Memory CD8⁺ T cells can outsource IFN-γ production but not cytolytic killing for antiviral protection

Abstract

Immunization with vaccinia virus (VACV), the virus comprising the smallpox vaccine, induces memory CD8(+) T cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ECTV) in mice. Memory CD8(+) T cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (IFN-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. We show that protection from ECTV infection after VACV immunization depends on the initial memory cell frequency and ability of expanded secondary effectors to kill infected targets in a perforin-dependent manner. Although IFN-γ is essential for antiviral protection, it can be produced by either secondary effectors or concomitant primary effector CD8(+) T cells recruited to the response. Thus, during lethal virus challenge, memory CD8(+) T cells are required for cytolytic killing of infected cells, but primary effectors can play important roles by producing IFN-γ.

Figure 1. M-WT but not M-IFN-γ^−/− CD8⁺ T cells efficiently protect IFN-γ^−/− mice from mousepox

A) IFN-γ^−/− mice received 5×10⁶ N-WT, M-WT or M-IFN-γ^−/− CD8⁺ T cells and infected with ECTV. Survival was monitored. The experiment is representative of three, where n=5 for every group except M-IFN-γ^−/− where n=6. B) The mice in A were weighed daily. C) IFN-γ^−/− mice that received 5×10⁶ N-WT, M-WT or M-IFN-γ^−/− CD8⁺ T cells were infected with ECTV. Seven days p.i., mice were killed and virus titers determined in liver. Data corresponds to five mice per group ^± SEM and is representative of two independent experiments. D) As in C but the virus titers were determined in spleen. Also see Figure S1 for liver pathology.

Figure 2. M-WT and M-IFN-γ^−/− CD8⁺ T cells respond strongly in the liver and spleen of IFN-γ^−/− mice

IFN-γ^−/− mice received 5×10⁶ N-WT, M-WT or M-IFN-γ^−/− CD8⁺ T cells. One day later, the mice were infected with ECTV and at 7 dpi the mononuclear cells infiltrating the livers (A-F) and splenocytes (G-L) were incubated for 5 h with TSYKFESV or without peptide and the indicated parameters were determined. Data corresponds to an experiment with five mice per group ^± SEM, with exception of N-WT that had 2 mice/group because three mice died at 7 dpi. Data are representative of two experiments. Data are represented as a mean ± SEM. Representative flow cytometry plots are shown in Figure S2.

Figure 3. Memory CD8⁺ T cells deficient in IFN-γ but not in Prf protect susceptible mice from lethal mousepox

CD8⁺ cells were magnetically purified from pooled LNs and spleens from donor naïve or VACV immune B6, IFN-γ^−/− or Prf^−/− mice. 10⁶, 2.5×10⁶ or 5×10⁶ CD8⁺ purified CD8⁺ T cells from each type of donor cell were transferred i.v. into groups of 5 B6.D2-D6 mice. One day later the mice were infected with ECTV and survival was monitored. A) The frequency of CD8⁺ T cells specific for the immunodominant determinant TSYKFESV was determined by staining with K^b- TSYKFESV dimers. B) Kaplan-Meier survival curve for mice transferred with ~65,000 K^b- TSYKFESV⁺ cells as determined from the results in A. C) Kaplan-Meier survival curve for mice transferred with ~130,000 K^b- TSYKFESV⁺ cells as determined from the results in A. All mice transferred with 10⁶ M-WT or M-Prf^−/− (~25,000 K^b-TSYKFESV⁺ cells) succumbed to the infection, all the mice transferred with 5×10⁶ M-IFN-γ^−/− (~270,000 K^b-TSYKFESV⁺ cells) survived and are not displayed graphically. D,E) Virus titers at 7 dpi in livers (D) and spleens (E) from B6.D2-D6-Thy1.1⁺ mice that received 5×10⁶ N-WT CD8⁺ T cells, or enough M-WT, M-IFN-γ^−/− or M-Prf^−/− CD8⁺ T cells to contain ~75,000 K^b-TSYKFESV⁺ cells. Data are represented as a mean ± SEM and are representative of two independent experiments.

Figure 4. Endogenous Prf but not IFN-γ in memory CD8⁺ T cells is required for the early control of ECTV LH spread

A-B) Virus titers at 7 dpi in livers (A) and spleens (B) from B6.D2-D6 mice that received 5×10⁶ N-WT CD8⁺ T cells, or enough MWT, M-IFN-γ^−/− or M-Prf^−/− CD8⁺ T cells to contain ~75,000 K^b-TSYKFESV⁺ cells. Data are representative of three independent experiments. C-J) N-WT, M-WT, M-IFN-γ^−/− or M-Prf^−/− CD8⁺ T cells were labeled with CFSE to identify divided donor cells. 5×10⁶ N-WT CD8⁺ T cells or a number of M-WT, M-IFN-γ^−/− or M-Prf^−/− CD8⁺ T cells that contained ~75,000 K^b-TSYKFESV⁺ cells were transferred into B6.D2-D6-Thy1.1⁺ mice. One day later, the mice were infected with ECTV and at 4 dpi D-LN cells were counted and analyzed by flow cytometry. The indicated parameters were analyzed. Data are represented as a mean ± SEM and are representative of two similar experiments. Representative flow cytometry plots are shown in (E). Also see Figure S3 for Cidofovir treated mice and confocal microscopy, respectively.

Figure 5. Endogenous Prf but not IFN-γ in memory CD8⁺ T cells is required for the late control of ECTV in the liver and spleen

B6.D2-D6-Thy1.1⁺ mice received 5×10⁶ N-WT CD8⁺ T cells, or enough M-WT, M-IFN-γ^−/− or M-Prf^−/− CD8⁺ T cells to contain ~75,000 K^b-TSYKFESV⁺ cells. One day later, the mice were infected with ECTV and at 7 dpi: A-I) Liver infiltrating mononuclear cells (J-R) and splenocytes were incubated for 5 h with TSYKFESV or without peptide and the indicated parameters were determined. Data correspond to five mice per group with exception of uninfected mice that had 4 mice/group and N-WT that had 3 mice/group because two mice died at 7 dpi. Data are represented as a mean ± SEM and are representative of two experiments using B6.D2-D6 Thy1.1⁺ recipients (shown) and a third experiment using B6.D2-D6 mice as recipients. See Figure S4 for representative flow cytometry plots and for higher ECTV doses and VACV and LCMV infection.

Figure 6. N-WT T cells can complement M-IFN-γ^−/− CD8⁺ T cells to protect IFN-γ^−/− mice from mousepox

IFN-γ^−/− mice were transferred with 2.5×10⁶ M-IFN-γ^−/− CD8⁺ T cells and/or with 5×10⁷ leukocytes (pooled splenocytes, LN cells and liver mononuclear cells) from naïve B6 mice that had been depleted or not of CD4⁺, CD8⁺, CD4⁺ and CD8⁺ T cells, or NK cells as indicated. One day after transfer, the mice were challenged with ECTV in the footpad. A) Survival. B) Weight loss. Data correspond to the mean of five mice per group ^± SEM and is representative of two independent experiments. Also see Figure S5.