Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection.

Most, if not all, autoantibodies specific for bromelain-treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti-PtC antibodies are produced by 5%-15% of CD5+ Ly-1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5- B cells. At 1 week of age PtC-specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC-specific Ly-1 B cells in B10.H-2aH-4bp/Wts mice predominantly express either of two heavy and kappa chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the V kappa-J kappa junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and V kappa-J kappa junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC-specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly-1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti-PtC antibodies of higher affinity during the formation of the Ly-1 B repertoire because the V region genes expressed by PtC-specific cells are unmutated. Our findings are consistent with an important, germ line-encoded function for the immunoglobulin products of these gene combinations.

Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline.

5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH-4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34.

Phosphatidyl choline is recognized by a series of Ly-1+ murine B cell lymphomas specific for erythrocyte membranes.

Cells from 6 of 14 different Ly-1+ murine B cell lymphomas bound to synthetic liposomes encapsulating fluorescein. The liposomes were made from distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG), and cholesterol. In all cases, liposome binding was due to recognition of phosphatidyl choline by the surface IgM on the tumor cells. Liposome binding could be inhibited by DSPC but not by DSPG, and the number of liposomes bound per cell was directly related to the cell surface concentration of IgM. The IgM secreted by a hybridoma derived from one of the lymphomas, CH12, was shown to agglutinate liposomes, and was used in a solid-phase immunoassay to study inhibition of liposome binding by pure phospholipids; DSPC and sphingomyelin both inhibited, whereas DSPG did not. The Ig borne by the six lymphomas that bind phosphatidylcholine also bind to both SRBC and bromelain-treated mouse erythrocytes. The idiotypic of CH12 IgM is similar to that expressed by Ly-1+ normal splenic B cells of the same specificity. The significance of these data in relation to other commonly studied autoantigens, and to the restricted specificity of normal Ly-1+ B cells is discussed.