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The hybridoma technology allows the production of large quantities of specific antibodies of a single isotype. Since different isotypes have special effector functions and are distributed distinctively throughout the body, it is often useful to have a library of switch variants from the original monoclonal antibody. We have shown previously that forced expression of activation induced cytidine deaminase (AID) in hybridomas increased their very low frequency of class switch recombination (CSR) in vitro only approximately 7-13 fold. Since we had previously identified rare hybridoma subclones that spontaneously switched at more than 100 times higher frequencies, we have now examined those higher switching variants to search for ways to further increase the frequency of isotype switching in vitro. AID was not responsible for the approximately 100 fold increase in CSR, so we used whole-genome gene expression profiling to provide a platform for studying candidate molecular pathways underlying spontaneous CSR in hybridomas.

T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.

We created mice expressing transgene-encoded BCRs with "dual reactivity" for the hapten Ars and nuclear autoantigens. Expression of transgene-encoded BCRs was not evident in the memory compartment despite observation of transgene-expressing B cells in germinal centers following Ars immunization. In contrast, dual reactive mAbs were readily obtained from mice with enforced expression of Bcl-2 following secondary Ars immunization. However, while these mAbs were hypermutated and displayed increased affinity for Ars, all had reduced avidity for DNA and intracellular autoantigens. Thus, Bcl-2 alters dominant-negative selection of dual reactive B cells during the Ars response, but this is restricted to those with lowered autoreactivity, demonstrating a hierarchy of peripheral tolerance during memory B cell development.

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain. However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.

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.

We investigated the role of apoptosis in the development of B cell memory by analyzing the (p-azophenylarsonate) Ars response in a line of A strain mice in which expression of human Bcl-2 was enforced in the B cell compartment. Previous studies of the Ars immune response in these A. Bcl-2 mice, demonstrated that a large percentage of the antibodies expressed by the Ars induced memory B cell compartment had accumulated point mutations via somatic hypermutation that increased their affinity for both Ars and the autoantigen DNA ("dual reactive" antibodies). This was in sharp contrast to normal A strain mice which displayed no dual reactive B cells in their Ars induced memory B cell compartment. These data suggested that interference with apoptotic pathways regulated by Bcl-2 allows developing memory B cells that have acquired autoreactivity to bypass a peripheral tolerance checkpoint. Further studies of these mice, reported here, demonstrate that enforced expression of Bcl-2 does not alter serum antibody affinity maturation nor positive selection of B cells expressing somatically mutated antibody with an increased affinity for Ars. Moreover, the somatic hypermutation process was unaffected in A. Bcl-2 mice. Thus, enforced expression of Bcl-2 in A. Bcl-2 mice appears to selectively alter a negative selection process that operates during memory B cell differentiation.

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.

We analyzed the participation of a predominant B cell clonotype in the anti-arsonate immune response of mice in which Bcl-2 expression was enforced in B cells. Many of the antibodies expressed by the arsonate-induced memory compartment of these mice were "dual-reactive," displaying increased affinity acquired via V region somatic hypermutation for both arsonate and the autoantigen DNA. The hypermutated antibodies expressed by the anti-arsonate memory B cell compartment of normal mice have increased affinity for arsonate but lack measurable affinity for DNA. Thus, interference with apoptotic pathways allows developing memory B cells that have acquired autoreactivity to bypass a peripheral tolerance checkpoint. These data demonstrate that both positive and negative selection, working in concert with V gene somatic hypermutation, result in the "specificity maturation" of the antibody response.

To examine the influence of Ag presentation by B cells on immune responses, we have used mice transgenic for an Ig heavy chain from a monoclonal anti-azobenzenearsonate (Ars) Ab to deliver Ag to B cells during immunization. A large proportion of transgene-expressing B cells in these mice binds Ars, while transgenic serum Ig shows poor Ars binding. Transgenic B cells present Ars proteins better than their nonhaptenated counterparts. This is associated with an increase in the proliferative responses of transgenic T cells to Ars protein immunization. Although B cell numbers in the transgenic mice are lower, many B cells in them show an activated phenotype, as identified by altered surface levels of peanut agglutinin reactivity, CD23, CD24, CD44, CD62L, and CD86. Even against nonhaptenated immunogens, transgenic responses show significant enhancement in the relative proportions of the Th1 cytokine IFN-gamma over the Th2 cytokines IL-4 and IL-10. Haptenated immunogens further enhance the predilection of transgenic mice to produce relatively more IFN-gamma. Consistent with this, there is an increase in IgG2a/IgG1 ratios in serum Abs in response to haptenated immunogens in transgenic mice. Adoptive transfer of primed hapten-specific secondary B cells into nontransgenic mice also induces an increase in relative levels of IFN-gamma in response to haptenated immunogens. Thus, presentation of immunogen in vivo by activated Ag-binding B cells contributes to enhanced immunogenicity and a Th1 cytokine bias.

The behavior of p-azophenylarsonate (Ars)-specific B cell clones during the primary T cell-dependent splenic response of A/J mice was investigated using an immunohistochemical approach. The earliest Ars-specific B cells were observed as isolated cells in the red pulp by day 3 after immunization with Ars-keyhole limpet hemocyanin, (KLH) and at day 6, large clusters of Ars-specific B cells were first detected in germinal centers, which continued to be observed for an additional 8 to 15 days. Surprisingly, no Ars-specific B cell foci were observed in or near the CD4 T cell-rich periarteriolar lymphoid sheath (PALS) during the entire primary response. Nevertheless, A/J mice immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma globulin (NP-CGG) or Ars-CGG mounted robust splenic (4-hydroxy-3-nitrophenyl)acetyl or CGG-specific PALS-associated focus reactions, respectively. In contrast, no Ars-specific PALS B cell foci were detected in A/J mice immunized with Ars-CGG. These data add to a growing body of evidence indicating that B cell proliferation and differentiation in CD4 T cell-rich microenvironments are not prerequisites for the GC reaction. Taken together with previous results obtained using other model Ags, the data suggest that the specificity of the B cell Ag receptor may strongly influence the lymphoid microenvironment in which a B cell clone first undergoes Ag-driven clonal expansion and differentiation.

