P182 Role of IRF8 as a molecular integrator that orchestrates CD8 T effector differentiation

Abstract

Compared to CD4 T cells, the differentiation pathway for CD8 T cells is still underexplored. We found that the CD8 activation/differentiation is accomplished by two ways, by high dose antigen-MHC/co-stimulation or by low-dose antigen-MHC/co-stimulation plus simultaneous gc-cytokine signaling. We asked if these two modes of activation are qualitatively different. Naïve OT-I CD8T cells were stimulated by an OVA257–264-peptide in the context of Class I MHC (on ex vivo maturated dendritic cells) in the presence or absence of exogenously added recombinant IL-15 (5 nM). Another set of cells were also stimulated by IL-15 alone. RNA extraction from these cells was followed by cDNA synthesis/labeling and hybridization to Affymetrix cDNA array was performed. Results from triplicate independent array analyses were statistically evaluated to identify induced genes that are specific for each type of stimulation. To validate the array results, qPCR was performed on the expression of selected genes. To further assess the relevance of the chosen one, IRF8, CD8 T cells were isolated from OT-ITg-IRF8 knockout mice and their effector differentiation was determined by intracellular cytokine staining (IFNg, TNFa), by qPCR (granzyme/perforin) and by cytotoxicity assay. Gene induction pattern was compared at 6 h and 12 h following stimulation. At 6hr, 3 modes of stimulation (cytokine alone, antigen alone, combination of both) showed rather overlapping gene expression pattern. However the pattern showed dramatic change as early as 12 h point. Antigen-stimulated gene pool has shrunk to <500 genes and that of IL-15 < 1200. In contrast the combinatorial stimulation induced >1600 genes and 769 of them were only seen with the combination of two stimulations. Thus the high-dose antigen/MHC/costimulation and low-dose antigen/MHC/co-stimulation + cytokine are qualitatively different stimuli. The list of unique genes that were induced upon the combinatorial stimulation contains a lot of known genes that are involved in the CD8 T cell activation, including IL-2, CD25, and IL-12Rbeta. We then focused our attention on transcription factors (TFs) in this gene pool because we were interested in delineating the transcription network during CD8 T cell differentiation. We identified c-jun and IRF8. We then chose IRF8 for subsequent studies because the role of IRF8 in CD8 T cell activation has been uncharacterized. So we tested if CD8 T cell differentiation is compromised in the absence of IRF8. Expression of the two known T-box TFs, T-bet and eomesdermin, was not affected in the absence or presence of IRF8, excluding the possibility that IRF8 operates upstream of the T-box TFs. In contrast, the depletion of IRF8 in OT-I T cells (generated by crossing OT-I Tg mice and IRF8 knockout mice) has functionally crippled the CD8 T cells because they failed to produce IRFg or exert cytotoxic activity againt OVA-expressing target cells upon stimulation. qPCR also validated that the induction of IRF8 mRNA requires the cooperation of gc-cytokine signal and antigen-MHC/co-stimulation stimulation. These results define a new role for IRF8 as an integrator of two distinct activation signal pathways (TCR and gc-cytokine), and uncover a potential novel intracellular pathways originated from IRF8 in the differentiation of CD8 T cells responding to suboptimal dose of antigen.