CD4+ T cells are required for the priming of CD8+ T cells following infection with herpes simplex virus type 1

Abstract

The role of CD4(+) helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4(+) T cells are needed for the generation of the protective HSV-1-specific CD8(+)-T-cell response. This study examined the contribution of CD4(+) T cells in the generation of the primary CD8(+)-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8(+)-T-cell response generated in the draining lymph nodes of CD4(+)-T-cell-depleted C57BL/6 mice and B6-MHC-II(-/-) mice is quantitatively and qualitatively distinct from the CD8(+) T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8(+) T cells express comparable levels of the activation marker CD44 in mice lacking CD4(+) T cells and normal mice. In contrast, CD8(+) T cells generated in the absence of CD4(+) T cells express the interleukin 2 receptor alpha-chain (CD25) at lower levels. Importantly, the CD8(+) T cells in the CD4(+)-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8(+) T cells is diminished in the absence of CD4(+)-T-cell help. These results suggest that CD4(+)-T-cell help is essential for the generation of fully functional CD8(+) T cells during the primary response to HSV-1 infection.

FIG. 1.

Lymphocyte cellularity in the draining PLN following acute infection with HSV-1. C57BL/6 (WT) mice, CD4-depleted B6 mice, or B6-MHC-II^−/− mice were infected in each hind FP with 2 × 10⁶ PFU HSV-1. Mice were sacrificed 5 days p.i., and the draining PLN cells were analyzed by flow cytometry. (A) Absolute number of HSV-1 gB-specific CD8⁺ T cells (four mice/group; *, P = 0.03). (B) Absolute number of HSV-1 gB-specific CD8⁺ T cells (three mice/group; *, P = 0.04). (C) Absolute number of total cells per PLN (four mice/group; *, P < 0.0001). (D) Absolute number of total cells per PLN (three mice/group; *, P = 0.0001). (E) Absolute number of CD8⁺ T cells per PLN (four mice/group; *, P < 0.0001). (F) Absolute number of CD8⁺ T cells per PLN (three mice/group; P = 0.2). For panels A and B, lymph node cells were simultaneously stained with anti-CD8α and H-2K^b-gB_498-505 tetramers to identify HSV-1-specific CD8⁺ T cells. Data are representative results from one of two independent experiments for each group (means ± SEM).

FIG. 2.

Analysis of expression of activation markers on HSV-1 gB-specific CD8⁺ T cells in CD4-depleted, B6-MHC-II^−/−, and WT mice in response to acute infection with HSV-1. Draining PLN cells were obtained from WT, CD4-depleted, or B6-MHC-II^−/− mice 5 days following infection of each hind FP with HSV-1 and stained using anti-CD8α, anti-CD44, anti-CD25, and H-2K^b-gB tetramers. (A and B) Expression of CD44 on HSV-1 gB-specific CD8⁺ T cells in WT compared to CD4-depleted B6 mice (A) and WT compared to B6-MHC-II^−/− mice (B). (C and D) Expression of CD25 in WT compared to CD4-depleted B6 mice (C) and WT compared to B6-MHC-II^−/− mice (D). Solid lines, WT; dotted lines, CD4-depleted or B6-MHC-II^−/− mice. Data are representative of two independent experiments for each group.

FIG. 3.

Assessment of IFN-γ-producing HSV-1 gB-specific CD8⁺ T cells during acute infection with HSV-1 in CD4-depleted, B6-MHC-II^−/−, and WT mice. Mice were infected with HSV-1 in the hind FP, and the draining PLN were harvested on day 5 p.i. The lymph node cells were stimulated in vitro with gB peptide or control (VSV) peptide in the presence of brefeldin A for 5 h. The stimulated lymphocytes were then surface stained for the expression of CD8 and CD25, fixed, permeabilized, and stained for intracellular expression of IFN-γ. (A) Representative dot plots show the frequency of CD8⁺ IFN-γ⁺ T cells in the draining lymph nodes. Numbers in the gated regions are the percentages of CD8⁺ IFN-γ⁺ T cells. Numbers beside the boxed area indicate the MFI for IFN-γ staining within the gated region. (B) (Top panels) Absolute numbers of IFN-γ producing CD8⁺ T cells (four mice/group; *, P = 0.01 [left]; three mice/group; *, P = 0.002 [right]). (Bottom panels) MFI of IFN-γ staining by gB-specific CD8⁺ T cells in the gated region (four mice/group; *, P = 0.004 [left]; three mice/group; *, P = 0.01 [right]). (C) Representative histograms showing expression of IFN-γ and CD25 on gated CD8⁺ T cells. Numbers in the quadrants are percentages of CD8⁺ cells which are IFN-γ⁺ CD25⁻ (quadrant 1) or IFN-γ⁺ CD25⁺ (quadrant 2). Numbers in parentheses are the MFI for IFN-γ staining. Data are representative of two independent experiments (means ± SEM). P values of <0.05 were considered significant.

