In T cells, the Tec kinases ITK and RLK are activated by TCR stimulation, and are required for optimal downstream signaling. Studies of CD4+ T cells from Itk−/− and Itk−/− Rlk−/− mice have indicated differential roles of ITK and RLK in TH1, TH2, and TH17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. Here, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro TH polarization experiments indicate that PRN694 is a potent inhibitor of TH1 and TH17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4+ T cells. Consistent with these findings, TH1 and TH17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in TH1-mediated inflammatory diseases.

This article provides in vitro data from which immunosuppressive regimens for the effective control of memory T cells in non-human preclinical experiments and in clinical trials are selected.

Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3(+)CD103(+) subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase-8 levels and the significant reduction in Bcl-2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble-factor-mediated T-cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down-regulated. A cytometry bead assay showed that the secretion of interleukin (IL)-2 decreased significantly, whereas that of IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells.

Innate lymphoid cells (ILC) are lymphocytes that lack an antigen-specific receptor and are preferentially localized in non-lymphoid tissues, such as mucosal barriers. In these locations ILC respond to tissue perturbations by producing factors that promote tissue repair and improve barrier integrity. We show that mice lacking the Tec kinase ITK have impaired intestinal tissue integrity, and a reduced ability to restore homeostasis after tissue damage. This defect is associated with a substantial loss of Type 2 ILC (ILC2) in the intestinal lamina propria. Adoptive transfer of bone marrow ILC2 precursors confirms a cell-intrinsic role for ITK. Intestinal ILC2 numbers in Itk-/- mice are restored by the administration of IL-2 complexes, also leading to improved intestinal tissue damage repair. Reduced Bcl-2 expression in intestinal Itk-/- ILC2 is also restored to WT levels after IL-2 complex treatment, indicating a tissue-specific role for ITK in ILC2 survival in the intestine.

The Tec kinase IL-2-inducible T cell kinase (ITK) regulates the expression of TCR-induced genes. Itk 2/2 T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice. To examine the function of ITK in T cell migration to the intestine, we examined the number of gut T cells in Itk 2/2 mice and then evaluated their expression of gut-homing receptors. Combined with in vitro murine T cell stimulation and in vivo migration assay using congenic B6 mice, we demonstrated an essential role for ITK in T cell migration to the intestine in mice. Reconstitution of Itk 2/2 mouse CD8 + T cells with IFN regulatory factor 4 restored gut-homing properties, providing mechanistic insight into the function of ITK-mediated signaling in CD8 + T cell migration to the intestinal mucosa in mice. ImmunoHorizons, 2020, 4: 57-71.

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4 1 Tregs have been identified, including Foxp3 1 , Tr1, Th3, and Foxp3neg iT(R)35 cells. In this study, we identified a new subset of CD4 1 VEGFR1 high Tregs that have immunosuppressive capacity. CD4 1 VEGFR1high T cells, which constitute approximately 1.0% of CD4 1 T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4 1 VEGFR1 high T cells are distinct from known Tregs. CD4 1 VEGFR1 high T cells suppressed the proliferation of CD4 1 CD25 2 T cell as efficiently as CD4 1 CD25 high natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4 1 VEGFR1 1 T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4 1 VEGFR1 high T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection with Citrobacter rodentium or enterohemorrhagic Escherichia coli, luminal C3 levels increase significantly and are required for protection. C. rodentium is remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis of C. rodentium functions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection.

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