*Purpose: The T-box transcription factors Eomesodermin (Eomes) and T-bet play a critical role in differentiation and function of CD8+ memory T cells (Tmem). We have previously shown, that in humans, high Eomes expression by allo-reactive CD8+T cells correlates with enhanced effector function. Our purpose was to evaluate the phenotypic and functional differences of alloreactive Eomes^hi CD8+ T cells in human and nonhuman primates.

*Methods: Healthy human and juvenile rhesus monkey T cells were purified followed by co-culture with allogeneic T-cell depleted peripheral blood mononuclear cells (PBMC). CD8+Tmem subsets were identified based on CD45RA/CD28 expression. Eomes expression by nonactivated and allo-activated CD8+ T cells was evaluated. The expression of T-bet, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), Programmed cell death-1 (PD-1) and effector cytokines (TNFα and IFNγ) were assessed.

*Results: Before allo-stimulation, human and monkey Eomes^hi CD8+T cells comprised high percentage of CD45RA-CD28+ central memory T cells, while Eomes^lo CD8+T cells comprised higher percentage of CD45RA+CD28+ naïve T cells. Additionally, human but not monkey Eomes^hi CD8+T cells exhibited higher percentages of TNFα/IFNγ double positive cells, than Eomes^lo CD8+T cells.T-bet expression was higher in human, but lower in monkey Eomes^hi, compared to Eomes^lo CD8+T cells. After allo-stimulation, the percentages of Tmem subsets was variable in both humans and monkeys CD8+ T cells. While both human and monkey Eomes^hi CD8+T-cells exhibited significantly higher TNFα/IFNγ double positive cells, than Eomes^hi CD8 T cells, only human but not monkey Eomes^hi CD8+T cells express significantly higher T-bet than Eomes^lo CD8+ T cells. In proliferating cells, high percentages of TNFα/INFγ double positive cells were observed in human Eomes^hi Tbet^hi and monkey Eomes^hiTbet^lo CD8+T cell subsets.

CTLA4 expression was significantly upregulated by CD8+T cells after allo-stimulation in both human and monkeys, where in monkeys, but not humans, an inverse correlation between Eomes and CTLA4 expression was observed by allo-stimulated CD8+ T cells. While co-expression of PD-1 and CTLA4 was comparable between Eomes^hi and Eomes^lo CD8+T cells in humans, the co-expression of PD-1 and CTLA4 was significantly lower in Eomes^hi compared to Eomes^lo monkey CD8+T cells

*Conclusions: In both humans and monkeys, higher Eomes expression correlates with effector T cell phenotype after allo-stimulation. However, concomitant expression of T-bet and the exhaustion/activation markers CTLA4 and PD-1 was different. The role of Eomes in the development and maintenance of allo-reactive Tmem may not be identical in human and nonhuman primates.

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