Parallel evolution of antibody variable regions by somatic processes: consecutive shared somatic alterations in VH genes expressed by independently generated hybridomas apparently acquired by point mutation and selection rather than by gene conversion.

We identified, in independently generated hybridoma antibodies, blocks of shared somatic alterations comprising four consecutive amino acid replacements in the CDR2s of their heavy chain variable regions. We found that the nucleotide sequences encoding the shared replacements differed slightly. In addition, we performed genomic cloning and sequencing analyses that indicate that no genomic sequence could encode the block of shared replacements in any one of the antibodies and thus directly serve as a donor by a recombinational process. Finally, in a survey of other somatically mutated versions of the same heavy chain variable gene, we found several examples containing one, two, or three of the shared CDR2 mutations in various combinations. We conclude that the shared somatic alterations were acquired by several independent events. This result, and the fact that the antibodies containing the four shared mutations were elicited in response to the same antigen and are encoded by the same VH and VK gene segments, suggests that an intense selection pressure has fixed the shared replacements by favoring the clonal expansion of B cells producing antibodies that contain them. The basis of this selection pressure is addressed elsewhere (Parhami-Seren, B., L. J. Wysocki, M. N. Margolies, and J. Sharon, manuscript submitted for publication).

Tracing the development of single memory-lineage B cells in a highly defined immune response.

To study the development of B lymphocyte memory, we identified and isolated splenic B cells expressing a highly defined antibody variable region that constitutes a reproducible and predominant component of the memory antibody response to p-azophenylarsonate (Ars). Isolation was achieved during the primary immune response by surface staining and flow cytometry using a specific anti-idiotypic antibody called E4, which recognizes this canonical V region, encoded by one set of V gene segments. The isolated E4+ cells displayed all of the phenotypic characteristics of germinal center centrocytes, including a low level of surface Ig, a lack of surface IgD, a high level of receptor for peanut agglutinin, and expression of mutated antibody V genes. E4+ B cells were first detected in the spleen 7-8 d after primary immunization, reached peak numbers from days 10-13, and waned by day 16. Surprisingly, at their peak, E4+ cells comprised only 40,000 of all splenocytes, and half of these failed to bind Ars. Using this number, we estimate the total number of Ars-specific memory-lineage cells in the spleen to be no more than 50,000 (0.1%) at any one time, and presumably far fewer that are committed to the memory pool. Chromosomal copies of rearranged V genes from single E4+ cells were amplified by nested PCR, and the amplified products were sequenced directly without cloning, using standardized conditions that disclose virtually no Taq polymerase errors. V gene sequence analyses of E4+ cells isolated from single mice confirmed their canonical nature and revealed that they were derived from few precursors. In the average mouse, the E4+ pool was derived from fewer than five canonical precursors. Somatic mutations were found within the V genes of almost all cell isolates. At day 13, a significant fraction of E4+ cells had mutations known to increase antibody affinity for Ars, suggesting they were products of at least one cycle of post-mutational antigen-driven selection. However, the lack of shared mutations by clonally related cells indicated that the selective expansion of mutant subclones typical of memory responses had not yet taken place. This was supported by the observation that half of the E4+ cells failed to bind Ars. Collectively, our results indicate that the memory compartment is a highly selected entity, even at relatively early stages of the primary immune response when somatic mutation and clonal selection are still in progress. If germinal centers are the source of memory B cells, our data suggest that B cell memory may be derived from only a small fraction of all germinal centers.

Single germline VH and V kappa genes encode predominating antibody variable regions elicited in strain A mice by immunization with p-azophenylarsonate.

We have cloned and sequenced the predominant germline V kappa gene segment expressed by B cells of strain A origin that synthesize antibodies with specificity for Ars. In hybridomas synthesizing anti-Ars antibodies, this V kappa gene segment (V kappa IdCR) has been found exclusively associated with the J kappa 1 gene segment without exhibiting junctional sequence variation. Sequence comparisons of the germline V kappa IdCR gene with expressed derivatives reveals that the latter frequently contain somatically introduced amino acid replacements. Taken together with results of previous structural analyses, these results show that the predominant population of IdCR+ V regions elicited in the secondary immune response is encoded by one or two combinations of V gene segments, has little junctional diversity, and is extensively diversified by somatic mutation in both heavy and light chains.

A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus.

