T cell-dependent production of IFN-gamma by NK cells in response to influenza A virus

Abstract

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA, IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells, as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA, while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells, which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab, while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together, these results suggest that at an early stage of recurrent viral infection, NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells.

Figure 1

IFN-γ production by T cells and CD56^dim and CD56^bright NK cell subsets in response to fluA. PBMCs from an adult donor were incubated with fluA (A–F) or SPG (negative control, G and H) for 17 hours, with brefeldin A added during the last 5 hours. The cells were stained for CD56, fixed and permeabilized, and then stained for CD3, IFN-γ, and perforin. See Methods for details. Displayed in the dot plots A–H are cells gated on different lymphocyte populations: (A) IFN-γ production of CD3^– and CD3⁺ lymphocytes (gated by forward scattering and side scattering) in response to fluA; (B) IFN-γ production of CD3⁺ lymphocytes in response to fluA; (C) IFN-γ production of CD3^– lymphocytes in response to fluA; (D) expression of CD56 and perforin by CD3^– lymphocytes; (E and F) IFN-γ production of CD56^dim perforin⁺ NK and CD56^bright perforin^– NK subsets, respectively, in response to fluA; (G and H) negative controls showing baseline levels of IFN-γ production by CD3^– and CD3⁺ lymphocytes in the absence of fluA. Numbers in the dot plots refer to percentage of IFN-γ⁺ cells in the gated population.

Figure 2

FluA-induced IFN-γ production by the CD3^–CD56^bright perforin^– NK cell subset (labeled as CD56^bright) and CD3^–CD56^dim perforin⁺ NK cell subset (labeled as CD56^dim) in 25 donors. PBMCs were incubated with fluA for 17 hours, followed by cytokine flow-cytometric analysis (see Methods). Lines connect pairs of observations from the same donor. Black bars mark the positions of groups’ means, which were compared using paired Student’s t tests. The attained significance levels (P values) are reported. For A and C, the t tests were performed on logarithmic-transformed data. (A) The percentage of IFN-γ⁺ cells in the 2 NK cell subsets. (B) The IFN-γ MFI of IFN-γ⁺ cells in the 2 NK cell subsets. (C) The total numbers of IFN-γ⁺ cells detected simultaneously in the 2 NK cell subsets from the same PBMC aliquot of each donor.

Figure 3

Purified NK cells do not produce IFN-γ in response to fluA. NK cells were isolated from PBMCs by negative selection and incubated under different conditions, followed by intracellular staining for IFN-γ. Displayed in the bar graphs are frequencies of IFN-γ⁺ cells in the CD56^bright perforin^– (left panels) and CD56^dim perforin⁺ (right panels) NK cell subsets. (A) PBMCs or purified NK cells from 3 donors (nos. 1–3) were incubated with fluA for 17 hours. (B) PBMCs or purified NK cells from a fourth donor were incubated with fluA for 17 hours. Purified NK cells were also incubated with recombinant IFN-γ (100 ng/ml) in the presence of fluA, or with recombinant IL-12 (100 ng/ml) and IL-2 (250 U/ml), for 17 hours.

Figure 4

Depletion of CD3⁺ cells from PBMCs reduces to background levels the IFN-γ response of the NK cells to fluA. CD3⁺ cells were depleted from the PBMCs of 8 donors (PBMC-CD3). PBMCs and CD3-deleted PBMCs were incubated with fluA for 17 hours, followed by intracellular staining for IFN-γ. Displayed in the bar graphs are frequencies of IFN-γ⁺ cells in the CD56^bright perforin^– (left) and CD56^dimperforin⁺ (right) NK cell subsets. Results of 3 donors (nos. 4–6) are presented in this figure, while those of other donors are presented in Figures 5 and 9 as part of other experiments.

Figure 5

IFN-γ response of NK cells to fluA required CD3⁺CD56^– cells but not CD3⁺CD56⁺ cells. (A) A blood sample was split into 2 fractions. CD3⁺ cells were depleted from fraction I. CD56⁺, CD36⁺, CD19⁺, and CD16⁺ cells were depleted from fraction II to yield enriched CD56^– T cells. Combination of fractions I and II resulted in a population depleted of CD3⁺CD56⁺ cells. (B) Flow-cytometric analysis of the unfractionated PBMCs, CD3-depleted PBMCs, CD56^– T cells, as well as CD3⁺CD56⁺ cell–depleted PBMCs. Displayed in the dot plots are cells gated on lymphocyte population by forward scattering and side scattering. (C) FluA-induced production of IFN-γ by NK cells in unfractionated PBMCs, CD3-depleted PBMCs, and CD3⁺CD56⁺ cell-depleted PBMCs. Cells were incubated with fluA for 17 hours. Displayed are cells gated on CD3^– lymphocyte population. The numbers in the dot plots are percentages of IFN-γ⁺ cells among CD3^–CD56⁺ NK cells. Similar results were obtained in experiments using blood samples from 2 other donors.

Figure 6

Level of IFN-γ response of the NK cell subsets correlates with frequency of fluA-specific T cells in PBMCs of 25 donors. PBMCs were incubated with fluA for 17 hours, followed by cytokine flow-cytometric analysis to determine the percentage of IFN-γ⁺ cells in the CD56^bright perforin⁺ NK subset (labeled as CD56^bright), CD56^dim perforin^– NK subset (labeled as CD56^dim), and CD3⁺ T cell subset of each donor. The percentage of IFN-γ⁺ cells in each NK subset was plotted against that in the T cell population from the same donor, respectively. The estimated Spearman correlation coefficient r_S and P value is reported in each plot.

Figure 7

IFN-γ and IL-2 production by NK cells and T cells in response to fluA. PBMCs from adult donors were incubated with fluA or SPG (negative control) for 12 hours, with brefeldin A added during the last 5 hours (A and B) or were incubated with fluA for 4–12 hours, with brefeldin A added for the last 4 hours (C). The cells were stained for CD56, fixed and permeabilized, and then stained intracellularly for CD3, IFN-γ, and IL-2. (A) Dot plots for a representative donor (no. 7) displaying cells gated on CD3^–CD56⁺ NK cell population (left panels) or CD3⁺ T cell population (right panels). Numbers in the dot plots refer to the percentage of cytokine-producing cells in each quadrant. (B) Summary of the levels of IFN-γ and IL-2 production by T cells from 4 donors (nos. 7–10). IL-2 was not detected in NK cells from any of these 4 donors. (C) Kinetics of IL-2 production by T cells and IFN-γ production by NK cells (donor no. 20).

Figure 8

Suppression of IFN-γ production of NK cells in response to fluA by IL-2–neutralizing Ab. PBMCs from 6 donors (nos. 11–16) were incubated with fluA for 17 hours in the presence of anti–IL-2 Ab or its isotype control (4 μg/ml), respectively, followed by intracellular staining for IFN-γ to determine frequencies of IFN-γ⁺ CD3^–CD56⁺ NK cells. Displayed in the bar graph is the percentage suppression for each donor, which is defined as [1 – (frequency of IFN-γ⁺ NK cells in the presence of anti–IL-2)/(frequency of IFN-γ⁺ NK cells in the presence of isotype control)] ×100.

Figure 9

The helper function of T cells for fluA-induced IFN-γ production in NK cells can be replaced by exogenous IL-2. PBMCs or CD3-depleted PBMCs from 3 donors (nos. 17–19) were incubated with fluA or control for 17 hours, with or without addition of recombinant IL-2 (250 U/ml). Displayed in the graphs are frequencies of IFN-γ⁺ cells in the CD56^bright or CD56^dim NK cell subsets under each condition.