Binding of antigen to Ia molecules on intact antigen presenting cells demonstrated by photoaffinity labeling.
We used a photoaffinity labeling technique to investigate whether a molecular interaction occurs between antigen and Ia molecules on antigen presenting cells (APC) in the absence of T lymphocytes. M.12.4.1 B lymphoma cells (Iad), which are able to present bovine insulin to Iad lymph node primed T cells, were given radioiodinated bovine insulin derivatized with the photoreactive group (2-nitro-4-azidophenylacetyl) at Lys 29 of the B chain of the insulin molecule. Processing of insulin was allowed by incubating the APC with antigen for increasing periods of time at 37 degrees C or 4 degrees C. The covalent coupling of the processed photoreactive antigen to any neighboring cellular protein was thereafter induced by u.v. irradiation. Immunoprecipitation of membrane proteins by monoclonal antibodies showed that under these conditions, the alpha and beta subunits of the Ia molecules were selectively photolabeled. Labeling was time- and temp-dependent as was the internalization of insulin. The apparent mol. wts of the antigen-Ia molecule complexes were not significantly different from that of native Ia molecules radioiodinated by surface labeling, indicating that only a small fragment of the antigen was covalently coupled to Ia molecules. Similar experiments performed with human B lymphoma cells (526 cells) gave similar results. These observations therefore indicate: (1) that Ia molecules expressed by intact APC are able to bind antigens in the absence of T lymphocyte antigen receptor; and (2) that this association, at least for insulin, requires uptake and a proteolytic fragmentation of the antigen by the APC.
Cousin
J L
JL
INSERM U210, Faculté de Médecine (Pasteur), Nice, France.
del Vesco
P
P
Samson
M
M
Brandenburg
D
D
Fehlmann
M
M
D000345:Affinity Labels; D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism*; D002460:Cell Line; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell; D013601:T-Lymphocytes; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D000949:Histocompatibility Antigens Class II; D007328:Insulin; D011948:Receptors, Antigen, T-Cell