These experiments tested the hypothesis that unmutated germline genes from normal mice can encode autoantibodies. We found that the unmutated VHIdCR gene segment, which encodes a large proportion of antiarsonate antibodies in A/J mice, also encodes antibodies with the ability to bind to DNA and cytoskeletal proteins. After Ars immunization, at a time when the VHIdCR gene segment mutates and antibody affinity for the hapten increases, reactivity with the autoantigens was lost. Six antibodies obtained after immunization with Ars bound both the Ars and DNA. Results of competitive inhibition assays suggested that the same variable region site in the antibodies bound to both Ars and DNA. The properties of the individual germline-encoded antibodies, which include reactivity to both DNA and cytoskeletal proteins, suggest that autoantibodies characteristic of SLE might be a subset of antibodies encoded by unmutated germline V genes.

Exposure of fish to high-intensity sonar does not induce acute pathology.

This study investigated immediate effects of intense sound exposure associated with low-frequency (170-320 Hz) or with mid-frequency (2.8-3.8 kHz) sonars on caged rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus and hybrid sunfish Lepomis sp. in Seneca Lake, New York, U.S.A. This study focused on potential effects on inner ear tissues using scanning electron microscopy and on non-auditory tissues using gross and histopathology. Fishes were exposed to low-frequency sounds for 324 or 628 s with a received peak signal level of 193 dB re 1 microPa (root mean square, rms) or to mid-frequency sounds for 15 s with a received peak signal level of 210 dB re 1 microPa (rms). Although a variety of clinical observations from various tissues and organ systems were described, no exposure-related pathologies were observed. This study represents the first investigation of the effects of high-intensity sonar on fish tissues in vivo. Data from this study indicate that exposure to low and midfrequency sonars, as described in this report, might not have acute effects on fish tissues.

The molecular basis of a VH gene polymorphism that determines the expression of a major idiotype.

The strain A immune response to a synthetic antigen (p-azophenylarsonate) is dominated by antibodies bearing an idiotype encoded by VH genes derived from a single germline VH gene segment called VHIdCR (a member of the J558 family). Balb/c mice fail to produce this idiotype. Southern blotting analyses with a probe derived from VHIdCR have shown that differences in patterns of hybridization and in intensity of bands are seen between the two strains. We demonstrate by DNA cloning and sequence analyses that Balb/c mice have no allelic version of VHIdCR. This result constrasts with that reported for interstrain comparisons of VH genes encoding antibodies to environmental pathogens where evolutionary conservation of VH sequence information is seen. We suggest, on the basis of these and earlier results, that domination of the anti-Ars immune response by antibodies encoded by VHIdCR is not the indirect consequence of evolutionary or somatic selection pressures acting on the VHIdCR gene segment.

Sequence heterogeneity in Ig kappa transcripts from single B lymphocytes.

Individually amplified kappa cDNA molecules from single B lymphocytes revealed sequence heterogeneity and aberrantly spliced products. The nature and frequency of the base changes and their absence from similarly amplified beta2 microglobulin transcripts indicate that they were not derived by Taq polymerase misincorporations or by a general infidelity in RNA polymerase. The trinucleotide sequences in which the base changes occurred are disfavored targets of the somatic hypermutation mechanism that modifies antibody variable (V) region genes during immunity. Taken together with the observation that the transcript alterations were absent from the kappa Ig gene, this suggests that somatic mutations were acquired by the kappa gene and rapidly repaired following limited transcription. Preferential repair of mutations located in specific trinucleotide contexts could be the basis for some of the microsequence-specific bias in mutation frequencies observed in antibody V region genes.

An early post-mutational selection event directs expansion of autoreactive B cells in murine lupus.

We report evidence for a strong selection event directing the outgrowth of autoreactive B cells in spontaneous murine lupus. The event occurred shortly following the induction of the somatic hypermutation process. This conclusion is derived from extensive sequence analyses of VH and VL loci expressed by hybridomas representing two large histone-specific clones (lineages) from an autoimmune (NZB x SWR)F1 mouse. To obtain unambiguous somatic mutational information, we devised a strategy to amplify and sequence the JH and JK clusters that flank expressed V genes. Somatic mutations in V flanking sequences of the two autoreactive clones revealed that in one clone the pattern was relatively simple: the frequency of mutation was low, and only one somatic mutation was shared by all clone members. Members of the second large histone-specific clone contained many somatic mutations in combinations that indicated numerous rounds of selection. Importantly, however, as observed with the first clone, one observed somatic mutation was shared by all clone members. Since, for each clone, all members shared only one visible mutation over extensive sequence tracts, we conclude that the autoreactive clones were derived from single precursors that had just begun to mutate their V genes. The data indicate that a strong selection event had occurred shortly after the initial acquisition of somatic mutation(s) in precursors to each clone, at a stage of development corresponding to that of the germinal center B cell approximately 1 week post immunization.

