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.

Predominance of a novel splenic B cell population in mice expressing a transgene that encodes multireactive antibodies: support for additional heterogeneity of the B cell compartment.

We generated IgHmudelta transgenic mice using a V(H) gene that in A/J mice encodes multireactive BCR in the preimmune B cell compartment and is predominantly expressed by a memory B cell subpopulation. Most primary splenic B cells in these mice have a size, cell-surface phenotype and in vitro response profile distinct from mature follicular (B2), marginal zone (MZ) or B1 B cells, but are long-lived and appear to be slowly cycling. They reside in conventional B cell areas of the spleen and mount robust foreign antigen-driven germinal center responses, but do not efficiently differentiate to secretory phenotype. We propose that these qualities result from ongoing, low-avidity BCR-self-ligand interactions and promote entry into the memory pathway. Given these data, and the enormous diversity and characteristic multireactivity of the preimmune antibody repertoire, we also suggest that it may be more appropriate to view the primary B cell compartment as a continuum of functional and phenotypic 'layers', rather than as a group of discrete B1, B2 and MZ subsets.

Activation and anergy in bone marrow B cells of a novel immunoglobulin transgenic mouse that is both hapten specific and autoreactive.

Available evidence indicates that B cell tolerance is attained by receptor editing, anergy, or clonal deletion. Here, we describe a p-azophenylarsonate (Ars)-specific immunoglobulin transgenic mouse in which B cells become anergic as a consequence of cross-reaction with autoantigen in the bone marrow. Developing bone marrow B cells show no evidence of receptor editing but transiently upregulate activation markers and appear to undergo accelerated development. Mature B cells are present in normal numbers but are refractory to BCR-mediated induction of calcium mobilization, tyrosine phosphorylation, and antibody responses. Activation marker expression and acquisition of the anergic phenotype is prevented in bone marrow cultures by monovalent hapten. In this model, it appears that induction of anergy in B cells can be prevented by monovalent hapten competing with autoantigen for the binding site.

A receptor presentation hypothesis for T cell help that recruits autoreactive B cells.

To uncover mechanisms that drive spontaneous expansions of autoreactive B cells in systemic lupus erythematosus, we analyzed somatic mutations in variable region genes expressed by a panel of (NZB x SWR)F(1) hybridomas representing a large, spontaneously arising clone with specificity for chromatin. A single mutation within the Jkappa intron that was shared by all members of the lineage indicated that the clone emanated from a single mutated precursor cell and led to the prediction that a somatic mutation producing a functionally decisive amino acid change in the coding region would also be universally shared. Upon cloning and sequencing the corresponding germline V(H) gene, we found that two replacement somatic mutations in FR1 and CDR2 were indeed shared by all seven clone members. Surprisingly, neither mutation influenced Ab binding to chromatin; however, one of them produced a nonconservative amino acid replacement in a mutationally "cold" region of FR1 and created an immunodominant epitope for class II MHC-restricted T cells. The epitope was restricted by IA(q) (SWR), and the SWR MHC locus is associated with systemic lupus erythematosus in (NZB x SWR)F(1) mice. These, and related findings, provoke the hypothesis that autoreactive B cells may be recruited by a "receptor presentation" mechanism involving cognate interactions between T cells and somatically generated V region peptides that are self-presented by B cells.

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.

Predicting regional mutability in antibody V genes based solely on di- and trinucleotide sequence composition.

Somatic mutations are not distributed randomly throughout Ab V region genes. A sequence-specific target bias is revealed by a defined hierarchy of mutability among di- and trinucleotide sequences located within Ig intronic DNA. Here we report that the di- and trinucleotide mutability preference pattern is shared by mouse intronic JH and Jkappa clusters and by human VH genes, suggesting that a common mutation mechanism exists for all Ig V genes of both species. Using di- and trinucleotide target preferences, we performed a comprehensive analysis of human and murine germline V genes to predict regional mutabilities. Heavy chain genes of both species exhibit indistinguishable patterns in which complementarity-determining region 1 (CDR1), CDR2, and framework region 3 (FR3) are predicted to be more mutable than FR1 and FR2. This prediction is borne out by empirical mutation data from nonproductively rearranged human VH genes. Analysis of light chain genes in both species also revealed a common, but unexpected, pattern in which FR2 is predicted to be highly mutable. While our analyses of nonfunctional Ig genes accurately predicts regional mutation preferences in VH genes, observed relative mutability differences between regions are more extreme than expected. This cannot be readily accounted for by nascent mRNA secondary structure or by a supplemental gene conversion mechanism that might favor nucleotide replacements in CDR. Collectively, our data support the concept of a common mutation mechanism for heavy and light chain genes of mice and humans with regional bias that is qualitatively, but not quantitatively, accounted for by short nucleotide sequence composition.

