Role of CD28/CD80-86 and CD40/CD154 costimulatory interactions in host defense to primary herpes simplex virus infection

Abstract

Dependence of the primary antiviral immune response on costimulatory interactions between CD28/CD80-86 and between CD40/CD154 (CD40 ligand) has been correlated with the extent of viral replication in two models of systemic infection, lymphocytic choriomeningitis virus and vesicular stomatitis virus. To determine the role of these costimulatory interactions in the context of an acute cytolytic, but locally replicating viral infection, herpes simplex virus (HSV) infection was assessed in mice that had the CD28/CD80-86 or CD40/CD154 interactions disrupted either genetically or with blocking reagents (CTLA4Ig and MR1, respectively). CTLA4Ig treatment greatly reduced paralysis-free survival during primary acute HSV infection. This reflected an almost total ablation of the anti-HSV CD4(+) and CD8(+) T-cell responses due to anergy and reduced cell numbers, respectively. Disruption of CD40/CD154 interactions impaired survival, but the effect was less severe than that observed in CTLA4Ig-treated mice, with reductions observed in the CD4(+) T-cell but not CD8(+) T-cell responses. These two costimulatory pathways functioned in part independently, since disruption of both further impaired survival. The dependence on these costimulatory interactions for the control of primary HSV infection may represent a more widespread paradigm for nonsystemic viruses, which have restricted sites of replication and which employ immunoevasive measures.

FIG. 1

Outcome of HSV-1 (KOS) infection in mice which CD28/B7, CD40/CD154, or both interactions have been disrupted. Outcome was measured as the fraction of mice surviving without neurological impairment (paralysis or gross motor ataxia) over time in days after mice were inoculated via dermal abrasion with 2.5 × 10⁶ (a and b) or 5 × 10⁵ (c) PFU of HSV/hindfoot.

FIG. 2

Representative IFN-γ intracellular staining of large activated cells from the draining LN of day 5 HSV-infected B6 mice in response to various stimuli. IFN-γ staining of cells stimulated in vitro with αCD3+PMA (A) or HSVgB_498–505 (B) are plotted in relation to CD8⁺ staining; cells stimulated with UVHSV (C) are shown with respect to CD4⁺ staining.

FIG. 3

Characterization of the cellular immune response to HSV over days 4 to 10 postinoculation in CD154^−/− (□) and control (●) mice. Mice were inoculated via intradermal injection with 5 × 10⁵ PFU of HSV/hindfoot, and draining LN cells were collected from three mice per group on each day. Absolute numbers of large activated CD4⁺ (a) and CD8⁺ (b) cells were determined via fluorescence-activated cell sorting analysis. The fraction and absolute number of HSV-specific CD4⁺ cells were determined via IFN-γ intracellular staining of cells in response to UVHSV as antigen (Ag) (c and e). The fraction and absolute number of HSV-specific CD8⁺ cells were determined from IFN-γ production of cells stimulated with HSVgB_498–505 (d and f). Insets in panels e and f depict IFN-γ staining of either CD4⁺ or CD8⁺ cells in response to maximal αCD3+PMA stimulus. ANOVA single-variant statistical analysis was used for determining significance between groups. P values: a, 0.055; b, 0.506; c, 0.004; d, 0.036; e, 0.010; f, 0.013.

FIG. 4

Characterization of the cellular immune response to HSV over days 4 to 10 postinoculation in CTLA4Ig-treated (□) and control (●) mice. Mice were treated and data are plotted as described for Fig. 3. ANOVA single-variant statistical analysis was used for determining significance between groups. P values: a, <0.0001; b, <0.0001; c, 0.17 (the variable CD4⁺ T-cell response at day 7 reflected one CTLA4Ig-treated mouse with high numbers of IFN-γ-producing cells [P < 0.0001 with censoring of this data point]; d, 0.0002; e, 0.0020; f, <0.0001.

FIG. 5

Fractions of cells from day 5 HSV-infected mice which produced IFN-γ in response to HSVgB_498–505 and which stained positive for MHC-I K^b/HSVgB_498–505 tetramer closely paralleled each other directly ex vivo (a) and after 3 days in culture with or without IL-2 (b). Tetramer staining was performed on unstimulated cells after 6 h of culture. IFN-γ staining was done on cells incubated with HSVgB_498–505 for 6 h.

FIG. 6

CTLA4Ig treatment causes anergy in HSV-specific CD4⁺ T cells but not in CD8⁺ T cells. Day 5 draining LN cells from CD154^−/− mice (), CTLA4Ig-treated mice (■), and corresponding controls () were analyzed directly ex vivo or after 3 days in culture with or without IL-2 (5 ng/ml) for HSV-specific IFN-γ production in response to UVHSV or HSVgB_498–505. Plots represent fractions of IFN-γ-producing HSV-specific CD4⁺ or CD8⁺ cells in large CD4⁺ or CD8⁺ populations in CD154^−/− (a) or CTLA4Ig-treated (b) mice.

FIG. 7

The effect of CTLA4Ig on HSV-specific cells is mediated through IL-2. Cells from day 5 HSV-infected mice were analyzed either directly or after 3 days in culture with or without (5 ng/ml), IL-2, CTLA4Ig, (20 μg/ml), and IL-2-specific blocking antibody (20 μg/ml). Plots represent fractions of IFN-γ-positive CD4⁺ or CD8⁺ cells in large CD4⁺ and CD8⁺ populations in response to UVHSV or HSVgB_498–505.

FIG. 8

(a) HSVgB_498–505-specific IFN-γ production correlates with HSV gB-specific lytic activity after 3 days in culture with or without IL-2 (5 ng/ml). Effector cells were from day 5 draining LN cells from HSV-infected control mice. EL4-HSVgB (●) and control EL4 (□) cells were used as targets in the CTL assay. (b) HSV gB-specific lytic activity of cells from CTLA4Ig-treated mice is reduced at later time points during infection compared to controls (left panel). Lytic activity at an effector/target (E:T) ratio of 12.5:1 was determined in day 5 cells. Symbols represent effector cells from CTLA4Ig-treated mice (squares), control effector cells (circles), EL4-HSVgB targets (solid symbols), and EL4 control targets (open symbols).