Previous work on the cis-acting elements that control heavy chain variable region (VH) gene somatic hypermutation has indicated the presence of an as yet unidentified element(s) 3' of the intron enhancer that is necessary for high rate mutation. Examination of cis-acting elements involved in kappa light chain V gene hypermutation has demonstrated a requirement for both the intronic and 3' kappa enhancers in this process. To examine whether the 3'alpha heavy chain enhancer [3'alpha E(hs1,2)] is required for somatic hypermutation of VH genes, we generated two types of transgenic mice. One type was generated using a construct containing a VH promoter, a rearranged VDJ, the heavy chain intronic enhancer, and the murine heavy chain 3'alpha E(hs1,2). The transgenes in the second lines were similar to the transgenes in the first with the addition of a second complete matrix attachment region (MAR) 3' of the heavy chain intronic enhancer, and splice acceptor and polyadenylation sites between the two enhancers. Analysis of both transgenes revealed levels of mutation at least 10-fold lower than endogenous VH genes. These data suggest that the 3'alpha E(hs1,2) does not play a role analogous to the 3' kappa enhancer in the regulation of the hypermutation process. Moreover, in one of the transgenes, the presence of the 3'alpha E(hs1,2) resulted in a lack of transcription in vivo, suggesting a negative regulatory role for this enhancer in certain contexts.

We showed previously that heavy chain gene junctional amino acid differences among unmutated p-azophenylarsonate (Ars) Abs that share a unique gene segment combination encoding these V regions, termed "canonical," alter affinity. To determine the contribution of junctional amino acid differences to binding, we introduced, by site-directed mutagenesis, various amino acids at position 100 and/or 107 (sequential numbering) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved or showed increased Ars binding (1-to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (< or = 0.5-fold). As much as a 150-fold difference in Ars binding was observed between 2 Abs with different sets of junctions (Asn100/Tyr107 and Val100/Lys107). Thus, amino acid replacements at D gene junctions can produce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially, can result in preferential selection of Abs containing certain junctions during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are known to be recurrent in vivo and are associated with increased Ars affinity. The mutant Abs all showed increased affinity, indicating that despite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. These additive effects reflect the "adaptability" of the canonical gene segment combination in sustaining somatic mutations leading to affinity maturation.

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.

A new assay system detecting antibody production and delayed-type hypersensitivity (DTH) responses to trinitrophenyl hapten in an individual mouse (AS-DAD) was established. BALB/c mice were immunized intraperitoneally with varying amounts of 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC) on day 0. Venous blood was collected on days 2, 4, 6, 8 and 10. Levels of anti-TNP IgM and IgG serum were assayed by enzyme-linked immunosorbent assay (ELISA). After series of bleeding the mice were challenged with 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution in the footpad on day 14. Footpad swelling was measured 24 or 48 h after the challenge. Peak responses of the anti-TNP IgM and IgG production were detected 4 or 6 days after the immunization with 10(9) TNP-SRBC. Maximum DTH response was also observed with 10(9) TNP-SRBC 24 h after the challenge on day 14. The antibody and DTH responses were also induced in other normal inbred strains such as C3H/He and DBA/1 but not BALB/c nu/nu mice. To evaluate AS-DAD in immunopharmacological studies, various immunomodulating agents were examined in BALB/c mice by subcutaneous administration on days 0, 1, 2 and 3. Cyclosporin or cyclophosphamide at 100 mg/kg/day completely inhibited not only the anti-TNP IgM and IgG production but also the TNP-specific DTH response. Prednisolone at 0.5 mg/kg/day had no significant effect on the IgM and IgG production, whereas it inhibited the TNP-specific DTH response. Interestingly, histamine-added mouse gamma-globulin at 150 MG/kg/day clearly enhanced the anti-TNP IgM and IgG production, while it showed a suppressive effect on the TNP-specific DTH response. Levamisole at 5.0 mg/KG/day showed suppressive effects on the anti-TNP IgG production without affecting the IgM production and the DTH response. These results suggest that AS-DAD is useful for evaluating the immunopharmacological action of various agents.

Most data available from in vivo sources regarding the impact of somatic hypermutation on Ab V region structure and function are heavily biased due to the influence of clonal selection. In an effort to address this issue directly, we "randomly" introduced point mutations throughout the length of the VH region of an anti-p-azophenylarsonate (Ars) Ab expressed as an Fab in the phage display format. This was accomplished by means of an error-prone PCR with two protocols, which resulted in two mutant libraries. The nature of the nucleotide substitutions obtained from each protocol differed from each other and resulted in different frequencies of phage clones that did not appear to contain Fab on their surfaces. However, the majority of mutants in both libraries lacked detectable Fab expression. Screening of the library containing the most expressed Fabs for those that had gained affinity for structurally related haptens yielded two independent mutants that lacked detectable affinity for Ars and had high affinity for p-azophenylsulfonate. These mutants both contained amino acid substitutions from Asn to Ser or Thr at VH CDR1 position 35, a putative Ars contact residue. In this paper, we discuss the significance of these data with regard to the frequencies of V region loss of function, gain of increased affinity, and gain of altered specificity that result from somatic hypermutation in vivo.

In the air pouch-type allergic inflammation model in rats, leukocytes collected from the pouch fluid 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils. The leukocytes from the immunized rats produced significantly higher amount of the chemotactic factors than that from the non-immunized rats. The major chemotactic factor, leukocyte-derived neutrophil chemotactic factor (LDNCF)-2, was purified and found to be identical with rat macrophage inflammatory protein (MIP)-2 by N-terminal amino acid sequence analysis. Expression of MIP-2 mRNA was higher in the leukocytes from the immunized rats than that from the non-immunized rats. Possible roles of LDNCF-2 (MIP-2) in neutrophil infiltration in the allergic inflammation is discussed.

