Supplementary Materials Supplemental material supp_92_11_e02103-17__index. mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV contamination. IMPORTANCE Recent data have shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV contamination in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines. = 0.0079) (Fig. 1D). Similarly, NK cell depletion showed little effect on the magnitude and frequency of the immunodominant GP33- or GP276-specific CD8+ interferon gamma-positive (IFN-+) T cell responses (Fig. 1E) and GP61-specific CD4+ T cell responses, as measured by intracellular cytokine staining (Fig. 1F) in tissues at week 8 postvaccination. These data suggest that NK cell depletion may have less of an impact on T cell responses induced by Ad vector vaccination than on those induced by viral contamination (22). Open in a separate window FIG 1 NK cell depletion has a minimal impact on CD4+ and CD8+ T cell responses elicited by Ad5-GP. Naive C57BL/6 mice received 500 g of anti-NK1.1 or isotype control antibody prior to immunization with Ad5-GP. (A) Schematic of the experimental setup. (B) GP33-specific CD8+ T cell responses measured via Db/AL11 tetramer ZL0454 binding assays. (C) Phenotypic differentiation of GP33-specific CD8+ T cells. (D) PD-1 (mean fluorescence intensity [MFI]) expression on GP33 tetramer-positive CD8+ T cells. (E) Intracellular cytokine staining of GP33- and GP276-specific CD8+ T cells. (F) Intracellular cytokine staining of GP61-specific CD4+ T cells. Error bars represent standard errors of the means for 5 mice per group with 1 sham-vaccinated control. Statistically significant values are indicated (**, 0.01 by a Mann-Whitney U ZL0454 test). NK cell modulation of CD8+ and CD4+ T cell responses in vaccinated versus unvaccinated mice following LCMV challenge. To address whether vaccine-elicited memory T cells are susceptible to NK cell rheostat regulation following LCMV challenge, we depleted NK cells from both Ad5-GP-vaccinated and unvaccinated animals prior to challenge with 2 106 PFU of LCMV Cl-13. We chose this dose of LCMV Cl-13 to establish Ctgf a chronic contamination given its ability to serve as a model of immune pathology with heightened T cell responses. We then evaluated the NK cell phenotype as well as ZL0454 the magnitudes and frequencies of antigen-specific CD4+ and CD8+ T cells (Fig. 2A). We observed that more NK cells were activated in unvaccinated animals, as marked by CD69 expression, with an increased upregulation of markers associated with activation (NKG2D) and inhibitory (2B4) activities (Fig. 2B) (3, 7). On day 5 postinfection, unvaccinated NK cell-depleted mice exhibited markedly higher GP33-specific ( 0.0001 for percent frequency and = 0.0035 for total numbers) and GP276-specific ( 0.0001 for percent frequency and = 0.0036 for total number) CD8+ T cell responses than did unvaccinated, isotype-treated mice, consistent with previous findings (Fig. 2C) (3, 5, 7, 11). In contrast, NK cell depletion had minimal to no impact on GP33- and GP276-specific CD8+ T cells in Ad5-GP-vaccinated mice following LCMV challenge (Fig. 2C). Moreover, the depletion of NK cells using the asialo-GM1 antibody recapitulated these results (data not shown). Open in a separate window FIG 2 ZL0454 NK cell modulation of T cell responses in vaccinated and unvaccinated mice following LCMV Cl-13 challenge. (A) Schematic outlining the experimental setup. (B) Vaccinated and unvaccinated mice were challenged with LCMV Cl-13. At day 3 following infection, animals were sacrificed, and NK cell responses in blood, spleen, and lymph node were evaluated as the percentage of activated NK cells, as marked by the upregulation of CD69, NKG2D expression on NK cells, and 2B4 expression on NK cells. Error.