The herpes simplex virus 1 latency-associated transcript promotes functional exhaustion of virus-specific CD8+ T cells in latently infected trigeminal ganglia: a novel immune evasion mechanism

Abstract

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB(498-505) tetramer(+)) CD8(+) T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT(+) virus compared to LAT(-) virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT(+) and LAT(-) viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT(+) virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT(-) TG having more functional HSV-gB(498-505) tetramer(+) CD8(+) T cells compared to LAT(+) TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8(+) T cells in latently infected TG, resulting in increased virus reactivation.

Fig. 1.

Higher percentages and numbers of total CD8⁺ T cells detected in LAT⁺ TG compared to LAT⁻ TG during latent herpesvirus infection. Three groups of B6 mice (n = 10) were either left uninfected (Mock) or were ocularly infected with 2 × 10⁵ PFU/eye of (i) LAT-null virus mutant dLAT2903 (LAT⁻) or with (ii) its marker-rescued virus, dLAT2903R (LAT⁺) (53). Both TG were harvested from each mouse either 11 days postinfection (5 mice) or 35 days postinfection (5 mice). The 10 TG were pooled and treated with collagenase I, and the numbers (n^br) and percentages of CD3⁺ T cells and CD3⁺ CD8⁺ T cells were determined in TG cell suspensions by FACS. (A). Dot plot showing percentages of CD3⁺ CD8⁺ T cells (left panel) and the average number ± SD/TG (right panel) of CD3⁺ CD8⁺ T cells per TG on day 11 postinfection. (B) Dot plot of the percentages of CD3⁺ CD8⁺ T cells (left panel) and the average number ± SD/TG (right panel) of CD3⁺ CD8⁺ T cells per TG on day 35 postinfection. Each histogram and dot plot represents seven consecutive independent experiments. The bar graphs show the means and SD of the seven independent experiments at each time point. *, P < 0.05 for cell numbers in LAT⁺ TG versus LAT⁻ TG.

Fig. 2.

The presence of increased CD8⁺ T cells in the TG of LAT⁺- versus LAT⁻-infected mice is independent of mutant and HSV-1 strain. Groups of B6 mice (n = 5) were mock infected or infected ocularly with 2 × 10⁵ PFU/eye of LAT⁺ or LAT⁻ viruses derived from (A) HSV-1 strain McKrae (LAT⁺ LAT3.3A versus LAT⁻ dLAT2903) or (B) HSV-1 strain 17 (LAT⁺ wt 17syn+ versus LAT⁻ 17N/H. On days 11 and 35 postinfection in panel A and on day 35 postinfection in panel B, the 10 TG in each group were pooled and processed as in Fig. 1. Each bar represents the mean and SD of five independent experiments. n^br, number. *, P < 0.05.

Fig. 3.

More exhausted HSV-specific CD8⁺ T cells are detected in LAT⁺ TG versus LAT⁻ TG. Groups of five B6 mice were ocularly infected as described above with wt McKrae (LAT⁺) or dLAT2903 (LAT⁻). Ten TG/group were harvested 35 days postinfection, pooled, and processed as described above. The cells were triple stained with FITC-labeled anti-PD-1, PerCP–anti-CD8 T cells, and PE-labeled HSV-gB_498–505/H-2^d tetramers. The numbers (n^br) and percentages of total CD8⁺ T cells and HSV-gB_498–505 epitope-specific CD8⁺ T cells expressing PD-1 and the PD-1 expression levels were determined by FACS. (A) Representative dot plot of total PD-1⁺ CD8⁺ T cells in latently infected LAT⁺ versus LAT⁻ TG. (B) Histograms of the average number of total PD-1⁺ CD8⁺ T cells (left) and the average mean fluorescence intensity (MFI) of PD-1 (right). (C) Representative dot plot of HSV-gB_498–505 epitope-specific CD8⁺ T cells expressing PD-1 in latently infected LAT⁺ versus LAT⁻ TG. (D) Average number/TG of HSV-gB_498–505 epitope-specific CD8⁺ T cells. (E) Percentage of HSV-gB_498–505 epitope-specific CD8⁺ T cells. (F) Average number of HSV-gB_498–505 epitope-specific CD8⁺ T cells expressing PD-1. (G) Percentage of HSV-gB_498–505 epitope-specific CD8⁺ T cells expressing PD-1. (H) Average number of HSV-gB_498–505 epitope-specific CD8⁺ T cells not expressing PD-1. Each bar in panels B to G represents the mean and SD of five independent experiments. *, P < 0.05.

Fig. 4.