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.

Offspring of mother mice treated immediately after delivery with deaggregated human gamma-globulins (dHGG) are unable to produce HGG-specific antibodies when challenged with immunogenic forms of HGG (HGG/CFA) in adulthood. Despite a defective antibody response, animals rendered tolerant to HGG as neonates retain tolerogen-specific T cells able to proliferate and secrete lymphokines. The pattern of IL-2 and IL-4 secretion by T cells isolated from tolerant animals could not be distinguished from the corresponding cells in control mice, suggesting that neonatal exposure to dHGG did not affect T cell reactivity or Th1/Th2 in vivo balance. Moreover, immunization of tolerant animals with haptenated HGG confirmed the presence of tolerogen-specific helper T cells in vivo. Functional T cell depletion by anti-CD3 mAbs during lactation failed to modify induction of B cell tolerance, suggesting that T cells are neither affected nor required to induce the selective tolerance status observed in this model. Based on the finding that antigen-presenting cell functions in secondary organs (spleen, peritoneal cavity) are a late acquisition during ontogeny and reach adult-like levels at weaning, we propose that most soluble proteins elude T cell recognition during lactation due to defective antigen presentation.

The immune response of A/J mice against p-azophenylarsonate (Ars)-keyhole limpet hemocyanin (KLH) is characterized by the dominance, late in primary and during the secondary, of a recurrent idiotype called CRIA, encoded by a canonical combination of Ig gene segments. In this study, A/J mice were given Ars coupled to deaggregated human gamma globulins (dHGG) within 24 h after delivery. The offsprings from these mice were then exposed as adults to Ars-KLH. These animals developed an unusual immune response. The level of anti-Ars antibodies was nearly normal but a dramatic shift in repertoire was observed: the cross-reactive idiotype which is the hallmark of the anti-Ars response in A/J mice was completely absent. The idiotype could be recovered by injection of anti-idiotypic antibodies alone, with no need of lipopolysaccharide coupling. Therefore the presence of antigen at birth can lead to a strong perturbation of idiotype selection. Similar results were obtained with neonatal treatment using anti-IgM antibodies. After recovery of suppression, A/J mice can mount an anti-arsonate response of normal level but devoid of the dominant idiotype.

We have used a well-defined idiotypic system, the cross-reactive idiotype of A strain (CRIA) (Ab1) idiotype generated in A/J mice injected with arsonate coupled to keyhole limpet hemocyanin (ARS-KLH), to determine the frequency of precursors for auto-anti-idiotypic antibodies (auto-Ab2) in naive and immunized A/J mice by limiting dilution analysis after polyclonal activation by lipopolysaccharide. In naive animals, the precursor frequencies of auto-Ab2 B cells were below the limit of sensitivity of the technique in the majority of A/J mice, and could be detected in only 20% of the animals. Upon immunization with ARS-KLH, a large increase in auto-Ab2 precursor frequency was observed. This shift in frequency was not found when A/J mice were injected with KLH alone, or when BALB/c mice, which do not express the CRIA idiotype, were injected with ARS-KLH. To study the functional role of the auto-Ab2 B cells, we injected neonatal A/J mice with polyclonal rabbit Ab3 antibodies directed against a recurrent idiotype of auto-Ab2. Thereafter, these mice were injected with ARS-KLH. Although the anti-arsonate response level was normal, the CRIA Ab1 expression was reduced tenfold. Thus, the suppression of auto-Ab2 affects Ab1 dominance. We further show that the presence of maternal Ab1 can strongly modify the immune response of the offspring by inducing higher levels of the idiotype after immunization. Furthermore, IgM anti-arsonate antibodies were detected before immunization with antigen. From these data, we conclude that the affinity of antigen alone cannot explain the dominance of CRIA. Network selection is important in the shaping of the available repertoire.

Antibody VH transgenes containing small amounts of natural 5' and 3' flanking DNA undergo nonreciprocal homologous recombination with the endogenous Igh locus in B cells. The resulting "hybrid" heavy chain loci are generated at a low frequency but are fully functional, undergoing somatic hypermutation and isotype class switching. We have used this recombination pathway to introduce a somatically mutated variable (V) region with an unusually high affinity for the hapten p-azophenylarsonate (Ars) into the preimmune antibody repertoire. The affinity of this V region for Ars is 100-fold higher than any unmutated anti-Ars antibody previously characterized. Expression of the transgene-encoded V region did not affect many aspects of antigen-driven B cell differentiation, including somatic hypermutation, in either Ars-specific transgene- or endogenous V gene-expressing clones. Thus, the regulation of these processes appears to operate in a "global" fashion, in that the mechanisms involved are imperceptive of the relative affinities for antigen of the antibodies expressed by B cell clones participating in the immune response. In contrast, the selection of V region mutants leading to affinity maturation and memory cell formation was found to be strongly influenced by the transgenic V region, but only in clones expressing this V region. Hybridomas derived from transgene- and endogenous V region-expressing memory cells were isolated at similar frequencies from individual transgenic mice. The V regions expressed by hybridomas in both of these groups had 2- to 30-fold greater affinity for Ars than their unmutated precursors, despite the fact that the transgene-encoded precursors had 100-fold higher affinity than their endogenous counterparts. These results show that the criterion for entry into the memory compartment is established not by the affinity of a B cell's V region relative to all other V regions expressed during the response, but by the affinity of this V region relative to its unmutated precursor. Thus, the development of B cell memory is regulated in a "clone-autonomous" fashion.

