Dynamic regulation of functionally distinct virus-specific T cells

Abstract

The functional capacities of CD8(+) T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4(+) T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8(+) T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-gamma, TNF-alpha, MIP1beta, and CD107a, showed that aAPC-generated CD8(+) T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8(+) T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8(+) T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8(+) T cell function.

Fig. 1.

Expansion of virus-specific CD8⁺T cells by in vitro stimulation with M1- and H576-loaded aAPCs. Enriched CD8⁺ T cells were ex vivo-stimulated with aAPCs loaded with either M1 peptide (A) or H576 peptide (B) and analyzed weekly by tetramer staining. Plots are gated on CD8⁺ tetramer-positive cells and show representative results for three healthy donors. (C) Antigen-specific CD8⁺ T cell function was analyzed after 3 weeks of expansion with ^H576aAPC. Frequencies of tetramer-positive, IFN-γ producing cells in response to stimulation with unloaded (Left) or peptide-loaded (Right) aAPC are indicated in the Upper Right quadrants.

Fig. 2.

Comparison of virus-specific CD8⁺ T cells expanded by aAPCs and moDCs. Enriched CD8⁺ T cells from three donors were cultured with (A) M1 peptide and (B) H576 peptide-loaded aAPCs (open bars) or moDCs (filled bars) for 3 weeks. Tetramer and intracellular cytokine staining was performed at the end of each 7-day stimulation period. Bars represent percentage of CD8⁺ T cells that were tetramer-positive minus background ± SEM averaged from three donors, each analyzed in triplicate. On day 21 expanded (C) M1-specific CD8⁺ T cells and (D) H576-specific CD8⁺ T cells were incubated with either unloaded or peptide-loaded aAPCs for 6 h, followed by intracellular cytokine staining for IFN-γ, TNF-α, and IL-2. Bars represent percentage of total CD8⁺ T cells expressing the cytokine minus background averaged from three donors ± SEM. Student’s unpaired t test was used for statistical analysis. * P < 0.05, *** P < 0.0001. Cytotoxicity was evaluated by staining for granule membrane protein CD107a (E).

Fig. 3.

Cytokine production by CD8⁺ T cells is determined by the type of APC used for stimulation. (A) To assess the effect and reversibility of APC stimulation on cytokine production, CD8⁺ T cells stimulated with ^M1aAPCs for 7 days were restimulated with ^M1moDCs for a further 7 days or CD8⁺ T cells initially stimulated with ^M1moDCs were restimulated with ^M1aAPCs in week 2. The proportion of cells producing two cytokines (IFN-γ and TNF-α) was assessed by intracellular cytokine staining on day 7 and day 14 (before and after switching APCs). The upper right quadrant represents the proportion of cells expressing IFN-γ and TNF-α. (B) To assess the effect of switching APCs on the cytokines produced, enriched CD8⁺ T cells were cultured for 3 weeks changing the type of APCs (aAPCs or moDCs) at the end of each 7-day stimulation cycle. The gated cells represent the proportion of cells expressing IFN-γ out of total CD3/CD8⁺ T cells. (C–G) The effect of switching the type of APC on cytokine, chemokine, and cytotoxicity is shown by a trend line that shows the proportion of cytokine-producing cells at the end of each 7-day stimulation cycle.

Fig. 4.

Generation of virus-specific multifunctional CD8⁺ T cells by aAPCs and moDCs. The multifunctional capacities of individual cells expanded for 3 weeks by ^M1aAPCs and ^M1moDCs were analyzed. (A) Every possible combination of cytokine production by M1-specific cells is shown on the x axis. Bars show the relative proportion of the total response contributed by CD8⁺ T cells with a given functional response. Blue bars show aAPC-stimulated cells. Green bars show moDC-stimulated cells. Red bars show cells stimulated with unloaded aAPCs. Pie charts (Left, aAPC; Center, no stimulation; Right, moDC) show the proportion of cells positive for different numbers of effectors: lavender, 0; yellow, 1; light blue, 2; green, 3; dark blue, 4; and red, 5. (B) Compiled IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a MFI of CD8⁺ T cells producing cytokines. Dots identify sample means.