Amplification of genes, single transcripts and cDNA libraries from one cell and direct sequence analysis of amplified products derived from one molecule.

We report a procedure to generate and amplify cDNA libraries and to amplify and sequence genes and single RNA transcript molecules from the same cell without cloning. An absence of cloning steps minimizes potential sources of contamination, which can be especially problematic when working at the single cell level. Potential contamination is further reduced by an absence of any purification step prior to PCR amplification. Amplifications are designed to minimize the production of aberrant molecules in favor of full-length products, which is especially advantageous when generating cDNA libraries. Genes are amplified from isolated single nuclei, which are segregated from cytoplasmic lysates by microcentrifugation. Specific cDNA, total cDNA or both are synthesized from aliquots of the cytoplasmic lysate, and single cDNA molecules are isolated from others of the same species by limiting dilution prior to PCR amplification. In this way, the frequency of amplified products provides for a direct calculation of cDNA copy number by a Poisson analysis. Incorporation errors by Taq DNA polymerase occur at a low frequency and can be eliminated by sequencing independently amplified cDNA molecules from the same cell. Single molecule amplifications provide sufficient material for numerous (approximately 150) direct DNA sequencing reactions. The limiting dilution approach also permits sequence information to be obtained from a single cDNA, when highly related transcripts derived from distinct genes are present in the same cell and simultaneously amplified with the same primers. In sum, this method provides for a maximum amount of nucleic acid information to be extracted from one cell. It has a wide range of applications to studies of the immune system where, to a first approximation, each lymphocyte has a unique receptor identity, where specific states of differentiation may be difficult to assess in a mixed cell population, and where cell immortalization procedures are not always possible nor practical.

NADPH diaphorase staining suggests localization of nitric oxide synthase within mature vertebrate olfactory neurons.

Nitric oxide, a simple gas which serves as a neurotransmitter in the CNS, has been proposed to serve as an interneuronal second messenger in olfactory transduction. However, the role of nitric oxide in olfaction has been questioned by experiments in which nitric oxide synthase, the enzyme that generates nitric oxide, could not be localized to the olfactory epithelium. We have localized nitric oxide synthase to the olfactory neurons in adult rat and catfish olfactory epithelia using a modified nicotinamide adenine dinucleotide phosphate diaphorase technique. In the rat, staining was also found in cells with morphology reminiscent of microvillar olfactory cells. In contrast, the respiratory epithelium and the sustentacular cells in the olfactory epithelium displayed no staining. The nicotinamide adenine dinucleotide phosphate diaphorase reaction, which has been shown to co-localize with immunohistochemical staining for nitric oxide synthase in the brain, was stimulated by addition of the nitric oxide synthase substrate L-arginine, and was inhibited by the nitric oxide synthase inhibitor L-NG-nitro arginine, indicating that staining was specific for nitric oxide synthase. Unilateral bulbectomy, which causes degeneration of mature olfactory neurons on the bulbectomized size, markedly reduced nicotinamide adenine dinucleotide phosphate diaphorase staining. These observations were substantiated by biochemical assays for nitric oxide synthase by monitoring the production of [3H]-L-citrulline from [3H]-L-arginine. This is the first demonstration of specific NADPH diaphorase staining of mature olfactory neurons in rat and catfish olfactory epithelial suggesting the presence of nitric oxide synthase in these cells. Our histological and biochemical findings, in conjunction with data from other research, are supportive of a role for nitric oxide synthase in olfactory function.

An ephemeral pheromone of female house mice: perception via the main and accessory olfactory systems.

