Cytokine-induced modulation of T lymphocyte responses to antigens

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Background: Activation of ‘naïve’ (antigen-inexperienced) CD8 T cells requires two signals. Signal 1 is delivered via the T cell antigen (Ag) receptor (TCR), and signal 2 is delivered via co-stimulatory receptors. Contrary to this two signal paradigm, we have shown that certain inflammatory cytokines produced during innate immune response (IL-6, IL-21) can synergize with homeostatic cytokines (IL-7, IL-15) to induce Ag non-specific proliferation of naïve CD8 T cells (Cell. Signal. 2007,19:806). We have also shown that a brief exposure of naïve CD8 T cells to IL-21 or IL-6 in the presence of IL-7 or IL-15 markedly decreases the TCR signaling threshold required for activation (J. Immunol. 2008,180: 7958). We refer to this cytokine-mediated increase in TCR responsiveness as ‘cytokine priming’, and have proposed that this pathway may bridge innate and adaptive immune responses (Crit. Rev. Immunol. 2009,29: 219). Progress: During the current grant period, we have shown that cytokine priming reduces the expression of CD5, a negative regulator of TCR signaling (Immunol. Cell. Biol. 2010,88: 451). We have also shown that cytokine-primed cells acquire the capacity to respond to weak TCR ligands, and that cytokine priming could occur in vivo using mouse models of autoimmunity (J. Immunol. 2010, 185:357; ibid. 2011,186:5131). Our recent findings indicate that (i) cytokine priming increases the lipid raft content and CD45, two key components of the TCR signaling machinery, (ii) cytokine priming induces a memory cell phenotype and attenuates Ag-induced exhaustion, (iii) cytokine-primed cells display show elevated spare mitochondrial respiratory capacity. Hypothesis: We hypothesize that the functional consequences of cytokine priming arise from spatial and temporal changes in TCR signaling along with modulation of differentiation pathways and cellular energy metabolism, and that specific changes in gene/protein expression underlie these alterations. Objectives: The long-term objective of this research program is to understand the mechanisms and the significance of cytokine priming in physiological and pathological immune responses. Our short-term goals for the next 5 years are: 1. Characterize the TCR signaling dynamics in cytokine-primed naive CD8 T cells. 2. Elucidate how cytokine priming modulates their energy metabolism. 3. Define the changes in gene/protein expression induced by the priming cytokines. 4. Determine the role of priming cytokines in CD8 T cell responses to weak TCR ligands in vivo. Methodology: As experimental system, we will use TCR transgenic mice to obtain a uniform population of naïve CD8 T cells. 1) We will use confocal microscopy to visualize the spatial and temporal distribution of lipid rafts, CD45 and key TCR signaling events at the immune synapse formed between T cells and Ag-loaded dendritic cells. 2) We will use the Seahorse® cell metabolism analyzer to measure oxygen consumption rate (a measure of ATP synthesis via mitochondrial electron transport chain) and extracellular acidification rate (a measure of glycolysis that generates building blocks needed for cell proliferation). 3) We will carry out gene expression analysis using microarrays, and proteomic analysis by mass spectrometry. 4) We will use a virus expressing either the cognate Ag recognized by the transgenic TCR, or a weak agonist, to infect wild type or specific cytokine deficient mice, and evaluate activation and functional differentiation of Ag-specific CD8 T cells in vivo. Significance: This research program will provide insight into the mechanisms by which inflammatory cytokines ‘prime’ naïve CD8 T cells for the ensuing encounter with Ags of invading pathogens.