Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules

Abstract

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble beta-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein-Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble beta-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I+ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcgammaRIIIA (CD16).

Fig. 1

Analysis of C3 fragments deposited onto target B cell lines using flow cytometry and MoAbs to specific C3 epitopes. Staining was carried out with a rabbit anti-C3c/C3d–FITC mixture (Rab anti-C3–FITC) or FITC-labelled MoAbs specific for C3bi, C3c, C3g, and C3d. Unopsonized target cells were used to assess non-specific staining. The upper panels compare the normal B cell-derived line on the left and Daudi cells on the right, with the solid lines representing the serum-opsonized cells and the dotted lines representing the unopsonized cells. The remaining four panels compare staining only with the two serum-opsonized cell types, with the solid lines representing the normal B cell-derived targets and the dotted lines representing Daudi cells.

Fig. 2

Reduced NK cell CR3-dependent cytotoxicity when an autologous class I⁺ Epstein–Barr virus (EBV)-transformed B cell line target is compared with class I⁻ Daudi cells. NK cells were tested for cytotoxicity in a 4-h assay at E:T cell ratios ranging from 5:1 to 20:1 and the lytic units (for 20% cytotoxicity) per 10⁷ effector NK cells were calculated. A B cell line generated from the NK cell donor's peripheral blood lymphocytes (PBL) was used as a target in comparison with class I⁻ Daudi cells. Priming of NK cell CR3 by addition of 2 μg/ml of soluble β-glucan to the reaction medium resulted in 625 ± 60 lytic units with the iC3b Daudi cells, but only 100 ± 9 lytic units with the iC3b autologous B cell line. Killing was blocked by OKM1 anti-CR3 but not by non-specific mouse IgG (MoIgG). No lytic units of cytotoxicity were detected with either target cell type in the media control lacking β-glucan.

Fig. 3

Enhancement of NK cell CR3-dependent cytotoxicity of autologous B cell line targets by blockade of target cell class I but not class II. A class I⁺ B cell line and class I⁻ Daudi cells were opsonized with equivalent amounts of iC3b as in Fig. 1 and tested for lytic sensitivity to NK cells isolated from the same donor as the B cell line as described in Fig. 2 An IgG1 MoAb to class I (DX17) increased the β-glucan/CR3-dependent lytic sensitivity of the autologous B cell line up to the same level as Daudi cells, while an IgG1 MoAb to class II (MEM 136), had no significant effect on cytotoxicity of either target cell.