Abstract:
Durable and effective immunity requires the generation of persistent and functional memory CD8+ T cells. The integration of a multitude of signals during naïve cell activation plays an important role in the programming of CD8+ T cells responses. However, knowledge of the regulation of naïve cell programming in humans is limited. Therefore, we aimed to expose naïve human CD8+ T cells to cytokines during their activation and measure their effects on the resultant memory populations. We first established a unique culture system for the stimulation and expansion of human naïve CD8+ T cells that allowed us to generate rested memory cells in vitro, supported by IL-7. Utilizing this system, we found that three-day cytokine exposure during priming distinctively programmed the cytokine secretion profiles and cellular phenotypes of the memory populations 21 days later. Priming in the presence of IL-12 was observed to programme enhanced effector cytokine production, and a more differentiated cell surface phenotype; while IL-21 exposure was found to programme reduced functional capacity, but a less differentiated phenotype. We then combined IL-12 and IL-21 exposure during priming, and found memory populations with an early differentiation status could be generated with superior effector function, establishing an expansion protocol with potential implications for adoptive immunotherapy. With this priming regime, a short duration of IL-2 supplementation was found to augment the expansion of naïve CD8+ T cells without significantly altering cellular attributes. Transcription factor expression was also examined in these experiments and novel changes were observed upon cytokine exposure, TCR stimulation and CD8+ T cell differentiation. Priming naïve cells with IL-4 strongly upregulated GATA-3 expression following secondary stimulation, confirming that the programming we observed in this model was acting via transcription factors. Transcription factor expression profile of the in vitro-generated memory cells also confirmed an early differentiation status with Tcf-1, Lef-1 and FoxO1 expression, and an absence of T-bet and Eomes. Collectively, we have established a culture system that enables the exploration of programming events during the priming of naïve human CD8+ T cells that can be further utilized to investigate human CD8+ T cell differentiation and optimal culture regimes for ACT.