CHARACTERIZATION OF THE EPITOPE RECOGNIZED BY A MAB THAT REACTS DIFFERENTIALLY WITH MURINE SUPPRESSOR T-CELLS

Although reliable antibodies are available that distinguish human supp ressor T (T-s) cells from CTL and other T cells, few are available for murine T-s cells. We have developed a mAb (984D4.6.5) that, in the pr esence of complement, depletes alloantigen-specific T-s cells but not CTL. This antibody recognizes activated T-s cells but not their precur sors. In these studies, flow cytometric analysis demonstrates that 984 D4.6.5 reacts with several T-s cell hybridomas, cloned T-s cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not dete cted with BW5147, T-h cell hybridomas, cloned T-h cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expressi on is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relat ively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhi bitors also reduce expression of this epitope. These observations sugg est that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a l arge panel of carbohydrates and glycolipids coupled to BSA. The only o ne that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Com parative analysis shows that both the sequence and the linkage of thes e sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of similar to 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to b e determined, but indirect evidence suggests that it is not CD45.