Dendritic cell-natural killer cell cross-talk modulates T cell activation in response to influenza A viral infection

Abstract

Influenza viruses lead to substantial morbidity and mortality including ~3-5 million cases of severe illness and ~290,000-650,000 deaths annually. One of the major hurdles regarding influenza vaccine efficacy is generating a durable, robust cellular immune response. Appropriate stimulation of the innate immune system is key to generating cellular immunity. Cross-talk between innate dendritic cells (DC) and natural killer (NK) cells plays a key role in activating virus-specific T cells, yet the mechanisms used by influenza A viruses (IAV) to govern this process remain incompletely understood. Here, we used an ex vivo autologous human primary immune cell culture system to evaluate the impact of DC-NK cell cross-talk and subsequent naïve T cell activation at steady-state and after exposure to genetically distinct IAV strains–A/California/07/2009 (H1N1) and A/Victoria/361/2011 (H3N2). Using flow cytometry, we found that exposure of DCs to IAV in co-culture with NK cells led to a decreased frequency of CD83+ and CD86+ cells on DCs and an increased frequency of HLA-DR+ on both DCs and NK cells. We then assessed the outcome of DC-NK cell cross-talk on T cell activation. At steady-state, DC-NK cell cross-talk increased pan T cell CD69 and CD25 expression while exposure to either IAV strain reduced pan T cell CD25 expression and suppressed CD4+ and CD8+ T cell IFN-γ and TNF production, following chemical stimulation with PMA/Ionomycin. Moreover, exposure to A/Victoria/361/2011 elicited lower IFN-γ production by CD4+ and CD8+ T cells compared with A/California/07/2009. Overall, our results indicate a role for DC-NK cell cross-talk in T cell priming in the context of influenza infection, informing the immunological mechanisms that could be manipulated for the next generation of influenza vaccines or immunotherapeutics.

Influenza A Virus Disruption of Dendritic Cell-Natural Killer Cell Crosstalk Impacts Activation of Helper and Cytotoxic T cell Subsets

“…To this end, T cells were cultured either alone (0:0:1), with MoDCs (1:0:1), with NK cells (0:4:1), or in triple co-culture at a MoDC: NK cell: T cell ratio of 1:4:1. This cell ratio was found to robustly activate pan naïve T cells in a prior study [25]. We found that both helper CD4 + (median = 62.5%) and cytotoxic CD8 + (median = 57.2%) T cell subsets exhibited significantly lower frequencies of the CD69 + population when co-cultured with MoDCs and NK cells, compared to only MoDCs, only NK cells, or solely T cells (Figure 1A-B ).…”

“…In our prior study, we found that DC-NK cell crosstalk triggered an increase in CD69 + expression on pan-naïve T cells [25]. The appearance of CD69 on the plasma membrane is a classic early marker of lymphocyte activation and has multifaceted roles including regulation of cytokine release and homing and migration of lymphocytes [28].…”

“…On day 2 and day 5 post-monocyte isolation, half of the medium volume was supplemented with fresh medium containing cytokines. MoDC differentation was confirmed by analytical flow cytometry measuring the downregulation of a monocyte surface marker (CD14) and upregulation of a DC surface marker (CD209) [25].…”

“…MoDC differentation was confirmed by analytical flow cytometry measuring the downregulation of a monocyte surface marker (CD14) and upregulation of a DC surface marker (CD209) [25].…”

“…Further, we aimed to test the hypothesis that active viral protein synthesis is required to curtail T cell activation by using replication-defective virus. These viral strains were selected based on our studies that demonstrated that NK cells secrete higher levels of IFN-γ response to Cal/09 compared to Vic/11, and this translates into heightened T cell IFN-γ production after DC-NK cell crosstalk [24,25]. We found that the DC-NK cell crosstalk heightened the expression of early (CD69) and late (CD25) markers of T cell activation while triggering the proliferation of both subsets.…”