Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes

Abstract

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4+ and CD8+ T cells, Thy-1+ T cells, or CD8+ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.

FIG. 1

Flow cytometric analysis of peripheral blood lymphocytes from nonimmune mice showing efficiency of T-cell depletion in vivo. Dot plots are of PE-stained CD4⁺ cells (vertical axis) and FITC-stained CD8⁺ cells (horizontal axis) in mice not treated with MAbs (A) and in mice treated with anti-CD4 MAb (B) or anti-CD8 MAb (C).

FIG. 2

Numbers of T and B lymphocytes in the vaginal mucosa of 5 nonimmune mice, 10 immune/challenged mice, and 10 immune/challenged mice that were treated with MAbs to deplete CD4⁺ and CD8⁺ T cells.

FIG. 3

Fluorescence micrographs showing immunolabeling of CD4⁺ T cells (a, arrows) and CD8⁺ T cells (b, arrows) in the vaginal stroma of immune mice 1 day after IVAG challenge with wild-type virus. Little or no labeling of CD4⁺ (c) or CD8⁺ T cells was observed in the vaginas of immune/challenged mice that were treated with MAb to CD4 or CD8 1 week before challenge. Magnifications in all panels, ×350.

FIG. 4

Fluorescence micrograph showing immunolabeling of HSV-2 (arrow) in a portion of the vaginal epithelium of a nonimmune mouse 1 day after challenge with wild-type virus (a). Adjacent regions of the epithelium (E) were negative. No labeling of HSV-2 was detected in the vaginal epithelium when FITC-conjugated normal rabbit IgG was used in place of FITC–rabbit anti-HSV-2 (b). The fluorescence in the stroma is due to the endogenous fluorescence of granulocytes. L, lumen. Magnification in both panels, ×250.

FIG. 5

Numbers of T and B lymphocytes in the vaginal mucosa of 5 nonimmune mice, 10 immune/challenged mice, and 10 immune/challenged mice that were treated with MAb to deplete Thy-1.2⁺ T cells.

FIG. 6

Numbers of T and B lymphocytes in the vaginal mucosa of 5 nonimmune mice, 10 immune/challenged mice, and 10 immune/challenged mice that were treated with MAb to deplete CD8⁺ T cells.

FIG. 7

Numbers of T and B lymphocytes in the vaginal mucosa of 5 nonimmune mice, 10 immune/challenged mice, and 10 immune/challenged mice that were treated with MAb to deplete CD4⁺ T cells. The CD4⁺ T cells were stained with the MAb (clone GK1.5) that was used for T-cell depletion. To confirm these data, we also used a second MAb to stain CD4⁺ (clone YTS191.1.2) and found 10.7 ± 2.2 CD4⁺ T cells per high-power field in intact immune mice and 0.38 ± 0.11 in depleted mice.

FIG. 8

Fluorescence micrographs showing immunolabeling of MHC class II antigens in vaginal mucosa 1 day after vaginal challenge with HSV-2. (a) Nonimmune mice showed labeling in dendritic cells in the epithelium (large arrow) and stroma (small arrow), but the vaginal epithelial cells were negative. (b) Immune/challenged mice showed bright staining in the vaginal epithelium. (c) Immune/challenged mice depleted of CD8⁺ or CD4⁺ T cells showed bright labeling in some regions of the epithelium and less bright staining in other regions. (d) Immune/challenged mice depleted of both CD8⁺ and CD4⁺ T cells or Thy-1⁺ T cells showed variable labeling in the vaginal epithelium within the same section of vagina, some regions being bright, some moderate, and some not labeled. L, lumen. Magnification in all panels, ×190.