Multiple receptors and ligands are involved in NK cell-mediated lysis of activated CD4⁺ T cells.

<p>Role of (A) activating and (B) inhibitory NK receptors in NK cell degranulation. Left column: representative histograms (of n≥3) for surface expression of ligands on activated (thick black line) and resting CD4⁺ T cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated CD4⁺ T) and filled histogram (resting CD4⁺ T). Middle- and right column: NK and CD4⁺ T cells were activated for 4 days <i>in vitro</i> as described, and co-cultured for 4 hours with 10 ug/mL mAb (or relevant isotype-matched control Ig). Degranulation is shown for CD56^dim (middle column) and CD56^bright (right column) NK cells. Representative histograms of surface expression of receptors on activated (thick black line) and resting NK cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated NK) and filled histogram (resting NK). * <i>P</i><0.05, ** <i>P</i><0.005, *** <i>P</i><0.001. (C) Sorted IL-2-activated CD56^dim and CD56^bright NK cells were co-cultured with ⁵¹Cr-labeled activated CD4⁺ T cells in a ⁵¹Cr-release assay with human IgG4 isotype control (•) or anti-NKG2A mAb (○). Data represents n = 3 experiments.</p

NK cells mediate TRAIL-dependent cytotoxicity towards activated CD4⁺ T cells.

<p>Autologous NK cells and CD4⁺ T cells were isolated and activated for 4 days as described. Resting NK and CD4⁺ T cells were unstimulated in media for 4 days. (A) Representative histograms for flow cytometric analysis of surface expression of TRAIL-R1 (DR4) and TRAIL-R2 (DR5) on activated CD4⁺ T cells (thick black line) and resting CD4⁺ T cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated CD4⁺ T) and filled histogram (resting CD4⁺). (B) Histograms are representative of TRAIL surface expression on activated NK cells (thick black line) and resting NK cells (thin black line). Isotype-matched control Ig are represented by dashed line (activated NK) and filled histogram (resting NK). (C) Sorted IL-2-activated CD56^dim and CD56^bright NK cells were co-cultured with ⁵¹Cr-labeled activated CD4⁺ T cells in a ⁵¹Cr-release assay with isotype-matched control Ig (•) or anti-TRAIL mAb (○). Experiment shown is representative of n = 3.</p

NKG2D and LFA-1 act synergistically in mediating NK cell degranulation.

<p>NK and CD4⁺ T cells were activated for 4 days <i>in vitro</i> as described, and co-cultured for 4 hours with FITC conjugated anti-CD107a+anti-CD107b mAbs. 10 ug/mL blocking antibodies (or relevant isotype-matched control Ig) were added where indicated. Flow cytometry was performed to assess degranulation of (A) CD56^dim and (B) CD56^bright NK cells. (C) CD56^dim NK cells were gated based on surface expression of CD94/NKG2A, and degranulation of each subset was evaluated. * <i>P</i><0.05.</p

Activated NK cells kill activated, but not resting, CD4⁺ T cells.

<p>(A) Representative gating strategy. NK cells were defined as viable, CD3⁻ singlets. NK cell subsets were defined based on expression of CD16 and CD56. (B) Activation of CD4⁺ T cells was confirmed by CD69 upregulation. CD4⁺ T cells were activated for 4 days with anti-CD3+anti-CD28 Dynabeads (propionate was added on day 3). Resting CD4⁺ T cells were unstimulated in media for 4 days. (C–D) NK cells were cultured for 4 days in IL-2, and CD4⁺ T cells were activated as described. Resting CD4⁺ T cells were unstimulated for 4 days in culture. Autologous NK cells and CD4⁺ T cells were co-cultured at an E∶T ratio of 1∶1 for 4 hours with FITC-conjugated anti-CD107a+anti-CD107b mAb. Flow cytometry was performed to determine CD107a/b expression on NK cell subsets. Data shown in (C) are for a representative donor, (D) are for n = 9. Data represent mean ± SEM. * <i>P</i><0.05; *** <i>P</i><0.001. (E) Sorted CD56^dim (□/▪) and CD56^bright (○/•) NK cells were cultured in media (□/○) or IL-2 (▪/•) for 4 days, and co-cultured with ⁵¹Cr-labeled activated CD4⁺ T cells in a ⁵¹Cr-release assay. Experiment shown is representative of n = 3.</p

CD56^bright NK cells have a higher cytotoxic potential towards activated CD4⁺ T cells.

<p>Autologous NK cells and CD4⁺ T cells were isolated, and CD4⁺ T cells were activated for 4 days as described. NK cells were activated as indicated: 200 IU/mL IL-2, 25 ng/mL IL-4, 25 ng/mL IL-7, 25 ng/mL IL-9, 5 ug/mL IL-15, 100 ug/mL IL-21, 50 ug/mL IL-12, 0.25 mg/mL IL-18 or 100 U/mL IFN-αA. NK cells and CD4⁺ T cells were co-cultured for 4 hours with FITC-conjugated anti-CD107a+anti-CD107b antibodies. Flow cytometry was performed to determine degranulation of (A) CD56^dim NK cells and (B) CD56^bright NK cells. Data represent mean ± SEM of n≥4 experiments. Statistical significance is calculated in comparison to resting NK cells (media) co-cultured with activated CD4⁺ T cells. * <i>P</i><0.05, ** <i>P</i><0.005, *** <i>P</i><0.001.</p