Influence of a V kappa 8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus.

A rearranged murine V kappa 8/J kappa 5 L chain gene that codes for the L chain of most antibodies generated in the primary response of BALB/c mice to the antigenic site, Sb, of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8) has been cloned. Three transgenic lines were generated by microinjecting the gene. Lines Ga and L each contain a single copy of the transgene whereas line Gb contains three complete copies. Mice of the Ga lineage showed increased V kappa 8-specific mRNA levels only in spleen, but not in nonlymphoid organs and therefore displayed apparently normal lymphoid-specific regulation of the Ig transgene. B cell hybridomas generated from these mice were analyzed for rearrangements of endogenous V kappa genes. Greater than 90% of the C kappa alleles were retained in germ-line configuration in the Ga line, compared with only 0 to 18% in the L line. Thus, a wide variation in the frequency of endogenous rearrangements is seen among mice of different lineages using the same transgene construct. None of more than 150 hybridomas derived from LPS-stimulated splenic B cells of Ga mice exhibited HA-binding activity although they expressed the transgene and, in most cases, excluded endogenous V kappa rearrangements. In contrast, a large fraction of hybridomas isolated after primary immunization with PR8 were HA(Sb)-specific. This indicated that the transgene was functional but formed HA-specific antibodies with a more restricted set of H chains than previously hypothesized. The primary anti-HA response to immunization with PR8 was diminished in all lines compared with normal mice except for a slightly accelerated but transient burst of anti-HA antibody formation in two out of three lines (Ga and Gb). This early response in G lineage mice was largely specific for HA(Sb) and thus appeared to be composed of transgene-expressing antibodies. No differences in serum titers were observed in the secondary anti-HA responses to booster inoculation with PR8 between transgenic and normal mice.

A large degree of functional diversity exists among helper T cells specific for the same antigenic site of influenza hemagglutinin.

The site-1 determinant of the hemagglutinin molecule of influenza virus (A/PR/8/34) is one of several immunodominant sites in the BALB/c Th cell response to Ha. A synthetic peptide comprising this T cell site (HA110-120), a panel of analogs containing single substitutions in this determinant, and homologs truncated at the amino- or carboxyl-terminal were used to examine the fine specificities of 15 T cells specific for site-1 in the context of I-Ed. The results indicate that every residue within the minimal determinant plays a role in the T cell recognition process, as single substitutions at any of these positions affected the ability of the peptide to stimulate at least some site 1-specific T cells. For the majority of the residues examined, substitutions had dissimilar effects on distinct T cells, indicating that the substituted residues were affecting recognition in a receptor-specific manner. Each of the 15 T cells examined had a distinct fine specificity pattern, suggesting that the BALB/c T cell repertoire for this site is likely to exceed 100 distinct clonotypes.

V region gene usage and somatic mutation in the primary and secondary responses to influenza virus hemagglutinin.

Most (80 to 90%) primary antibodies specific for the Sb site of influenza virus (A/PR/8/34) hemagglutinin share an Id (designated C4). Secondary antihemagglutinin(Sb) antibodies also exhibit the C4 Id although less frequently (10 to 15%). We have analyzed the V region nucleotide sequences of primary and secondary antibodies with the C4 Id. Primary C4 antibodies are encoded by the same Vk gene, belonging to the Vk8 group, usually rearranged to Jk5. The H chains are diverse, encoded by VH genes belonging to at least four different VH families, a variety of DH genes, and either JH2, JH3, or JH4. There is only one somatic mutation among seven Vk and two VH genes encoding primary C4 antibodies. Secondary C4 antibodies are also encoded by the same Vk8-Jk5 gene segment and by diverse VH genes. Additional heterogeneity in the secondary response is caused by somatic mutation.

Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes.

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-expressing APC. This indicates that HA endogenously synthesized within these cell lines is available for Ag processing by an exogenous route.

Enumeration of antigenic sites of influenza virus hemagglutinin.

The antigenic sites on the hemagglutinin of X-31 (H3) influenza virus have been defined by using a competitive radioimmunoassay with a panel of monoclonal antibodies which includes those known to select variants with substitutions of particular amino acids. The capacity of each monoclonal antibody to block the binding of other radioiodinated monoclones to purified hemagglutinin permitted classification of the panel into four separate groups, each of which defined a particular antigenic site on the hemagglutinin molecule. Three of these are located on the polypeptide backbone and correspond to the "hinge," the "loop," and the "tip/interface" of the X-ray crystallographic model of Wiley et al. (Nature [London] 289:373-378, 1981). Nonreciprocal blocking of certain anti-interface antibodies by anti-loop antibody suggests that much of the exposed surface of the head of the hemagglutinin molecule extending from the loop to the interface may be a continuum of epitopes. A fourth antigenic site is carbohydrate in nature, presumably situated on the antigenic oligosaccharide side chains. These four domains are in addition to two antigenic sites defined by monoclonal antibodies that inhibit neither hemagglutination nor infectivity (Breschkin et al., Virology 113:130-140, 1981;' Yewdell et al., Nature [London] 279:246-248, 1979).

A monoclonal antibody to viral glycoprotein blocks virus-immune effector T cells operating at H-2Dd but not at H-2Kd1.

A particular monoclonal antibody that binds to the influenza virus HA molecule inhibits HA-specific thymus-derived lymphocytes mediating cytotoxicity in the context of H-2Dd but not of H-2Kd. Another monoclonal antibody blocks both sets of HA-specific effector T cells. This observation, together with related findings from other laboratories, is considered to support the idea that T cell recognition is directed against some association of viral and H-2 glycoproteins, as proposed in the original formulation of the "altered self" concept.

Cell-mediated cytotoxicity against murine cells infected with 6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue.

Maximum cytotoxicity to parainfluenza type 1 viral determinants occurs on day 5 post-immunization in mice with and without preexisting anti-6/94 virus antibody. Animals with preexisting anti-6/94 serum antibody, however, exhibited a markedly higher cytotoxic response. Syngeneic, allogeneic, and xenogeneic virus-infected cells induce virus-specific cytotoxicity, but only infected cells histocompatible with the effector cells can serve as susceptible target cells. The effector cell is a T cell, and hence cytotoxicity is thymus dependent. Coincident with the maximum cytotoxic activity on day 5 is the occurrence of pathological lesions, the sites of which are dependent on the route of inoculation.