CD40-deficient, influenza-specific CD8 memory T cells develop and function normally in a CD40-sufficient environment

Abstract

Two models have been proposed to explain the requirement for CD40 signaling in CD8 T cell responses. The first model suggests that CD4 T cells activate antigen-presenting cells (APCs) through CD40 signaling (APC licensing). In turn, licensed APCs are able to prime naive CD8 T cells. The second model suggests that CD154-expressing CD4 T cells activate CD40-bearing CD8 T cells directly. Although the requirement for CD40 in APC licensing can be bypassed by inflammatory responses to pathogens that activate APCs directly, the second model predicts that CD8 responses to all antigens will be dependent on CD40 signaling. Here we determined which model applies to CD8 responses to influenza. We demonstrate that optimal CD8 T cell responses to influenza are dependent on CD40 signaling, however both primary and secondary responses to influenza require CD40 expression on non-T cells. Furthermore, CD40-/- CD8 T cells proliferate and differentiate to the same extent as CD40+/+ CD8 T cells in response to influenza, as long as they have equal access to CD40+/+ APCs. Thus, CD4 T cells do not activate influenza-specific CD8 cells directly through CD40 signaling. Instead, these data support the classical model, in which CD4 T cells provide help to CD8 T cells indirectly by activating APCs through CD40.

Figure 1.

CD40 is required for optimal CD8 T cell responses to influenza. (A) CD40^−/− and WT mice were infected with PR8 and cells from the lungs were analyzed on days 9 and 12. The percentage of NP-specific CD8 cells is indicated. (B) Cells from the lungs of infected animals were stimulated with NP_366–374 peptide and the production of IFNγ was measured by intracellular staining. The percentage of IFNγ⁺ CD8 cells is indicated. (C) The total numbers of NP-specific, tetramer-binding CD8 cells (top) and NP-specific, IFNγ-producing CD8 cells (bottom) are shown for both WT mice (black bars) and CD40^−/− mice (gray bars). This experiment contained five mice per group per time point and was performed three times.

Figure 2.

Generation of chimeric mice. Irradiated TCRβ^−/−δ^−/− recipient mice were reconstituted with (A) TCRβ^−/−δ^−/− BM and C57BL/6 BM, (B) CD40^−/− BM, (C) TCRβ^−/−δ^−/− BM and CD40^−/− BM, or (D) TCRβ^−/−δ^−/− BM and CD154^−/− BM. The proportion of B cells, DCs, and T cells of each genotype that are expected to repopulate the chimeras is indicated. Cells from the LNs (top) and lungs (bottom) of chimeric mice were analyzed on day 10 after infection to determine the actual frequency of CD40-expressing B cells and DCs. The percentage of B cells or DCs expressing CD40 is indicated.

Figure 3.

Optimal CD8 T cell responses to influenza require CD40 expression on hematopoietic cells, but not on T cells. (A) Chimeric mice were infected with PR8 and the frequency of PNA^hi FAS^hi GC B cells (left) as well as the frequency of CD138^hi plasma cells and IgG2⁺ B cells (right) in the draining LNs of chimeric mice was analyzed on day 10. All plots show cells gated on CD19 expression. The numbers refer to the percentage of B cells that have the indicated phenotype. (B) NP-specific CD8 T cells were enumerated on days 10 and 49. Memory mice were infected on day 50 with X31, and the responding memory CD8 T cells were analyzed on day 56. All plots show cells gated on CD8 expression. The numbers refer to the percentage of NP-specific CD8 cells. (C) The total numbers of NP-specific CD8 T cells in the lungs of chimeric mice were calculated for each time point. This experiment contained five mice per group per time point and was performed two times. The samples in A are from the same mice as the day 10 samples in B.

Figure 4.

B cells require CD40 expression to respond to influenza. Chimeric mice were infected with PR8 and the B cell response in the LNs was analyzed on day 10. (A) The contribution of Ly5.1 and Ly5.2 donor cells to the total CD19⁺ B cell population is shown. (B) GC B cells were identified by coexpression of peanut agglutinin and FAS. The percentage of B cells with a GC phenotype is indicated next to the circled areas. (C) The contribution of Ly5.1 and Ly5.2 donor cells to the GC B cell population is shown. (D) The contribution of Ly5.1 and Ly5.2 donor cells to the CD138⁺ plasma cell population is shown. This experiment contained five mice per group and was performed two times.

Figure 5.

CD40^−/− CD8 T cells develop into memory cells with normal proliferative and functional capacity. Chimeric mice were infected with PR8 on day 0 and analyzed on days 10 and 49. Memory mice were infected with X31 on day 50 and analyzed on day 56. (A) T cells were gated on CD8 and the contribution of Ly5.1 and Ly5.2 cells to the CD8 population was determined by Ly5.1 expression. The numbers in the upper corners of each plot refer to the percentage of total CD8 cells that are either LY5.1⁺ or Ly5.2⁺. (B) The contribution of Ly5.1 and Ly5.2 cells to the NP tetramer-binding CD8 population was determined by Ly5.1 expression. The numbers in the upper corners of each plot refer to the percentage of NP-specific CD8 cells that are either LY5.1⁺ or Ly5.2⁺. (C) The contribution of Ly5.1 and Ly5.2 cells to the NP-specific, IFNγ-producing CD8 population on day 56 was determined by Ly5.1 expression. The numbers in the upper corners of each plot refer to the percentage of NP-specific CD8 cells that are derived from Ly5.2 donor (left) and the Ly5.1 donor (right). This experiment contained five mice per group per time point and was performed two times. The day 10 samples in this experiment are from the same mice as the samples in Fig. 4.