ovalbumin and 2-phenyloxazolone

We examined the immune response of Balb/c mice to antigens prepared by conjugating 2-phenyloxazolone (phOx) to a foreign protein, ovalbumin (OVA), or a self-protein, mouse serum albumin (MSA), in order to study how these chemical modifications would affect immune recognition. We found that anti-OVA antibodies and CD4(+) T cells produced by OVA immunization reacted with OVA as well as with phOx-OVA. Anti-phOx antibodies were produced by phOx-OVA immunization and, interestingly, T cells from these mice reacted only with phOx-OVA but not with the intact OVA. These results suggested that the classical model of hapten-carrier immunization, in which B cells specific to hapten are activated with assistance from T cells specific to a carrier protein, might not be a major route for production of anti-hapten antibodies in hapten-carrier immunization. Furthermore, phOx-MSA immunization induced production of anti-phOx antibodies, which could not be accounted for in terms of the assistance of carrier-specific T cells because of the absence of MSA-specific T cells. Therefore, we proposed a new model in which anti-hapten B cells are assisted by T cells specific to the haptenated carrier.

Topics: Animals; Antibodies; Antibody Formation; Antigens; B-Lymphocytes; CD4-Positive T-Lymphocytes; Haptens; Immunization; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Ovalbumin; Oxazolone

We have examined the immunoglobulin variable gene usage and antibody affinities during the memory maintenance phase of the immune response to the hapten phenyl-oxazolone. Hapten-specific hybridomas representing the memory population were generated 4-6 months postimmunization. The V-gene expression of these hybridomas was determined by reverse transcriptase-polymerase chain reaction screening and antibody affinities were estimated by biointeraction analysis (BIA) using the BIAcore biosensor. Our results show that the V-gene repertoire has already been shifted during the memory maintenance phase of the immune response, i.e. prior to a second antigenic challenge, and did not entail any advantages in terms of antigen-binding capacities. Our results concur with the view that antibody affinities are modulated mainly through differences in dissociation rates rather than in association rates, and the implications of this with respect to affinity maturation is discussed.

Topics: Animals; Antibody Affinity; B-Lymphocyte Subsets; Binding Sites, Antibody; Gene Rearrangement, B-Lymphocyte; Genes, Immunoglobulin; Haptens; Immunoglobulin Variable Region; Immunologic Memory; Mice; Mice, Inbred BALB C; Ovalbumin; Oxazolone

The molecular mechanism behind affinity maturation is the introduction of point mutations in immunoglobulin (Ig) V genes, followed by the selective proliferation of B cells expressing mutants with increased affinity for antigen. An in vitro culture system was developed in which somatic hypermutation of Ig V genes was sustained in primed B cells. Cognate T cell help and cross-linking of the surface Ig were required, whereas the addition of lipopolysaccharide or a CD40 ligand to drive proliferation was insufficient. This system should facilitate understanding of the molecular and cellular mechanisms that regulate somatic mutation and B cell selection.

Topics: Amino Acid Sequence; Animals; B-Lymphocytes; Base Sequence; CD40 Antigens; CD40 Ligand; Cells, Cultured; Coculture Techniques; Genes, Immunoglobulin; Haptens; Hybridomas; Immunoglobulin Variable Region; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutation; Ovalbumin; Oxazolone; Receptors, Antigen, B-Cell; Th2 Cells; Transfection

We have studied somatic mutation activity early in a response to 2-phenyl-5-oxazolone coupled to ovalbumin (phOx-OVA). Although the V kappa Ox1 gene rearranged to J kappa 5 is known to predominate in this response, other closely related V kappa genes are involved. We compared the introduction of point mutations into V kappa Ox1 genes and into a set of related V kappa genes rearranged to the same J kappa segment at two time points after primary immunization. The result showed that quantitation of mutations in a single rearrangement substrate leads to an underestimation of the total mutational activity. There is pronounced somatic mutation activity early within genes that may be absent later in the response. We also show that multiple somatic mutations can be detected in B cells from draining lymph nodes after foot-pad injection with phOx-OVA already at day 7 after immunization. The data suggest a system in which mutation acts early in the response on a wide range of substrates and that selection and expansion of high affinity paratopes occurs later.

Topics: Animals; B-Lymphocytes; Base Sequence; DNA; DNA Mutational Analysis; Gene Rearrangement, B-Lymphocyte, Light Chain; Genes, Immunoglobulin; Immunization; Immunoglobulin kappa-Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Ovalbumin; Oxazolone; Point Mutation