Antibodies that are specific for a single amino acid interchange in a protein epitope use structurally distinct variable regions.

We have analyzed how the immune system generates antibodies that are specific for analogues of an epitope on the influenza virus hemagglutinin (HA) that differ solely by the presence of Asp or Gly at amino acid 225. Most antibodies induced in response to HA(Asp225) use one of a few closely related variable (V) region structures that are encoded by characteristic VH/Vk gene segment combinations. Remarkably, none of these VH/Vk combinations was induced in response to HA(Gly225). Instead of modifying the HA(Asp225)-specific V regions by junctional variation or somatic mutation to recognize the altered epitope, new VH/Vk combinations were used. The expression of unique VH/Vk combinations appears to confer exquisite specificity to the selection of HA-specific B cells from the pre-immune repertoire.

Activation and negative selection of functionally distinct subsets of antibody-secreting cells by influenza hemagglutinin as a viral and a neo-self antigen.

We have compared transgenic mice that express the influenza virus PR8 hemagglutinin (PR8 HA) as a membrane-bound neo-self antigen (HA104 mice) with nontransgenic (non-Tg) mice for their ability to generate HA-specific B cell responses after primary immunization with PR8 virus. HA-specific, IgM-secreting B cells were induced with similar frequencies in HA104 and non-Tg mice. In addition, a B cell clonotype (C4) that is characteristic of anti-HA immune responses of BALB/c mice was identified among HA-specific IgM hybridomas from HA104 mice. A subset of HA-specific, IgG-secreting B cells that arises rapidly after primary virus immunization in non-Tg mice, however, was substantially reduced in HA104 mice. Likewise, a B cell clonotype (C12) that dominates HA-specific IgG hybridomas generated after primary immunization of non-Tg mice was present at greatly reduced frequencies among hybridomas from HA104 mice. Because HA-specific, IgG-secreting B cells were generated by HA104 mice in response to a mutant HA containing an amino acid interchange in a B cell antigenic site, we conclude that these PR8 HA-specific, IgG-secreting B cells are negatively selected in HA104 mice as a result of their specificity for the neo-self PR8 HA. The findings demonstrate that HA-specific B cells that display distinct phenotypic potentials in non-Tg mice also differ in their susceptibility to negative selection from the primary B cell repertoire of HA104 mice: a subset of B cells that undergo rapid differentiation to become HA-specific IgG antibody-secreting cells (ASC) after activation in non-Tg mice is negatively selected in HA104 mice. By contrast, a subset that gives rise to HA-specific, IgM-secreting ASC persists in the primary repertoire of HA104 mice and can be activated by virus immunization.

Transgenic mice that express different forms of the influenza virus hemagglutinin as a neo-self-antigen.

We have generated transgenic mouse lineages that express the influenza virus hemagglutinin in different physical forms. One kind expresses the full-length hemagglutinin molecule as a cell surface glycoprotein and can be recognized by hemagglutinin-specific B and T cells. The other expresses a truncated polypeptide corresponding to the N-terminal third of the hemagglutinin molecule. This polypeptide encodes known hemagglutinin-specific T-cell determinants; however, it contains no native B-cell epitopes, since these depend on the conformation of the fully folded protein. In each case, the hemagglutinin transgenic mice display ubiquitous expression of transgenic messenger RNA and induce T-cell tolerance to the transgene-encoded T-cell determinant site 1. Thus, the hemagglutinin is a neo-self-antigen in both kinds of hemagglutinin transgenic mice and should provide a useful system for understanding the factors and mechanisms that govern tolerance and autoimmunity to self-antigens.

Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin.

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.