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. 2005 Nov;116(3):354-61.
doi: 10.1111/j.1365-2567.2005.02235.x.

Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity

Elena G Addison  1 , Janet North, Ismail Bakhsh, Chloe Marden, Sumaira Haq, Samia Al-Sarraj, Reza Malayeri, R Gitendra Wickremasinghe, Jeffrey K Davies, Mark W Lowdell
Affiliations

Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity

Elena G Addison et al. Immunology. 2005 Nov.

Abstract

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha form are more cytotoxic than their CD8- counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8- NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8alpha+ cells after lysis of the same targets. We report that cross-linking of the CD8alpha chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8alpha+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8- subset, CD8alpha+ NK cells are capable of sequential lysis of multiple target cells.

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Figures

Figure 1
Figure 1
CD8+ NK cells from normal donors and AML patients in remission are more cytotoxic than CD8– NK cells against K562 cells and autologous AML blasts, respectively. Freshly isolated CD8+ or CD8– NK cells from normal donors (n = 5) or patients with AML in complete remission after chemotherapy (n = 4) were incubated in triplicate with PKH-26-labelled K562 cells (a) or autologous AML blasts (b), respectively, in 4-hr assays at 37°. Cell death was determined by flow cytometric measurement of influx of To-Pro iodide into PKH26+ target cells. Results are presented as paired results for 5 : 1 effector : target ratios. Paired Student's t-test showed consistent significant increases (P < 0·05; Student's paired t-test) in cytotoxicity of CD8+ NK subset compared to the CD8– subset from the matched donor in all cases.
Figure 2
Figure 2
Co-incubation of NK cells with target cells initiates apoptosis in the CD8– subset which is not apparent in the CD8+ cells. Purified NK cells from normal donors (n = 5) were incubated with equal numbers of PKH-26-labelled K562 cells for 1 and 3 hr. Cells were labelled with anti-CD8 APC and target cells were excluded from the analysis by virtue of PKH26 expression. CD8– NK cells (a) showed a marked increase in the proportion of annexin V+/FSClow (apoptotic) cells within 1 hr of co-incubation which was absent in the CD8+ subset even at 3 hr (c). The apoptosis of the CD8– NK cells at 1 hr was confirmed by the detection of activated Caspase 3 (b) as determined by PhiPhiLux which was not apparent in the CD8+ subset (d). The figures in each plot represent the proportion of apoptotic cells. The paired changes in annexin V expression in CD8– and CD8+ cells for the five donors at 1 and 3 hr is shown in (e).
Figure 3
Figure 3
Induction of intracellular Ca2+ in purified CD8+ ve NK cells by anti-CD8 MAb. Purified CD8+ ve (OKT8) NK cells were loaded with the fluorescent calcium indicator Fluo-3/AM and stimulated with RPA-T8 in the presence of extracellular calcium (A) or sequentially with goat-antimouse Ig and RPA-T8 in the absence of extra-cellular calcium (B). Stimulation with anti-CD56 was used as a negative control (C). Calcium ionophore was used as a positive control (D). Ligation of CD8 with RPA-T8 induces surface expression of CD69 within 4 h (E). Asterisks indicate results which are significantly greater (P < 0·05) than the untreated cells.
Figure 4
Figure 4
CD8 is not a cytotoxic triggering molecule on freshly purified or IL-2-stimulated NK cells. Neither freshly isolated NK cells at various effector : target ratios (a) nor IL-2-activated NK cells at an effector : target ratio of 5 : 1 (b) were triggered by either anti-CD8 (RPA-T8) or anti-CD8 (OKT8) to mediate reverse ADCC of P815 target cells (a kind gift from Prof. M. Glennie, University of Southampton). Anti-CD16 (3G8), was used as a positive control. Asterisks indicate results which are significantly greater (P < 0·05) than the background NK lysis in the absence of mAb.
Figure 5
Figure 5
NK cells conjugate simultaneously to target cells and to other NK cells and provide CD8 ligation. Freshly isolated NK cells (CD8+ and CD8–) were co-incubated with K562 cells for 30–240 min. At 60 min NK–NK and NK–target cell conjugates formed (a). (b) shows a 90-minute conjugate of a single K562 target cell (i) and a CD8+ NK cell (ii) with capping of the CD8 molecule (Leu2 FITC) at the cell–cell synapse between two CD8+ NK cells (ii and iii). Furthermore, the second CD8+ NK cell has formed synapses with two CD8– NK cells (iv). The insert (v) shows the homogeneous expression of CD8 on a non-conjugated NK cell. Smaller conjugates were apparent involving a CD8+ NK cell (ii) and a target cell (i) with CD8 ligation by a single CD8– NK cell (iii) (c). Apoptosis was measured at 240 min by flow cytometric assessment of annexin V expression on CD8+ and CD8– NK cells in the presence or absence of W632 (d). CD8+ NK showed low expression of annexin V (shaded histogram) in the absence of W632 in contrast to the high expression on the CD8– cells (dashed histogram) and coincident reduction in forward angle light scatter (not shown) indicative of apoptosis. In the presence of the MHC Class I blocking mAb W632 the CD8+ NK cells showed equivalent levels of apoptosis to the CD8– cells (solid line). In 1-hr K562 conjugation assays with five normal donors (e), blockade of the CD8–MHC Class I interaction with W632 (solid bars) increased the activation-induced apoptosis within the CD8+ NK subset significantly (P < 0·001; paired t-test; n = 5) to levels equivalent to those of CD8– NK cells in the same culture and this could be prevented by direct ligation of CD8 on the NK-cell surface with RPA-T8 mAb (hatched bars). Neither mAb had any effect on the CD8– NK cells in the culture (e).
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