Two experiments examined the chemosensory modalities by which males detect an ephemeral sex pheromone in the freshly voided urine of female mice. Experiment 1 examined the interaction of deafferenting the accessory olfactory system (vomeronasal organ removal) and subsequent sexual experience upon ultrasonic vocalizations by male mice to freshly voided female urine. In general, sexually experienced males vocalized substantially more than sexually naive males. In addition, males possessing a vomeronasal organ vocalized slightly more than those without. Nonetheless, a functioning vomeronasal organ clearly was not essential for vocalizing to fresh female urine. Experiment 2 examined the effects of deafferenting the main olfactory system (ZnSO4 nasal irrigation) and/or the accessory olfactory system (vomeronasal removal) in sexually experienced males. Males with both olfactory systems functioning vocalized at high levels to fresh urine, while males with only one functioning system vocalized at intermediate levels. Males with neither system functioning did not vocalize at all to fresh urine. In contrast, when female mice themselves served as stimuli, all groups of males vocalized at high levels. We conclude that adult male mice can detect the ephemeral pheromone via either the main olfactory system or the accessory olfactory system. However, vocalizations to the female herself can be mediated by other sensory systems as well.

Spontaneous autoimmunity in mice that carry an IghV partial transgene: a required arginine in VHCDR3.

We describe a unique spontaneous mouse model of autoimmunity, which occurs on a non-autoimmune-prone SWR genetic background. In this model, SWR mice carry an IghV partial transgene (pTg) encoding only the heavy chain variable domain of an antibody directed against chromatin. Autoimmune disease in pTg mice was manifested by some of the features of systemic lupus erythematosus (SLE), including the presence of serum anti-nuclear antibodies, splenomegaly, skin lesions and a moderate degree of kidney pathology, in various combinations among individuals. Autoimmunity was observed in three independent transgenic lines, but not in three control lines carrying a nearly identical pTg, in which a VHCDR3 codon for Arg was replaced by one for Ser to ablate chromatin reactivity. Various features of disease were often but not always accompanied by anti-chromatin antibodies. Unexpectedly, the anti-chromatin antibodies detected in seropositive animals were not encoded by the pTg. These observations strongly implicate a role for the transgene product in disease initiation but not necessarily for end-state pathology, and they raise the possibility that autoreactive B cells may play a previously unappreciated role in initiating the development of systemic autoimmunity.

T cell recognition of somatically-generated Ab diversity.

During an immune response, specific Abs and B cells that are infrequently represented in the preimmune repertoire become amplified to abundance. Novel structures are also created through the physiological process of somatic hypermutation in Ab genes. This presents a challenge for T cell self-tolerance, because many potential V region epitopes are either rare or nonexistent during the maturation of the T cell repertoire in the thymus. To explore the potential for T cell recognition of Ab V regions, we immunized A/J mice with two somatically mutated mAbs (mAb36-71 and mAb45-49) derived from A/J mice and produced 13 mAb-specific T cell hybridomas. All of the T cell hybridomas express alpha beta receptors and CD4, and their responses to the mAb are restricted in the context of class II MHC glycoproteins. In presentation studies with fixed APCs and whole or trypsinized mAb, we found that processing of the mAb is necessary for stimulation of the T cell hybridomas. Therefore each of the hybridomas recognizes the mAbs in a conventional class II MHC-restricted manner. We also found that each of the 13 T cell hybridomas responded to the somatically-mutated light chains of mAb45-49 and mAb36-71. In contrast, none of them responded to unmutated versions of the same light chains. These results show that the T cell repertoire includes members able to recognize syngeneic Abs containing somatic mutations that were physiologically acquired during the course of an immune response.

The ontogenetic development of auditory sensitivity, vocalization and acoustic communication in the labyrinth fish Trichopsis vittata.

The anabantoid fish Trichopsis vittata starts vocalizing as 8-week-old juveniles. In order to determine whether juveniles are able to detect conspecific sounds, hearing sensitivities were measured in six size groups utilizing the auditory brainstem response-recording technique. Results were compared to sound pressure levels and spectra of sounds recorded during fighting. Auditory evoked potentials were present in all size groups and complete audiograms were obtained starting with 0.18 to 0.30 g juveniles. Auditory sensitivity during development primarily increased between 0.8 kHz and 3.0 kHz. The most sensitive frequency within this range shifted from 2.5 kHz to 1.5 kHz, whereas thresholds decreased by 14 dB. Sound production, on the other hand, started at 0.1 g and sound power spectra at dominant frequencies increased by 43 dB, while dominant frequencies shifted from 3 kHz to 1.5 kHz. Comparisons between audiograms and sound power spectra in similar-sized juveniles revealed no clear match between most sensitive frequencies and dominant frequencies of sounds. This also revealed that juveniles cannot detect conspecific sounds below the 0.31 to 0.65 g size class. These results indicate that auditory sensitivity develops prior to the ability to vocalize and that vocalization occurs prior to the ability to communicate acoustically.

