Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon gamma production from CD4(+) versus CD8(+) T cells

Abstract

CD4(+) and CD8(+) T cells exhibit important differences in their major effector functions. CD8(+) T cells provide protection against pathogens through cytolytic activity, whereas CD4(+) T cells exert important regulatory activity through production of cytokines. However, both lineages can produce interferon (IFN)-gamma, which can contribute to protective immunity. Here we show that CD4(+) and CD8(+) T cells differ in their regulation of IFN-gamma production. Both lineages require signal transducer and activator of transcription (Stat)4 activation for IFN-gamma induced by interleukin (IL)-12/IL-18 signaling, but only CD4(+) T cells require Stat4 for IFN-gamma induction via the TCR pathway. In response to antigen, CD8(+) T cells can produce IFN-gamma independently of IL-12, whereas CD4(+) T cells require IL-12 and Stat4 activation. Thus, there is a lineage-specific requirement for Stat4 activation in antigen-induced IFN-gamma production based on differences in TCR signaling between CD4(+) and CD8(+) T cells.

Figure 1

CD4⁺ T cells exhibit strict requirement for IL-12 and Stat4 in antigen-induced IFN-γ production. (A) Splenocytes from DO11.10 or DO11.10 × Stat4-deficient (Stat4−/−) were cultured with IL-2 and OVA, and either IL-12 (Th1) or IL-4 (Th2) as indicated for 7 d. Cells were restimulated with irradiated BALB/c splenocytes and OVA for 40 h and supernatants were assayed for IFN-γ. Data shown are the mean ± SD of four replicate determinations and is representative of three similar experiments. (B) Cells were treated as above except Brefeldin A was added for the final 4 h of a 19-h restimulation. Samples were stained with FITC-conjugated anti-CD4 and biotin-conjugated KJ1-26 followed by cychrome-streptavidin and PE-conjugated anti–IFN-γ as described in Materials and Methods. Analysis gates were set on live KJ1-26⁺ cells. Quadrants are based on isotype control staining. (C) Sorted DO11.10 CD4⁺ T cells were stimulated with OVA/APCs, IL-2, and either IL-12 (black bars) or anti–IL-12 (gray bars). Sorted CD8⁺ T cells from 2C mice were stimulated with APCs, and either IL-12 (black bars) or anti–IL-12 (gray bars). After 6 d, equal numbers of DO11.10 and 2C T cells were reactivated in the absence of cytokines for 40 h and IFN-γ was measured. Data are the mean ± SD of IFN-γ production represented as a percentage of the IL-12–treated condition (% maximum) from four independent experiments.

Figure 2

CD8⁺ T cells exhibit Stat4-independent IFN-γ production. CD4⁺ (black bars) and CD8⁺ (gray bars) T cells were sort-purified from pooled lymph nodes and spleens of wild-type or Stat4-deficient (Stat4 −/−) mice and stimulated with IL-2, IL-12, and irradiated allogeneic splenocytes (H-2^b) (left and middle) or plate-bound anti-CD3 and anti-CD28 (right). After 6 d, cells were harvested and reactivated at 4 × 10⁵/ml with irradiated allogeneic APCs (left) or plate-bound anti-CD3 (middle and right), and IFN-γ was measured after 40 h. Data in left and middle panels are the mean ± SD pooled from six independent experiments. Data in right panel are the mean ± SD from one of two experiments.

Figure 3

CD4⁺ and CD8⁺ T cells have distinct requirements for Stat4 in TCR-induced IFN-γ production. CD4⁺ and CD8⁺ T cells were sorted from lymph nodes and spleens of wild-type or Stat4-deficient mice and stimulated in the presence of IL-2 and IL-12 with either irradiated allogeneic splenocytes (A) or plate-bound anti-CD3 and anti-CD28 (B). After 5 d, cells were restimulated with irradiated allogeneic APCs (A) or plate-bound anti-CD3 (B) for 17 h with Brefeldin A added for the final 5 h. Samples were stained with FITC-conjugated anti-CD4 or anti-CD8, fixed, permeabilized, and stained with PE-conjugated anti–IFN-γ. Syngeneic splenocytes were used as stimulators for a specificity control and no IFN-γ⁺ cells were detected (data not shown). Gates for analysis excluded dead cells and quadrants were set based on isotype control staining. The percentages displayed indicate the frequency of cells positive for IFN-γ in the CD4⁺ (top) or CD8⁺ (bottom) population, rather than the percentage of total cells. Data shown are representative of three independent experiments.

Figure 4

CD8⁺ T cells possess both Stat4-independent (TCR) and Stat4-dependent (IL-12/IL-18) pathways for IFN-γ induction. CD4⁺ (top) and CD8⁺ (bottom) T cells were sorted from lymph nodes and spleens of wild-type (black bars) or Stat4-deficient (gray bars) mice and stimulated with irradiated allogeneic splenocytes, IL-2, and IL-12. After 5 d, cells were harvested and equal cell numbers were restimulated by the addition of IL-12 and IL-18 or plate-bound anti-CD3 for 40 h, and IFN-γ was measured by ELISA. Data are the mean ± SD of four independent experiments.