PD-1 expression in LAT3.3A and in LAT⁺ and LAT⁻ HSV-1 strain 17syn+ viruses. Groups of five B6 mice were ocularly infected as described above with 17syn+ (LAT⁺), 17N/H (LAT⁻), LAT3.3A (LAT⁺), or dLAT2903 (LAT⁻). Ten TG/group were harvested 35 days postinfection, pooled, and processed as described above. The cells were triple stained with FITC-labeled anti-PD-1, PerCP–anti-CD8, and PE-labeled HSV-gB_498–505/H-2^d tetramers and analyzed by FACS as described above. The average numbers (n^br) of HSV-gB_498–505 epitope-specific CD8⁺ T cells expressing PD-1 are shown. (A) McKrae-derived LAT⁺ LAT3.3A (expresses LAT nt 1 to 1499) versus McKrae-derived LAT⁻ dLAT2903. (B) 17syn+-derived LAT⁺ wt versus 17syn+-derived LAT⁻ 17N/H. Each bar shows the mean and SD of three independent experiments. *, P < 0.05.

Fig. 5.

HSV-gB_498–505-specific CD8⁺ T cells from LAT⁺ TG have reduced cytokine production and cytolytic activity. Two groups of B6 mice (n = 5) were ocularly infected with wt McKrae (LAT⁺) or dLAT2903 (LAT⁻) as described above. At 35 days postinfection, the 10 TG in each group were pooled, and single-cell suspensions were prepared as described above. For intracellular cytokine staining, the cells were stimulated with gD_498–505 peptide (10 μg/ml) or PMA + Iono (1 μg/ml) (positive control) or mock stimulated (negative control) at 37°C for 6 h in the presence of Golgi plug (BD Biosciences). The cells were then stained with PerCP-conjugated anti-mouse CD8 for 30 min, permeabilized, and stained with PE-labeled anti-mouse IFN-γ or TNF-α antibodies, as recommended by the manufacturer. For the CD107 degranulation assay, the cells were mock stimulated or stimulated with HSV-gB_498–505 peptide or PMA + Iono (positive control) at 37°C for 6 h in the presence of 0.7 μl/ml of BD Golgi-Stop and 10 μl of CD107^a/b-FITC, washed with FACS buffer, and stained with PerCP-conjugated anti-mouse CD8 MAb at 4°C. The percentages and average numbers (N^br)/TG of HSV-gB_498–505 epitope-specific CD8⁺ T cells producing IFN-γ, TNF-α, or expressing CD107^a/b were determined by FACS. (A) Representative dot plots of intracellular IFN-γ⁺ CD8⁺T cells. (B) Percentage of HSV-gB_498–505 epitope-specific CD8⁺ T cells producing IFN-γ, TNF-α, or CD107^a/b per TG. (C) Average number/TG of HSV-gB_498–505-epitope-specific CD8⁺ T cells producing IFN-γ, TNF-α, or CD107^a/b. In panels B and C, each bar represents the average ± SD of five experiments. *, P < 0.05.

Fig. 6.

Upregulation of MHC-I and PD-L1 in neuroblastoma cell lines expressing LAT. (A) Parental Neuro2A cells, JWLAT (LAT⁺) cells, and JW-ΔLAT (LAT⁻) cells were stained with FITC-labeled anti-mouse MHC-I MAb (clone M1/42) (bold line) or an FITC-labeled isotype control (light line). The mean fluorescence intensities (MFI) of MHC-I are shown. (B) Parental C1300 cells or DC-LAT6 (LAT⁺) or DC-ΔLAT311 (LAT⁻) cells were treated as in panel A. The results are presented as overlaid histogram plots. (C) The histogram bars represent the mean and SD of the RNA fold increase compared to the parental control cell lines. (D) Western blot of PD-L1 in total cell lysates of JWLAT LAT⁺-expressing Neuro2A cells and JW-ΔLAT LAT⁻ Neuro2A cells. (E) Relative PD-L1 expression in JWLAT LAT⁺-expressing Neuro2A cells and JW-ΔLAT LAT⁻ Neuro2A cells as determined by FACS. (F) Mean fluorescent intensity of PD-L1 in the Neuro2A cell lines from panel E. Each bar is the mean ± SD of three independent experiments each done in triplicate. (G) PD-L1 from total TG cells from LAT⁺ versus LAT⁻ latently infected mice. Each bar represents the mean ± SD of the fluorescence intensity from five experiments each done in triplicate. *, P < 0.05. The experiments shown in panels C to G were done using “bulk cultures” (rather than cloned cells) expressing LAT, as described in the text.