Differentiation

Signaling through the IL-4R α-chain (IL-4Rα) is crucial for the development of Th2 cells, central effectors in atopic disease. Alleles of the IL-4Rα have been identified that have been variably associated with increased incidence of allergic disease, but there is little direct evidence that any variant is sufficient to alter a target that determines allergic pathophysiology or susceptibility. Variants of IL-4Rα encoding isoleucine instead of valine at position 50 (I50 vs V50, respectively) can signal increased Stat6-dependent transcriptional activity, whether in an I50, Q551 or I50, R551 haplotype. Strikingly, signaling through these receptors did not increase the efficiency of Th2 development or the IL-4 mediated repression of Th1 development or a target gene, IL-18Rα. Further, IL-4-induced proliferation was similar for Th2 cells independent of the variant expressed. Together these findings indicate that IL-4Rα variants that exhibit gain-of-function with respect to Stat6 do not act directly through alterations in Th2/Th1 induction after Ag exposure. The data further suggest that for such variants, any mechanistic involvement is based on a role in cellular targets of Th2 cytokines.

New Programming of IL-4 Receptor Signal Transduction in Activated T Cells: Stat6 Induction and Th2 Differentiation Mediated by IL-4Rα Lacking Cytoplasmic Tyrosines

Signaling by the IL-4 receptor α-chain (IL-4Rα) is a key determinant of the development of the Th2 lineage of effector T cells. Studies performed in tissue culture cell lines have indicated that tyrosines of the IL-4Rα cytoplasmic tail are necessary for the induction of Stat6, a transcription factor required for Th2 differentiation. Surprisingly, we have found that in activated T cells, IL-4Rα chains lacking all cytoplasmic tyrosines promote induction of this IL-4-specific transcription factor and efficient commitment to the Th2 lineage. Mutagenesis of a tyrosine-free cytoplasmic tail identifies a requirement for the serine-rich ID-1 region in this new program of IL-4R signal transduction observed in activated T cells. Additional findings suggest that an extracellular signal-regulated kinase pathway can be necessary and sufficient for the ability of such tyrosine-free IL-4Rα chains to mediate Stat6 induction. These results provide novel evidence that the molecular mechanisms by which a cytokine specifically induces a Stat transcription factor can depend on the activation state of T lymphoid cells. Furthermore, the data suggest that one pathway by which such new programming may be achieved is mediated by extracellular signal-regulated mitogen-activated protein kinases.