Investigation of calcium signatures in human CD4+ T cells with a focus on modulation of differentiation by plasma membrane ATPases

Abstract

The immune system is optimized to provide protection against pathogens while maintaining immune tolerance to self-antigens. During the establishment of an immune response, components of the innate system are essential for the early constraint of the infection and for orchestrating activation, expansion and differentiation of cells of the adaptive immune system. T cells of the adaptive immune system represent a stronger mean of defense with increased protection during recall responses. In this regard, the establishment of the adaptive immune response relies on the activation and programing of naïve T cells into effector cells after proper cognate antigen recognition, which allows the primed T cells to proliferate and differentiate into cells with a variety of functions needed to fight against the pathogen. Following pathogen clearance, the majority of effector cells die, leaving behind different memory T cell subsets. These memory cells are different in their functions and migratory properties, thereby mediating rapid and specific immune responses upon re-infection. One of the most important mediators of T cell activation is the second messenger Calcium ion (Ca2+), which control complex and essential effector functions such as proliferation, differentiation, metabolism, cytokine secretion and cytotoxicity. In this regard, inadequate Ca2+ regulation can lead to various autoimmune, inflammatory and immunodeficiency syndromes. In the first part of this work, we studied the Ca2+ profiles of human T helper subtypes and proposed different mechanisms that may contribute to their Ca2+ differences in phenotypes following store operated Ca2+ entry (SOCE). We also investigated the Ca2+ profiles of regulatory T cells and found out that in vivo regulatory T cells, as the in vitro regulatory T cells, showed the highest Ca2+ SOCE peak. Our results indicate that a lower expression of Orai2 in regulatory T cells than in conventional T cells may contribute to the higher Ca2+ peak. Interestingly, memory T cells polarized into Th17 cells showed the highest expression of ATP2B4 among the CD4+ T helper subtypes, which lead us to investigate the role of PMCAs in fate decisions within the CD4+ T cell compartments. In the second part of this work, we show how plasma membrane Ca2+ ATPase isoform 4 (PMCA4) regulates the levels of intracellular Ca2+ within the CD4+ T cell compartment. Modulation of PMCA either by allosteric inhibition of PMCA4 pump activity through pharmacological inhibition or siRNA-mediated downregulation increased SOCE in memory but not in naïve CD4+ T cells. Importantly, downregulation of PMCA4 in naïve CD4+ T cells, followed by polyclonal stimulation, decreased the percentage of primed cells expressing effector markers upon activation indicating a role of PMCA during the effector phase of the T cell response. Furthermore, we showed for the first time that PMCA4 is differentially expressed in human CD4+ T cell compartments which resulted in distinct store operated Ca2+ entry profiles. Along this line, PMCA4 was increasingly up-regulated in the order naïve, central memory (CM) T cells, effector memory (EM) and effector memory RA positive (EMRA+) T cells, which corroborated its role in memory fate decisions during T cell responses. On the other hand, analysis of calcium signals after disturbing mitochondrial function in naïve and memory T cells in both resting and under activation with a polyclonal stimulus, showed that memory cells rely more on PMCA4 for controlling the amplitude and dynamic of SOCE, while naïve T cells make preferential use of mitochondrial Ca2+ uptake to shape SOCE.