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Picogram quantities of dinitrophenylated (DNP) dextran, a thymus-independent antigen or lipopolysaccharide, a B cell mitogen, signal down B lymphocytes to unresponsiveness. Down signals were detected by a decision test in which signaled lymphocytes were allowed one hour to react to an immunogenic pulse of DNP-dextran. Depletion of T cells or macrophages did not interfere with the generation of down signals. The signaling can proceed entirely at 4 degrees C and it its negative effect is transitory. Down signals which could be detected after 15 min at 37 degrees C were reversed by 60 min. It is suggested that down regulation by low dose antigenic signals provides a means of distinguishing background noise from true antigenic stimuli.

BALB/c mice immunized with phosphorylcholine-conjugated keyhole limpet hemocyanin (PC-KLH) produce two types of anti-PC antibodies, designated group I and group II, which differ in their fine specificity and idiotype expression. Group II hybridomas can utilize VH genes and VL genes (in particular, V kappa 1-3) distinct from those expressed in the group I-like anti-PC myelomas. Here we have analyzed additional anti-PC hybridomas from BALB/c and (CBA/N X BALB/c)F1 male mice and also anti-PC antibodies purified from BALB/c and C57BL/6N antisera. Isoelectric focusing indicates that one of the group II hybridomas utilizes V kappa 1-3 and that related L chains are expressed in a major portion of group II serum antibodies. Other group II antibodies in antisera express different L chains, some of which are presently unidentified. However, isoelectric focusing analysis also indicates that the L chains of some group II hybridomas and serum antibodies are related to those found in the group I anti-PC myelomas and group I hybridoma and serum antibodies. In addition, one hybridoma was found to utilize lambda 2. Thus, it appears that the anti-PC antibodies with group II-like fine specificity can utilize a variety of VL genes related to, or distinct from, those expressed in group I antibodies.

Three murine anti-phosphorylcholine (PC) hybridomas with group II-like fine specificity patterns isolated during a memory response to PC-keyhole limpet hemocyanin (KLH) are examined at the molecular level to determine the origins of the VH and VL used by these antibodies. Southern blots of Hind III cut DNA were hybridized with a probe specific for the V1 gene of the T15 VH family. The V1 germ-line configuration is retained in these hybridomas indicating that this gene which encodes the VH gene product expressed by most group I anti-PC hybridomas is not used for antibody production. Southern blots of Eco RI cut DNA hybridized to a probe specific for JH1-JH4 indicated that all three hybridomas PCG1-2, PCG1-3 and PCM-23 share a 5.2-kb rearranged JH band, suggesting utilization of a common VH gene segment. N-terminal amino acid sequence analysis of the heavy chains of two of the hybridoma proteins PCG1-2 and PCG1-3 indicates that they belong to mouse heavy chain subgroup II and are closest in sequence to a VH-12 isotype anti-PC hybridoma protein, HPC-104, derived from BALB/c mice suppressed for the T15 idiotype; PCG1-2 and PCG1-3 each differed from HPC-104 at only 1/20 residues. In addition, these proteins have in common a lysine at position 1 which has not been found previously in 203 other heavy chain sequences reported. N-terminal sequences of the light chains of PCG1-2 and PCG1-3 are each shown to differ at only 1/22 residues from V kappa 24, and PCM-23 had previously been shown to use V kappa 8; both of these have been associated previously with heavy chains derived from the V1 gene in anti-PC antibodies. These results indicate that the VH-12 isotype can be used during a normal antibody response to PC and thus that heavy chains derived from both subgroup II and subgroup III (the T15 heavy chain) contribute to the molecular heterogeneity observed in memory responses to PC-KLH.