CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection

Abstract

Immunization in the absence of CD4(+) T cell help results in defective CD8(+) T cell memory, deficient recall responses and diminished protective immunity. Here we investigated at what stage during the immune response to pathogen CD4(+) T cells are essential in the promotion of functional CD8(+) T cell memory. Memory CD8(+) T cell numbers decreased gradually in the absence of CD4(+) T cells despite the presence of similar numbers of memory cell precursors at the peak of the effector phase. Adoptive transfer of effector or memory CD8(+) T cells into wild-type or CD4(+) T cell-deficient mice demonstrated that the presence of CD4(+) T cells was important only after, not during, the early CD8(+) T cell programming phase. In the absence of CD4(+) T cells, memory CD8(+) T cells became functionally impaired and decreased in quantity over time. We conclude that in the context of an acute infection, CD4(+) T cells are required only during the maintenance phase of long-lived memory CD8(+) T cells.

Figure 1

Gradual decrease in memory CD8⁺ T cell numbers in MHC class II–deficient versus wild-type mice. Wild-type and MHC class II–deficient mice containing 5 × 10⁴ P14 cells (Thy-1.1) were immunized with rLmGP (a) or LCMV (b), and CD8⁺ T cell responses specific for GP(33–41) (gp33) were measured by MHC class I tetramer staining as well as by intracellular IFN-γ staining. The percentage of antigen-specific cells in the total CD8⁺ T cell population (left) and absolute number of antigen-specific cells in whole splenocyte population (right) were determined at various time points after infection (horizontal axes). Numbers above symbols indicate the percentage of CD8⁺ T cells from MHC class II–deficient mice versus wild-type mice. Data are presented as the mean ± standard deviation of three to five mice per group at each time point and are representative of three independent studies.

Figure 2

Changes in IL-7Rα expression on CD8⁺ T cells during the programming and memory phases in wild-type and MHC class II–deficient mice. (a) Surface IL-7Rα expression on naive, CD8⁺ T cell–enriched P14 cell populations. Number beside boxed area indicates the percentage of IL-7Rα^hi cells in the CD8⁺ T cell population. (b) Wild-type and MHC class II–deficient mice containing 5 × 10⁴ P14 cells were immunized with LCMV, and day-8 effector and day-100 memory CD8⁺ T cells specific for GP(33–41) (using anti-Thy-1.1 staining) were analyzed for surface expression of IL-7Rα. Plots are gated on CD8⁺ splenocytes; numbers beside boxed areas indicate the percentage of IL-7Rα^hi cells within the GP(33–41)-specific CD8⁺ T cell populations (top number) and in the total spleen population (bottom number in parentheses). Data are representative of three independent experiments, with three to five mice per group at each time point.

Figure 3

Transfer of effector CD8⁺ T cells demonstrates that CD4⁺ T cells are required after, but not during, the programming phase for the development of stable CD8 memory. (a,b) LCMV-specific effector CD8⁺ T cells at 8 d after infection from wild-type (WT) mice (a) or MHC class II–deficient (II KO) mice (b) were adoptively transferred into (→) wild-type or MHC class II–deficient secondary recipients. In the primary host, naive CD8⁺ T cell populations underwent programming and expanded to effector cell populations, which were then isolated and enriched for CD8⁺ T cells. Approximately 5 × 10⁶ to 7 × 10⁶ effector cells were adoptively transferred to all secondary recipients to undergo contraction and memory maintenance. Secondary recipients receiving transferred cells were analyzed 3, 30 or 60 d later, and absolute numbers of antigen-specific cells in the whole splenocyte population were calculated. Data are presented as the mean ± standard deviation of two to four mice per group at each time point. (c,d) Percentage of memory CD8⁺ T cells in MHC class II–deficient versus wild-type secondary recipients at different days after receiving effector CD8⁺ T cells from wild-type (c) or MHC class II–deficient (d) primary hosts. Numbers above bars indicate percentage of CD8⁺ memory T cells in MHC class II–deficient versus wild-type mice. Results are representative of three independent studies.

Figure 4

Effector CD8⁺ T cells transferred to MHC class II–deficient mice become functionally impaired. LCMV-specific effector CD8⁺ T cells at 8 d after infection from wild-type (a,c) or MHC class II–deficient (b,d) primary hosts were adoptively transferred into wild-type or MHC class II–deficient secondary recipients. Transferred cells were analyzed at days 3 and 60 after transfer for production of IFN-γ and IL-2 after 5 h of stimulation with GP(33–41) peptide. (a,b) Mean fluorescence intensity (MFI) for IFN-γ staining in gated region. (c,d) Percentages of IL-2-producing cells in the total IFN-γ-producing CD8⁺ T cell population. Data are representative of three independent studies, with two to four mice per group at each time point.

Figure 5

Effector CD8⁺ T cells transferred to MHC class II–deficient mice are unable to confer protection against bacterial challenge. Effector CD8⁺ T cells generated in wild-type primary hosts were transferred into wild-type or MHC class II–deficient secondary recipients and, on day 60 after transfer, mice were challenged with 2 × 10⁵ rLmGP. Naive mice (no effector CD8⁺ T cells transferred) were challenged as controls. Bacterial clearance was measured by determination of the average colony-forming units (Log₁₀ CFU) 72 h after rLmGP challenge in spleen (a) and liver (b). Data are presented as the arithmetic mean ± standard deviation of two to four mice per group and are representative of two independent studies.

Figure 6

Transfer of memory cells demonstrates that CD4⁺ T cells are required for the maintenance of CD8⁺ T cell memory. LCMV-specific memory CD8⁺ T cells at day 35 after infection from wild-type mice (a) or MHC class II–deficient mice (b) were adoptively transferred into wild-type or MHC class II–deficient secondary recipients. In the primary host, naive CD8⁺ T cell populations underwent programming and contraction and became memory cell populations, which were then isolated and enriched for CD8⁺ T cells. Memory cells (1 × 10⁶ to 2 × 10⁶) were adoptively transferred into secondary recipients for examination of the maintenance of CD8⁺ T cell memory. Secondary recipients receiving transferred memory cells were analyzed 3, 30 or 60 d later, and absolute numbers of antigen-specific cells in the whole splenocyte population were calculated. Data are presented as the mean ± standard deviation of two to four mice per group at each time point. (c,d) Percentage of memory CD8⁺ T cells in MHC class II–deficient versus wild-type secondary recipients at different days after receiving memory CD8⁺ T cells from wild-type (c) or MHC class II–deficient (d) primary hosts. Numbers above bars indicate percentage of CD8⁺ memory cells in MHC class II–deficient versus wild-type mice. Results are representative of two independent studies.