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. 2010 Aug;84(16):8241-9.
doi: 10.1128/JVI.00784-10. Epub 2010 Jun 9.

CD4 T-cell help programs a change in CD8 T-cell function enabling effective long-term control of murine gammaherpesvirus 68: role of PD-1-PD-L1 interactions

Peter Dias  1 , Francesca Giannoni, Lian Ni Lee, Dongun Han, Sorah Yoon, Hideo Yagita, Miyuki Azuma, Sally R Sarawar
Affiliations

CD4 T-cell help programs a change in CD8 T-cell function enabling effective long-term control of murine gammaherpesvirus 68: role of PD-1-PD-L1 interactions

Peter Dias et al. J Virol. 2010 Aug.

Abstract

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II(-/-) (CII(-/-)) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII(-/-) mice caused a significant reduction in lung viral titers, in contrast to those from control CII(-/-) mice. Anti-CD40 treatment also greatly prolonged survival of infected CII(-/-) mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII(-/-), CD40(-/-), or CD80/86(-/-) mice, compared with that in wild-type or CD28/CTLA4(-/-) mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.

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Figures

FIG. 1.
FIG. 1.
Anti-CD40 treatment induces a change in CD8 T-cell function. Groups of donor or recipient MHC class II−/− (CII−/−) mice or donor wild-type (WT) mice were infected with MHV-68 (5 × 104 PFU intranasally [i.n.]). Donor class II−/− mice were treated with either 100 μg/mouse anti-CD40 or isotype control antibody i.v. on days 1 and 15 postinfection. At day 18 after infection, donor mice were killed, and CD8 T cells were purified by negative selection from pooled lymph node and spleen cells from each group. A total of 3 × 106 purified CD8 cells were administered intravenously to the recipient mice, which had been lightly irradiated (300 rads) 1 day earlier. Virus titers in the lungs were determined by plaque assay at day 42 after infection. Data are pooled from two independent experiments, and each symbol represents the titer for an individual mouse. There was a highly significant difference between the lung virus titers in mice that had received cells from control antibody-treated and anti-CD40-treated CII−/− donors (P < 0.0001, Mann-Whitney rank sum test) or wild-type donors (P = 0.003). The dashed line represents the limit of detection.
FIG. 2.
FIG. 2.
Anti-CD40 treatment increases survival of MHV-68-infected MHC class II−/− mice. Groups of 5 MHC class II−/− mice were infected with MHV-68 and treated with either 100 μg/mouse anti-CD40 or isotype control antibody i.v. on days 1 and 15 postinfection. Control groups of wild-type mice were also infected with MHV-68. Mice were observed for 125 days after infection. Data are representative of two independent experiments that gave similar results. There was a highly statistically significant difference in survival between anti-CD40-treated mice and control class II−/− mice (P < 0.001, log rank [Mantel Cox] test).
FIG. 3.
FIG. 3.
Absence of CD94/NKG2A does not prevent viral reactivation in CD4 T-cell-deficient mice. Groups of C57/BL/6 (B6) mice and DBA/1 and DBA/2 mice (lacking surface expression of NKG2A) were infected with MHV-68 intranasally and depleted of CD4 T cells by treatment every 2 or 3 days with monoclonal antibody (MAb) GK1.5 (0.5 mg/ml intraperitoneally [i.p.]) for 14 days and weekly thereafter. Control groups of infected mice were not depleted of CD4 cells. Viral titers in lung homogenates were determined by plaque assay on NIH 3T3 cell monolayers. The symbols show the titers from individual animals, and the black bar shows the mean for each group of mice. The dashed line represents the limit of detection. The data are representative data from two independent experiments that gave similar results.
FIG. 4.
FIG. 4.
Increased expression of the inhibitory receptor PD-1 on the surfaces of CD8 T cells in the lungs of mice lacking CD4 T cells and its role in viral reactivation. MHC class II−/− mice (which are CD4 T cell deficient) or wild-type C57BL/6 mice were infected i.n. with MHV-68. Infiltrating cells were harvested from the lung by bronchoalveolar lavage via the trachea at day 50 postinfection and dual stained with FITC-conjugated anti-CD8 and PE-conjugated anti-PD-1. (A) Representative dot plots of PD-1 and CD8 expression in BAL fluid samples from wild-type and MHC class II−/− (CII−/−) mice. (B) Mean percentage of CD8 T cells that were positive (+ve)for PD-1 in BAL fluid samples from wild-type and MHC class II−/− mice. Mean percentages plus standard deviations (SDs) (error bars) are shown. Similar results were obtained in two independent experiments. The values for the two groups of mice were statistically highly significantly different (P < 0.001, Student's t test) (***). (C) Blocking PD-1-PD-L1 interactions reduces reactivation of MHV-68 in MHC class II−/− mice. Groups of 4 or 5 MHC class II−/− mice were infected intranasally with MHV-68 and treated with 0.2 mg/mouse i.p. of either anti-PD-1 (RMP114), anti-PD-L2 (TY25), or anti-PD-L1 (MIH5) antibody every 2 or 3 days from day 35 postinfection onwards. Control groups were treated with rat Ig (0.2 mg/mouse every 2 or 3 days). Groups of wild-type mice were also infected as additional controls. Lung virus titers were determined at day 50 after infection by plaque assay. The symbols represent titers for individual mice, and the horizontal bar shows the mean for each group. The dashed line indicates the detection limit. The data are combined from two independent experiments that gave similar results. There was a statistically significant difference between lung viral titers in control and anti-PD-1-treated class II−/− mice (P = 0.02, Mann-Whitney rank sum test), anti-PD-L1-treated class II−/− mice (P = 0.04) or wild-type mice (P < 0.0001). There was no significant difference between lung virus titers in control and anti-PD-L2-treated class II−/− mice.
FIG. 5.
FIG. 5.
PD-1 expression on CD8 T cells and lung virus titers in wild-type, class II−/−, CD40−/−, CD80/86−/−, and CD28/CTLA4−/− mice. Groups of 3 to 5 mice, wild-type MHC class II−/−, CD40−/−, CD80/86−/−, and CD28/CTLA4−/− mice, were infected with MHV-68. Infiltrating cells were harvested from the lung by bronchoalveolar lavage via the trachea at day 50 postinfection and dual stained with FITC-conjugated anti-CD8 and PE-conjugated anti-PD-1. (A) Representative dot plots of PD-1 and CD8 expression in BAL fluid samples. The percentage of BAL cells positive for each marker is shown. (B) Lung virus titers were determined at day 50 after infection by plaque assay. Each symbol represents the virus titer for an individual mouse, and the horizontal bar shows the mean. (C) Mean percentages of CD8 T cells that were positive for PD-1 in BAL fluid samples. The mean percentage plus standard error of the mean (SEM) is shown. There was a statistically highly significant difference between the percentage of CD8 T cells positive for PD-1 in the BAL fluid samples from CII−/−, CD40−/−, and CD80/86−/− mice compared with that of wild-type mice (P < 0.001 [***]; P < 0.01 [**]). There was no significant difference in the percentages for wild-type and CD28/CTLA4−/− mice.
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