Quantitative cytometry of MHC class I digestion from living cells.
Digestion of crude membrane preparations with papain releases the extracellular portion of major histocompatibility complex (MHC) class I molecules. MHC class I molecules are integral membrane glycoprotein complexes formed by the noncovalent association of 2 invariant molecules, the heavy chain and the beta2-microglobulin (beta2-m), to a wide array of peptides. The cleaved soluble moiety retains the antigenic properties of the intact membrane-bound complex. Here we show that MHC class 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 results in time- and dose-dependent disappearance of membrane MHC-associated-fluorescence as detected by FACS analysis with MHC-specific monoclonal antibodies (mAbs). beta2-m and peptides became detectable by HPLC analysis and western blotting in the digestion buffer and were quantitated by comparison with purified standards. The cytometric assessment of the digestion allows one to simultaneously monitor efficacy and toxicity of the treatment. The procedure we describe allows to selectively retrieve by affinity chromatography MHC from the cell membrane, avoiding any contamination due to intracellular, "immature" MHC molecules.
Galati
G
G
Laboratori di Immunologia dei Tumori e di Tecniche Separative, Istituto Scientifico H.S. Raffaele and Università di Milano, Milan, Italy.
Arcelloni
C
C
Paroni
R
R
Heltai
S
S
Rovere
P
P
Rugarli
C
C
Manfredi
A A
AA
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry*; D002469:Cell Separation; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D051379:Mice; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D014407:Tumor Cells, Cultured; D001613:beta 2-Microglobulin / Q000032:analysis
D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D001613:beta 2-Microglobulin; D010206:Papain