The earliest intracellular signals determined in T cell activation are local, sub-second Ca2+ microdomains (1). Here we identify a Ca2+ entry component involved in Ca2+ microdomain formation in both non-stimulated and stimulated cells. In non-stimulated cells, spontaneous small Ca2+ microdomains depend on expression of ORAI1, STIM1, and STIM2. Using T cells stably transfected with ORAI1 fused to a genetically encoded Ca2+ indicator for optical imaging spontaneous Ca2+ microdomains depending on ORAI1 were also detected. Super resolution microscopy of non-stimulated T cells resulted in identification of a circular subplasmalemmal region with a diameter of approx. 300 nm with preformed patches of co-localized ORAI1, ryanodine receptors (RYR), and STIM1. Preformed complexes of STIM1 and ORAI1 in non-stimulated cells were confirmed by co-immunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second of T cell receptor (TCR) stimulation, Ca2+ microdomain numbers increase in the subplasmalemmal space, an effect not observed upon genetic deletion of Orai1, Stim2 or Ryr1 or upon antagonism of the Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Taken together, while preformed clusters of STIM and ORAI1 allow for local Ca2+ entry events in non-stimulated cells, upon TCR activation, NAADP-evoked Ca2+ release via RYR1, in tight interplay with Ca2+ entry via ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

Overwhelming activation of T cells in acute malaria is associated with severe outcomes. Thus, counter-regulation by anti-inflammatory mechanisms is indispensable for an optimal resolution of disease. Using Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice, we performed a comprehensive analysis of co-inhibitory molecules expressed on CD4 + and CD8 + T cells using an unbiased cluster analysis approach. We identified similar T cell clusters co-expressing several co-inhibitory molecules like programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) in the CD4 + and the CD8 + T cell compartment. Interestingly, despite expressing co-inhibitory molecules, which are associated with T cell exhaustion in chronic settings, these T cells were more functional compared to activated T cells that were negative for co-inhibitory molecules. However, T cells expressing high levels of PD-1 and LAG-3 also conferred suppressive capacity and thus resembled type I regulatory T cells. To our knowledge, this is the first description of malaria-induced CD8 + T cells with suppressive capacity. Importantly, we found an induction of T cells with a similar co-inhibitory rich phenotype in Plasmodium falciparum-infected patients. In conclusion, we demonstrate that malaria-induced T cells expressing co-inhibitory molecules are not exhausted, but acquire additional suppressive capacity, which might represent an immune regulatory pathway to prevent further activation of T cells during acute malaria.

Overwhelming activation of T cells in acute malaria is associated with severe outcomes. Thus, counter-regulation by anti-inflammatory mechanisms is indispensable for an optimal resolution of disease. Using Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice, we performed a comprehensive analysis of co-inhibitory molecules expressed on CD4+ and CD8+ T cells using an unbiased cluster analysis approach. We identified similar T cell clusters co-expressing several co-inhibitory molecules like programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) in the CD4+ and the CD8+ T cell compartment. Interestingly, despite expressing co-inhibitory molecules, which are associated with T cell exhaustion in chronic settings, these T cells were more functional compared to activated T cells that were negative for co-inhibitory molecules. However, T cells expressing high levels of PD-1 and LAG-3 also conferred suppressive capacity and thus resembled type I regulatory T cells. To our knowledge, this is the first description of malaria-induced CD8+ T cells with suppressive capacity. In conclusion, we demonstrate that malaria-induced T cells expressing co-inhibitory molecules are not exhausted but acquire additional suppressive capacity, which might represent an immune regulatory pathway to prevent further activation of T cells during acute malaria.