FIG. 4.

Analysis of TNF-α-producing HSV-1 gB-specific CD8⁺ T cells following acute infection with HSV-1. WT and CD4-depleted mice were infected with HSV-1 in the FP, and draining PLN cells were harvested on day 5 p.i. and stimulated in vitro with gB peptide in the presence of brefeldin A for 5 h. The stimulated lymphocytes were then surface stained for the expression of CD8 and CD25, fixed, permeabilized, and stained for intracellular expression of TNF-α and IFN-γ. (A) Representative dot plots show the frequency of CD8⁺ TNF-α⁺ T cells in the draining lymph nodes. Numbers are the percentages of CD8⁺ TNF-α⁺ T cells. Numbers in parentheses are the MFI for TNF-α staining. (B) (Left) Absolute number of TNF-α-producing CD8⁺ T cells (three mice/group; *, P = 0.02). (Right) MFI of TNF-α staining by CD8⁺ T cells (three mice/group; *, P < 0.0001). Data shown are representative of two independent experiments (means ± SEM). (C) Representative histograms show percentages of gated CD8⁺ T cells which express IFN-γ only (upper left quadrant), IFN-γ and TNF-α (upper right quadrant), or TNF-α only (lower right quadrant). P values of <0.05 were considered significant.

FIG. 5.

Analysis of in vivo cytolytic activity in WT, CD4-depleted, and B6-MHC-II^−/− mice. WT, CD4-depleted, and B6-MHC-II^−/− mice were infected in each hind FP with 2 × 10⁶ PFU HSV-1 and examined for HSV-1-specific cytolytic function in vivo using a 4-h CFSE-based assay as described in Materials and Methods. For acute responses to HSV-1 infection, cytolytic activity was measured on day 5 p.i., which corresponds to the peak of the response in the draining PLN. (A) Representative histogram plots of lymph node cells obtained from naïve control and day 5 infected WT, CD4-depleted, and B6-MHC-II^−/− mice 4 h after transfer of CFSE-labeled target cells. Numbers in the plots are percent specific lysis of target cells. (B) Histograms show lysis of CFSE-labeled target cells in naïve control and day 5 infected WT and CD4/CD8-depleted mice 4 h after transfer. Numbers in the plots are percent specific lysis of target cells.

FIG. 6.

Kinetic analysis of in vivo cytolytic activity in WT and CD4-depleted mice. WT, CD4-depleted, and B6-MHC-II^−/− mice were infected in each hind FP with 2 × 10⁶ PFU HSV-1 and examined for HSV-1-specific cytolytic function in vivo using a CFSE-based assay as described in Materials and Methods. The presence of CFSE^hi and CFSE^lo cells remaining 1, 2, and 4 h after i.v. transfer of target cells was determined. No significant difference was apparent between the two experimental groups at any time point examined.

FIG. 7.

CD4 depletion does not affect CTL activity in vivo. (A) Representative histograms from the PLN of naïve, CD4-depleted, and nondepleted B6 mice, 5 days post-HSV-1 infection. (B) Percent specific lysis as a function of peptide concentration (means ± SEM). Percent specific lysis was calculated as described in the text. Squares, naïve mice; triangles, CD4-depleted mice; circles, nondepleted mice. Statistical analysis using the Wilcoxon sum of ranks test revealed no significant differences in the percentage of HSV-1-specific lysis between CD4-depleted and nondepleted mice at any of the concentrations examined.

FIG. 8.

Perforin and granzyme B levels in CD8⁺ T cells were not affected in mice lacking CD4⁺ T cells during primary responses to HSV-1 infection. Draining cells from CD4-depleted B6, MHC class II^−/−, and WT mice were harvested on day 5 p.i. with HSV-1 and stained for expression of CD8, perforin, and granzyme B as described in Materials and Methods. Expression of perforin (A) and granzyme B (B) on CD8⁺ T cells is shown. Data are representative of two independent experiments for each group.

FIG. 9.

Increased viral titers in CD4-depleted and B6-MHC-II^−/− mice. Virus levels in the FP tissues of WT, CD4-depleted, and B6-MHC-II^−/− mice were determined by virus plaque assay of samples from five to seven mice per group at 5 days after infection with HSV-1. *, P = 0.01 (n = 5) for CD4-depleted versus WT mice and 0.03 (n = 7) for B6-MHC-II^−/− versus WT mice. Each symbol represents one individual animal. Horizontal bars indicate means. A P value of <0.05 is considered significant.