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HLA and Related Genes, Phenotyping by FACS

Surface expression in cultured cells was determined by indirect immunofluorescence using the appropriate anti HLA class I mAb and FITC- labeled rabbit anti-mouse Ig F(ab)2. Indirect Immunofluorescence has been most widely used to identify and quantify different markers in the same cell populations. Fluorescence was analyzed with a FACsort flow cytometer (Becton-Dickinson). A total of 1 x 10e4 cells were analyzed for each immunoflourescence profile.

HLA class I molecules: The MHC class I complex is a ternary complex composed of MHC class I heavy chain (HC), beta-2 microglobulin (b2m) and peptide. The cell-surface presentation of antigenic peptides by class I MHC molecules to CD8+ T-cell receptors is part of an immune surveillance mechanism aimed at detecting foreign antigens.

HLA-ABC: mAbs W6/32 is employed. Specific for HLA-A, HLA-B and HLA-C.
HLA-A: HLA molecules coded by HLA-A locus genes
HLA-B:HLA molecules coded by HLA-B locus genes
HLA-Bw4, HLA Bw6: HLA epitopes defining a diallelic systems of HLA B molecules

HLA class II molecules:The class II molecules consist of a MW 34000 a chains non covalently associated with a MW 29000 b chain, both of which are polymorphic.

HLA-DR, HLA-DP HLA-DQ: Class II HLA antigen found on B lymphocytes and their precursors, monocyte macrophage lineage cells and activated mature T cells as well as their neoplastic counterparts and some melanoma.

HLA class Ib Non classical MHC proteins which are structurally similar to class I or class II but have different roles in immunity

HLA-E: This molecule bind peptides derived from the signal sequences of the classical HLA-A,.-B and C molecules, creating complexes that are recognized by receptor on natural killer cells (NK).
HLA-G: Is encoded in the class I region and acts to control immune responses at the fetal-maternal interface.

Other

MIC-A, MIC-B Molecules induced by cellular stress
B2m: 12 kd chain non polymorphic polypeptide

Key to results

The fluorescence was quantified by evaluation of fluorescence mean channel

NDNot Determined
-1-5
+6-100
++101-500
+++501-1000
++++>1000