Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome

Producción CientíficaBackground: Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott- Aldrich syndrome (WAS). The transcription factors nuclear factor-kB (NF-kB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. Objective: To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). Methods: T cells from WASP2/2 mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kB–dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. Results: Nuclear translocation and activity of NF-kB were normal in T cells from WASP2/2 mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. Conclusion: These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS

WIP and WASP play complementary roles in T cell homing and chemotaxis to SDF-1a

Producción CientíficaHoming of lymphocytes to tissues is a biologically important multistep process that involves selectindependent rolling, integrin-dependent adhesion and chemokine-directed chemotaxis. The actin cytoskeleton plays a central role in lymphocyte adhesion and motility. Wiskott–Aldrich syndrome protein (WASP), the product of the gene mutated in Wiskott–Aldrich syndrome, and its partner, the Wiskott–Aldrich syndrome protein-interacting protein (WIP), play important roles in actin re-organization in T lymphocytes. We used mice with disruption of the WASP and WIP genes to examine the role of WASP and WIP in T cell homing. T cell homing to spleen and lymph nodes in vivo was deficient in WASP / and WIP / mice and severely impaired in WASP / WIP / double knockout (DKO) mice. Deficiency of WASP, WIP or both did not interfere with selectin-dependent rolling or integrin-dependent adhesion of T cells in vitro. Chemotaxis to stromal cell-derived factor-1a (SDF-1a) in vitro was mildly reduced in T cells from WASP / mice. In contrast, it was significantly impaired in T cells from WIP / mice and severely reduced in T cells from DKO mice. Cellular F-actin increase following SDF-1a stimulation was normal in WASP / and WIP / T cells, but severely reduced in T cells from DKO mice. Actin re-organization and polarization in response to SDF-1a was abnormal in T cells from all knockout mice. Early biochemical events following SDF-1a stimulation that are important for chemotaxis and that included phosphorylation of Lck, cofilin, PAK1 and extracellular regulated kinase (Erk) and GTP loading of Rac-1 were examined in T cells from DKO mice and found to be normal. These results suggest that WASP and WIP are not essential for T lymphocyte rolling and adhesion, but play important and partially redundant roles in T cell chemotaxis in vitro and homing in vivo and function downstream of small GTPases

Mechanism of recruitment of WASP to the immunological synapse and of its activation following TCR ligation

Producción CientíficaF-actin polymerization following engagement of the T cell receptor (TCR) is dependent on WASP and is critical for T cell activation. The link between TCR and WASP is not fully understood. In resting cells, WASP exists in a complex with WIP, which inhibits its activation by Cdc42. We show that the adaptor protein CrkL binds directly to WIP. Further, TCR ligation results in the formation of a ZAP-70-CrkL-WIP-WASP complex, which is recruited to lipid rafts and the immunological synapse. TCR engagement also causes PKCtheta-dependent phosphorylation of WIP, causing the disengagement of WASP from the WIP-WASP complex, thereby releasing it from WIP inhibition. These results suggest that the ZAP-70-CrkL-WIP pathway and PKCtheta link TCR to WASP activation

Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function

Lrrc8a is a ubiquitously expressed gene that encodes a leucine-rich repeat (LRR)–containing protein detected at higher levels on the surface of thymocytes than on other immune cells. We generated Lrrc8a−/− mice to investigate the role of LRRC8A in lymphocyte development and function. Lrrc8a−/− mice had increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities. Lrrc8a−/− mice displayed a modest block in B cell development but intact intrinsic B cell function. In contrast, both Lrrc8a−/− mice and Lrrc8a−/−→Rag2−/− bone marrow chimeras exhibited a severe cell-intrinsic block in early thymic development, with decreased proliferation and increased apoptosis of thymocytes, and impaired peripheral T cell function. Thymic epithelial cells expressed an LRRC8A ligand that was critical for double-negative to double-positive thymocyte differentiation and survival in vitro. LRRC8A constitutively associated with the GRB2–GAB2 complex and lymphocyte-specific protein tyrosine kinase (LCK) in thymocytes. LRRC8A ligation activated AKT via the LCK–ZAP–70–GAB2–PI3K pathway, and AKT phosphorylation was markedly reduced in the thymus of Lrrc8a−/− mice. These findings reveal an essential role for LRRC8A in T cell development, survival, and function

RORα-expressing T regulatory cells restrain allergic skin inflammation

Atopic dermatitis is an allergic inflammatory skin disease characterized by the production of the type 2 cytokines in the skin by type 2 innate lymphoid cells (ILC2s) and T helper 2 (TH2) cells, and tissue eosinophilia. Using two distinct mouse models of atopic dermatitis, we show that expression of retinoid-related orphan receptor α (RORα) in skin-resident T regulatory cells (Tregs) is important for restraining allergic skin inflammation. In both models, targeted deletion of RORα in mouse Tregs led to exaggerated eosinophilia driven by interleukin-5 (IL-5) production by ILC2s and TH2 cells. Expression of RORα in skin-resident Tregs suppressed IL-4 expression and enhanced expression of death receptor 3 (DR3), which is the receptor for tumor necrosis factor (TNF) family cytokine, TNF ligand–related molecule 1 (TL1A), which promotes Treg functions. DR3 is expressed on both ILC2s and skin-resident Tregs. Upon deletion of RORα in skin-resident Tregs, we found that Tregs were no longer able to sequester TL1A, resulting in enhanced ILC2 activation. We also documented higher expression of RORα in skin-resident Tregs than in peripheral blood circulating Tregs in humans, suggesting that RORα and the TL1A-DR3 circuit could be therapeutically targeted in atopic dermatitis

A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton

Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor–driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.United States. Public Health Service (RO1AI114588)United States. Public Health Service (K08AI114968

RIP Links TLR4 to Akt and Is Essential for Cell Survival in Response to LPS Stimulation

Receptor-interacting protein (RIP) has been reported to associate with tumor necrosis–associated factor (TRAF)2 and TRAF6. Since TRAF2 and TRAF6 play important roles in CD40 signaling and TRAF6 plays an important role in TLR4 signaling, we examined the role of RIP in signaling via CD40 and TLR4. Splenocytes from RIP−/− mice proliferated and underwent isotype switching normally in response to anti-CD40–IL-4 but completely failed to do so in response to LPS–IL-4. However, they normally up-regulated TNF-α and IL-6 gene expression and CD54 and CD86 surface expression after LPS stimulation. RIP−/− splenocytes exhibited increased apoptosis and impaired Akt phosphorylation after LPS stimulation. These results suggest that RIP is essential for cell survival after TLR4 signaling and links TLR4 to the phosphatidylinositol 3 kinase–Akt pathway

DOCK8 Functions as an Adaptor that Links TLR–MyD88 Signaling to B Cell Activation

DOCK8 and MyD88 have been implicated in serologic memory. Here we report antibody responses were impaired and $CD27^+$ memory B cells were severely reduced in DOCK8-deficient patients. Toll-like receptor 9 (TLR9)- but not CD40-driven B cell proliferation and immunoglobulin production were severely reduced in DOCK8-deficient B cells. In contrast, TLR9-driven expression of AICDA, CD23 and CD86, and activation of NF-κB, p38 and Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. Following TLR9 ligation, DOCK8 became tyrosine phosphorylated by Pyk2, bound the Src family kinase Lyn and linked TLR9 to a Src-Syk-STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells