Migratory properties of Treg are

Migratory properties of Treg are extremely important for the potential in vivo application. Therefore, the observed expression of CXCR3 on almost all obtained insulin-specific Treg is crucial for directing sphingosine kinase inhibitor into the inflamed tissue (in this case pancreatic islets). CXCR3 responds to CXCL9, CXCL10 and CXCL11 that are highly expressed during inflammation (Groom and Luster, 2011). Further expansion of pure insulin-specific Treg was dependent upon the presence of direct cell contact with DC. The ever-present problem for in vitro expanded or generated Treg is their potential reversion to the inflammatory phenotype (Ren and Li, 2017). The observed destabilization of insulin-specific Treg phenotype in the absence of supporting cells was partly ameliorated by the addition of STAT3 inhibitor that blocks Th17 differentiation (Chen and Laurence, 2007). Treg conversion into Th17 is well-documented and it happens because these cells share the same progenitor and the cytokine requirements for their differentiation are very similar (Ren and Li, 2017). The absence of the supporting effect for rapamycine and 5-aza 3’deoxycytdine was in contrast to the previously published studies. Specifically, rapamycine is known to stimulate Treg proliferation by blocking mTOR pathway (Battaglia et al., 2005; Hou et al., 2018), while 5-aza 3′deoxycytdine through demethylation of FoxP3 promotor stabilizes long-term FoxP3 expression (Freudenberg et al., 2018). This discrepancy may be a result of already optimal engaged signalling pathways (through TCR and IL-2) that lead to FoxP3 expression, and therefore the addition of stimulators has no further impact on proliferation or gene expression. In conclusion, in vitro generation of insulin-specific Treg is a three-stage protocol and it consists of: enrichment stage where insulin-specific CD4+ cells are expanded in the presence of cellular signal from mDC, purification stage where sorted insulin-specific CD4+ are subjected to TCR signal, IL-2 and TGF-β and the only population that survives is insulin-specific Treg population, and finally expansion stage where again in the presence of mDC, insulin-specific Treg proliferate (Fig. 6). The obtained insulin-specific Treg are fully functional and this protocol repeatedly provided sufficient numbers of Treg needed for the future in vivo application in the mouse model of T1D.
Introduction Genetic researches are fundamental for understanding the molecular basis of host defense against pathogen and tumor. Even though targeted mutations in mammalian models such as mice give rise to insights into the immune homeostasis and response, forward genetic screening is of particular interest to randomly identify novel genes or alleles that are important for development and function of the immune system (Beutler, 2016). In this study, we performed ENU mutagenesis coupled to multi-parameter flow cytometric analysis in mice. Using exome capture and next generation sequencing, we identified a novel mutant allele of Cd4 gene that was responsible for the complete absence of CD4 T cell development in thymus. During T cell development in thymus multiple highly coordinated signaling pathways are required for proper thymocytes proliferation and elimination of autoreactive T cells, which include those via T cell receptor, costimulatory molecules, and the co-receptors such as CD4 (Malissen and Bongrand, 2015, Rahemtulla et al., 1991). Structural studies showed that multiple extracellular domains of CD4 exist on T cell surface, including the first extracellular domain that interacts with its ligand MHCII (Wang et al., 2001a). However functional studies elucidating how the extracellular domain maintain its interaction with MHCII, on the basis of in vivo mouse models, are still limited. Our data for the first time showed experimentally in mice the critical role of first extra-cellular domain, and a loss of function mutation from Ile to Asn at the position 99 of CD4. Interestingly, this novel CD4I99N mutation was not structurally located in the known interface where CD4 and its ligand interact, but presumably destroyed the three-dimensional structure from the center through alteration of the hydrophobic interaction among those critical amino acids. We further sought to determine whether the mutant allele affected gene expression or function of the protein. Our data showed that such CD4I99N mutant protein can be expressed on the surface of human cells, suggesting that absence of CD4 T cells in mice rooted in the deficiency in function and expression of CD4. In further experiments, we used this novel CD4 T cell deficient model as recipient mice for adoptive transfer study, and the results showed that it could be an optimal model for study of CD4 T cells.