Cell-mediated immune mechanisms have long been suspected of playing an important role in the pathogenesis of various renal diseases. An animal model of active nephritis secondary to an exogenous antigen that requires antigen presentation to immune-competent T cells has not been developed. Consequently, the potential of kidney cells to serve as effective antigen presenting cells after an exposure to a therapeutic, biological, or environmental agent in the intact animal has not been documented. The present experiments were designed to demonstrate the capacity of the kidney to become the target for cell-mediated immune injury. A model system has been developed whereby a chemically reactive form of the hapten azobenzenearsonate is introduced directly into the left kidney of pre-immunized Brown Norway rats. Previous studies have shown that this form of the hapten requires active antigen presentation but no intracellular processing, since the reactive form of the hapten modifies directly surface expressed proteins. Delayed hypersensitivity was demonstrated in the actively immunized animals by standard lymphocyte stimulation index and by in vivo skin testing. Peak foot pad swelling of 220 +/- 13 x 10(-2) mm in response to the hapten was observed between days 11 and 14 as compared to < 10 x 10(-2) mm in the contralateral foot injected with vehicle alone and < 20 x 10(-2) mm in response to azobenzenearsonate injection in animals immunized with adjuvant alone. The exposure of the kidney to the hapten in the primed animal results in an active unilateral granulomatous nephritis with marked destruction of tubules and glomeruli. On average, 71.5 +/- 5.2% of the renal cortex is affected by the inflammatory process in the actively immunized animals, compared to only 8.1 +/- 3.8% in controls. The disease can be reproduced qualitatively by adoptive transfer of T cells but not by passive antibody administration to naive recipients. These studies demonstrate that intrinsic kidney cells can act as effective antigen presenting cells in the intact animal and that the kidney can become the target of a cell-mediated immune injury.

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.

It has been proposed that the autoantibody-secreting cells active during autoimmune diseases are derived from B cells initially responding to environmental antigens. In order to test the relationship between the antigen-induced and autoimmune repertoires, we monitored the fate of antigen-activated idiotypically defined B cells present in mice that developed the systemic lupus erythematosus (SLE)-like syndrome associated with the lpr mutation. Mice homozygous for both the A/J-derived Igh and Ig kappa region haplotypes and the lpr mutation were bred. Immunization of these mice with p-azophenylarsonate (Ars)-protein conjugates elicited the idiotypic components (IdCR) characteristic of the A/J anti-Ars response and did not interfere with the spontaneous development of the lpr-mediated autoimmune disease. These Id/lpr mice provided an ideal system for studying the relationship between the exogenously and endogenously induced responses because: (1) VHIdCR antibodies have been shown to bind autoantigens in vitro; and (2) serological and molecular reagents exist which can identify and monitor VHIdCR antibody production as disease progresses. Serum samples and hybridoma cell lines derived from non-immune as well as Ars-keyhole limpet haemocyanin (KLH)-immunized Id/lpr mice were monitored for idiotype expression as well as Ars and ssDNA reactivity at various stages of disease progression. We found that antibodies utilizing the VHIdCR gene segment did not preferentially contribute to the autoantibody pool. Moreover, even when IdCR B-cell clones were expanded by deliberate immunization with Ars-KLH, Ars non-binding variants were only rarely detected among the activated B-cell populations of diseased mice. These results indicate that there is only minimal overlap between the VHIdCR conventional and autoimmune repertoires.

Antigen-specific molecules secreted by murine T cell hybrids (TABM) specific for 4-hydroxy 3-nitrophenyl (NP) or azobenzenearsonate (ABA) were purified from ascitic fluid by ion exchange chromatography and/or affinity for antigen. Partial amino acid sequence of reduced Mr 72,000 NP-specific polypeptides and Mr 20,000 peptides prepared by treatment of the ABA-specific immunoprotein with cyanogen bromide was obtained and a septapeptide of the NP-specific TABM shared 3/7 residues with the ABA-specific TABM. Both TABM shared residues present in 95% T cell receptor for antigen (TCR) V alpha subgroup I and 83-96% murine immunoglobulin V kappa Fr3. These results provide evidence that extracellular antigen-specific T cell proteins are soluble analogues of TCR alpha chains and belong to the immunoglobulin supergene family.

We have elicited anti-arsonate antibodies bearing the major cross-reactive idiotype (CRIA) in a double congenic idiotype-negative strain (C.C58.AL-20) bearing a light chain polymorphism that has previously been shown serologically not to complement idiotype-positive heavy chains. Using the idiotype cascade (Ab1-->Ab2-->Ab3-->-->Ab1'), CRIA-positive antibodies were raised and monoclonal antibodies were isolated and characterized serologically and by nucleotide sequence analysis. Two types of idiotype-positive anti-arsonate antibodies were generated in the C.C58.AL-20 strain. One group of hybridomas used the canonical VH1.8 heavy chain gene segment with V kappa 10 variant light chains. A second group used a VHGAM3.8 heavy chain with V kappa 10 variant light chains. This latter heavy-light pairing has been observed in CRIA-like responses previously in BALB/c mice after idiotypic manipulation (or rarely after antigen alone). These studies demonstrate the plasticity of the immune response when manipulated with idiotype reagents as well as its structural variability. Additionally, they provide important insights into the potentials of idiotype vaccines.

Neonatal germfree (GF) colostrum-deprived and conventional (CV) colostrum-fed piglets were immunized IP with p-azo-phenyl-arsonate-bovine gamma globulin (Ars-BGG) in Freund's adjuvant to study the development of the immune response in the absence or presence of maternal antibodies and environmental antigens. Overall, the immune response varied greatly within each group but did not differ in GF from CV piglets statistically. Affinity immunoblot analysis suggested that anti-Ars antibody was more restricted in GF than CV piglets and clonotype shifts occurred more in GF than CV piglets after each antigenic stimulation. In contrast, the clonotype pattern of the anti-BGG antibody was similarly heterogeneous in the two groups. Based on the affinity immunoblot data the antibodies generated to the Ars-haptenic group in CV piglets are more heterogeneous than GF piglets and suggest that clonotype generation is influenced by maternal antibodies and environmental antigens.