Characteristics of the strong antibody response to mycobacterial Hsp70: a primary, T cell-dependent IgG response with no evidence of natural priming or gamma delta T cell involvement.

Despite its high degree of evolutionary conservation, hsp70 is a surprisingly robust Ag, to such a degree that it is under consideration as a potential substrate in vaccine development. The cellular basis of the strong humoral response, however, is unknown, although it is often hypothesized to derive from restimulation of memory T cells that have been primed by hsp of intestinal flora. In this study, we tested this hypothesis and performed additional studies on the immune response to hsp70 of Mycobacterium tuberculosis. Superficially, the primary Ab response to this protein resembles a T cell-dependent secondary one, constituted almost exclusively by IgG. However, there is no evidence of natural priming, as revealed both by in vitro stimulation experiments and by immunity in germfree mice. Although hsp70 stimulates gammadelta and alphabeta T cells from unprimed mice to proliferate in vitro, gammadelta cells are not required for the strong humoral response, which is indistinguishable in normal and gammadelta T cell-deficient mice. Thus, the unusual immunogenicity of this protein in eliciting a humoral response appears to be due to a strong alphabeta T cell response with no evidence of natural priming or a gammadelta T cell involvement.

Somatic origin of T-cell epitopes within antibody variable regions: significance to monoclonal therapy and genesis of systemic autoimmune disease.

During an immune response, specific antibody variable region genes are diversified by a somatic point mutation process that generates de novo "foreign" V-region sequences. This creates an interesting problem in immune regulation because B cells are highly proficient at self-presenting V-region peptides in the context of class II MHC. Though our studies indicate that the corresponding T-cell repertoire attains a state of tolerance to germline-encoded antibody V-region diversity, it is presently unknown whether the same is true of mutationally generated diversity. On the basis of immunoregulatory considerations, we hypothesize that contact exclusion or tolerance normally precludes T cells from helping B cells via self-presented mutant V-region peptides. The lack of recurrent somatic mutations that create known T-cell epitopes in antibody V regions lends some support to this idea. In contrast, our studies of spontaneously autoreactive B cells in systemic autoimmune disease strongly suggest that precursors of such cells are recruited by T-cell help directed to self-presented mutant idiopeptides. Failures in tolerance or contact exclusion mechanisms may be responsible for this apparently abnormal event. In addition to their importance in immune regulation, somatic mutations or other differences from germline-encoded V-region sequence may be largely responsible for undesirable patient responses to therapeutic monoclonal antibodies. These reactions might be averted or diminished by inducing tolerance in the T-cell repertoire with synthetic peptide correlates of non-germline-encoded V-region sequences in humanized antibodies.

T cell recognition and tolerance of antibody diversity.

The capacity of B cells to self-present their Ab variable regions in the context of class II MHC structures suggests a potential regulatory problem. If T cells were able to recognize self-presented Ab, then T cell help might be delivered to B cells independently of a foreign carrier epitope, resulting in a chronic state of unregulated Ab synthesis. For this reason, we have proposed that T cells normally attain a state of tolerance to Ab V region diversity. Here, we tested this idea by performing direct immunizations with unmutated isologous mAb. We also identified and analyzed epitopes recognized by class II MHC-restricted T cell hybridomas that were originally generated against two physiologically mutated isologous mAb. Our results indicate that the class II MHC-restricted T cell repertoire is tolerant of germ-line-encoded Ab diversity and that the physiologic somatic hypermutation process creates immunogenic epitopes in Ab V regions, in some cases by producing class II MHC-binding peptides. In agreement with these findings, we found that germ-line-encoded Ab V regions are presented by endogenous splenic APC at a level that is physiologically significant.

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.

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.

Di- and trinucleotide target preferences of somatic mutagenesis in normal and autoreactive B cells.

