Type I IFNs and IL-18 Regulate the Antiviral Response of Primary Human γδ T Cells against Dendritic Cells Infected with Dengue Virus

Primary γδ T cells in PBMC respond to autologous DC infected with DV. DC were infected with DV at MOI = 1 and, at 24 h postinfection, were cocultured in vitro for an additional 4 h with autologous PBMC. The response of CD3⁺ γδ TCR⁺ cells was analyzed by flow cytometry. The gating strategy used to define γδ T cells is shown in Supplemental Fig. 1A. DC exposed to heat-inactivated DV (i.e., mock-infected DC) or DC that had not been exposed to any DV (i.e., uninfected DC) served as experimental controls. The response of γδ T cells in PBMC not cocultured with DC (PBMC only) is also shown. (A) The frequency of IFN-γ⁺ cells among CD3⁺ γδ TCR⁺ cells. Contour plots depict representative IFN-γ responses of γδ T cells in PBMC cocultured with DV-infected DC or with mock-infected DC. (B) Frequency of CD69⁺ γδ T cells and representative CD69 expression on γδ T cells in PBMC cocultured with DV-infected DC or mock-infected DC. (C) Summary of CD107a mean fluorescence intensity (MFI) values and representative expression of this marker on γδ T cells in PBMC cocultured with DV-infected DC or mock-infected DC. Data in (A)–(C) are the mean of six independent experiments, each using PBMC from different donors. Error bars denote the SD. *p < 0.05, ****p < 0.0001, one-way ANOVA with Holm–Sidak post hoc test. (D) The Vδ2⁺ subset represents the majority of IFN-γ–producing γδ T cells in PBMC that respond to DV-infected DC. PBMC were cocultured with autologous DV-infected DC, as described for (A)–(C), using individuals with a balanced distribution of Vδ2⁺ and non-Vδ2⁺ γδ T cells in their blood. The FACS gating strategy used and the distribution of Vδ2⁺ and non-Vδ2⁺ γδ T cells are depicted. Results from four independent experiments using PBMC from different donors are depicted. p = 0.72, paired t test. (E) The frequency of IFN-γ⁺ cells among CD3⁺ γδ TCR⁺ Vδ2⁺ cells in PBMC. Contour plots depict representative responses to DV-infected DC or mock-infected DC. (F) The frequency of IFN-γ⁺ cells among CD3⁺ γδ TCR⁺ Vδ2⁻ cells in PBMC. Contour plots depict representative responses to DV-infected DC or mock-infected DC. (G) Relative proportion of Vδ2⁺ versus non-Vδ2⁺ cells among CD3⁺ γδ TCR⁺ cells in PBMC that produced IFN-γ when cocultured with DV-infected DC. The FACS gating strategy and contour plot showing representative response are depicted in Supplemental Fig. 1B. Data in (E)–(G) are the mean of four independent experiments using PBMC from different donors. The error bars denote SD. *p < 0.05, **p < 0.01 paired t test. ns, not significant.

The IFN-γ response of γδ T cells to DV-infected DC is dependent on type I IFN, IL-18, and the ATP-P2X₇ receptor pathway. DC that were infected with DV (as described in Fig. 2) were pretreated for 30 min with the indicated cytokine inhibitors, blocking Abs, or isotype-control Abs and then cocultured with autologous PBMC for an additional 4 h. For blocking the P2X₇ receptor, DC were pretreated with 100 μM of 15D for 30 min prior to coculture with PBMC, and the final concentration of 15D in coculture was maintained at 100 μM. The IFN-γ response of CD3⁺ γδ TCR⁺ cells was analyzed by flow cytometry using the gating strategy depicted in Supplemental Fig. 1B, unless otherwise stated. (A) Blockade of type I IFN with recombinant B18R protein derived from vaccinia virus or Ab-mediated neutralization of IL-18 or TNF-α. (B) Expression of IL-18Rα and IFN-γ on γδ T cells in PBMC cocultured with DV-infected DC. (C) Antagonizing the P2X₇ receptor with 15D. The IFN-γ response of γδ T cells was analyzed by flow cytometry using the gating strategy depicted in Supplemental Fig. 4A. (D) Blockade of IL-1 with rIL-1R antagonist. (E) Ab-mediated blockade of IL-7. (F) Ab-mediated blockade of IL-12 or IL-15. (G) Inhibition of the mevalonate pathway of IPP synthesis with mevastatin. Data in (A), (B), and (D)–(G) are the mean value obtained from three independent experiments using blood from different donors, whereas data in (C) are from four independent experiments using blood from different donors. Error bars denote the SD. **p < 0.01, ****p < 0.0001 one-way ANOVA with Holm–Sidak post hoc test. ns, not significant.

The IFN-γ response of purified γδ T cells to DV-infected DC is diminished compared with the response of γδ T cells within unfractionated PBMC, which is associated with diminished IL-18 levels in the former cultures; conversely, exogenous IL-18 augments the anti-DV responses of purified γδ T cells. (A) DC infected with DV, as described in Fig. 2, were cocultured with purified autologous primary γδ T cells for 4 h. The IFN-γ response of γδ T cells was analyzed by flow cytometry. Mock-infected DC or uninfected DC served as experimental controls. Data shown are the mean obtained from five independent experiments, each using cells from different donors. Error bars represent the SD. (B) Less IL-18 is present in the supernatant of cocultures containing DV-infected DC and purified γδ T cells compared with cocultures of DV-infected DC with PBMC. DV-infected DC were cocultured with purified autologous γδ T cells or unfractionated PBMC. Culture supernatant was harvested after 24 h for the measurement of IL-18 by ELISA. Cocultures containing mock-infected DC served as experimental controls. (C) Exogenous IL-18 is sufficient to increase the IFN-γ response of purified γδ T cells to DV-infected DC. DV-infected DC were cocultured with purified autologous γδ T cells for 4 h in the presence of either rIL-18 (100 ng/ml) or rIFN-α (1,000 or 10,000 U/ml), added at the time of coculture. Cocultures containing mock-infected DC served as experimental controls. (D) γδ T cells primed overnight with IL-18 showed increased ability to kill DV-infected DC. DV-infected DC were loaded with BATDA reagent and cocultured for 2.5 h with either purified autologous γδ T cells that had been primed for 16 h with IL-18 (100 ng/ml) or γδ T cells that had not been exposed to exogenous IL-18, both at an E:T ratio of 10. The percentage of specific lysis was determined as described in Materials and Methods. Mock-infected DC cocultured with γδ T cells served as experimental controls. Data in (B)–(D) are the mean obtained from four independent experiments, each using cells from different donors. Error bars represent the SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 one-way ANOVA with Holm–Sidak post hoc test. ns, not significant.

Depletion of monocytes from PBMC diminished the IFN-γ response of γδ T cells to DV-infected DC and was associated with reduced IL-18 levels in culture supernatant. DC infected with DV (as described in Fig. 2) were cocultured with autologous PBMC or PBL for 4 h. Mock-infected DC cocultured with PBMC served as experimental controls. (A) The IFN-γ response of γδ T cells was analyzed by flow cytometry using the gating strategy depicted in Supplemental Fig. 1B. Data are mean values obtained from four independent experiments, each using cells from different donors, Error bars represent the SD. (B) DV-infected DC were cocultured with autologous PBMC or PBL. After 24 h, culture supernatant was harvested for the measurement of IL-18 by ELISA. Mock-infected DC cocultured with PBMC served as controls. The mean result from 13 experiments is depicted, each using cells from different donors. Error bars represent the SD. *p < 0.05, **p < 0.01 one-way ANOVA with Holm–Sidak post hoc test.