Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.
Class IV semaphorin family members, Sema4A and Sema4D, displayed opposite effects on human Treg cells in vitro. In PBMC cultures, Sema4A potentiated the CD4+CD25+Foxp3+ cell numbers and increased CD25/Foxp3 expression while Sema4D inhibited them. Sema4A downregulated lung inflammation in allergen-induced experimental asthma whereas Sema4D was necessary for optimal allergic response. Allergen treatment decreased lung local and lymphoid tissue Treg cell numbers in Sema4A−/− mice and increased them in Sema4D−/− mice as compared to similarly treated WT counterparts. Sema4A and Sema4D competitively bind to Plexin B1 in microplate-based binding studies. Using Bayesian Partitioning with Pattern Selection (BPPS) and Structurally Interacting Pattern Residues’ Inferred Significance (SIPRIS), we identified Sema4A residues likely responsible for its functional specificity. We mutated the Sema4A residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. We evaluated their stimulatory activity toward human Treg cells and assayed them for binding to Plexin B1. The F223A mutant was unable to stimulate Treg stability in the in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to WT and M198A proteins. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing novel immunotherapeutics for asthma. This work was supported by NIH grant R01AI 122631 and the Merit Review Award Number I01 BX001850 from the US Department of Veterans Affairs Biomedical R&D Service (both awarded to A.D.K.) and by NIH-NIGMS grant R01GM125878 to A.F.N.
TCR signal strength regulates T cell fate at various stages of thymocyte development and the differentiation of peripheral T cells into distinct T cell subsets. We previously showed that TCR engagement forms a heterotetrametric complex composed of LAT, Gads, SLP-76, PLC-γ1 and that this complex is essential to activate PLC-γ1 to further transduce the TCR signal. The interaction of the SLP-76 proline-rich region (PRR) with the PLC-γ1 SH3 domain is relatively weak, and these two domains are highly conserved amongst many mammalian species. Therefore, we hypothesized that the SLP-76 PRR sequence with low affinity for PLC-γ1 might be evolutionarily favored to prevent excess TCR signals. To test this hypothesis, we developed mice carrying a conditional T cell-specific knock-in mutation in the SLP-76 PRR with a 2.5-fold increased affinity for PLC-γ1 by CRISPR/Cas9-mediated gene editing and Cre/LoxP system (cKI). Notably, cKI increased the expression of active caspase-3 in signaled SP4 and SP8 thymocytes and decreased the numbers of mature medullary thymocytes, suggestive of enhanced negative selection. Under steady-state conditions, cKI enhanced the generation of CD8 +central memory T cells, implying an increased sensitivity to self-Ag/MHC I in the periphery. Moreover, cKI rendered CD8 +T cells more sensitive to OVA altered peptide ligands with a weaker affinity for OT-I TCR. Finally, cKI enhanced priming of OT-I CD8 +T cells that had not undergone massive cell divisions for IFNγ-producing effectors in vivo, implying an increased TCR signal strength in weakly activated CD8 +T cells by cognate Ag. Our data indicate that the SLP-76 PRR/PLC-γ1 SH3 interaction plays a critical role in fine-tuning TCR signal strength to avoid excess signals via TCR. NCI/NIH Intramural Support
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.