Regulatory T cells (Tregs) control immune responses and are essential to maintain immune homeostasis and self-tolerance. Hence, it is no coincidence that autoimmune and chronic inflammatory disorders are associated with defects in Tregs. These diseases have currently no cure and are treated with palliative drugs such as immunosuppressant and immunomodulatory agents. Thereby, there is a great interest in developing medical interventions against these diseases based on enhancing Treg cell function and numbers. Here, we give an overview of Treg cell ontogeny and function, paying particular attention to mucosal Tregs. We review some notable approaches to enhance immunomodulation by Tregs with therapeutic purposes including adoptive Treg cell transfer therapy and discuss relevant clinical trials for inflammatory bowel disease. We next introduce ways to expand mucosal Tregs in vivo using microbiota and dietary products that have been the focus of clinical trials in various autoimmune and chronic-inflammatory diseases.

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Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Human rhinovirus (RV) is the most common cause of upper respiratory infections and exacerbations of asthma. In this work, we selected 14 peptides (6 from RV A and 8 from RV C) encompassing potential CD4 T cell epitopes. Peptides were selected for being highly conserved in RV A and C serotypes and predicted to bind to multiple human leukocyte antigen class II (HLA II) molecules. We found positive T cell recall responses by interferon gamma (IFNγ)-ELISPOT assays to eight peptides, validating seven of them (three from RV A and four from RV C) as CD4 T cell epitopes through intracellular cytokine staining assays. Additionally, we verified their promiscuous binding to multiple HLA II molecules by quantitative binding assays. According to their experimental HLA II binding profile, the combination of all these seven epitopes could be recognized by >95% of the world population. We actually determined IFNγ responses to a pool encompassing these CD4 T cell epitopes by intracellular cytokine staining, finding positive responses in 29 out of 30 donors. The CD4 T cell epitopes identified in this study could be key to monitor RV infections and to develop peptide-based vaccines against most RV A and C serotypes.

Human Rhinovirus (HRV) is the most common cause of upper respiratory infections and exacerbations of asthma. In this work, we selected 14 peptides (6 from HRV A and 8 from HRV C) encompassing potential CD4 T cell epitopes. Peptides were selected for being highly conserved in HRV A and C serotypes and predicted to bind to multiple HLA II molecules. We found positive T cell recall responses by IFNγ-ELISPOT assays to 8 peptides, validating 7 of them (3 from HRV A and 4 from HRV C) as CD4 T cell epitopes through intracellular cytokine staining assays. Additionally, we verified their promiscuous binding to multiple HLA II molecules by quantitative binding assays. According to their experimental HLA II binding profile, the combination of all these 7 epitopes could be presented and recognized by > 95 % of the world population. We actually determined IFNγ responses to a pool encompassing these CD4 T cell epitopes by intracellular cytokine staining, finding positive responses in 29 out of 30 donors. The CD4 T cell epitopes identified in this study could be key to monitor HRV infections and to develop peptide-based vaccines against most HRV A and C serotypes.