Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Abstract

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.

FIG. 1.

CD8⁺ T cells control the frequency of cells that reactivate or carry the viral genome. Data points reflect the mean of all experiments ± the standard error (n = number of experiments; five mice per experiment). The horizontal line indicates 63%, which was used to calculate the frequency of cells reactivating virus or containing viral DNA by the Poisson distribution. (A) Ex vivo reactivation. The frequencies of cells reactivating in serial dilutions of PEC or splenocytes from B6 and CD8^−/− mice at various days (d) postinfection with γHV68 are shown. To detect preformed infectious virus, duplicate samples were mechanically disrupted and plated (disrupt). On the y axis is the percentage of wells positive for a cytopathic effect (CPE; 24 wells per dilution). For PEC, the statistical significances of differences between CD8^−/− and B6 mice were P = 0.004, 0.0001, and 0.0001 at 16, 28, and 42 days, respectively. For the spleen, the statistical significances of differences between CD8^−/− and B6 mice were P = 0.06 and 0.03 at 28 and 42 days, respectively. At 28 days, the number of PEC reactivating from latency was 1 in 600 and the maximum number of persistently infected cells was 1 in 150,000 (see Fig. 2 results for calculations). (B) Frequency of cells containing viral DNA. PCR was used to detect the γHV68 genome in several dilutions of PEC or splenocytes harvested at 42 days postinfection. On the y axis is the percentage of reaction mixtures positive for viral DNA at each cell dilution (12 wells per dilution per experiment). For both PEC and the spleen, the statistical significance of the difference between CD8^−/− and B6 mice was P < 0.0001.

FIG. 2.

IFN-γ controls γHV68 latency in PEC but not in the spleen. Experiments with IFN-γ^−/− mice are identical to those described in the legend to Fig. 1. B6 data (from Fig. 1) are included for comparison. (A) Ex vivo reactivation. On the y axis is the percentage of wells positive for a cytopathic effect (CPE). For PEC, statistical significances of differences between IFN-γ^−/− and B6 mice were P = 0.007, 0.0001, and 0.001 at 16, 28, and 42 days (d), respectively. (B) Frequency of cells containing viral DNA at 42 days postinfection. On the y axis is the percentage of reactions positive for viral DNA at each cell dilution. For both PEC and spleens, the statistical significance of the difference between IFN-γ^−/− and B6 mice was P < 0.0001.

FIG. 3.

Perforin controls γHV68 latency in the spleen. Experiments with Pfp^−/− mice are identical to those described in the legend to Fig. 1. B6 data (from Fig. 1) are included for comparison. (A) Ex vivo reactivation. On the y axis is the percentage of wells positive for a cytopathic effect (CPE). For PEC, statistical significances of differences between Pfp^−/− and B6 mice were P = 0.008, 0.0005, and 0.001 at 16, 28, and 42 days (d), respectively. For the spleen, statistical significances of differences between Pfp^−/− and B6 mice were P = 0.005, 0.0001, and 0.06 at 16, 28, and 42 days, respectively. (B) Frequency of cells containing viral DNA at 42 days postinfection. On the y axis is the percentage of reaction mixtures positive for viral DNA at each cell dilution. For the spleen, the statistical significance of the difference between Pfp^−/− and B6 mice was P < 0.0001.

FIG. 4.

Summary of γHV68 infection in B6, CD8^−/−, Pfp^−/−, and IFN-γ^−/− mice at 42 days postinfection. (A) Latent infection. LD reactivation data from nondisrupted samples from Fig. 1 to 3 are summarized. (B) Persistent infection. LD reactivation data from disrupted samples from Fig. 1 to 3 are summarized. (C) Frequencies of reactivation and frequencies of cells harboring γHV68 DNA. Symbols: ∗, frequencies calculated on the basis of the Poisson distribution of results from Fig. 1 to 3; †, number of cells reactivating = total number of cells recovered × frequency of cells reactivating; ‡, number of γHV68⁺ cells = total number of cells × frequency of cells γHV68⁺;. §, reactivation efficiency = (frequency of reactivation/frequency of cells γHV68⁺) × 100; ¶, presence of persistent virus as detected by LD reactivation of disrupted samples; ∗∗, quantity of persistent virus is 1 PFU/200,000 cells; ‡‡, reactivation efficiency could not be calculated because LD reactivation curve did not reach 63.2%. CPE, cytopathic effect.