Characterization of a surface membrane molecule expressed by natural killer cells in most inbred mouse strains: monoclonal antibody C9.1 identifies an allelic form of the 2B4 antigen

Abstract

A newly generated monoclonal antibody (mAb C9.1) described in this study identifies a surface membrane molecule that is involved in the lytic programme of activated natural killer (NK) cells. This conclusion is based on the facts that, first, this antigen was expressed on the vast majority of surface immunoglobulin (sIg)- CD3- CD4- CD8- spleen lymphocytes, albeit it was also present on minor subsets of sIg+ B (approximately 7%) and CD3+ T (approximately 2%) lymphocytes; second, that all splenic NK activity was contained within the C9.1+ cell population, and was almost totally abolished by treatment of spleen cells with mAb C9.1 and complement; third, that mAb C9.1 was capable of increasing interleukin-2-cultured and in vivo polyinosinic:polycytidylic acid-activated, NK cell-mediated, antibody-redirected lysis, but not freshly isolated NK cell-mediated killing. Furthermore, the strain distribution of the C9.1 antigen was shown to be antithetical to that of the 2B4 antigen already described as a molecule associated with major histocompatibility complex-unrestricted killing mediated by activated NK cells. The gene encoding C9.1 antigen was linked to the Akp1 isozyme locus on chromosome 1 close to the 2B4 gene. Although C9.1 and 2B4 were monomeric glycoproteins of 78 000 MW and 66 000 MW, respectively, removal of N-linked sugars from both antigens by endoglycosidase F yielded identical protein backbones of 38 000 MW. Thus, all of these results suggest that mAb C9.1 recognizes an allelic form of the 2B4 antigen. However, the detection of mAb C9.1-reactive antigen on a minor subset of B cells may suggest a possible reactivity of mAb C9. 1 with some product of other members of the 2B4 family genes.

Figure 1

Two-colour immunofluorescence (flow cytometry) analyses of surface expression of an antigen recognized by mAb C9.1 on C3H mouse spleen lymphocytes. Spleen lymphocytes (a and b) or B-cell-depleted NWNA spleen lymphocytes (c–g) were stained with FITC-labelled mAb C9.1 or biotinylated mAb C9.1 and simultaneously with biotinylated or FITC- or PE-conjugated anti-sIg (a), anti-CD45 (B220) (b), anti-CD3 (c), anti-TCRγδ (d), anti-TCRαβ (e), anti-CD4 (f), or anti-CD8 (g). Biotinylated antibodies were detected with PE– Streptavidin.

Figure 2

Lysis of YAC-1 cells by C9.1⁺ and C9.1⁻ spleen cells. NWNA spleen cells prepared from C3H/He mice pretreated with polyI:C 16 hr before ⁵¹Cr-release assays were sorted into C9.1⁺(•) and C9.1⁻ (□) cells and were used as effector cells; unsorted NWNA cells (▪).

Figure 3

Complement-dependent NK-inhibitory activity of mAb C9.1. PolyI:C-activated spleen lymphocytes from a variety of inbred mouse strains were incubated with mAb C9.1, washed once, and further incubated with anti-rat κ light-chain mAb, and then treated with rabbit complement as described in the Materials and Methods. Per cent inhibition was determined by the following formula [(lytic units of control assay – lytic units of test assay)/(lytic units of control assay)]×100.

Figure 4

SDS–PAGE of ¹²⁵I-labelled antigen recognized by mAb C9.1. (a) A-LAK cells derived from C3H/He mice were iodinated with Na¹²⁵I by the lactoperoxidase method. ¹²⁵I-labelled membrane proteins were incubated with mAb C9.1 (lanes 1 and 3) or mAb FD441.8 (anti-LFA-1) (lanes 2 and 4), followed by incubation with anti-rat IgG mAb-bound protein G–Sepharose 4B. The immune complexes were analysed under non-reducing (lanes 1 and 2) or reducing (lanes 3 and 4) conditions by SDS–PAGE on 7·5% gels and subjected to autoradiography. (b) A-LAK cells from (C57BL/6×C3H) F₁ mice were radioiodinated and immunoprecipitated with mAb C9.1(lanes 1 and 3) or anti-2B4 mAb (lanes 2 and 4) as in (a). Immune complexes were treated with (lanes 3 and 4) or without (lanes 1 and 2) endoglycosidase F, analysed under reducing conditions by SDS–PAGE on 10% gels and subjected to autoradiography.

Figure 5

mAb C9.1-induced, redirected cytotoxicity. A-LAK cells derived from C3H/He mice (a), spleen cells from C3H/He mice pretreated with polyI:C (polyI:C-activated NK cells) (b), or freshly isolated NK cells from normal C3H/He mice (c) were used as effector cells in standard 4-hr ⁵¹Cr-release assays against ⁵¹Cr-labelled FcγR⁺ Daudi target cells at the indicated ratios. Assays were performed in the presence of mAb C9.1 (□), or mAb H12 (◊), or in their absence (•). As a positive control, ⁵¹Cr-labelled YAC-1 cells were included in each assay as target cells (▪).