During Ag-driven development of memory B cells, Ab V genes are modified by somatic mutagenesis. Although V gene somatic mutations have important biologic consequences in both physiologic and autoimmune Ab responses, little is known about the mechanism of mutation, or whether it operates normally in autoreactive B cells. To approach these issues, we analyzed somatic mutations in Ab genes for evidence of sequence-specific target preferences. Our analysis was confined to noncoding segments of V genes so that the intrinsic characteristics of the somatic mutation process could be reliably dissociated from the indirect but substantial influences of cellular selection. We consistently observed that some dinucleotides, GC and TA in particular, mutated at frequencies that were higher than expected based on their frequency of occurrence. Most of the dinucleotide mutation preferences could not be extrapolated directly from mononucleotide mutation preferences. Specific trinucleotides, including AGC, TAC, and their inverse repeats (GCT, GTA), also mutated more frequently than expected. These and other mutation characteristics were virtually indistinguishable in V genes of normal and autoreactive B cells. An analysis of mutations in published flanking sequences confirmed the target preferences, as did an examination of reported "hot spots" within coding V sequences. The shared preferences in coding and noncoding regions of V genes suggests that somatic mutations are generated de novo. Collectively, our findings indicate that the somatic mutation process exhibits sequence-specific preferences, consistent with an untemplated mechanism, and appears to operate similarly in normal and autoreactive B cells.

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.

A single engineered amino acid substitution changes antibody fine specificity.

During the acquisition of humoral immunity, the process of somatic hypermutation introduces nucleotide substitutions into expressed antibody (Ab) V region genes. Studies employing in vitro mutagenesis have shown that recurrent mutations observed in vivo often enhance the affinity of the target Ab for Ag. Here we show that a single amino acid replacement at position 35 in the H chain of an unmutated Ab with specificity for p-azophenylarsonate (Ars) confers specificity for the structurally related hapten p-azophenylsulfonate (Sulf) while abolishing specificity for Ars. The mutant Ab binds Sulf with an affinity characteristic of Ab produced by memory B cells. The same mutation in the somatically mutated anti-Ars Ab 36-71, for which the Fab crystal structure is known, resulted in a significant shift in fine specificity from Ars to Sulf. Examination of the crystal structure suggests that the specificity change is caused by a decrease in binding site size and/or new hydrogen bond geometry. Because the mutation at position 35 had been observed in somatically mutated Ab elicited by immunization with Ars followed by Sulf, the results confirm that somatic mutation in vivo can alter Ab specificity. The results also support the potential of Ab engineering to alter antigenic specificity.

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.

Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.

We have devised a protocol to obtain accurate and complete sequences of the immunoglobulin heavy- and light-chain variable-region (VH and VL) genes of single B-hybridoma cells that express defined V genes. The amplification achieved ranges from 2 x 10(13)- to 1 x 10(14)-fold. Only one potential Taq DNA polymerase error was observed in 7590 nucleotides of sequence, thus permitting the identification of naturally occurring somatic mutations. The two-step nature of the amplification protocol provides sufficient DNA for a minimum of 160 sets of sequencing reactions of both the VH and VL genes from one cell without cloning. The amplification of relatively long segments of DNA in the first step of the protocol permits second-step amplification and sequencing of regions that flank VH and VL codons. Fractionating cellular lysates prior to the first step of amplification permits the separate amplification of V genes on opposite sister chromatids and possibly on opposite strands of the same DNA duplex. Accurate sequencing of VH and VL genes of defined germ-line origin that are expressed by single B cells taken directly from the animal is thus made feasible by this approach.

B-cell proliferation initiated by Ia cross-linking and sustained by interleukins leads to class switching but not somatic mutation in vitro.

Somatic mutations that are acquired by antibody V genes of antigen-stimulated B cells ultimately provide the clonal diversity from which memory B cells are selected during immune responses to T-cell-dependent antigens. Somatic mutations apparently are not acquired when B cells are stimulated by mitogens nor when they participate in immune responses to T-cell-independent antigens. Since the basis of T-cell-dependent humoral immunity is T-cell recognition of processed antigen in the context of class II major histocompatibility glycoproteins (Ia) on the B-cell surface, we sought to determine whether the ligation of Ia on B cells induces somatic mutation. B cells were stimulated in vitro by a procedure in which their proliferation was dependent upon ligation of surface Ia with antibody. Sequences of hybridoma V genes derived from these B cells revealed no somatic mutations despite prolonged stimulation in vitro and the induction of immunoglobulin secretion and switching to isotypes characteristic of T cell-dependent humoral immunity. We infer that Ia-mediated signalling and isotype switching are not causally related to somatic mutation. The avenue of differentiation that leads to somatic mutation in memory B cells is apparently separable from that leading to proliferation, immunoglobulin secretion and switching.

Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype.

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.

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).