"Panning" for lymphocytes: a method for cell selection.

We have developed a simple method for the fractionation of T and B lymphocytes. Plastic dishes coated with antibodies specific for mouse Ig selectively bind splenic B lymphocytes. The adherent cells are easily removed by gentle pipetting; both adherent and nonadherent populations retain immunologic function. In a typical experiment, when 3 X 10(7) splenic lymphocytes were added to a 100 X 15 mm plastic dish coated with microgram quantities of anti-Ig, 98 % of the nonadherent cells were Ig negative and 97% of the adherent cells were Ig positive. The method is sufficiently sensitive to allow detection and separation of cell types comprising as little as 2% of the total population and can be modified to allow the selection of cells by a double-antibody procedure. We believe that the plastic dish method will be generally useful for fractionating cells on the basis of their cell surface antigens.

The strain A anti-p-azophenylarsonate major cross-reactive idiotypic family includes members with no reactivity toward p-azophenylarsonate.

The sera of immunized A/J mice contain low but detectable levels of immunoglobulin, bearing previously described cross-reactive idiotypic (CRI) determinants diagnostic of the strain A anti-p-azophenylarsonate (Ars) response. Such molecules from nonimmune sera cannot be adsorbed onto affinity columns coupled to a high density with Ars. After extensive immunization with Ars-coupled proteins, Ars-nonbinding CRI is found at the same low level, even though substantial Ars-binding CRI appears. On the other hand, immunization with a monoclonal rat anti-CRI, which was originally raised against Ars-binding CRI, elicits high concentrations of Ars-binding as well as Ars-nonbinding CRI immunoglobulin. Three hybridoma proteins produced from an A/J mouse immunized with the rat anti-CRI react with all tested anti-idiotypic sera from three species of animals, but show no reactivity toward Ars in several different assays. One hybridoma protein from the same fusion demonstrates Ars-binding capacity.

Recruiting memory B cells with changed antigenic specificity.

During T cell-dependent antibody responses, the V region genes of responding B lymphocytes are physiologically mutated at a high rate. An intense selection process expands subclones of B cells producing mutant antibodies that bind Ag optimally. This implies that most mutant B cells and their antibody products are unselected and not often observed by conventional hybridoma or serum sampling procedures. Herein we show that the pool of mutant B cells includes unselected members that have acquired new antigenic specificities. Mice were immunized with a haptenated carrier protein to recruit and somatically diversity hapten-specific B cells producing antibodies with a defined V region bearing a major idiotype. During the primary immune response, the mice were given booster injections with a second related hapten conjugated to the same carrier. Hybridomas were isolated that produced idiotypic antibodies binding the second hapten but not the first. V gene sequencing analyses conclusively demonstrated that one of these was derived from a precursor B cell that expressed the defined unmutated V region with specificity for the first hapten. Sequence of the V genes expressed by the remaining six hybridomas supported this interpretation. In essence, single B cell clones were mutationally diversified to include members that had lost an original antigenic specificity while acquiring a new one, and the mutants were recruited into the memory compartment by antigenic selection. These results support the view that selection processes in vivo normally reveals only a small fraction of a mutationally diversified B cell clone. They also suggest a potential route by which antibodies of differing antigenic specificities can be generated from a single B cell clone of predefined origin and antigenic specificity.

The amino acid residues at the VH-D-JH junctions affect the affinity of anti-p-azophenylarsonate antibodies.

Murine A/J anti-p-azophenylarsonate (Ars) antibodies sharing a predominant idiotype are encoded by a single combination of germ-line V region gene segments. The dominance of this idiotype among secondary immune response anti-Ars antibodies has been explained by the Ag-driven selection of favorable somatic mutants of this gene segment combination, associated with an intrinsic Ars-affinity of the germ-line V region higher than that of other possible combinations. To determine the effect of junctional diversity upon affinity for Ag, independently of somatic mutation, we determined the V region sequences and affinity for Ars of five primary response antibodies. These antibodies share identical unmutated V regions but differ only at the D gene junctions. Among the five antibodies, Ars-affinity differed up to 10-fold depending upon the identity of the amino acid residues at the VH-D and the D-JH junctions. The combination of junctional residues observed in two primary response antibodies with relatively low Ars-affinity has not been observed among secondary response antibodies. Thus the identity of junctional residues resulting from gene rearrangement prior to antigen stimulation must be taken into account in hypotheses which account for idiotype dominance by selection on the basis of affinity.