Signal 3 cytokines as modulators of primary immune responses during infections: the interplay of type I IFN and IL-12 in CD8 T cell responses

Abstract

Signal 3 cytokines, such as IL-12 or type I IFN, support expansion and differentiation of CD8 T cells in vivo. If and how these two signal 3 cytokines compensate each other in T cell activation during different infections is so far unknown. Using CD8 T cells lacking receptors for IL-12, type I IFN or both, we show that the expansion of CD8 T cells depends on type I IFN (LCMV infection), type I IFN and IL-12 (Listeria and vesicular stomatitis virus infection) or is largely independent of the two cytokines (vaccinia virus infection). Furthermore, we show that CD8 T cells lacking IL-12 and type I IFN signals are impaired in cytokine production and cytolytic activity in the context of VSV and Listeria infection. These effector CD8 T cells fail to express KLRG1, thereby exhibiting a memory-like phenotype which correlated with lower expression of the transcription factor T-bet and higher expression of Eomes. This indicates that the variable interplay of both signal 3 cytokines is mandatory for cell fate decision of CD8 T cells in the context of different infections. Furthermore our results demonstrate that the pathogen-induced overall inflammatory milieu and not the antigen load and/or the quality of antigen presentation critically determine the signal 3 dependence of CD8 T cells.

Figure 1. Expansion of CD8 T cells shows a complex pattern of dependency on signal 3 cytokines.

10⁵ (Fig. 1 A–D) Thy1.1⁺ P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice (Thy1.2⁺) followed by infection with rVSV_GP, rVV_GP, LCMV or rListeria _GP33. Kinetics of P14.WT (•) and P14.IFNARKO, P14.IL12RKO as well as P14. DOKO (○) T cells gated on CD8⁺ T cells in the blood at indicated time points. Values are expressed as mean ± SEM (n = 3). Results are representative of three independent experiments. IL-12 and type I IFN levels in sera of infected mice as determined by ELISA (for IL-12) or VSV protection assay (for type I IFN) are indicated (Fig. 1 E).

Figure 2. Analysis of P14 CD8 T cells in the context of a rVSV_GP infection.

P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice followed by infection with rVSV_GP. Analysis of spleen cells at day 5 after infection is shown. A, Relative and total P14 T cell numbers per spleen is shown. B, Analysis of cell division by BrdU incorporation. After adoptive transfer, recipient mice were treated with 2 mg of BrdU i.p. on day 4. BrdU staining of P14.WT or P14.DOKO T cells gated on CD8⁺ T cells on day 5 in the spleen is shown. Numbers in brackets indicate percentages of BrdU⁺ cells of total P14 T cells. C, Analysis of KLRG1 and CD127 expression on P14 T cells. Plots are gated on CD8⁺, Thy1.1⁺ (P14) cells. D, Statistical analysis of T cell differentiation. Percentages of KLRG1 and CD127 positive P14 T cells of total P14 T cells are depicted. E, Cytolytic activity of P14 T cells was determined five days post infection by a ⁵¹Cr-release assay. Gp33-41 peptide-loaded or adeno peptide-loaded EL-4 cells (control) were used as targets. Equal numbers of purified P14 T cells were used as effectors. Expression of IFN-γ (F) and TNF-α (G) of P14 T cells. Percentages of cytokine positive P14 T cells of total P14 T cells, as well as MFI of cytokine expression are indicated. Values are expressed as mean ± SEM (n = 3). *p<0,01. Results are representative of three independent experiments.

Figure 3. Analysis of P14 CD8 T cells in the context of a rListeria_GP33 infection.

P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice followed by infection with rListeria_GP33. Analysis of spleen cells at day 5.5 after infection is shown. A, Relative and total P14 T cell numbers per spleen is shown. B, Analysis of cell division by BrdU incorporation. After adoptive transfer, recipient mice were treated with 2 mg of BrdU i.p. on day 4. BrdU staining of P14.WT or P14.DOKO T cells gated on CD8⁺ T cells on day 5 in the spleen is shown. Numbers in brackets indicate percentages of BrdU⁺ cells of total P14 T cells. C, Analysis of KLRG1 and CD127 expression on P14 T cells. Plots are gated on CD8⁺, Thy1.1⁺ (P14) cells. D, Statistical analysis of T cell differentiation. Percentages of KLRG1 and CD127 positive P14 T cells of total P14 T cells are depicted. E, Cytolytic activity of P14 T cells was determined five days post infection by a ⁵¹Cr-release assay. Gp33-41 peptide-loaded or adeno peptide-loaded EL-4 cells (control) were used as targets. Equal numbers of purified P14 T cells were used as effectors. Expression of IFN-γ (F) and TNF-α (G) of P14 T cells. Percentages of cytokine positive P14 T cells of total P14 T cells, as well as MFI of cytokine expression are indicated. Values are expressed as mean ± SEM (n = 3). *p<0,01. Results are representative of three independent experiments.

Figure 4. Expression of T-box transcription factors T-bet and Eomes after infections with rVSV_GP and rListeria_GP33 infection.

P14.WT, P14.IL-12RKO, P14.IFNARKO or P14.DOKO T cells were transferred into B6 mice followed by infection with A, rVSV_GP, or B, rListeria_GP33. Expression of T-bet and Eomes was analyzed in the spleen on day 5 after infection. Histograms show intracellular expression of T-bet or Eomes in P14.WT (black line), P14.DOKO (red line) and naïve P14 CD8 T cells (filled curve) directly ex vivo. Percentages of T-bet or Eomes positive P14 T cells of total P14 T cells are indicated (middle panels). Mean fluorescence intensity (MFI) of T-bet or Eomes expression of P14 T cells is shown (right panels). p<0,01. Results are representative of two independent experiments.

Figure 5. The pathogen-induced inflammatory milieu determines signal 3 dependence of CD8 T cells.

P14.WT or P14.IFNARKO T cells were transferred into B6 mice followed by infection with A, rVV_GP, rVV_GP and LCMV8.7 or LCMV8.7 alone or, B, rVSV_GP, rVSV_GP and LCMV8.7 or LCMV8.7 alone. Analysis of peripheral blood lymphocytes at day 5 after infection is shown. Numbers in brackets indicate percentages of Thy1.1 positive of total CD8 T cells. Results are representative of two independent experiments.

Figure 6. Infection independent T cell expansion can be suppressed by the LCMV-induced inflammatory milieu.

A timeline of the experiment is shown in A. P14.WT or P14.IFNARKO T cells were transferred into H8 mice at day −1, followed by B, treatment with anti-CD40 antibody alone (day 0 and day 2), C, treatment with anti-CD40 antibody and infection with LCMV8.7 or D, infection with LCMV8.7 alone. Expansion of transferred CD8 T cells at day 5 and day 7 after treatment and/or infection is shown in the blood. Numbers in brackets indicate percentages of Thy1.1 positive of total CD8 T cells. Kinetics in the right panels indicates the expansion of the transferred Thy1.1 T cells of total CD8 T cells from day 3 to day 7 in the blood. Values are expressed as mean ± SEM (n = 3). Results are representative of two independent experiments.