Figure 7. Innate immune protection against a vaccinia virus challenge is conferred on naïve RAG1^ko mice by the adoptive transfer of primed Thy1⁺ liver-resident NK cells.

Six months after priming, 1×10⁵ Thy1⁺ liver NK cells, 1×10⁵ Thy1⁻ liver NK cells, or 5×10⁶ NK cell-depleted liver mononuclear cells from vaccinia virus-primed or naïve mice were adoptively transferred into RAG1^ko mice. At the same time, mice were administered either control immunoglobulins (those mice that received NK-depleted cell transfers) or T cell-depleting monoclonal antibodies (those mice that received Thy1^- and Thy1⁺ NK cell transfers). One week later the mice were challenged with 1×10⁵ pfu rVV-luc intraperitoneally, and viral loads were monitored by IVIS imaging. (a) IVIS images of representative mice from each group over time following challenge are shown. (b) The ability of RAG1^ko mice to resolve vaccinia virus infection by 3 weeks post-challenge is represented as the percent clearance within each group. Results were compiled from 4 separate experiments. RAG1^ko mice that received primed Thy1⁺ liver NK cells showed enhanced clearance of vaccinia virus as compared to mice that received naïve Thy1⁺ NK cells, naïve Thy1-depleted NK cells, and primed Thy1-depleted NK cells (two-tailed Fischer test between groups). All statistics compare the test group relative to the group that received primed Thy1⁺ liver-resident NK cells. (c) The ability of RAG1^ko mice to clear vaccinia virus infection throughout the course of challenge is represented as the percent clearance within each group. Results represent 4 separate experiments. RAG1^ko mice that received primed Thy1⁺ liver NK cells showed enhanced protection against vaccinia virus challenge as compared to mice that received naïve Thy1⁺ NK cells, naïve Thy1⁻ NK cells, and primed Thy1⁻ NK cells. Statistical analysis was performed to compare Kaplan-Meier survival curves using GraphPad Prism 4 software. All statistics compare the test group relative to the group that received primed Thy1⁺ liver-resident NK cells. Protection provided by transferred primed Thy1⁺ hepatic NK cells surpassed the protection observed in mice that received hepatic naïve Thy1⁻ NK cells (using the log rank test; p = 0.0168), primed Thy1⁻ NK cells (p = 0.0043), and naïve Thy1⁺ NK (p = 0.0033). (d) Kaplan-Meier curves showing the survival of naïve RAG1^ko recipients of the indicated adoptively transferred cell populations. Results represent the compilation of 4 separate experiments. RAG1^ko mice that received primed Thy1⁺ liver NK cells showed enhanced survival of vaccinia virus challenge as compared to mice that received naïve Thy1⁺ NK cells, naïve Thy1⁻ NK cells, or primed Thy1⁻ NK cells. Statistical analysis was performed as in (c), demonstrating that host protection provided by transferred primed Thy1⁺ hepatic NK cells surpassed the protection observed in mice that received hepatic naïve Thy1⁻ NK cells (log rank test; p = 0.013), primed Thy1⁻ NK cells (p = 0.0049), or naïve Thy1⁺ NK (p = 0.0004). (e) Kaplan-Meier curves showing the survival of naïve RAG1^ko recipients of the indicated adoptively transferred cell populations isolated from livers of naïve and MVA-primed mice. RAG1^ko mice that received MVA-primed Thy1⁺ liver NK cells showed no evidence of enhanced survival following rVV-luc challenge as compared to mice that received naïve Thy1⁺ NK cells, naïve Thy1⁻ NK cells, or MVA-primed Thy1⁻ NK cells. Statistical analysis was performed as in (c and d). Results shown are representative of 2 independent experimental replicates.