Persistent Antigen and Prolonged AKT-mTORC1 Activation Underlie Memory CD8 T Cell Impairment in the Absence of CD4 T Cells

Abstract

Recall responses by memory CD8 T cells are impaired in the absence of CD4 T cells. Although several mechanisms have been proposed, the molecular basis is still largely unknown. Using a local influenza virus infection in the respiratory tract and the lung of CD4(-/-) mice, we show that memory CD8 T cell impairment is limited to the lungs and the lung-draining lymph nodes, where viral Ags are unusually persistent and abundant in these mice. Persistent Ag exposure results in prolonged activation of the AKT-mTORC1 pathway in Ag-specific CD8 T cells, favoring their development into effector memory T cells at the expense of central memory T cells, and inhibition of mTORC1 by rapamycin largely corrects the impairment by promoting central memory T cell development. The findings suggest that the prolonged AKT-mTORC1 activation driven by persistent Ag is a critical mechanism underlying the impaired memory CD8 T cell development and responses in the absence of CD4 T cells.

FIGURE 1

CD8 T cell recall defects differ in various organs of CD4^−/− mice. (A) Scheme of experimental procedures. (B–C) Numbers of 2C cells quantified with a clonotypic antibody (1B2) in the indicated sites in CD4^−/− and WT mice at 7 dpi and 30 dpi, respectively. (D–E) Number in BAL fluid in recall responses of adoptively transferred 2C cells (D) and endogenous Thy1.2⁺ SIY/K^b-specific CD8 T cells (SIY/K^b-dimer⁺ cells) (E). In D and E, the donor tissues are labeled. Error bars: SEM from 3–4 mice per group from one of two independent experiments. * P<0.05.

FIGURE 2

Memory CD8 T cell development differs in WT and CD4^−/− mice. The experimental procedures were the same as in Fig. 1A. (A–B) Percentage of T_EMP (CD27^hi CD62L^lo KLRG1^lo) 2C cells (A) and T_CMP (CD27^hi CD62L^hi KLRG1^lo) 2C cells (B) in the indicated organs in CD4^−/− and WT mice at 7 dpi. (C–D) Percentage of 2C T_EMP (CD27^hi CD62L^lo KLRG1^lo) cells (C) and 2C T_CMP (CD27^hi CD62L^hi KLRG1^lo) cells (D) in the indicated organs in CD4^−/− and WT mice at 9 dpi. (E–F) Absolute numbers of 2C T_EMP cells (E) and 2C T_CMP cells (F) in the indicated organs in CD4^−/− and WT mice at 9 dpi. (G-H) Percentage (G) and number (H) of 2C T_CM (CD62L^hi) cells in the indicated organs in CD4^−/− and WT mice at 30 dpi. Error bars: SEM from 4 mice per group from one of two experiments. * P<0.05.

FIGURE 3

Comparison of virus persistence and AKT phosphorylation in WT and CD4^−/− mice. (A) WT and CD4^−/− mice were infected intranasally with 100 pfu of WSN-SIY virus. BAL was harvested at the indicated dpi and virus titer measured by plaque assays. ND, not detected. (B–G) Naive 2C T cells were adoptively transferred into WT and CD4^−/− mice followed by intranasal infection with WSN-SIY. At 7 and 9 dpi, cells from the lung and spleen were stained for CD8, 2C TCR, CD27 and pAKT308. Shown are CD27 vs. pAKT308 staining profiles gating on CD8⁺ 2C TCR⁺ cells at 7 dpi (B) and 9 dpi (E), percentages of CD27^hi pAKT308⁺ 2C cells at 7 dpi (C) and 9 dpi (F), and MFI of pAKT308 of CD27^hi 2C cells at 7 dpi (D) and 9 dpi (G). (H) Naïve OTI and 2C cells were isolated from OTI rag1^−/− Thy1.1⁺ and 2C rag1^−/− Thy1.2⁺ mice, respectively. These cells were mixed at the 1 to 1 ratio and activated by either SIY or SIINFEKL peptides. Two days later, cells were stained for Thy1.1, Thy1.2, CD8 plus CD69 or pAKT308 or pAKT473. Shown are Thy1.1 vs. CD69 or pAKT308 or pAKT473 staining profiles gating on CD8⁺ cells. Error bars: SEM from 4 mice per group in one of two experiments. * P<0.05.

FIGURE 4

Rapamycin treatment enhances T_CM development. (A) Scheme of experimental procedures. CD4^−/− and WT mice were infected i.n. with 100 pfu of WSN-SIY virus. The SIY/K^b-specific endogenous memory CD8 T cells in the indicated tissues were analyzed 37 dpi by staining for CD8, CD62L and SIY/K^b-dimer. (B–E) The percentage of CD8⁺ CD62L^hi SIY/K^b-specific T_CM cells among total CD8⁺ cells at 37 dpi in the lung (B), DLN (C), spleen (D), NDLN (E) in CD4^−/− and WT mice with and without rapamycin treatment. Error bars: SEM from 5–6 mice per group from one of three representative experiments. * P<0.05.

FIGURE 5

Rapamycin treatment partially corrects CD8 T cell recall defects in CD4^−/− mice. The mice were injected i.v. with bone marrow derived dendritic cell pulsed with SIY peptides (BMDC-SIY) 37 dpi (Fig. 4A). The numbers of T cells specific for SIY/K^b and expressing IFN-γ were analyzed 5 days later. (A–E) The number of SIY/K^b-specific CD8 T cells in the lung (A), spleen (B), mediastinal LN (C) and inguinal LN (D) of CD4^−/− and WT mice with or without rapamycin treatment. (E) The number of IFN-γ⁺ CD8 T cells in the spleen of CD4^−/− and WT mice with or without rapamycin treatment. Error bars: SEM from 5–6 mice per group from one of three representative experiments. * P<0.05.

FIGURE 6

Schematic comparison of memory CD8 T cell development and responses in WT and CD4^−/− mice and how rapamycin restores memory CD8 T cell development and responses in CD4^−/− mice.