PMC

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.

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.

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.

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.

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.

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. . 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.

(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).

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.