Multiple-cytokine-producing antiviral CD4 T cells are functionally superior to single-cytokine-producing cells

Abstract

Virus-specific CD4 T cells are endowed with multiple functions, such as cytokine production, CD40 ligand (CD40L) expression (associated with the costimulation of CD8 and B cells), and degranulation (associated with cytotoxic potential). Here, we used antiviral CD4 T cells present in human blood to evaluate the relationship between cytokine production and other functions of CD4 T cells. Antiviral CD4 T cells specific for a virus causing persistent infection, cytomegalovirus (CMV), and two viruses causing nonpersistent infections, influenza virus and the smallpox vaccine virus (vaccinia virus), were studied. CD4 T cells specific for each of the viruses produced all seven possible combinations of the cytokines gamma interferon (IFN-gamma), interleukin-2, and tumor necrosis factor alpha. Cells producing three or two cytokines (triple producers and double producers) represented nearly 50% of the total response to each of the viruses. Triple producers expressed the highest levels of cytokines per cell, and single producers expressed the lowest. Following stimulation, higher frequencies of triple producers than single producers expressed CD40L. Only CMV-specific CD4 T cells underwent degranulation. However, higher frequencies of CMV-specific triple producers than single producers showed this functional characteristic. In contrast to the functional phenotypes, the memory phenotypes of triple producers and IFN-gamma single producers did not differ. These results demonstrate a strong positive association between the cytokine coproduction capacity of a virus-specific CD4 T cell and its other functional characteristics and suggest that vaccines should aim to elicit T cells that coproduce more than one cytokine.

FIG. 1.

Magnitude of virus-specific CD4 T cells expressing IFN-γ, IL-2, or TNF-α. PBMC were stimulated with viral lysates, and the virus-specific CD4 T-cell responses were measured using an intracellular cytokine staining assay. The percentage of total cytokine-positive (+ve) CD4 T cells specific for the indicated virus and positive for each cytokine is plotted.

FIG. 2.

Schematic for the analysis of cytokine coexpression profiles of CD4 T cells following stimulation with SEB. Lymphocytes were gated based on forward scattering (FSC) and side scattering (SSC), and CD4 T cells (CD3⁺, CD8⁻, and CD4⁺) were then analyzed for IFN-γ, IL-2, and TNF-α expression. Cytokine coexpression profiles were determined using the Boolean gating function of FlowJo software. I, IFN-γ; L, IL-2; and T, TNF-α.

FIG. 3.

Cytokine coexpression profiles of vaccinia virus-, flu virus-, and CMV-specific CD4 T cells. PBMC were stimulated with viral lysates, and cytokine coexpression profiles were determined using the Boolean gating function of FlowJo software. (A) The proportions of subsets of virus-specific CD4 T cells positive for specific cytokines were expressed as percentages of total cytokine-positive (+ve) virus-specific CD4 T cells and plotted using GraphPad Prism. I, IFN-γ; L, IL-2; and T, TNF-α. (B) The pie charts present the mean frequencies of the T-cell subsets positive for the indicated cytokines in each group. TP, triple producers; DP, double producers; and SP, single producers.

FIG. 4.

Triple producers produce higher levels of cytokine per cell than single producers. (A) Representative plots showing the fluorescence intensities for IFN-γ, IL-2, and TNF-α from triple producers (TP) and single producers (SP). Dotted lines represent data for the isotype control. (B) Summary of mean fluorescence intensities (MFI) for vaccinia virus-, flu virus-, and CMV-specific triple producers, double producers (DP), and single producers. I, IFN-γ; L, IL-2; and T, TNF-α. Note that for double and single producers, only the dominant subsets in each virus-specific group are shown. Asterisks, significantly higher than single producers.

FIG. 5.

Higher proportions of triple producers than single producers express CD40L. (A) Representative plots showing the levels of surface expression of CD40L (CD154) by triple and single producers. Bars indicate CD154-positive cells, and numbers indicate CD154-positive cells as a percentage of total cytokine-positive cells. (B) Summary of the proportions of CD40L-positive cells as percentages of the respective cytokine coexpression subsets. Bars indicate median value for the group. TP, triple producers; DP, double producers; SP, single producers; I, IFN-γ; L, IL-2; and T, TNF-α. (C) Summary of mean fluorescence intensity (MFI) data. Note that for double and single producers, only the dominant subsets in each virus-specific group are shown.

FIG. 6.

Higher proportions of CMV-specific triple producers than single producers degranulate. (A) Representative flow charts showing the degranulation potential of vaccinia virus-, flu virus-, and CMV-specific IFN-γ-producing CD4 T cells. Percentage of total CD4 cells is indicated. (B) Representative flow charts showing the overlay of total CD4 T cells (gray) with CMV-specific triple producers, IFN-γ and TNF-α double producers, and IFN-γ single producers (black). Numbers on the graphs indicate the frequencies of virus-specific CD4 T cells as percentages of the respective cytokine coexpression subsets. (C) Summary of CD107a/b and CD154 expression in different cytokine coexpression subsets. TP, triple producers; DP, double producers; SP, single producers; I, IFN-γ; L, IL-2; T, TNF-α; and +ve, positive. (D) Coexpression of CD107a/b and granzyme B by CMV-specific CD4 T cells.

FIG. 7.

Memory phenotypes of cytokine coexpression subsets. (A) Representative data showing the overlay of symbols for total CD4 T cells (light gray) with symbols for cytokine coexpression subsets (black) for different virus-specific cells. The numbers on the graphs represent the frequencies of different subsets as percentages of the respective total cytokine coexpression subsets. I, IFN-γ; L, IL-2; and T, TNF-α. (B) Summary of levels of CCR7 expression (shown as percentages of CCR7-positive cells among the respective cytokine coexpression subsets) by different cytokine coexpression subsets specific for vaccinia virus (V), flu virus (F), and CMV (C). Black bars represent triple producers, gray bars represent double producers, and white bars represent single producers. +ve, positive.