Quantifying SIV Env-specific NK Cell Activity

K562 cells devoid of MHC-I stably (ATCC) expressing HLA-E^*0101 (K562-E^*0101; Applied Biological Materials Inc.) cells were incubated with 50 µM of SIVmac_239/251 Env peptide (NQLLIAILL) at 26 °C for 15–20 h^{7 (link)}. Isolated NK cells were cultured for 6 h in the presence of an anti-CD107a-PE-Cy5 mAb (clone HA43, 5 μL, cat. 555802; BD Biosciences), either alone (NK), or co-cultured at a 5:1 ratio with 2 × 10⁴ K562-E^*0101 cells that were either unpulsed (NK+K562^*E) or loaded with SIVmac_239/251 Env peptide (NK+K563^*E+ENV). GolgiStop and GolgiPlug (BD Biosciences) was added 1 h following culture initiation. The frequency of NK cells expressing surface CD107a was measured by flow cytometry per each culture condition to assess the background (%CD107a⁺_NK), the maximum (%CD107a⁺_NK+K562*E), and the peptide-specific degranulation activity (%CD107a⁺_{NK+K562*E+ENV}; Supplementary Fig. 6). From these measurements the Env-specific NK cell activity was calculated as previously described^{30 (link),55 (link)}: ${\log }_{10}\left[100\times \frac{\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}+\mathrm{K}562*\mathrm{E}+\mathrm{ENV}}^{+}-\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}}^{+}}{\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}+\mathrm{K}562*\mathrm{E}}^{+}-\%\mathrm{CD}107{\mathrm{a}}_{\mathrm{NK}}^{+}}\right]=\mathrm{Env\; -\; specific}\mathrm{NK}\mathrm{cell}\mathrm{activity}$