The framework regions of antibodies fold into a conserved beta-sheet structure that acts as scaffolding for the antigen-contacting complementarity-determining regions (CDR). To test the structural equivalence of the frameworks between two antibodies with widely different combining sites, we created chimeric H and L chains by grafting the CDR of an alpha(1-->6)dextran specific antibody onto the framework of a p-azophenylarsonate (Ars) specific antibody through oligonucleotide-directed mutagenesis of the anti-Ars variable region genes. Antibodies consisting of various chain combinations of the chimeric, anti-dextran, and anti-Ars derived H and L chains were generated in transfectomas and tested for binding to dextran and Ars. Of the newly created chimeric and/or hybrid antibodies, an antibody with the chimeric H chain and the anti-dextran L chain bound to dextran with the same association constant as the parental anti-dextran antibody, and like the anti-dextran antibody was shown by immunochemical mapping to have a site complementary to six glucose residues. None of the other new variable region combinations, including the all-chimeric combination, showed binding to either dextran or Ars. These results indicate that the H chain but not the L chain anti-dextran and anti-Ars frameworks are functionally equivalent. Attempts to confer dextran binding on the H and L chain chimeric antibody, by mutagenizing selected framework residues, were unsuccessful. This study demonstrates the important role of the frameworks in the precise alignment of the CDR for Ag binding.

The hapten- and carrier-specific T lymphocyte clone D5 and a T hybridoma (D5h) derived from D5 cells recognize several different protein Ag conjugated with p-azobenzenearsonate (arsonate) presented by the class II MHC protein I-Ad. We show here that the ligand recognized by the D5 TCR is a complex of a haptenated peptide bound to I-Ad. We have identified a peptide fragment generated by enzymatic cleavage of arsonate-conjugated OVA (Ars-OVA), which stimulates D5 cells when presented by I-Ad-bearing APC. A synthetic peptide corresponding to this fragment, OVA(36-50), forms a ligand for D5h cells when it is conjugated with arsonate and presented by cells bearing I-Ad. Paraformaldehyde-fixed, I-Ad-bearing cells present Ars-OVA(36-50), or the longer stimulatory peptide Ars-OVA(33-49), to D5h cells, demonstrating that haptenated synthetic peptides can substitute for naturally processed antigenic peptides. The peptide Ars-OVA(33-49) binds to the major peptide-binding site of I-Ad because it competitively inhibited presentation of the peptide OVA(323-339), previously demonstrated to bind to I-Ad directly in vitro, to the OVA/I-Ad-specific T cell hybridoma 3DO-54.8. The unconjugated OVA(33-49) peptide failed to inhibit the presentation of OVA(323-339), demonstrating that the hapten facilities binding of the peptide to I-Ad. Conversely, the peptide OVA(323-339) competitively inhibited the presentation of Ars-OVA(33-49) to D5h cells, indicating that the two peptides Ars-OVA(33-49) and OVA(323-339) bind to overlapping sites on I-Ad. Amino acid substitutions introduced into the beta 1 domain of I-Ad that affected recognition of OVA(323-339) by 3DO-54.8 cells also affected recognition of Ars-OVA(33-50) by D5h cells, demonstrating that similar regions on I-Ad are required for TCR recognition of conventional as well as haptenated peptides. These results represent the first demonstration that the ligand recognized by a hapten- and carrier-specific T cell clone restricted to an MHC class II protein is a haptenated peptide Ag bound to the MHC molecule.

Murine A/J anti-p-azophenylarsonate mAb that express a dominant cross-reactive Id are encoded by a single set of germ-line VH and VL region genes. The crystal structure of the Fab of antibody 36-71, which uses this canonical set of genes but is somatically mutated, was previously determined. An Fab 36-71:phenylarsonate complex was modeled, identifying amino acid side chains that were proposed as contact residues to hapten. The remarkable conservation of these residues among canonical anti-p-azophenylarsonate antibodies suggested that the overall binding site geometry was maintained among somatically mutated antiarsonate monoclonal antibodies. To test this hypothesis, we used the germ-line-encoded antibody 36-65 to construct mutant antibodies, using oligonucleotide-directed mutagenesis, which differed only at the putative H chain hapten-contacting residues, and measured their hapten binding. A framework residue at H chain position 47 involved in a hydrogen bond network with CDR residues was also mutated. Substitution of several amino acids at each position permitted evaluation of the stereochemical requirements for binding. The results indicate the importance of aromatic stacking of two H chain tyrosine residues against the phenyl ring of the hapten in maintaining affinity, as well as strict complementarity at H chain position 35. The results are consistent with the crystal model of the combining site, and provide further evidence for conservation of the three-dimensional binding site motif among antiarsonate antibodies that bear a dominant heritable ld.

Using two polyclonal (rabbit) and two monoclonal anti-idiotype (anti-Id) reagents, we investigated structural correlates of the Id of mAb 36-71, a somatically mutated member of the CRIA Id family that has an exceptionally high affinity for the p-azobenzenearsonate (Ars) hapten. The two monoclonal anti-Ids reacted principally with the L chain of 36-71. The polyclonal anti-Ids interacted with both the H and L chain. The amino acid sequences of the VH and VL regions of 36-71 differ in eight and 11 positions respectively from those of the anti-Ars mAb 36-65, an unmutated prototype of the CRIA family. In the presence of 36-71L only three substitutions in 36-65 VH, introduced by mutagenesis, sufficed to restore full expression of the 36-71 Id. The same three substitutions had previously been shown to increase the affinity of 36-65 by a factor of 200, to a level equivalent to that of 36-71. X-ray crystallography had indicated that two of these substitutions introduce conformational changes consistent with the increase in affinity. We propose that these conformational changes may also account for the critical role of the three amino acids in Id expression. We also found that 36-65 is a very poor inhibitor of the interaction of 36-71 with its polyclonal anti-Ids, despite identity of the hapten-contacting residues in the two mAbs and evidence (from hapten inhibition) that the hapten-binding region is part of an important Id. Again, a difference in conformation at the binding site of the two mAbs could account for these observations.

