Immunogenetics (2000) 51 : 246–248 Q Springer-Verlag 2000 BRIEF COMMUNICATION Zihai Li 7 Veronika Groh 7 Roland K. Strong Thomas Spies A single amino acid substitution causes loss of expression of a MICA allele Received: 4 August 1999 / Revised: 27 September 1999 Z. Li 1 7 V. Groh 7 R.K. Strong 7 T. Spies (Y) Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA e-mail: tspies6fred.fhcrc.org Tel.: c1-206-6676940 Fax: c1-206-6675978 Present address: 1 Center for Immunotherapy, University of Connecticut Health Center, MC 1601, Farmington, CT 06030, USA Key words MHC 7 MICA 7 Tumor cell lines 7 Null allele The human major histocompatibility complex (MHC) class I homologues MICA and MICB are stress-induci- ble surface molecules that are not associated with b 2 - microglobulin and peptides, are expressed in intestinal epithelium and epithelial tumors, and are recognized by V d 1 gd T cells, a subset of gd T cells that is enriched in epithelial sites (Bahram et al. 1994; Groh et al. 1996, 1998, 1999). Analogous to the interactions of MHC class I molecules with inhibitory or activating natural killer (NK) cell receptors that are expressed on NK cells and T cells (Lanier 1998), MICA/B are ligands for an activating receptor, NKG2D, which is expressed on most NK cells, CD8 c ab T cells and gd T cells (Bauer et al. 1999). MICA/B are variably expressed in some lung, breast, kidney, ovary, prostate, and colon carcinomas (Groh et al. 1999). While exploring this heterogeneity using cell lines, we identified two gastric epithelial tu- mor lines that lacked expression of MICA protein de- spite the presence of abundant mRNA. Analysis of this deficiency showed that both cell lines express a single allele of MICA (MICA010), which is defective because of a single amino acid substitution in the a1 domain that can be predicted to interfere with a stable protein fold. Epithelial tumor cell lines were screened by flow cy- tometry for lack of expression of MICA and/or MICB, using monoclonal antibodies (mAbs) specific for MICA (mAbs 2C10 and 3H5) or MICA and MICB (mAbs 6D4 and 6D5) (Groh et al. 1996, 1998). Two gastric tumor cell lines, AGS and KATO III, had little or no surface MICA, compared with the positive con- trol HCT116 and Lovo colon carcinoma lines, although they contained equal or larger amounts of MICA mRNA. AGS cells expressed MICB mRNA and stained with the mAbs 6D4 and 6D5, whereas KATO III cells had little or no MICB mRNA (Fig. 1A,B; all cell lines were from the American Type Culture Collec- tion). We examined whether the lack of MICA surface expression was due to a defect in protein processing in the secretory pathway. Immunoprecipitation with the mAbs 2C10 and 3H5 from lysates of metabolically la- beled cells, N-glycanase treatment of immunocom- plexes, and sodium docecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detected the 43,000 M r MICA polypeptide in HCT116 but not in AGS and KATO III cells (Fig. 1C). However, transfection of cDNA or of a cosmid encoding MICA (allele 004) res- tored surface MICA on KATO III cells (Fig. 2A). This indicated that expression of the endogenous gene was not impaired by a post-transcriptional or protein-proc- essing defect, but was likely caused by a deleterious se- quence alteration. A substantial number of allelic variants of MICA have been described (Fodil et al. 1996), which have so far not been associated with differences in function. Neither the distribution of amino acid substitutions nor the crystal structure of MICA have offered potential explanations (Li et al. 1999). Using specific primers and reverse transcription-polymerase chain reaction, MICA mRNA sequences were derived from KATO III and AGS cells. Direct sequencing of amplicons identified a single sequence that was identical in both cell lines and corresponded to the MICA010 allele, which has a prol- ine for arginine substitution at position 6 in the first b strand of the a1 domain (Fig. 2B) (Fodil et al. 1996). Arginine at this position is conserved in all MICA and MICB alleles, as well as in homologous sequences from diverse nonhuman primate species (Ando et al. 1997;