Genital HSV-2 infection induces short-term NK cell memory

Abstract

NK cells are known as innate immune cells that lack immunological memory. Recently, it has been shown that NK cells remember encounters with chemical haptens that induce contact hypersensitivity and cytomegalovirus infection. Here, we show the existence of NK cell memory following HSV-2 infection. Stimulation with HSV-2 Ags led to higher IFNγ production in NK cells that were exposed 30 days previously to HSV-2, compared to NK cells from naïve mice. More importantly, this increased production of IFNγ in NK cells was independent of B- and T- lymphocytes and specific for the HSV-2 Ags. We also showed that previously exposed NK cells in a B- and T-lymphocyte free environment mediate protection against HSV-2 infection and they are necessary for the protection of mice against HSV-2 infection. Collectively, NK cells remember prior HSV-2 encounters independent of B- and T- lymphocytes leading to protection against HSV-2 mediated morbidity and mortality upon re-exposure.

Figure 1. Prior exposure of NK cells to HSV- 2 Ags increases IFNγ production upon re-exposure.

Six to eight weeks old female C57BL/6 mice in diestrus were immunized intra-vaginally with 1×10⁵ PFU per animal of TK^−/− HSV-2 (n = 3–5 per group). Thirty and sixty days following immunization, splenocytes were isolated and stimulated with HSV-2 lysate for 18–21 hours. The resultant culture supernatants were collected to assay IFNγ concentration by ELISA and the NK cells were stained for intracellular IFNγ production. (a and d): representative FACS plots showing CD3-NK1.1+IFN-γ+ cells in the spleens of HSV-2 exposed and naive mice, (b and e): percentage of CD3-NK1.1+IFN-γ+ cells in the spleen of HSV-2 exposed and naive mice, (c and f) IFN-γ production in HSV-2 exposed and naive splenocytes upon stimulation with HSV-2 Ags or control cell lysate.

Figure 2. Prior exposure to HSV-2 Ags increases IFNγ production in NK cells even in the absence of B- and T- lymphocytes.

Female RAG1^−/− mice were immunized intra-vaginally with 1×10⁴ PFU per animal TK^−/− HSV-2 (n = 3–5). Thirty or sixty days following immunization, splenocytes were isolated and stimulated with HSV-2 lysate for 18–21 hours. The resultant culture supernatants were collected for the determination of IFNγ concentration by ELISA and the NK cells were stained for intracellular IFNγ production. (a and d): representative FACS plots showing CD3-NK1.1+IFN-γ+ cells in the spleens of HSV-2 exposed and naive mice, (b and e): percentage of CD3-NK1.1+IFN-γ+ cells in the spleens of HSV-2 exposed and naive mice, (c) IFN-γ production in HSV-2 exposed and naïve splenocytes 30 days post-immunization following stimulation with HSV-2 or control cell lysate. The experiment was repeated twice with similar results.

Figure 3. NK cells previously exposed to HSV-2 Ags reduce the consequence of secondary HSV-2 infection in mice that lack B- and T- lymphocytes.

A group of female RAG1^−/− mice (n = 4–5) were immunized intra-vaginally with TK^−/− HSV-2 (1×10⁴ PFU per animal) alongside unimmunized controls. Both groups were infected 3 weeks following immunization with a lethal dose of wild type HSV-2 (5×10³ PFU per animal) intra-vaginally. The mice were observed for genital lesions and vaginal washes were collected 1–3 days post-infection. (a), (b) and (c) illustrate the percentage survival, the vaginal lesion scores and viral titers in vaginal washes. The experiment was repeated twice with similar results.

Figure 4. Response of NK cells that experienced HSV-2 was specific for HSV-2.

(a) Six to eight weeks old female C57BL/6 mice in diestrus were immunized intra-vaginally with 1×10⁵ PFU per animal of TK^−/− HSV-2 (n = 5) alongside unimmunized controls (n = 5). Thirty days following immunization, splenocytes were isolated and stimulated with poly I:C (10 µg/ml) for 18–21 hours. The NK cells were stained for intracellular IFNγ production and percentage of CD3-NK1.1+IFN-γ+ cells in the spleen of HSV-2 exposed and naive mice are presented. (b) Six to eight weeks old female C57BL/6 mice in diestrus were immunized intra-vaginally with 1×10⁵ PFU per animal of TK^−/− HSV-2 (n = 9). Three weeks following immunization, the mice were challenged with B16F10 cells (1×10⁶ cells/mouse) i.v. alongside unimmunized controls (n = 9). Counts of tumor nodules in the lung two weeks following the tumor challenge are presented.

Figure 5. Depletion of previously exposed NK cells affects the B- and T-lymphocyte independent but not dependent protection against HSV-2 infection.

A group of female RAG1^−/− (n = 4–5 per group) and C57BL/6 mice (n = 4 per group) were immunized intra-vaginally or intra-nasally with TK^−/− HSV-2 (1×10⁴ PFU and 1×10⁵ PFU per animal respectively). One of the groups of RAG1^−/− and C57BL/6 mice, received anti NK1.1 antibody (200 µg) on day −2, −1, +2 and +5. NK cell depleted and undepleted control groups were infected 3 weeks following immunization (day 0) with a lethal dose of wild type HSV-2 (5×10³ PFU or 1×10⁵ PFU per animal respectively) intra-vaginally. The mice were observed for genital lesions (all groups) and vaginal washes were collected 1–3 days post-infection for RAG1^−/− mice. (a) representative FACS plots showing the efficiency of NK cell depletion, (b), (c), (d) and (e) illustrates the percentage survival, the vaginal lesion scores, viral titers and concentration of IFN-γ in vaginal washes of RAG1^−/− mice. (f) representative FACS plots showing the efficiency of NK cell depletion, (g) and (h) illustrates the percentage survival of C57BL/6 mice immunized intra-vaginally and intra-nasally, respectively. NK cell depleted and control C57BL/6 mice immunized intra-vaginally were free of any vaginal pathology (data not shown).

Figure 6. HSV-2 Ag experienced and naive NK cells are phenotypically similar.

Six to eight week old female C57BL/6 (n = 3–5) and RAG1−/− mice (n = 4–6) in diestrus were immunized intravaginally with 1×10⁵ PFU per animal of TK−/− HSV-2 alongside unimmunized controls. Two days post-immunization cells (lymph node and spleen) from three C57BL/6 immunized and three C57BL/6 control mice were isolated and stained to compare the CD3-NK1.1+ cell numbers in immunized and control mice. Thirty days following immunization, C57BL/6 and RAG1−/− splenocytes were isolated from immunized (n = 5) and unimmunized controls (n = 5) and stained to analyze NK cell phenotype via CD27 and CD11b. (a) representative FACS plots showing CD3-NK1.1+ cells in the lymph node and spleen of HSV-2 exposed and naive mice 2 days following immunization, (b and d) representative FACS plots showing CD3-NK1.1+ cells in the spleens of HSV-2 exposed and naive mice on a CD27 and CD11b cross-gate, (c and e) percentage of CD27−CD11b+, CD27+CD11b−, CD27+CD11b+, and CD27−CD11b+ NK cells in HSV-2 exposed and naive mice.