We review here our attempts to achieve a better understanding of the structure--function relationship of antibody combining sites, and to gain insights into the engineering of antibodies with desired specificity and affinity. We have focused on a model system--antibodies to the hapten p-azophenylarsonate (Ars) derived from A/J mice. Oligonucleotide-directed mutagenesis was used to alter the sequence of the variable region genes of such anti-Ars antibodies. Mutant antibodies were generated in hybridoma cells following transfection of the altered genes, and the effects of the primary structure changes on antibody specificity, affinity, and idiotypic expression were assessed. These studies suggest that an antibody combining site with basic specificity for an antigen could be created by introducing a set of a few amino acid residues in the complementarity determining regions, and that the affinity of such a site could be improved one substitution at a time in a sequential manner.

The three-dimensional structure of FabR19.9 from a well-characterized anti-p-azobenzenearsonate monoclonal antibody has been determined by x-ray diffraction techniques in two crystalline forms (I and II) to a resolution of 2.8 and 2.7 A, respectively. Essentially the same tertiary and quaternary structure of the Fab is observed in the two forms. The major difference resides in the intermolecular contacts, which are interpreted to favor an irreversible transition from the metastable form I to the more stable form II. The third complementarity-determining region of the heavy chain (H3) folds back over the combining site and requires rearrangement for hapten binding. This dynamic requirement on H3 is consistent with its mobility in the structure and can explain hapten binding to an otherwise inaccessible antibody combining site.

Antigen-specific molecules secreted by a murine T cell hybrid specific for azobenzene arsonate (ABA) were purified from ascites fluid by ion exchange chromatography and affinity for antigen. The antigen-specific proteins were purified 250 fold and were resolved predominantly as Mr 110,000 polypeptides by reduction and SDS-polyacrylamide gel electrophoresis. The ability of these molecules to bind antigen was analyzed by an ELISA using antigen-coated microtiter trays. Binding of the T cell proteins to antigen was detected with antisera specific for the proteins. Antigen binding to ABA-ovalbumin but not ovalbumin was optimal at 37 degrees C and protein derived from another T cell hybrid did not bind ABA-ovalbumin. Solid phase antigen binding was inhibited specifically by soluble ABA-ovalbumin, indicating that these T cell-derived proteins bind nominal antigen in the solid or liquid phase. It is suggested that these proteins represent a soluble, antigen specific manifestation of some T cell function.

The elucidation of the structural basis for expression of the cross-reactive Id of the A strain mouse (CRIA) in response to the hapten p-azophenylarsonate has been the object of considerable research effort. Most conclusions regarding the amino acids involved in Id expression have been inferential, based on comparisons of amino acid sequences, chain recombination experiments, or chemical modification of particular amino acids. To more rigorously designate the amino acids critical to the phenotype of the CRIA, a system for the expression and directed mutation of antibody molecules was developed. Based on the baculovirus expression of foreign proteins in cultured insect cells, functional antibodies can be produced at very high levels in vitro. By the process of oligonucleotide-directed mutagenesis, a series of specific amino acid changes were introduced into both the H and L chains of a prototype CRIA expressing antibody molecule. Analysis of the gain or loss of polyclonal Id and each of several monoclonal idiotopes has allowed us to identify more precisely the amino acids responsible for expression of the CRIA. Thus we have shown that expression of the CRIA is equally dependent on amino acids in the second and third CDR of the H chain. Furthermore, the idiotopes studied surround the Ag-binding site and clearly involve multiple interactions in several of the L and H chain CDR.

Some T cells produce membrane-associated or soluble molecules which bind nominal antigen specifically (TABM) and effect immunoregulation or events similar to cell-mediated hypersensitivity. We have used polyclonal antisera raised against an azobenzene arsonate (ABA)-specific TABM secreted by an ABA-specific T cell hybrid or against TNP-specific polypeptides produced by immunoregulatory T cells to identify the expression of soluble (secreted) or membrane-associated TABM. Ascites fluid or culture medium containing a T cell hybrid or T cell lines, respectively, contain TABM recognized only by an antiserum specific for the secreted T cell hybrid (ABA-specific) derived TABM. Conversely, an antiserum that recognized the TNP-specific polypeptides detected cell-membrane associated TABM but did not bind TABM secreted by the T cell hybrid or cell lines.

The structure of the antigen-binding fragment (Fab) of an anti-p-azophenylarsonate monoclonal antibody, 36-71, bearing a major cross-reactive idiotype of A/J mice has been refined to an R factor of 24.8% at a resolution of 1.85 A. The previously solved partial structure of this Fab at a resolution of 2.9 A (Rose et al., 1990) was used as an initial model for refinement against the high-resolution data. The complex with hapten has been modeled by docking the small-molecule crystal structure of phenylarsonic acid into the structure of the native Fab on the basis of a low-resolution electron density map of the complex. In this model, residue Arg-96 in the light chain and residues Asn-35, Trp-47, and Ser-99 in the heavy chain contact the arsonate moiety of the hapten; an additional bond is found between the arsonate group and a tightly bound water molecule. The phenyl moiety of the hapten packs against two tyrosine side chains at positions 50 and 106 in the heavy chain. Residue Arg-96 in the light chain had been implicated as involved in hapten binding on the basis of previous experiments, and indeed, this residue appears to play a crucial role in this model. Experiments employing site-directed mutagenesis directly support this conclusion. The heavy-chain complementarity-determining regions have novel conformations not previously observed in immunoglobulins except for the recently solved anti-p-azophenylarsonate Fab R 19.9 (Lascombe et al., 1989).

