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 approximate ly 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-immunoprecip itation experiments demonstrated that CD3.5 could be immunoprecipitate d from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 coul d be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag comple xes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in diges tion profiles that clearly distinguished the glycoproteins. Additional ly, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sial oglycoprotease, 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 mo lecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitel y sensitive to very low-dose chymotrypsin treatment. Therefore, our da ta suggest that GD3.5 Ag is a previously uncharacterized, lineage-spec ific gamma delta T cell Ag. Furthermore, we show that CD3.5 and WC1 ar e sialomucins, which provides important clues to their function.