CD4+ T helper cells use CD154-CD40 interactions to counteract T reg cell-mediated suppression of CD8+ T cell responses to influenza

Abstract

CD4(+) T cells promote CD8(+) T cell priming by licensing dendritic cells (DCs) via CD40-CD154 interactions. However, the initial requirement for CD40 signaling may be replaced by the direct activation of DCs by pathogen-derived signals. Nevertheless, CD40-CD154 interactions are often required for optimal CD8(+) T cell responses to pathogens for unknown reasons. Here we show that CD40 signaling is required to prevent the premature contraction of the influenza-specific CD8(+) T cell response. CD40 is required on DCs but not on B cells or T cells, whereas CD154 is required on CD4(+) T cells but not CD8(+) T cells, NKT cells, or DCs. Paradoxically, even though CD154-expressing CD4(+) T cells are required for robust CD8(+) T cell responses, primary CD8(+) T cell responses are apparently normal in the absence of CD4(+) T cells. We resolved this paradox by showing that the interaction of CD40-bearing DCs with CD154-expressing CD4(+) T cells precludes regulatory T cell (T reg cell)-mediated suppression and prevents premature contraction of the influenza-specific CD8(+) T cell response. Thus, CD4(+) T helper cells are not required for robust CD8(+) T cell responses to influenza when T reg cells are absent.

Figure 1.

CD40 signaling prevents premature contraction of influenza-specific CD8⁺ T cells. (A–D) B6 and Cd40^−/− mice were infected with influenza, and the frequency (A and C) and number (B and D) of NP-specific CD8⁺ T cells in the mLNs (A and B) and lungs (C and D) were determined by flow cytometry at the indicated times. A–D are representative of three independent experiments (mean ± SD of five mice per time point). (E–H) B6 mice were infected with influenza and treated with either 250 µg of the CD154-blocking antibody MR1 or control antibody, and the frequency (E and G) and number (F and H) of NP-specific CD8⁺ T cells in the mLNs (E and F) and lungs (G and H) were determined by flow cytometry on day 10 after infection. Plots were gated on CD8⁺ lymphocytes. E–H are representative of five independent experiments (mean ± SD of three to five mice per group). P-values were determined using a two-tailed Student’s t test.

Figure 2.

CD40 is required on DCs but not T cells or B cells for efficient CD8⁺ T cell responses to influenza. (A–D) B-WT and B-Cd40^−/− chimeras were infected with influenza, and the frequency (A) and number (B) of GC B cells as well as the frequency (C) and number (D) of NP-specific CD8⁺ T cells were determined 12 d later. Plots were gated on CD19⁺ lymphocytes (A) or CD8⁺ lymphocytes (C). (E–G) B6:Cd40^−/− mixed BM chimeras were infected with influenza, and the frequency of CD45.1⁺ and CD45.2⁺ cells in the total CD8⁺ T cell population (E) and in the NP-specific CD8⁺ T cell population (F), as well the number of CD45.1⁺ and CD45.2⁺ NP-specific CD8⁺ T cells (G) were determined by flow cytometry on day 12 after infection. A–G are representative of two (A–D) or three (E–G) independent experiments (mean ± SD of five mice per group). (H and I) Irradiated B6 mice were reconstituted with CD11c-DTR BM, infected with influenza 8 wk later, and treated with either PBS or 60 ng DT on day 6 after infection, and the frequency (H) and number (I) of NP-specific CD8⁺ T cells in mLNs were determined by flow cytometry on day 12. (J and K) DC-WT, DC-Cd40^−/−, and DC-Cd154^−/− BM chimeras were infected with influenza and treated with DT on days 0, 3, 5, and 7 after infection, and the frequency (J) and number (K) of NP-specific CD8⁺ T cells were determined by flow cytometry on day 10. H–K are representative of four (H and I) or two (J and K) independent experiments (mean ± SD of four to five mice per group). P-values were determined using a two-tailed Student’s t test.

Figure 3.