Comparison between the structures and solvent-accessible surfaces of the antigen-binding fragments of two murine anti-p-azophenylarsonate monoclonal antibodies, one bearing a major cross-reactive idiotype of A/J strain mice (36-71) and one lacking the idiotype (R19.9; Lascombe et al., 1989), highlight the structural basis for the determination of hapten affinity and idiotypy. Since the sequence of R 19.9 is identical with the germline-encoded sequence at 16 positions in both heavy-chain and light-chain variable regions where somatic mutations and junctional differences have occurred to produce the 36-71 sequence, the structure of R 19.9 can be used to model the structure of the germline-encoded antibody (36-65) in the regions around these sites. These 16 sequence differences exclude the third heavy-chain complementarity-determining region because R 19.9 utilizes a D gene segment not associated with the predominant idiotype, which is 4 residues longer than the canonical D gene segment utilized in the sequences of 36-71 and 36-65. This difference between the structures of R 19.9 and 36-71 does not affect the validity of using the structure of R 19.9 to model the structure of 36-65 since the third heavy-chain complementarity-determining region is highly solvent-exposed in both 36-71 and R 19.9, and does not interact with any of these 16 sites. Comparing the structures of 36-71 and R 19.9 suggests that only three of the differences in the heavy-chain sequences, and three of the differences in the light-chain sequences of 36-71 and 36-65, increase the affinity for hapten.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutant mAb with increased Ag binding were generated from a hybridoma cell line, 36-65, that secretes an IgG1,kappa anti-p-azophenylarsonate-(Ars) specific antibody. The mutant antibodies were identified using an Ars-specific ELISA and sib selection so that approximately 10(6) cells could be analyzed. The ELISA used as Ag a low ratio of Ars coupled to BSA and was set up so that only those antibodies that had higher binding than the parent would be detected. Seven mutant producing cell lines were isolated from five independent clones of 36-65. The mutant antibodies bind Ag 20 to more than 200-fold better than the parent and have wild type V region sequences. All have C region mutations that result in an increased avidity. At least five different genetic events are responsible for the C region mutations.

p-azophenylarsonate-specific antibodies of A/J mice which bear the Ars-A crossreactive idiotype utilize the V kappa-Ars-A gene segment, a member of the V kappa 10 family. Southern hybridization of genomic DNA from several inbred strains using a probe from the 5' flanking region of the V kappa-Ars-A gene demonstrated three patterns of restriction fragment length polymorphisms (RFLP). Six genes corresponding to hybridizing bands were obtained from DNA libraries of C.AKR, PERU and A/J mice, and nucleotide sequence comparisons revealed two allelic groups: AKR1 (Igk-V10.1a), AJ1 (Igk-V10.1b) and PERU1 (Igk-V10.1c); AKR2 (Igk-V10.2a), AJ2 (Igk-V10.2b), and PERU2 (Igk-V10.2c). The Igk-V10.1b gene of the A/J strain is the V kappa-Ars-A gene used in Ars-A idiotype-positive antibodies. The product of the C.AKR allele (Igk-V10.1a) contained four amino acid substitutions in CDR3 as compared with Igk-V10.1b. These substitutions probably explain the failure of AKR mice and other strains with the same V kappa 10 RFLP pattern to provide in genetic crosses a L chain which, together with the A/J VH-ArsA gene product, form Ars-A idiotype-positive antibodies. Also, the nucleotide sequence identity between the Igk-V10.1c and Igk-V10.1b alleles and the Igk-V10.2c and Igk-V10.2b alleles is significantly greater than that seen in comparisons with the Igk-V10.1a and Igk-V10.2a alleles, respectively, suggesting an evolutionary pathway similar to that of the linked Igk-J locus. BALB/c antibodies bearing the A48 regulatory idiotype contain L chains encoded by the BALB/c Igk-V10.1b and Igk-V10.2b alleles. Strongly A48 idiotype-positive antibodies utilize the Igk-V10.1b chain, and weakly A48-positive antibodies use the Igk-V10.2b L chain. The possible effects of amino acid substitutions specified by the Igk-V10.1a, Igk-V10.1c, Igk-V10.2a, and Igk-V10.2c alleles on their ability to provide L chains used in A48 idiotype-positive antibodies are discussed.

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.

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).

The basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence of the lower-affinity antibody into that of the higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data showed that out of the 19 amino acid differences between the two hybridoma antibodies, the affinity increase could be reproduced by three heavy-chain substitutions that are present in the high-affinity antibody. The combined effect on affinity of amino acid substitutions was generally found to reflect their individual effects. Although the light chain of the high-affinity antibody did not seem to play a major role in the affinity increase, its contribution varied with the kind and number of heavy-chain substitutions. The results hold promise for antibody engineering and are consistent with a stepwise acquisition of somatic hypermutations in which the existing structural context of an antibody most likely influences the affinity-based selection of later substitutions. They further suggest that many substitutions may be tolerated in vivo during the antigen-driven selection process, even though they confer on the antibody no affinity increase.

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.

Antibody V region structural diversity in the mouse is generated, in part, by the combinatorial joining of different gene segments, as well as by the "imprecision" of these joining events. The same two gene segments can be joined at different locations, and nucleotides can be deleted or added de novo to the segment junction. While it is clear that such junctional processes are a major contributor to V region diversity, the mechanisms that generate this diversity are poorly understood. Here I present sequences in the VH-D-JH region of 34 VH genes that are composed of the same three VH gene segments. In combination with a single V kappa-J kappa pair, these VH genes encode a family of V regions that are recurrently expressed in the immune response of A/J mice to p-azophenylarsonate (Ars). The germline sequences of the three constituent gene segments for these VH genes are known, making it possible to determine the origin of the nucleotides in junctional regions. An examination of the frequency and type of nucleotides present in these regions provides insight into the properties of the segment joining mechanism. In addition, the data suggest that recurrent expression of the anti-Ars V regions which these VH genes partially encode is due not only to antigenic selection, but to the high probability with which these VH genes are formed during B cell differentiation.

