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.

Keywords: H1N1; H3N2; T cells; cross-talk; dendritic cells; influenza; natural killer cells; pandemic.

Figure 1

Lineage gating schematic for MoDCs and NK cells. Diagram of lineage gating tree used to identify MoDCs and NK cells by flow cytometry. MoDCs and NK cells were separated into gates by size and granularity using forward and side scatter. MoDCs were further identified as CD7^- while NK cells were identified as CD7⁺CD56⁺. Cal/09 and Vic/11 infection of MoDCs at an MOI of 3 at 24 HPI is shown using an antibody specific to IAV nucleoprotein (FluA-NP). AViD, LIVE/DEAD Fixable Violet Dead Cell Stain; FSC, forward scatter; SSC, side scatter.

Figure 2

IAV exposure leads to the downregulation of CD83 and CD86 and upregulation of HLA-DR on MoDCs in co-culture with NK cells. (A) Summary plot of the frequency of CD83⁺ Cal/09- or Vic/11-exposed MoDCs (MOI = 3) cultured alone (1:0, 4:0) or after a 7 h (n = 4) or 23 h (n = 6) co-culture with NK cells at indicated cell ratio as assessed by flow cytometry using an antibody specific to CD83. (B) Representative flow plot of CD83⁺ frequency and median fluorescence intensity (MdFI) on MoDCs at a MoDC to NK cell ratio of 4:1 (top panel) or 1:4 (bottom panel) at 24 HPI (MOI = 3). (C) Summary plot of the frequency of CD86⁺ Cal/09- or Vic/11-exposed MoDCs (MOI = 3) cultured alone (1:0, 4:0) or after a 7 h (n = 5) or 23 h (n = 7) co-culture with NK cells at indicated cell ratio as assessed by flow cytometry using an antibody specific to CD86 (D) Representative flow plot of CD86⁺ frequency and MdFI on MoDCs at a MoDC to NK cell ratio of 4:1 (top panel) or 1:4 (bottom panel) at 24 HPI (MOI = 3). (E) Summary plot of the frequency of HLA-DR⁺ Cal/09- or Vic/11-exposed MoDCs (MOI = 3) cultured alone (1:0, 4:0) or after a 7 h (n = 5) or 23 h (n = 7) co-culture with NK cells at indicated cell ratio as assessed by flow cytometry using an antibody specific to HLA-DR. (F) Representative flow plot of HLA-DR⁺ frequency and MdFI on MoDCs at a MoDC to NK cell ratio of 4:1 (top panel) or 1:4 (bottom panel) at 24 HPI (MOI = 3). Poly (I:C) treatment served as a positive control. The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, Wilcoxon signed-rank test.

Figure 3

NK cells express HLA-DR after co-culture with IAV-exposed MoDCs. (A) Summary plot of the frequency of HLA-DR⁺ NK cells after culture alone or after 6 h (n = 7) or 23 h (n = 6-9) co-culture with mock-treated or Cal/09- or Vic/11-exposed MoDCs at an MOI of 3 as assessed by flow cytometry using an antibody specific to HLA-DR. In the NK cell-only conditions (0:1, 0:4), influenza virions were added concurrently with the NK cells. (B) Representative flow plot of the percentage of HLA-DR⁺ NK cells and MdFI of HLA-DR on NK cells after 23 h co-culture with Cal/09- or Vic/11-exposed MoDCs (MOI = 3) at a MoDC to NK cell ratio of 4:1 (top panel) or 1:4 (bottom panel). PMA/I treatment for 6 h served as a positive control. The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, Wilcoxon signed-rank test.

Figure 4

Direct contact is required for HLA-DR expression on NK cells after co-culture with IAV-exposed MoDCs. (A) Percentage of HLA-DR⁺ NK cells and HLA-DR MdFI after 23 h co-culture with mock-treated or IAV-exposed MoDCs at an MOI of 3 as assessed by flow cytometry using an antibody specific to HLA-DR. MoDCs were cultured in direct contact with NK cells (contact), with MoDCs seeded in the dish and NK cells seeded in the transwell with fresh medium (Trans-well) or with conditioned medium (Trans-well (conditioned medium) (n = 3). (B) Schematic of experimental set-up shown in (A). (C) Representative flow histograms of the percentage of the maximum count of HLA-DR on NK cells after exposure to supernatant from mock-treated (top), Cal/09- (middle), or Vic/11- (bottom) exposed MoDCs cultured alone (left panel) or with NK cells (right panel) at a MoDC to NK cell ratio of 4:1 (n = 3). (D) Relative HLA-DR mRNA levels in NK cells re-purified after 23 h co-culture with mock-treated or IAV-exposed MoDCs when compared to NK cell control with RT-qPCR (n = 3). The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, ns p > 0.05. Two-way ANOVA, Prism V9.0.0.