The NP-specific CD8⁺ T cell response requires CD154 expression on T cells but is normal in the absence of CD4⁺ T cells. (A and B) T-WT, T-Cd154^−/−, and T-85% chimeric mice were infected with influenza, and the frequency (A) and number (B) of NP-specific CD8⁺ T cells were determined on day 12. (C and D) B6 mice were infected with influenza and treated with 250 µg anti-CD4 or control IgG, and the frequency (C) and number (D) of NP-specific CD8⁺ T cells were calculated by flow cytometry on day 10. (E and F) B6 and Iab^−/− mice were infected with influenza, and the frequency (E) and numbers (F) of NP-specific CD8⁺ T cells were calculated by flow cytometry on day 10. A–F are representative of three (A, B, E, and F) or four (C and D) independent experiments (mean ± SD of four to five mice per group). P-values were determined using a two-tailed Student’s t test.

Figure 4.

NP-specific CD8⁺ T cell response requires CD154 expression on CD4⁺ T cells but not on NKT or CD8⁺ T cells. (A and B) B6 and Cd1d^−/− mice were infected with influenza, and the frequency (A) and number (B) of NP-specific CD8⁺ T cells were calculated by on day 10. (C–E) CD4⁺ and CD8⁺ T cells purified from spleens of naive B6 and Cd154^−/− mice were adoptively transferred to Tcrβδ^−/− recipients, which were infected with influenza the next day, and the frequency (C) and number (D) of NP-specific CD8⁺ T cells and total CD8⁺ T cells (E) were determined by flow cytometry 14 d later. A–E are representative of three independent experiments (mean ± SD of four to five mice per group). P-values were determined using a two-tailed Student’s t test.

Figure 5.

The NP-specific CD8⁺ T cell response does not require CD40 signaling in the absence of CD4⁺ T cells. (A–D) B6 mice were treated with combinations of control IgG, anti-CD4, and anti-CD154 and infected with influenza, and the frequency (A and C) and number (B and D) of NP-specific CD8⁺ T cells in the mLNs (A and B) or lung (C and D) were determined by flow cytometry on day 10. (E–H) B6 and Iab^−/− mice were treated with anti-CD154 or control IgG and infected with influenza, and the frequency (E and G) and absolute number (F and H) of NP-specific CD8⁺ T cells in the mLNs (E and F) and lung (G and H) were calculated on day 10. (I–L) B6 and Cd154^−/− mice were treated with anti-CD4 or control IgG and infected with influenza, and the frequency (I and K) and number (J and L) of NP-specific CD8⁺ T cells in the mLNs (I and J) and lung (K and L) were calculated by flow cytometry on day 10. A–L are representative of three independent experiments (mean ± SD of four to five mice per group). All plots were gated on CD8⁺ lymphocytes. P-values were determined using a two-tailed Student’s t test.

Figure 6.

The NP-specific CD8⁺ T cell response does not require CD40 signaling in the absence of T reg cells. (A–C) B6 and FoxP3-DTR mice were infected with influenza and treated with DT on days 0, 4, and 7, and the frequency of FoxP3⁺CD4⁺ T cells (A) as well as the frequency (B) and number (C) of NP-specific CD8⁺ T cells were determined in the mLN by flow cytometry on day 10. (D and E) B6 and FoxP3-DTR mice were infected with influenza, treated with anti-CD154 or control IgG on days 0 and 4, and treated with DT on days 0, 4, and 7, and the frequency (D) and number (E) of NP-specific CD8⁺ T cells were determined in the mLNs by flow cytometry on day 10. (F and G) B6 and FoxP3-DTR mice were infected with influenza and treated with DT on days 6 and 9, and the frequency (F) and absolute number (G) of NP-specific CD8⁺ T cells were determined in the mLN by flow cytometry on day 12. F and G are representative of two independent experiments (mean ± SD of four to five mice per group). (H and I) B6 and FoxP3-DTR mice were infected with influenza, treated with anti-CD154 or control IgG on days 6 and 9, and treated with DT on days 6 and 9, and the frequency (H) and number (I) of NP-specific CD8⁺ T cells were determined in the mLN by flow cytometry on day 12. (J and K) B6 and FoxP3-DTR mice were infected with influenza, treated with anti-CD154 or control IgG on days 6 and 9, and treated with DT on days 6 and 9, and the frequency (J) and number (K) of NP-specific CD8⁺ T cells were determined in the lung by flow cytometry on day 12. A–E and H–K are representative of four (A–C), three (D and E), or two (H–K) independent experiments (mean ± SD of three to five mice per group). Plots are gated on CD8⁺ lymphocytes. P-values were determined using a two-tailed Student’s t test.