Search

In the heterozygous T lymphoma line LDHB, variants which have lost the expression of individual H‐2 class I genes are spontaneously generated in vitro at a frequency of 10(‐1)‐10(‐2). A cell line (HK13) in which the class I gene Kk is stably expressed (frequency of loss variants less than 10(‐4) was selected from LDHB cells by fluorescence activated cell sorting. Further selection of HK13 cells for high Kk expression led to the isolation of the HK22 line which expresses twice as much Kk as HK13. From HK13 and HK22 cells, spontaneous structural variants of Kk having lost individual serological determinants of the Kk wild‐type molecule, were isolated by fluorescence activated cell sorting. Such variants occur at a frequency of 10(‐6)‐10(‐7) per cell per generation. The analysis of these variants indicates that they carry mutations in the Kk structural gene and that HK13 cells express a single Kk gene which appears to be duplicated in HK22 cells. We did not find evidence for the generation of variants expressing ‘alien’ class I products in the LDHB cell line. The instability of class I gene expression in LDHB cells and the transition to stable expression may represent steps of T‐cell differentiation in the thymus.

We describe here a method for the isolation of somatic cell variants that express a structurally altered gene product on their cell surface. The method makes it possible to select positively for cells of the variant phenotype which is defined by loss of a given antigenic determinant. As an example, we present the isolation of a cloned cell line expressing a structurally altered H–2Kkmolecule, designated H-2KkS1. Cells of this phenotype were found to be present in the wild-type population at a frequency of 10−5–10−6and were isolated in three rounds of selection in the fluorescence-activated cell sorter (FACS) and subsequent growth in vitro.