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We previously identified a nuclear variant of bone morphogenetic protein 2 (BMP2), named nBMP2, that is translated from an alternative start codon. Decreased nuclear localization of nBMP2 in the nBmp2NLStm mouse model leads to muscular, neurological, and immune phenotypes—all of which are consistent with aberrant intracellular calcium (Ca2+) response. Ca2+ response in these mice, however, has yet to be measured directly. Because a prior study suggested impairment of macrophage function in nBmp2NLStm mutant mice, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from wild type and nBmp2NLStm mutant mice. Immunocytochemistry revealed that nuclei of both BMD and splenic macrophages from wild type mice contain nBMP2, while the protein is decreased in nuclei of nBmp2NLStm mutant macrophages. Live-cell Ca2+ imaging and engulfment assays revealed that Ca2+ response and phagocytosis in response to bacterial supernatant are similar in BMD macrophages isolated from naïve (uninfected) nBmp2NLStm mutant mice and wild type mice, but are deficient in splenic macrophages isolated from mutant mice after secondary systemic infection with Staphylococcus aureus, suggesting progressive impairment as macrophages respond to infection. This direct evidence of impaired Ca2+ handling in nBMP2 mutant macrophages supports the hypothesis that nBMP2 plays a role in Ca2+ response.

The adaptive immune response is orchestrated by T helper cells and their function is dependent upon interactions between the T cell receptor (TCR), peptide MHC (pMHC) and co-receptors. TCR-pMHC interactions initiate calcium signaling cascades which determine T cell activation, survival, proliferation and differentiation. CD5 is a co-receptor that plays an important role in regulating T cell signaling and fate during thymocyte education. CD5 surface expression on mature single positive thymocytes correlates with the TCR signal strength for positive selecting self-ligands. CD5 also plays a role in T cell function after thymic development is complete. Peripheral T cells with higher CD5 expression respond better to foreign antigen than those with lower CD5 expression and CD5-high T cells are enriched in memory populations. In our study, we examined the role of CD5 expression and calcium signaling in the primary response of T cells using two Listeria monocytogenes specific T helper cells (LLO118 and LLO56). These T cells recognize the same immunodominant epitope (LLO190-205) of L. monocytogenes and have divergent primary and secondary responses and different levels of CD5 expression. We found that each T cell has unique calcium mobilization in response to in vitro stimulation with LLO190-205 and that CD5 expression levels in these cells changed over time following stimulation. LLO56 naïve T helper cells, which expresses higher levels of CD5, have higher calcium mobilization than naïve LLO118 T cells. Three days after in vitro stimulation, LLO118 T cells had more robust calcium mobilization than LLO56 and there were no differences in calcium mobilization 8 days after in vitro stimulation. To further evaluate the role of CD5, we measured calcium signaling in CD5 knockout LLO118 and LLO56 T cells at these three time points and found that CD5 plays a significant role in promoting the calcium signaling of naïve CD5-high LLO56 T cells.