Herpes simplex virus (HSV)-specific T cells activated in the absence of IFN-gamma express alternative effector functions but are not protective against genital HSV-2 infection

Abstract

Interferon gamma (IFNgamma) is important for immune resistance to herpes simplex virus (HSV) infection. To examine the influence of IFNgamma on the development of HSV-specific immune responses and test for IFNgamma-independent adaptive immune mechanisms of protection, IFNgamma-deficient mice (IFNgamma(-/-)) were immunized with thymidine kinase-deficient HSV-2 (HSV-2 333tk(-)). HSV-specific cellular and humoral responses were elicited in immunized IFNgamma(-/-) mice resulting in increased resistance relative to non-immune C57BL/6J (B6) mice following challenge with fully virulent HSV-2. CD8(+) T cells from IFNgamma(-/-) mice displayed cytotoxic activity and secreted TNFalpha. HSV-specific CD4(+) T cells from immunized IFNgamma(-/-) mice secreted IL-4, TNFalpha, and IL-17, but unlike T cells from HSV-immune B6 mice, could not clear virus from genital tissue following adoptive transfer. HSV-immune IFNgamma(-/-) mice produced predominantly IgG(1) HSV-specific antibodies while immune B6 mice produced predominantly IgG(2c) antibodies. Transfer of equivalent amounts of HSV-specific antibodies from either strain to naïve mice imparted equivalent early resistance against infection of the genital epithelia. However, protection against neurological symptoms mediated by immune B6 antibodies was superior late in infection. Taken together, these results demonstrate that the limited resistance of HSV-immune IFNgamma(-/-) mice to HSV-2 infection resulted from the action of HSV-specific Ab rather than IFNgamma-independent effector functions of T cells. Further, protection against neurological manifestations of HSV-2 infection was superior in mice receiving Ab from immune B6 mice suggesting that Ab-mediated protective mechanisms involving IFNgamma-induced IgG subclasses were more effective once virus had spread to neural tissues.

Fig. 1

HSV-2 challenge of HSV-immune B6 and IFNγ^−/− mice. A) Clearance of HSV-2 333tk⁻ from the genital epithelium non-immune of B6 or IFNγ^−/− mice. Mice were inoculated intravaginally with 2 × 10⁵ PFU HSV-2 333tk⁻ (n = 9; * P < 0.05; ** P < 0.0001; ANOVA). B) Survival of HSV-immune B6, -immune IFNγ^−/−, and naïve B6 mice after intravaginal challenge with 10⁵ PFU HSV-2 186 (n = 10; * P < 0.0001 compared to naïve B6 mice; ** P < 0.05 compared to immune B6; Logrank test). C) Failure of HSV-immune IFNγ^−/− mice to clear HSV-2 from the vagina. HSV-immune B6, -immune IFNγ^−/− mice, and naïve B6 mice were challenged with HSV-2 186. Vaginal titers were obtained on the indicated days (n = 9; * P < 0.001; ** P < 0.0001 compared to immune B6 mice; ANOVA). HSV-2 titers in lumbosacral ganglia (D) and adjacent spinal cords (E) of HSV-immune B6, -immune IFNγ^−/−, and naïve control mice after challenge with 10⁴ PFU HSV-2 186. Mice were sacrificed on days 5 or 7 after challenge and infectious HSV-2 was quantified from the sensory ganglia and spinal cords. Day 5 lumbosacral ganglia and spinal cord titers from naïve B6 mice were significantly greater than HSV-immune B6 and -immune IFNγ^−/− mice (P < 0.001; ANOVA).

Fig. 2

Cytokine secretion by HSV-specific CD8⁺ T cells from HSV-immune B6 and IFNγ^−/− mice. HSV-immune B6, -immune IFNγ^−/−, and naïve B6 mice were challenged intravaginal with 10⁴ PFU HSV-2 186. Six days later, CD8⁺ T cells isolated from spleens and iliac lymph nodes were stimulated with syngeneic spleen cells pulsed with HSV gB_498–505 peptide, and IFNγ-secreting cells and IL-4-secreting cells from the spleen (A) or iliac lymph nodes (B) were quantified by ELISPOT (*P < 0.001; ANOVA). (C) An in vivo CTL assay was performed on the indicated days after challenge of HSV-immune B6 and IFNγ^−/− mice (*P < 0.005; ** P < 0.0001; Student’s t test). Results are representative of 2 experiments performed.

Fig. 3

Cytokine secretion by HSV-specific CD4⁺ T cells from HSV-immune B6 and IFNγ^−/− mice (A). CD4⁺ T cells were isolated and pooled from iliac lymph nodes and vaginal tracts of HSV-immune B6, -immune IFNγ^−/− and naïve B6 mice on the indicated day after challenge with 10⁴ PFU HSV-2 186. Results are expressed as the total number of cells obtained from the tissues from 4 mice and are representative of 3 experiments performed (*P < 0.05; ** P < 0.01; *** P < 0.001 for comparisons between immune B6 and immune IFNγ^−/− mice; Student’s t test). Adoptive transfer of CD4⁺ T cells from HSV-immune B6, but not HSV-immune IFNγ^−/− mice, results in clearance of HSV-2 333tk⁻ from the genital epithelium (B). Lymphocytes from HSV-immune B6, -immune IFNγ^−/−, and naïve OT-II mice were activated in culture for 4 days with specific antigen. CD4⁺ T cells were isolated and transferred to irradiated B6 recipients as described in Methods. Mice were immediately challenged with 5 × 10³ PFU HSV-2 333tk⁻ and virus was quantified on the indicated days. (B) Virus titers following HSV-2 challenge (*P < 0.001 compared to HSV-immune B6 mice; ANOVA). (C) Percent of mice shedding virus. Results are pooled from 2 experiments of identical design.

Fig. 4

Differential protection by passive transfer of HSV-specific antibodies from HSV-immune B6 and IFNγ^−/− mice. B6 mice received 23.0 μg HSV-specific IgG from immune B6 or IFNγ^−/− mice or non-immune mouse serum and were challenged 3 days later with 10⁵ PFU HSV-2 186. Mice were swabbed for virus on days 1 and 2 post-challenge and were observed 16 days for disease symptoms. A) HSV-specific IgG subclasses in IgG-enriched Ab preparations from HSV-immune B6, -immune IFNγ^−/−, or naïve mice. B) HSV-2 titers in the genital tracts of HSV-immune B6 IgG-, -immune IFNγ^−/− IgG-, or non-immune serum recipients (*P < 0.01 compared to non-immune serum-recipients; ANOVA). Results are representative of 2 experiments performed. C) Survival of mice following transfer of HSV-immune IgG preparations or non-immune serum (*P < 0.0001 compared to non-immune serum recipients; ** P = 0.039 compared to immune IFNγ^−/− serum recipients; Logrank test). Results are pooled from 2 experiments of identical design.