Regulation of innate CD8+ T-cell activation mediated by cytokines

Abstract

Virus-specific CD8(+) T cells develop the ability to function in an "innate" capacity by responding to a remarkable array of cytokines in a TCR-independent manner. Although several cytokines such as IL-12 and IL-18 have been identified as key regulators of CD8(+) T-cell activation, the role of other cytokines and the ways in which they interact with each other remain unclear. Here, we have used an unbiased, systematic approach to examine the effects of 1,849 cytokine combinations on virus-specific CD8(+) T-cell activation. This study identifies several unexpected cytokine combinations that synergize to induce antigen-independent IFNγ production and CD69 up-regulation by CD8(+) T cells in addition to cytokines that exhibit differential regulatory functions, with the ability to either enhance or inhibit T-cell IFNγ production, depending on which cytokine partner is present. These findings underscore the complexity of cytokine interactions while also providing insight into the multifaceted regulatory network controlling virus-specific CD8(+) T-cell functions.

Fig. 1.

Differential regulation of IFNγ production by virus-specific CD8⁺ T cells. At 8 d post-LCMV infection, splenic CD8⁺ T cells were stimulated in vitro with the indicated cytokines at 100 ng/mL for 6 h before intracellular staining for IFNγ and analysis by flow cytometry. Numbers in the Upper Right quadrant of each dot plot represent the percentage of CD11a^highNP118-tetramer⁺ CD8⁺ T cells expressing IFNγ, after background subtraction of medium controls (Upper Left dot plot). Numbers in parentheses represent the percent increase or decrease of IFNγ⁺ T cells following incubation with each cytokine pair relative to incubation with IL-12, IL-15, or IL-18 alone. Data are representative of six BALB/c mice from three independent experiments.

Fig. 2.

Direct versus indirect activation of virus-specific CD8⁺ T cells by cytokines. Splenocytes from BALB/c mice at 8 d after LCMV infection were stimulated with the indicated cytokines directly ex vivo, or CD8⁺ T cells were purified by MACS (>95% purity) before direct ex vivo stimulation. Numbers in the Upper Right quadrant of each dot plot represent the percentage of IFNγ⁺ CD11a^highNP118-tetramer⁺ CD8⁺ T cells. Data are representative of four mice from two independent experiments.

Fig. 3.

Cytokine-mediated IFNγ production by effector T cells during acute LCMV infection. At 8 d postinfection with LCMV, MACS-purified CD8⁺ T cells from BALB/c mice were stimulated with the indicated cytokine combinations at 100 ng/mL. Bars labeled “unsorted” represent the IFNγ response of NP118-tetramer⁺ CD8⁺ T cells to the indicated single cytokine in a population of bulk splenocytes. All other responses represent the results observed with purified CD8⁺ T-cell populations. Open bars represent IFNγ responses to the unpartnered individual cytokines on the x axis, and the corresponding solid bars represent IFNγ responses to each cytokine in combination with the cytokine labeled at the Top of each panel. Spontaneous production of IFNγ in medium-only controls was typically <0.2%, which was subtracted before preparing the graphs. Data represent the mean ± SD of four to eight mice per group. IFNγ responses to each cytokine pair were compared with responses after stimulation with the individual cytokines using an unpaired two-tailed t test. Cytokine pairs that induced T-cell responses that were significantly different (P < 0.05) from both responses to the individual cytokines within the pair are marked with an asterisk (*). Note that different y axis scales are used for each cytokine.

Fig. 4.

IFNγ production by memory T cells following exposure to defined cytokine combinations. MACS-purified CD8⁺ T cells from LCMV-immune BALB/c mice (>60 d postinfection) were stimulated as described in Fig. 3. Spontaneous production of IFNγ in medium-only controls was typically <0.1%, which was subtracted before preparing the graphs. Data represent mean ±SD of four to eight mice. Note that different y axis scales are used for each cytokine.

Fig. 5.

Differential cytokine-mediated induction of CD69 and IFNγ expression. To determine whether virus-specific CD8⁺ T cells can be activated without producing IFNγ, MACS-purified CD8⁺ T cells from BALB/c mice were stimulated directly ex vivo with the indicated cytokine combinations at 100 ng/mL. Each data point represents the percentage of NP118-tetramer⁺ effector T cells that up-regulated CD69 or produced IFNγ in response to the indicated cytokine combinations. Spontaneous production of IFNγ in medium-only controls was typically <0.2%, and CD69 was typically expressed on ∼15–20% of NP118-tetramer⁺ CD8⁺ T cells directly ex vivo. Data represent the average ±SD of three to six mice per group.