Activation, coactivation, and costimulation of resting human natural killer cells

Abstract

Natural killer (NK) cells possess potent perforin- and interferon-gamma-dependent effector functions that are tightly regulated. Inhibitory receptors for major histocompatibility complex class I display variegated expression among NK cells, which confers specificity to individual NK cells. Specificity is also provided by engagement of an array of NK cell activation receptors. Target cells may express ligands for a multitude of activation receptors, many of which signal through different pathways. How inhibitory receptors intersect different signaling cascades is not fully understood. This review focuses on advances in understanding how activation receptors cooperate to induce cytotoxicity in resting NK cells. The role of activating receptors in determining specificity and providing redundancy of target cell recognition is discussed. Using Drosophila insect cells as targets, we have examined the contribution of individual receptors. Interestingly, the strength of activation is not determined simply by additive effects of parallel activation pathways. Combinations of signals from different receptors can have different outcomes: synergy, no enhancement over individual signals, or additive effects. Cytotoxicity requires combined signals for granule polarization and degranulation. The integrin leukocyte function-associated antigen-1 contributes a signal for polarization but not for degranulation. Conversely, CD16 alone or in synergistic combinations, such as NKG2D and 2B4, signals for phospholipase-C-gamma- and phosphatidylinositol-3-kinase-dependent degranulation.

Fig. 1. Inhibitory receptors expressed on human peripheral blood NK cells

Inhibitory receptors expressed by freshly isolated, resting NK cells and their ligands are listed. KIR, NKG2A, LIR-1, KLRG1, NKR-P1, Siglec-7, and Siglec-9 are only expressed by subsets of NK cells.

Fig. 2. Activating receptors expressed on human peripheral blood NK cells

Activating receptors expressed by freshly isolated, resting NK cells and their ligands are listed. KIR, NKG2C, and CD2 are only expressed by subsets of NK cells.

Fig. 3. Regulation of LFA-1–mediated adhesion

Inside-out signals from NK cell activating receptors may promote conformational changes leading to a high affinity, ligand binding conformation of LFA-1. They may also promote LFA-1 avidity through signals for clustering of LFA-1. Upon ligand binding, LFA-1–mediated outside-in signals are conveyed into the cell.

Fig. 4. Regulation of granule polarization

In resting NK cells, (A) LFA-1 alone or (B) a synergistic combination of signals from CD16 and 2B4 promote granule polarization.

Fig. 5. Pharmacological inhibitors of PLC-γ abrogate degranulation by resting NK cells

Resting NK cells were pre-incubated with vehicle (DMSO), a Src kinase inhibitor (PP2), a PLC-γ inhibitor (U73122), or an inactive analog of the PLC-γ inhibitor (U73343), for 30 min. Thereafter, NK cells were incubated for 2 hours either alone or with P815 cells and mAbs in addition to inhibitors as specified. Cells were stained with flourochrome-conjugated anti-CD56 and anti-CD107a mAbs and analyzed by flow cytometry. NK cells were gated on forward scatter/side scatter plots and the percentage of CD56⁺ CD107a⁺ NK cells was calculated. One representative experiment is shown.

Fig. 6. Summary of NK cell activation by insect cells expressing ligands for human receptors

Ligands expressed on insect SC2 cells are indicated in yellow (CD48), blue (ICAM-1), and green (IgG). The outcome of interaction with NK cells is listed on the right.

Fig. 7. Co-activation receptors co-stimulate degranulation for antibody-dependent cellular cytotoxicity

Engagement of CD16 is sufficient to induce Ca²⁺ mobilization and degranulation in resting NK cells. Ca²⁺ mobilization and degranulation is enhanced by co-engagement of co-stimulatory receptors such as NKG2D and 2B4.

Fig. 8. Co-activation of resting NK cells

(A) Schematic representation of synergies among co-activation receptors for Ca²⁺ mobilization among receptors expressed on resting NK cells. (B) Co-engagement on non-ITAM–associated receptors can synergistically induce Ca²⁺ mobilization and degranulation in resting NK cells.

Fig. 9. Pharmacological inhibition of PI3K abrogates NKG2D–mediated co-activaiton of degranulation, but not Ca²⁺-flux in resting NK cells

(A) NK cells were pre-incubated on ice with inhibitor and mAbs to the receptors indicated on the left, loaded with Fluo-4 and Fura Red, resuspended in HBSS 1% FBS, and pre-warmed at 37°C in the presence of inhibitor as indicated. Cells were analyzed by flow cytometry. After 30 seconds, secondary F(ab’)₂ goat anti-mouse IgG was added to each sample. Changes in Fluo-4 (FL-1)/Fura (FL-3) ratios are shown as a function of time. Black lines represent activation with isotype control mAb; blue lines represent activation by receptors in the presence of vechicle (DMSO); red lines represent activation by receptors in the presence of PI3K inhibitor (wortmannin). (B) NK cells were pre-incubated with vehicle (dark bars) or inhibitor (shaded bars), and incubated for 2 hours either alone or with P815 cells and the indicated mAbs. Cells were stained with fluorochrome-conjugated anti-CD56 and anti-CD107a mAbs, and analyzed by flow cytometry. NK cells were gated on forward scatter/side scatter, and the percentage of CD56⁺ CD107a⁺ NK cells was calculated. One representative experiment is shown.