Figure 5

MoDC-NK cell cross-talk increases the frequency of an early marker of T cell activation. Summary plots of the frequency of CD69⁺ T cells after culture with mock-treated or Cal/09 or Vic/11-exposed MoDCs (MOI = 3) after 95 h co-culture with MoDCs and NK cells (n = 6) at a MoDC to NK cell to T cell ratio of (A) 1:1:1, (C) 4:1:1, and (E) 1:4:1 as assessed by flow cytometry using an antibody specific to CD69. Representative flow plots of the frequency of CD69⁺ T cells expression and MdFI after 47 h co-culture with Cal/09- or Vic/11-exposed MoDCs (MOI = 3) at a MoDC to NK cell to T cell ratio of (B) 1:1:1, (D) 4:1:1, and (F) 1:4:1. PMA/I treatment for 12 h served as a positive control. The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, Wilcoxon signed-rank test.

Figure 6

MoDC-NK cell cross-talk increases the frequency of a late marker of T cell activation. Summary plots of the frequency of CD25⁺ T cells after culture with mock-treated or Cal/09- or Vic/11-exposed MoDCs (MOI = 3) after 95 h co-culture with MoDCs and NK cells (n = 8) at a MoDC to NK cell to T cell ratio of (A) 1:1:1, (C) 4:1:1, and (E) 1:4:1 as assessed by flow cytometry using an antibody specific to CD25. Representative flow plots of the frequency of CD25⁺ T cells and MdFI after 95 h co-culture with Cal/09- or Vic/11-exposed MoDCs (MOI = 3) at a MoDC to NK cell to T cell ratio of (B) 1:1:1, (D) 4:1:1, and (F) 1:4:1. PMA/I treatment for 12 h served as a positive control. The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, Wilcoxon signed-rank test.

Figure 7

MoDC-NK cell cross-talk curtails the production of a broad array of cytokines as a result of influenza exposure. (A) MAGPIX data showing the mean fluorescence intensity (MFI) of (A) IFN-γ, (B) TNF, and (C) IL-10 present in the supernatant of either mock-treated or virion-exposed T cells (0:0:1), T cells co-cultured with mock or virus-exposed MoDCs (MOI = 3, 96 HPI) (1:0:1), T cells co-cultured with mock or virion-exposed NK cells (0:4:1) or T cells co-cultured with both virus-exposed and mock MoDCs (MOI = 3, 96 HPI) and NK cells (1:4:1) (n = 3). The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, ***p < 0.0001, ****p < 0.0001. Two-way ANOVA, Prism V9.0.0.

Figure 8

IAV-exposed MoDCs in co-culture with NK cells suppress the production of IFN-γ and TNF from CD4⁺ and CD8⁺ T cells. Summary plots of CD4⁺ and CD8⁺ T cell IFN-γ⁺ (A) or (C) TNF⁺ expression after culture with mock-treated or Cal/09- or Vic/11-exposed MoDCs (MOI = 3) after 7-day co-culture with MoDCs and NK cells at a MoDC to NK cell to T cell ratio of 0:0:1, 4:0:1, 0:1:1 or 4:1:1 followed by stimulation with PMA/I for 4 h and assessed by intracellular cytokine staining and flow cytometry using an antibody specific to IFN-γ or TNF (n = 7). Representative flow plots of the frequency of IFN-γ⁺ (B) or (D) TNF⁺ CD4⁺ T cells (top) and CD8⁺ T cells (bottom) after 7-day co-culture with Cal/09- or Vic/11-exposed MoDCs (MOI = 3) at a MoDC to NK cell to T cell ratio of 4:1:1 followed by stimulation with PMA/I for 4 h and assessed by flow cytometry using an antibody specific to IFN-γ or TNF. The same shape between conditions indicates that the data point is derived from the same donor. *p < 0.05, Wilcoxon signed-rank test.