Antiviral memory CD8 T-cell differentiation, maintenance, and secondary expansion occur independently of MyD88

Abstract

Inflammatory signals induced during infection regulate T-cell expansion, differentiation, and memory formation. Toll-like receptors (TLRs) are inflammatory mediators that allow innate immune cells to recognize and respond to invading pathogens. In addition to their role in innate immune cells, we have found that signals delivered through the TLR adapter protein myeloid differentiation protein 88 (MyD88) play a critical, T cell-intrinsic role in supporting the survival and accumulation of antigen-specific effector cells after acute viral infection. However, the importance of MyD88-dependent signals in regulating the generation and maintenance of memory T cells remained unclear. To address this, we used a novel, inducible knockout system to examine whether MyD88 is required for optimal memory CD8 T-cell generation and responses after lymphocytic choriomeningitis virus infection. We show that whereas MyD88 is critical for initial T-cell expansion, it is not required for the subsequent differentiation and stable maintenance of a memory T-cell population. Furthermore, in contrast to naive CD8 T cells, memory CD8 T cells do not depend on MyD88 for their secondary expansion. Our findings clarify the importance of MyD88 during distinct phases of the antiviral T-cell response and establish differential dependence on MyD88 signaling as a novel characteristic that distinguishes naive from memory CD8 T cells.

Figure 1

Myd88ΔT mice mount greatly reduced CD8 T-cell responses to LCMV infection. (A) Splenocytes were isolated from a Myd88ΔT mouse, T and B cells were FACS purified, and MyD88 expression was examined by Western blot. (B-C) Myd88^flox/flox, Myd88ΔT, and Myd88^−/− mice were examined 8 days after infection with LCMV-Arm. Data represent the means ± SD of 4 mice per group. (B) The number of splenic CD8 T cells was determined. (C) The frequency of H2-D^b-gp33–specific (left panel) and H2-D^b-np396–specific (right panel) cells among gated CD8 T cells from the indicated tissues was measured using tetramers. (D) Naive Myd88^flox/flox and Myd88ΔT mice and mice that had been primed with LCMV-Arm 60 days earlier were infected with LCMV-CL13. Six days after CL13 infection, splenocytes were restimulated with the indicated peptides and antigen-specific IFNγ production was assessed. Data represent the means ± SD of at least 3 mice per group.

Figure 2

Tamoxifen treatment induces deletion of MyD88 in YFP⁺ cells in cKO but not cHet mice. (A) cHet and cKO mice were treated with tamoxifen or left untreated, and YFP expression in the indicated splenocyte populations was determined 5 days later. (B) YFP⁺ and YFP⁻ splenocytes were FACS purified from tamoxifen-treated or untreated cHet and cKO mice, and MyD88 expression was determined by Western blot. (C) YFP⁺ and YFP⁻ CD4 and CD8 T cells were FACS purified from treated or untreated cKO mice, and MyD88 expression was determined by Western blot.

Figure 3

YFP⁺ T cells in cKO mice exhibit reduced expansion in response to LCMV infection. cKO and cHet mice were treated with tamoxifen and infected with LCMV-Arm 5 days later. Splenocytes were examined 8 days after infection. (A) The frequency of CD8 T cells in the YFP⁺ (left panel) and YFP⁻ (right panel) splenocyte populations was determined. (B) Splenocytes were restimulated with the indicated peptides and the frequency of IFNγ-producing YFP⁺ (left panel) and YFP⁻ (right panel) CD8 T cells was determined by intracellular staining. (C) The frequency of LCMV-specific YFP⁺ CD8 T cells was determined using tetramers. A representative flow cytometry plot is shown and all graphs represent the means ± SD of at least 4 mice.

Figure 4

MyD88 is not required for the development and maintenance of memory T cells after LCMV infection. (A) The frequency of tetramer-specific CD8 T cells in the blood of cKO and cHet mice was determined 8 days after LCMV infection. (B) cKO and cHet mice were treated with tamoxifen 10 days after LCMV infection, and the frequency of YFP⁺ cells was measured in blood lymphocytes by serial bleeds. (C) The frequency of H2-D^b-gp33–specific (left panel) and H2-D^b-np396–specific (right panel) cells within YFP⁺ blood lymphocytes of cKO and cHet mice was determined by tetramer staining. Data represent the means ± SD of at least 8 mice. (D) The frequency of H2-D^b-gp33–specific cells within the YFP⁺ CD8 T cells from the indicated tissues of cKO and cHet mice was assessed by tetramer staining 60 days after infection. (E) The expression of the indicated phenotypic markers was determined on gated gp33-specific YFP⁺ cells from the spleens of cKO and cHet mice 60 days after infection.

Figure 5

MyD88 is not required for the rapid production of effector cytokines by memory T cells. cKO and cHet mice were infected with LCMV and then treated with tamoxifen as in Figure 4. Splenocytes were isolated 60 days after LCMV infection and restimulated with the indicated LCMV-derived peptides. (A) IFNγ, TNFα, and IL-2 production by YFP⁺ CD8 T cells was assessed by intracellular staining. (B) The frequency of YFP⁺ CD8 T cells producing the indicated combinations of cytokines in response to stimulation with pooled LCMV peptide was determined. Data represent the means ± SD of 3 mice.

Figure 6

Memory T-cell expansion in response to secondary LCMV infection is MyD88 independent. cKO and cHet mice were infected with LCMV-Arm, treated as in Figure 4, and reinfected with LCMV-CL13 60 days after primary infection. (A-B) The frequency of CD44⁺ (A) and H2-D^b-np396–specific (B) YFP⁺ CD8 T cells was measured in the peripheral blood at the indicated time points. Data are the means ± SD of 9 mice per group. (C) The frequency of antigen-specific YFP⁺ CD8 T cells in the spleens of cKO and cHet mice was assessed 6 days after reinfection by tetramer staining. Data are representative of 9 mice per group. (D) YFP⁺ and YFP⁻ CD44⁺CD8 T cells were sorted from the spleens of reinfected cKO and cHet mice and MyD88 expression was measured by Western blot. (E) The viability of gated np396-specific YFP⁺ CD8 T cells from cKO and cHet mice was assessed by annexin V staining either directly ex vivo or after the indicated 18 hours of in vitro culture as indicated. Data are representative of 3 mice per group. (F) Splenocytes were restimulated with the indicated LCMV-derived peptides, and IFNγ production by YFP⁺ CD8 T cells was assessed by intracellular staining. (G) The frequency of YFP⁺ CD8 T cells producing the indicated combinations of IFNγ, TNFα, and IL-2 in response to stimulation with pooled LCMV peptide was determined. Data represent the means ± SD of 3 mice per group.