Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells

Abstract

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.

Keywords: CD8 T cells; apoptosis; apoptosis inhibition; cytomegalovirus; immune evasion.

Fig. 1.

CMVs lacking antiapoptotic proteins UL36/M36 are susceptible to CD8 T cells' control. (A and B) MRC-5 cells were infected with indicated HCMV variants at a multiplicity of infection (MOI) of 0.1 and cocultured with HLA-A2–restricted pp65-specific CD8 T cells at 3:1 (E:T). (A) Still frames from live-cell imaging of coculture. Reporter viruses expressed mNeonGreen (green). Propidium iodide (PI, purple) was used to label dead cells. Time 00:00 is 24 h after T cell addition. (Scale bar, 50 μm.) (B) Time-lapse imaging was used to quantify fraction of dead cells in the infected population for each frame by counting the dead cells within the infected population. The cumulative n of such events for 48 h post T cells’ coculture is plotted. Each symbol represents one time-lapse imaging field. Data are compiled from at least three independent experiments. (C–E) BFF2 skin fibroblast cells were infected with HCMV at an MOI of 0.1 and cocultured with autologous HLA-B35–restricted pp65-specific CD8 T cells at 4:1 (E:T). (C) Representative images from the isogeneic coculture show HCMV-infected BFF2 cells 6 d post CD8 T cell coculture (control = without T cells). (Scale bar, 400 μm.) (D) HCMV-infected BFF2 cells were analyzed 6 d post CD8 T cell coculture with flow cytometric analysis. (E) Supernatants were collected from infected BFF2 cocultured with autologous CD8 T cells 6 d post infection (dpi), and titrated with standard plaque assay on BFF2 cells. (F and G) BL/6 MEF cells were infected with indicated viruses and cocultured at 4:1 (E:T) with syngeneic CD8 T cells (CD44+CD62L−) sorted from spleen of BL/6 animals latently infected with MCMV. (F) MEFs were infected at an MOI of 0.2, 24 h prior to CD8 T cell coculture. Fraction of dead cells in the infected population for initial 48 h post T cells’ coculture is plotted. Each symbol represents one time-lapse imaging field. Data are compiled from at least four independent experiments. (G) MEFs were infected at an MOI of 0.1 and cocultured with CD8 T cells. Cells were analyzed 6 d post CD8 T cell coculture with flow cytometric analysis to quantify mCherry positive (infected) cells. Panels represent typical data from at least two independent experiments. Data with error bars depict mean ± SD. B and F show statistical significance calculated with Dunn’s post test following Kruskal-Wallis test. **P < 0.01, ****P < 0.0001, P > 0.05 not significant (n.s.).

Fig. 2.

Control of MCMV.∆M36 by different epitope-specific CD8 T cells. MEFs were infected with indicated viruses at an MOI of 0.1 and cocultured with epitope-specific CD8 T cells from latently infected BL/6 mice. (A) MEFs were infected with MCMV^r (filled bars) or MCMV^r.∆M36 (empty bars) and cocultured with CD8 T cells at 1:1 (E:T) or in their absence (control). (B) Representative fluorescent microscopy images of coculture wells from A at 6 dpi. (C–H) MEFs were infected with indicated viruses at an MOI of 0.1 and cocultured with either OT-I cells (C–F) or M38-specific CD8 T cells (G and H). Both OT-I and M38-specific CD8 T cells were isolated from BL/6 animals latently infected with an MCMV-inducing inflationary responses against either epitope. D, F, and H show representative images at 4 dpi from C, E, and F, respectively. (Scale bar, 3.4 mm.) Panels represent typical data from at least two independent experiments. Data with error bars depict mean from biological triplicates ± SD.

Fig. 3.

In vivo control of ∆M36 mutant by CD8 T cells. (A and B) OT-I cells (10⁴) were adoptively transferred to RAG2^−/−γc^−/− host and activated with SIINFEKL peptide plus Poly(I:C). Animals were infected with MCMV via i.p. route and sacrificed 3 dpi. (A) Prior to sacrification, animals were bled to ensure activation and proliferation of OT-I cells. (B) Virus replication in spleen and liver was assayed with plaque assay. Each symbol represents one mouse, and the horizontal line represents the median value and error bars show SD. Data pooled from two experiments. (C–F) OT-I cells were adoptively transferring to BL/6 host and activated by infection with MCMV-IE2ova. Then, spleens from donors were isolated 7 dpi, and OT-I cells were sorted. After sorting, 10⁶ primed OT-I cells were transferred to RAG2^−/−γc^−/− host 24 h prior to infection with 10⁶ pfu of MCMV via i.v. route. Animals were sacrificed 24 h post infection (hpi), and the spleen was isolated. Flow cytometric analysis was performed to quantify percent infected cells among spleen endothelial cells (C and D) and spleen mesenchymal cells (E and F). Data with error bars depict mean ± SD. Statistical significance was calculated with Kruskal-Wallis test followed by Dunn’s postanalysis. *P < 0.05, **P < 0.01, P > 0.05 not significant (n.s.).

Fig. 4.

CD8 T cells control the MCMV lacking M36 by inducing apoptosis via caspase-8. MEFs were infected at an MOI of 0.1 and cocultured with CD8 T cells at 4:1 (E:T). (A) Supernatant collected 6 dpi from wells treated with either vehicle or zVADfmk. (B) MEFs from Casp8^+/−Ripk3^−/− or Casp8^−/−Ripk3^−/− mice were infected with indicated viruses and cocultured with CD8 T cells. (C) MEFs were infected with indicated MCMV-expressing FADD^DN and cocultured with CD8 T cells. (D) Supernatants were collected 6 dpi from coculture of infected MEF and either Prf1^−/− or Prf1^+/+ CD8 T cells stimulated with IL2. All of the experiments were performed at least twice, and typical data are shown. Error bars represent mean from biological triplicates ± SD.