Cx43-CT Contributes to T Cell Receptor Signaling

The diverse set of effector T helper (Th) cells maintains immune homeostasis and activates the proper adaptive immune response. Activated T cells undergo proliferation and clonal expansion and acquire effector function. Self-reactive Th have been found in multiple sclerosis where self-reactive clones can be detected years apart. In the tumor microenvironment Th cells known as T regulatory cells support tumor growth by suppression cytotoxic T cells. Understanding the molecular components of T cell activation and suppression will advance the field and discover novel therapies. Naive CD4+ T cells are activated by interaction of the T cell receptor (TCR) signaling complex with antigenic peptide presented by an antigen presenting cell. A structure forms between the cells called the immunological synapse where the phosphorylation of kinases and adaptor molecules associated with the TCR form macromolecular signaling complexes. This complex includes CD3 and zeta chains, phospholipase C, LAT (linker of activated T cells). As the signal propagates away from the immunological synapse multiple signaling pathways are activated including Ca2+ flux causing nuclear factor of activated T cells (NFAT) to enter the nucleus. Our work along with others have shown a functional role for the gap junction, Connexin 43 (Cx43), at the immunological synapse. Cx43’s N-terminus (NT) has transmembrane domains that form hexamers, which can then interact with an adjacent cell to form a gap junction allowing ions such as Ca2+ and small molecules to pass from cell to cell. The cytoplasmic Cx43’s C-terminus (CT) can be phosphorylated and thus function as an adaptor or scaffolding complex. Cx43 is knocked out (KO) in mice expressing CRE during T cell development, which excises any “floxed”. Previous data from T cells isolated from Cx43 KO mice have reduced proliferation to TRC stimulus and downstream signaling deficits supporting a hypothesis that Cx43’s CT scaffolding role at the immunological synapse may predominate over the NT gap junction. This can be tested by replacing the CX43’s NT with rCD2’s transmembrane domain, allowing us to isolate the function of Cx43’s CT. Using CRISPR gene editing technology to specifically excise and replace the WT Cx43 with the rCD2-Cx43-CT construct can be done in immune precursor cells isolated from the bone marrow. These chimeric bone marrow cell lines can be perpetuated indefinitely by injecting them into lymphopenic mice and then harvesting the bone marrow. The calcineurin-NFAT pathway can be tested by NFAT western blot and by flow cytometry analysis of Indo-I fluorescence, which provides a ratiometric read out of calcium flux after TCR stimulation. In addition we can use the autoimmune mouse model to see if these cells regain their ability to activate. This is a novel project that fully utilizes gene editing technology and mouse models of disease. It has important application in CART therapy that suffers from T cell exhaustion.