CD8(+) T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons

Abstract

Recurrent herpes simplex virus type 1 (HSV-1) disease usually results from reactivation of latent virus in sensory neurons and transmission to peripheral sites. Therefore, defining the mechanisms that maintain HSV-1 in a latent state in sensory neurons may provide new approaches to reducing susceptibility to recurrent herpetic disease. After primary HSV-1 corneal infection, CD8(+) T cells infiltrate the trigeminal ganglia (TGs) of mice, and are retained in latently infected ganglia. Here we demonstrate that CD8(+) T cells that are present in the TGs at the time of excision can maintain HSV-1 in a latent state in sensory neurons in ex vivo TG cultures. Latently infected neurons expressed viral genome and some expressed HSV-1 immediate early and early proteins, but did not produce HSV-1 late proteins or infectious virions. Addition of anti-CD8alpha monoclonal antibody 5 d after culture initiation induced HSV-1 reactivation, as demonstrated by production of viral late proteins and infectious virions. Thus, CD8(+) T cells can prevent HSV-1 reactivation without destroying the infected neurons. We propose that when the intrinsic capacity of neurons to inhibit HSV-1 reactivation from latency is compromised, production of HSV-1 immediate early and early proteins might activate CD8(+) T cells aborting virion production.

Figure 1

HSV-1 replication in day 7 TG cultures. TGs were excised 7 d after HSV-1 corneal infection, and single cell suspensions were prepared and pooled. Cultures (1 TG equivalent of TG cells) were incubated with 300 μg/ml of control mAb (○), 150 μg/ml of rat anti–mouse CD4 mAb (▿), 150 μg/ml of rat anti–mouse CD8α mAb (⋄), 150 μg each of anti-CD4 and anti-CD8 mAbs (•), or exogenous CD8⁺ T cells obtained from the LNs of Balb/c mice 7 d after HSV-1 corneal infection (□). On alternate days, 150 μl of culture supernatant fluid was removed from each culture and replaced by fresh medium of the same composition. The supernatant fluids were assayed for infectious HSV-1 with a standard virus plaque assay. Pooled data from three assays (n = 10) are presented as the mean PFU/TG ± SEM.

Figure 2

CD8⁺ T cells present in the TG 14 d after infection can block HSV-1 reactivation from latency. TGs were excised 14 d after HSV-1 corneal infection, and single cell suspensions were prepared and pooled. Cultures (1 TG equivalent) were incubated in culture medium containing 150 μg/ml of control mAb (▴), 150 μg/ml of anti-CD8α mAb (•); or cultures were incubated for 5 d in culture medium, followed by 4 d of incubation in medium containing 150 μg of anti-CD8α mAb (▵). Samples of culture supernatant fluids were assayed for infectious HSV-1 titers and scored as positive (reactivation) or negative. Pooled data from three assays (n = 15) are presented as the percentage of cultures that reactivated.

Figure 3

The CD8⁺ T cells present in the TG 14 d after HSV-1 corneal infection can control viral gene expression in latently infected neurons. TGs were excised 14 d after HSV-1 corneal infection, and single cell suspensions were prepared and pooled. Cultures (1 TG equivalent) were incubated for 5 d in culture medium followed by 3 d incubation with control mAb (first two pictures in each row) or anti-CD8α mAb (last two pictures in each row). The cultures were then stained simultaneously for HSV-1 genome by FISH (green), and by immunofluorescence (red) with a polyspecific anti–HSV-1 antibody (b and d), or with monospecific antibody to the HSV-1 IE protein ICP4 (f and h), the HSV-1 early protein ICP8 (j and l), or the HSV-1 late protein gC (n and p). The cultures were examined by confocal microscopy and representative fields are depicted in phase–contrast and fluorescence in sequential photomicrographs (i.e., a and b, c and d, etc.). Areas that were positive for both viral genome and viral protein appear yellow. Neurons from TGs of noninfected mice did not stain by either the FISH or the immunofluorescence techniques (not shown). Bar (p), 10 μm.

Figure 4

CD8⁺ T cells present in the TG 14 d after HSV-1 corneal infection prevent the expression of mRNA for the HSV-1 late gene gC. TGs were excised 14 d after HSV-1 corneal infection, and single cell suspensions were prepared and pooled. 13 cultures were prepared (1 TG equivalent/culture) and incubated for 5 d in culture medium. The cells were removed from five cultures, and RNA was extracted. The remaining cultures were incubated for an additional 3 d with control mAb or with anti-CD8α mAb. RNA was extracted from these cultures on day 8. All RNA preparations were treated with DNase. A portion of the RNA from each culture was reverse transcribed (RT+), and HSV gC cDNA was amplified through 35 cycles of PCR, or HPRT cDNA was amplified through 26 cycles of PCR as described in Materials and Methods. To control for contaminating genomic DNA, PCR was performed on a portion of the RNA from each culture without RT (RT−). HSV-1 genomic DNA contamination was heaviest in cultures treated with anti-CD8α mAb, probably due to virus replication in these cultures.

Figure 5

CD8⁺ T cells remaining in the TG 34 d after HSV-1 corneal infection can delay HSV-1 reactivation but require supplementation to block reactivation. TGs were excised 34 d after HSV-1 corneal infection, and single cell suspensions were prepared and pooled. Cultures (1 TG equivalent) were incubated in medium containing 150 μg/ml of control mAb (•), or 150 μg/ml of rat anti–mouse CD8α mAb (○). Other cultures received exogenous, highly enriched CD8⁺ T cells from HSV-1–immunized mice and were incubated in medium alone from day 1 to day 5, followed by control mAb (▴) or anti-CD8α mAb (▵) from day 5 to day 10. Samples of culture supernatant fluids were assayed for infectious HSV-1 titers and scored as positive (reactivation) or negative. Pooled data from two assays (n = 16) are presented as the PFU/culture (A) or percentage of cultures in which HSV-1 reactivated (B).

Figure 5

CD8⁺ T cells remaining in the TG 34 d after HSV-1 corneal infection can delay HSV-1 reactivation but require supplementation to block reactivation. TGs were excised 34 d after HSV-1 corneal infection, and single cell suspensions were prepared and pooled. Cultures (1 TG equivalent) were incubated in medium containing 150 μg/ml of control mAb (•), or 150 μg/ml of rat anti–mouse CD8α mAb (○). Other cultures received exogenous, highly enriched CD8⁺ T cells from HSV-1–immunized mice and were incubated in medium alone from day 1 to day 5, followed by control mAb (▴) or anti-CD8α mAb (▵) from day 5 to day 10. Samples of culture supernatant fluids were assayed for infectious HSV-1 titers and scored as positive (reactivation) or negative. Pooled data from two assays (n = 16) are presented as the PFU/culture (A) or percentage of cultures in which HSV-1 reactivated (B).