The number of neutrophils, eosinophils, and mononuclear cells that had migrated into pouch fluid were measured after injecting an antigen (azobenzenearsonate-conjugated acetyl bovine serum albumin) solution into air pouches made on the dorsum of immunized rats. The number of neutrophils began to increase 4 hr after the antigen challenge, reaching a maximum at 16-24 hr. Increases in the number of eosinophils and mononuclear cells were quite poor. The pouch fluid supernatant fraction was fractionated to hydrophilic and lipophilic fractions with the aid of an octadecylsilyl silica cartridge, and the chemotactic activity in each fraction was measured by the modified Boyden chamber method. The chemotactic activity in the pouch fluid supernatant fraction was the highest at 2-4 hr and decreased with time after the antigen challenge. At 16 and 24 hr, chemotactic activity was decreased to a very low level although there was a large number of neutrophils in the pouch fluid. At 4 hr, the chemotactic activity in the lipophilic fraction was significantly higher than that in the hydrophilic fraction. However, at 8 hr, the chemotactic activity in the lipophilic fraction was changed to a significantly lower level than that in the hydrophilic fraction. The chemotactic activity in the lipophilic fraction had decreased to a very low level at 8 hr. Heat treatment of the pouch fluid supernatant fraction at 92 degrees C for 5 min both at 4 and 8 hr significantly increased the chemotactic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

We have compared by functional assays the relative preference among Ia molecules for their ability to present tyrosine-azobenzenearsonate (ABA-Tyr) to T cells. Immunization of B10.BR mice (IAk, IEk) with ABA-Tyr resulted in the induction of IAk-restricted T cells only. Immunization of B10.A(5R) mice (IAb, IEb/k) gave only IEb/k-restricted T cell clones even though IAb-restricted responses could be induced in C57BL/6 mice (IAb). These results indicated that IAk was preferred over IEk and IEb/k was preferred over IAb for presentation of ABA-Tyr. A comparison between IAk and IEb/k made by immunizing [B10.BR x B10.A(5R)]F1 mice (IAk, IEk, IAb, IEb/k), showed that IEb/k was favored over IAk. No IAb- or IEk-restricted response was seen. Further attempts were made to compare Ia preference for ABA-Tyr presentation by competitive inhibition assays. It could be shown that the presence of IEb/k molecules on an accessory cell interfered with the ability of IAb molecules on the same cell to present ABA-Tyr to an IAb-restricted T cell clone by direct competition. Such a competition was not observed between IAk and IEk. Finally, it could be shown that addition of ABA-Tyr inhibited the presentation of moth cytochrome-c peptide (81-103) by IEb/k but did not influence its presentation by IEk. From these functional studies we suggest that the binding affinity of ABA-Tyr with the Ia molecules will fall in the order: IEb/k greater than IAk greater than IAb greater than IEk.

Using Ia antigen-containing lipid vesicles, we investigated by stopped-flow fluorometry the requirements for helper T cell recognition and activation. When azobenzenearsonate-L-tyrosine (ABA-L-Tyr)-specific, I-Ak-restricted helper T cell hybridomas 2-45-12 were mixed with ABA-L-Tyr and purified I-Ak molecules on lipid vesicles, an increase of intercellular calcium ion concentration ([Ca2+]i) in the T cells were detected within 1-2s. The average increases of [Ca2+]i were not much different when the lipid vesicles were composed of dimyristoylphosphatidylcholine or of egg phosphatidylcholine, but they were dependent on the density of I-Ak molecules on the liposomes. The increase of [Ca2+]i was inhibited in the presence of anti-I-Ak monoclonal antibody 10.2.16, but not by the addition of anti-L3T4 monoclonal antibody GK1.5. However, the addition of anti-L3T4 antibody during the first 3 h of cultivation completely inhibited the T cell activation [interleukin (IL-2) production]. In our experimental system, IL-2 production was observed either when L3T4-positive T cell hybridomas 2-45-12 were stimulated with ABA-L-Tyr and Ia molecules on the vesicles in the presence of phorbol 12-myristate 13-acetate, or when L3T4-negative T cell hybridomas 3H60.12 were incubated with ABA-L-Tyr and Ia molecules on the planar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The T cell response to L-tyrosine-azobenzenearsonate (ABA-tyr) has been studied using T cell lines and clones derived from three different mouse strains, B10.BR, B10.A (5R) and C57B1/6. In all cases, the arsonate group in conjunction with the amino group of tyrosine formed the functional T cell epitope. Molecules without any one or both of these groups are non-stimulatory. The hydrophobic moiety consisting of the azo-linked benzene rings forms the agretope of the molecule, as is evident from competitive inhibition of T cell stimulation by non-stimulatory analogues lacking the epitope. Substitutions on the benzene ring at ortho or meta positions resulted in decreases in ability to compete, indicating the likelihood of steric inhibition of binding of the agretope with the Ia molecule. This pattern was observed for clones and lines restricted by IAk, IAb and IEb/k MHC class II molecules. Peptides from lambda repressor protein, P84-98 and P73-88, showed haplotype specificity in their ability to inhibit ABA-tyr-induced proliferation of T cell clones, BRTC-4 and B6TC, respectively. The binding constants of ABA-tyr analogues were considered to be comparable to those of lambda repressor peptides because equimolar concentrations resulted in similar levels of competition. A cluster of aromatic amino acids on the floor of most MHC class II molecule binding sites might provide strong hydrophobic interaction with azo-linked benzene rings of ABA-tyr, thus accounting for its immunogenicity in all strains of mice studied.