QUANTITATIVE CYTOMETRY OF MHC CLASS-I DIGESTION FROM LIVING CELLS

Digestion of crude membrane preparations with papain releases the extr acellular portion of major histocompatibility complex (MHC) class I mo lecules. MHC class I molecules are integral membrane glycoprotein comp lexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the beta(2)-microglobulin (beta(2)-m), to a wide a rray of peptides, The cleaved soluble moiety retains the antigenic pro perties of the intact membrane-bound complex. Here we show that MHC cl ass I digestion may be carried out on living cells, and we quantitate the surface expression of MHC complexes by a combined cytometric/high performance liquid chromatographic (HPLC) approach. Papain digestion r esults in time- and dose-dependent disappearance of membrane MHC-assoc iated-fluorescence as detected by FAGS analysis with MHC-specific mono clonal antibodies (mAbs). beta(2)-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were qu antitated by comparison with purified standards, The cytometric assess ment of the digestion allows one to simultaneously monitor efficacy an d toxicity of the treatment. The procedure we describe allows to selec tively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, ''immature'' MHC mol ecules. (C) 1997 Wiley-Liss, Inc.