Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family.
In this report, we describe GD3.5, a new lineage-specific gamma delta T cell marker that is distinct from TCR and known Workshop Cluster 1 (WC1). FACS analysis indicated that GD3.5Ag is expressed on approximately 90% of the peripheral blood gamma delta T cell population and GD3.5 specifically stained gamma delta T cells and not alpha beta T cells, B cells, neutrophils, or monocytes. Also, a significant portion of the GD3.5-positive population was WC1-negative. Nonreducing Western blot analysis and immunoprecipitation experiments revealed a single 220- to 240-kDa glycoprotein recognized by GD3.5 compared with two WC1 bands at 200 kDa and 300 kDa recognized by the IL-A29 Ab. Cross-immunoprecipitation experiments demonstrated that GD3.5 could be immunoprecipitated from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 could be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag complexes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in digestion profiles that clearly distinguished the glycoproteins. Additionally, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sialoglycoprotease, and neuraminidase, indicating differences in N- and O-linked sugars and the presence of sialic acid residues. Both GD3.5 Ag and WC1 appeared to be sialomucin-like molecules that share similar O-sialoglycoprotein endopeptidase sensitivity with other cell surface molecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitely sensitive to very low-dose chymotrypsin treatment. Therefore, our data suggest that GD3.5 Ag is a previously uncharacterized, lineage-specific gamma delta T cell Ag. Furthermore, we show that GD3.5 and WC1 are sialomucins, which provides important clues to their function.
Jones
W M
WM
Department of Veterinary Molecular Biology, Montana State University, Bozeman 59717, USA.
Walcheck
B
B
Jutila
M A
MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry*; D000954:Antigens, Surface / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D003429:Cross Reactions; D010450:Endopeptidases; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology*; D011233:Precipitin Tests; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000276:immunology*; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C086500:DMBT1 protein, Bos taurus; D008562:Membrane Glycoproteins; D016692:Receptors, Antigen, T-Cell, gamma-delta; D010450:Endopeptidases