*Purpose: Human CD4⁺CD8^loT cells have been reported in patients with organ allograft rejection. We evaluated the incidence, phenotype, and function of CD4⁺CD8^loT cells in nonhuman primates (NHP).

*Methods: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of naïve rhesus monkeys. The percentages of CD4⁺CD8^loT cells were evaluated. In comparison to CD4⁺CD8^negT cells, memory T cell (Tmem) subsets were evaluated based on their expression of CD45RA, CCR7, CD28, and CD95. Effector function was assessed by the production of interferon-gamma (IFNg) and tumor necrosis factor-alpha (TNFa). Cytokine production was also assessed after stimulation with allogeneic PBMC for 5 days. The mean fluorescence intensity (MFI) of T-box transcription factors T-bet and Eomesodermin (Eomes) was also assessed.

*Results: In naïve rhesus monkeys, the percentage of CD4⁺CD8^loT cells in peripheral blood was 1.54±0.32%. In CD4⁺CD8^loT cells, CD45RA^–CCR7^– effector Tmem (Tem) were 17.9±2.0%, compared to 11.3±1.1% in CD4⁺CD8^negT cells (p<0.01). CD45RA⁺CCR7^– terminally-differentiaed effector Tmem (Temra) were 25.3±4.2%, compared to 5.8±0.8% in CD4⁺CD8^neg T cells (p<0.01). CD95⁺CD28^– Tem were 33.4±5.5%, compared to 4.0±0.8% in CD4⁺CD8^negT cells (p<0.01).The percentage of IFNg⁺TNFa⁺ T cells in CD4⁺CD8^loT cells was 28.6±3.1%, compared to 8.2±1.8% in CD4⁺CD8^neg T cells (p<0.01) Notably, following allogeneic stimulation, IFNg⁺TNFa⁺ T cells were significantly higher in CD4⁺CD8^loT cells (22.7±3.3%) compared to CD4⁺CD8^negT cells (8.7±0.8%) (p<0.01). Additionally, CD4⁺CD8^loT cells exhibited significantly higher Eomes and T-bet expression (MFI= 95.6±3.7 and 588.7±87, respectively), compared to CD4⁺CD8^negT cells (MFI= 66.1±3.2, and 232.3±3.5, respectively) (p<0.05).

*Conclusions: We show for the first time that under steady-state conditions, CD4⁺CD8^loT cells exhibit a higher memory profile and effector function in comparison to CD4⁺CD8^negT cells. These findings justify the evaluation of the role of CD4⁺CD8^loT cells in organ allograft rejection in NHP and their response to therapeutic intervention.

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