Use of somatic cell genetics to study chromosomes contributing to antigen plus I recognition by T cell hybridomas.

Abstract

Keyhole limpet hemocyanin (KLH)/I region-specific T cell hybridomas have been prepared by fusing KLH/I-specific T cell blasts from mice with single pairs of metacentric chromosomes to the inducible, interleukin 2 (IL-2)-secreting T cell hybridoma FS6-14.13.AG2.1. T cell hybridomas with KLH/I receptors were identified by their ability to secrete IL-2 in response to KLH and the appropriate antigen-presenting cells. After cloning and subcloning, KLH/I reactivity was correlated with the presence or absence of metacentric chromosomes derived from the KLH/I-specific T cell blast parent. Hybridomas were identified that had lost all chromosomes 4 and 6 or 16 and 17 derived from their normal T cell parent, but retained the ability to respond to KLH/I. This suggested that products of genes on these chromosomes did not contribute to the specific portions of T cell Ag/I receptors. These gene products would include, of course, kappa and lambda chains and H-2. We did not obtain any T cell hybridomas that had lost both metacentric (8.12) chromosomes derived from T cells of the Robertsonian mouse strain Rb(8.12)5, so we could not draw any conclusions about the contributions of products of genes on these chromosomes. T cell hybridomas with KLH/I reactivity were found that contained only one metacentric (8.12) chromosome derived from this strain. Moreover, a T cell hybridoma was found that retained both metacentric (8.12) chromosomes from its normal T cell parent, but had lost KLH/I reactivity. These results suggested that neither two chromosomes 8 nor two chromosomes 12 were required for antigen/I reactivity in normal T cells and that antigen/I reactivity was controlled, at least in part, by genes mapping on chromosomes other than 8 or 12.

Murine T cell receptor beta chain is encoded on chromosome 6.

“…The results localizing this gene for the T cell receptor to mouse chromosome 6 conflict with those of Marrack and Kappler (17). In their experiments, Robertsonian fusions between chromosomes 4 and 6 were used to make T cell hybridomas.…”

T cell receptors for responses to Mls determinants and allo-H-2 determinants appear to be encoded on different chromosomes.

“…Experimental Approach. T cell hybridomas are karyotypicaily unstable and lose chromosomes for some period of the time after fusion (6). Hence, if the recognition structures for T cell responses to Mls and H-2 determinants were encoded on the same chromosome, then loss of reactivity to Mls determinants by chromosome loss should also be accompanied by loss of H-2 responsiveness, and vice versa.…”

The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody.

“…Table I lists IL-2 Production and Assay. The ability of T cell hybridomas to be stimulated to produce IL-2 was assessed as previously described with a few modifications (24,28,32). Briefly, 250-#1 cultures were prepared containing limiting numbers of hybridoma T cells (1.25 × 104/cuhure), 100-200 #~ of antigen, and either 1 × 106 irradiated (4,000 rad from a laTCs source) spleen cells or 1 × 10 A20-2J BALB/c B lymphoma cells (33,34) as Ag-presenting cells.…”