Vaginal NK cell effector activity in IL-21R KO and WT mice day 2 p.i. with HSV-2.

<p>Vaginas were harvested on day 2 p.i. with 6.7×10⁴ PFUs HSV-2 intra-vaginally. (<b>A</b>) Example of intracellular IFN-γ and granzyme B staining in vaginal NK cells. (<b>B</b>) IFN-γ⁺ vaginal NK cells in % per mouse in IL-21R KO and WT mice. (<b>C</b>) Total number of IFN-γ⁺ vaginal NK cells per mouse in IL-21R KO and WT mice. (<b>D</b>) CD107a+b expression on FACS sorted viable CD45⁺ vaginal cells from WT mice, cultured in the presence or absence of MHC class 1 deficient YAC-1 cells, and gated on NK cell marker NK1.1. (<b>E</b>) CD107a+b⁺ NK cells in %, of FACS sorted viable CD45⁺ vaginal cells, cultured in the absence or presence of YAC-1 cells, and gated on NK1.1, in IL-21R KO and WT mice. (<b>A-C</b>) <i>n</i>  =  12 mice per group. (<b>D-E</b>) <i>n</i>  =  6 mice per group. (<b>B-C</b>) Horizontal bars indicate median. Each dot represents one animal. All mice were 8-9 weeks old. ns  =  not significant.</p

Mice lacking the IL-21R are more susceptible to HSV-2 infection than WT mice.

<p>(<b>A-D</b>) 10 weeks old mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2. Vaginal fluids were collected on day 1-3 p.i. for quantification of (<b>A</b>) vaginal viral load day 1-3 p.i. and (<b>B</b>) IFN-α/β protein levels in vaginal fluids day 2 p.i. The dashed line indicates the lower detection limit of the assay. (<b>C</b>) After HSV-2 infection mice were scored daily for disease development. Data are shown as median disease scores for WT and IL-21R KO mice. (<b>D</b>) When reaching score 4 (hind limb paralysis) mice were euthanized and recorded. Data are shown in a Kaplan Meier plot of survival, <i>p</i><0.001. (<b>A-B</b>) Horizontal bars indicate median. Each dot represents one animal. (<b>A-D</b>) Data are representative of two independent experiments (<i>n</i>  =  7 and 13 mice per group respectively, in A data are pooled). * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, # <i>p</i>  =  0.058 when using a Mann-Whitney test but when applying an unpaired Students t-test <i>p</i>  =  0.02. ns  =  not significant. (<b>E</b>) 20×10⁶ CD4⁺ splenocytes from Thy1.2 mice were adoptively transferred i.p. to Thy1.1 mice. Spleens were harvested the following day and splenocytes were analysed by flow cytometry for Thy1.2 and CD4. (<b>F-H</b>) 10 weeks old IL-21R KO mice were injected i.p. with 20×10⁶ splenocytes from 10 weeks old WT or IL-21R KO mice respectively. One day after the transfer, mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2. (<b>F</b>) Vaginal fluids were collected on day 1-3 p.i. for quantification of vaginal viral load day 1-3 p.i. Horizontal bars indicate median. Each dot represents one animal. (<b>G</b>) After HSV-2 infection mice were scored daily for disease development. Data are shown as median disease scores for IL-21R KO mice transferred with WT or IL-21R KO splenocytes. Differences in median disease score between the two groups did not reach statistical significance on any of the days. (<b>H</b>) When reaching score 4 (hind limb paralysis) mice were euthanized and recorded. Data are shown in a Kaplan Meier plot of survival. ns  =  not significant. Data (<b>E-H</b>) represent one experiment.</p

IL-21R is expressed by vaginal epithelial cells in WT mice and IL-21R expression is increased by HSV-2 infection.

<p>8 weeks old C57BL/6 mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2 and vaginas were harvested on day 1-3 post infection (p.i.) (<b>A</b>) Quantitative RT-PCR analysis of mRNA encoding IL-21R in vaginal tissue in uninfected (u.i.) and HSV-2 infected WT mice on day 1-3 p.i. (<b>B-C</b>) IHC staining with IL-21R primary antibodies and HRP-linked goat anti-rabbit secondary antibodies on 2 µm sections of vaginal tissue in (<b>B</b>) u.i. and HSV-2 infected WT mice day 1 p.i. and in (<b>C</b>) HSV-2 infected WT and IL-21R KO mice day 1 p.i. as control. Scale bar  =  20 µm. (<b>A</b>) IL-21R is normalized to β-Actin in each sample. Horizontal bars indicate median. Each dot represents one animal. Data are representative of two individual experiments (<i>n</i>  =  10 mice per group respectively). ** <i>p</i><0.01, *** <i>p</i><0.001.</p

mIL-21 treatment has anti-viral effect in the innate immune response.

<p>8 weeks old WT mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2. mIL-21 treated mice were inoculated intra-vaginally with 5 µg mIL-21 on day -1 to 3 p.i., and untreated mice received PBS. Vaginal fluids from WT mice infected with HSV-2 and untreated (-) or treated (+) with mIL-21, were collected on day 1-3 p.i. for quantification of (<b>A</b>) vaginal viral load day 1-3 p.i. and (<b>B</b>) IFN-α/β protein levels in vaginal fluids day 2 p.i. The dashed line indicates the lower detection limit of the assay. (<b>C</b>) After HSV-2 infection mice were scored daily for disease development. Data are shown as median disease scores in HSV-2 infected, untreated or mIL-21 treated WT mice. (<b>D</b>) When reaching score 4 (hind limb paralysis) mice were euthanized and recorded. Data are shown in a Kaplan Meier plot of survival, p<0.001. (<b>A-B</b>) Horizontal bars indicate median. Each dot represents one animal. (<b>A-D</b>) Data are representative of three individual experiments (<i>n</i>  =  10 mice per group). * <i>p</i><0.05, ** <i>p</i><0.01. ns  =  not significant.</p

Cytokine levels in vaginal fluids from HSV-2 infected IL-21R KO and WT mice.

<p>Vaginal fluids were collected on day 1-3 p.i. with 6.7×10⁴ PFUs HSV-2 intra-vaginally. (<b>A-F</b>) IL-6, IFN-γ, TNF-α, MCP-1 (CCL-2), KC (CXCL-1, murine homologue of IL-8) and IL-10 protein levels in vaginal fluids from HSV-2 infected IL-21R KO and WT mice, quantified by Luminex. Horizontal bars indicate median. Each dot represents one animal. Data are representative of two individual experiments (<i>n</i>  =  7 and 13, 10 weeks old mice, per group respectively). Statistical analysis has been performed on all data sets. Where differences between WT and IL-21R KO mice reached statistical significance it is indicated in the graph by * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. When differences did not reach statistical significance there is no indication in the graph.</p