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L.H. Mason^*, S.K. Anderson, W.M. Yokoyama, H.R.C. Smith, R. Winkler-Pickett^*, and J.R. Ortaldo^*

From the ^* Laboratory of Experimental Immunology, Division of Basic Sciences, and Intramural Research Support Program, SAIC Frederick, National Cancer Institute-FCRDC, Frederick, Maryland 21702-1201; Division of Rheumatology, Washington University, St. Louis, Missouri 63110

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D⁺ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D⁺ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D⁺ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D⁺ NK cells can augment lysis of FcR⁺ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D⁺ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Animal care was provided in accordance with the procedures outlined in the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 86-23, 1985).

¹Abbreviations used in this paper: ab+c', antibody plus complement; FCA, flow cytometry analysis; ITIM, immunoreceptor tyrosine-based inhibitory motif; IP, immunoprecipitation; NWNAD, nylon wool–nonadherent (cells); PI, propidium iodide; RADCC, reverse antibody-dependent cellular cytotoxicity.

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