Engagement of NKT cells during cross-priming enhances the CD8⁺ T cell response via GATA3 and IL-4/TNFα

Abstract

CD8⁺ T cells play an important role in the defense against intracellular pathogens and malignantly transformed cells, making them a promising target for vaccination and therapy approaches. Naïve CD8⁺ T cells are not activated directly by their target cells but by antigen-presenting immune cells (APCs) such as dendritic cells (DCs) that cross-present their cognate antigen and in turn need to interact with further immune cells. To improve CD8⁺ T cell activation during vaccination, different adjuvants are used. The glycolipid alpha-galactosylceramide (αGC) has been found to be especially effective. In contrast to classical adjuvants such as CpG, αGC engages Natural Killer T (NKT) cells instead of CD4⁺ T cells in the activation of the CD8⁺ T cells. As the differentiation of CD8⁺ T cells is influenced by factors such as TCR signaling strength, costimulation and cytokines, the involvement of different immune cells during αGC-mediated alternative and CpG-mediated classical cross-priming suggests differences in the differentiation of the activated CD8⁺ T cells. Here, alternatively cross-primed CD8⁺ T cells were characterized in depth by comparing them directly to classically cross-primed cells. <br> Effector CD8⁺ T cells activated under the influence of NKT cells were found to be more abundant, produced higher amounts of effector molecules and presented a greater cytotoxicity on a per cell basis than classically cross-primed cells. Early after activation, alternatively cross-primed CD8⁺ T cells exhibited increased metabolic activity and were characterized by the high expression of both Tbet and GATA3. Knockdown experiments confirmed the importance of the high expression of GATA3 early after activation for the effector phenotype of alternatively cross-primed CD8⁺ T cells. The cytokines IL-4 and TNFα were determined as factors involved in the differentiation of these CD8⁺ T cells. IL-4 was required for the high expression of GATA3. It was found to increase the cytotoxicity of CD8⁺ T cells when being present during priming and to improve their ability to cope with glucose deprivation. TNFα appeared to be required for the Tbet^high GATA3^high phenotype of alternatively cross-primed CD8⁺ T cells by preventing the IL-4-mediated downregulation of Tbet. It was also found to be involved in the regulation of CD25 expression and to overall increase the effects of IL-4. Both cytokines, especially in combination, were involved in the metabolic phenotype of alternatively cross-primed CD8⁺ T cells. The identified cytokine combination was applied to prophylactic and therapeutic vaccination approaches, and showed promising results for the improvement CD8⁺ T cell-based treatments.