Blood, Vol. 114, Issue 18, 3822-3830, October 29, 2009

The decreased expression of Siglec-7 represents an early marker of dysfunctional natural killer–cell subsets associated with high levels of HIV-1 viremia
Blood Brunetta et al. 114: 3822

Supplemental materials for: Brunetta et al

CD107a degranulation assay
Freshly purified PBMCs were activated with recombinant IL-2 (rIL-2) at 100 UI/ml for 24 hours. 2 × 10⁵ rIL-2 activated PBMCs were then washed twice and cultured in a 96 flat bottom well plate with 2 × 10⁵ K562 cell line in 200 µl of complete medium. Cells were mixed by gentle pipetting, spun down for 3 minutes at 300 rpm, and incubated for 3 hours at 37°C in 5% C0₂ in the presence of a PE-labeled CD107a mAb (BD Pharmigen). Thereafter, cells were washed, stained with the previously mentioned CD56, CD3, CD14, CD19, and CD16 directly conjugated mAbs and incubated in PBS 2% FBS for 45 minutes on ice. Cells were then washed and analyzed by flow cytometry.³¹

Intracellular IFN-γ production
Freshly purified PBMCs were activated with rIL-2 at 100 UI/m for 24 hours. In order to trigger an optimal NK cell activation within PBMCs, 96 flat bottom well plate were previously coated overnight with an anti-human CD16 IgM mAb (Immunological Sciences) at 5 µg/ml in PBS 2% FBS. r-IL2-activated PBMCs were plated at a concentration of 1 × 10⁶ cells/ml in flat bottom 96 well plate in 200 µl of complete medium and incubated for 3 hours at 37°C in 5% C0₂. 0.3 µg of monensin (Golgistop, BD Pharmigen) was added after 3 hours of incubation. PBMCs were then harvested, washed, and stained with the previously mentioned CD56, CD3, CD14, CD19, and CD16 directly conjugated mAbs for 30 minutes at 4°C. Cells were then washed with in PBS 2% FBS, fixed, and permeabilized by cytofix/cytoperm solution and washed with perm-wash solution 1× (BD-Pharmigen) according to the protocol provided from the manufacturer. Cells were then incubated for 30′ with a PE-labeled IFN-γ mAb (BD Pharmigen), washed, and analyzed by flow cytometry.

Statistical analysis
The distributions of each immune parameter between healthy donors and all HIV-1 infected patients at different stages of diseases were compared using the Mann-Whitney test. The functional differences between the three different NK cell subsets in each single group of uninfected and HIV-1 infected individuals were evaluated using the Wilcoxon signed ranks test. All p-values are 2-sided and unadjusted. The statistical differences of surface levels of NK cell receptors between the baseline (time 0) and the different time points of viral suppression in patients undergone ART (Times 6, 12, 18, and 24) were calculated using the one-way analysis of variance (ANOVA with tukey correction).