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S H Clarke

Publications and source records attributed to S H Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1996

Interdependence of N nucleotide addition and recombination site choice in V(D)J rearrangement.

Diversity in the Ag binding receptors of B and T cells is achieved through a process of genomic rearrangement involving selection of recombination sites and, in adult mice, addition of nontemplated (N) nucleotides. We have analyzed 543 Ig heavy chain nonproductive rearrangements, involving a single variable region gene segment, from adult and perinatal mice. We infer several fundamental and novel features of the recombination mechanism. N regions are formed predominantly from the DNA plus strand or from the DNA minus strand polymerizations, rather than as a concatenation of the two. Homologous overlaps of as few as one nucleotide between gene segments cause significant skewing of recombination sites. The V(H) recombination site spectrum differs in perinatal and adult mice, with sites representing overlap between V(H) and D over-represented in the perinatal mice, and sites representing overlaps between V(H) and the N strand polymerized onto the D segment over-represented in the adult mice. Thus, in V(D)J joining, N nucleotide addition and recombination site choice are highly interdependent events.

Animals↗
PubMed1996

The transition of pre-BI to pre-BII cells is dependent on the VH structure of the mu/surrogate L chain receptor.

We have demonstrated previously that the majority ( > 90%) of VH12 B cells are absent from the adult peripheral repertoire, and that most that remain have the fourth position at the D-J function (designated 10/G4). We report here that most VH 12-expressing pre-B cells are lost during the transition from the pre-BI to the pre-BII cell stage in normal mice, and that pre-BII cell productive (P) rearrangements ar enriched in 10/G4 CDR3. This coincides with the initial expression of H chain and the generation of the mu/surrogate L chain (SL) receptor. In contrast, there is not enrichment for 10/G4 CDR3 in mu MT mice, and the frequency of P rearrangements is as expected from a random rearrangement mechanism, ruling out a biased rearrangement mechanism unique to VH12. We have also demonstrated that non-10/G4 mu chains can associate with SL and be expressed on the cell surface, suggesting that they are available on the cell surface for selection. Thus, transition of pre-BI to pre-BII cells is dependent on the structure of the VH domain.

Animals↗
PubMed1996

Both Sm and DNA are selecting antigens in the anti-Sm B cell response in autoimmune MRL/lpr mice.

More than half of the anti-Sm hybridomas isolated from MRL/Mp-lpr/lpr (MRL/lpr) mice produce Abs that also bind ssDNA, and half of these bind dsDNA. Intraclonal comparisons indicate that DNA is a selecting Ag for at least some dual-binding clones. To determine whether Sm itself is a selecting Ag for anti-Sm, we have identified the somatic mutations within the expressed VH and V kappa genes of eight anti-Sm hybridomas, six of which do not bind DNA. We find these V genes have between 0 and 12 somatic mutations each, and that four hybridomas possess a higher number of heavy or light chain CDR replacement (R) mutations than expected by chance, suggesting that these anti-Sm-producing B cells have undergone Ag selection. To demonstrate directly the effect of somatic mutation on Sm binding, we have engineered the unmutated counterpart of Ab 2-12, an Sm-specific hybridoma Ab with a nonrandom distribution of V kappa CDR R mutations, and compared its ability to bind Sm and ssDNA with that of the originally isolated 2-12 Ab. We find that the unmutated Ab has a much lower avidity for Sm than the mutant, but, unlike the mutant, it binds ssDNA. We conclude that Sm can drive clonal expansion in the anti-Sm response, and that Sm-only binding B cells can arise from Sm/DNA dual-binding B cell clonal precursors. These data also suggest that dual binding is not necessary to sustain clonal expansion. Thus, this response is unique in that it can be driven by either of two Ags.

Amino Acid Sequence↗
PubMed1996

Comparison of protein S functional and antigenic assays in normal pregnancy.

OBJECTIVE: Our purpose was to determine the effect of pregnancy on the protein S functional assay (clot based), which is used to screen for all subtypes of protein S deficiency states, and to compare its behavior in pregnancy with antigenic assays. STUDY DESIGN: This was a cross-sectional study of 37 normal pregnant women without thromboembolic risks who were tested by both functional and antigenic protein S assays during the first, second, and third trimesters. RESULTS: Mean protein S functional levels decline strikingly from the first to the third trimester, all 10 third-trimester patients had functional protein S levels well below the lower limit of the reference range. In contrast, only 3 of 10 third-trimester and none of the second-trimester patients had free protein S antigenic levels below the reference range. CONCLUSIONS: The protein S functional assay should not be used in pregnancy to screen for the subtypes of protein S deficiency; misdiagnosis and inappropriate treatment could result.

Adolescent↗
PubMed1995

Sm and DNA binding by dual reactive B cells requires distinct VH, V kappa, and VH CDR3 structures.

We have previously demonstrated an overlap of the anti-Sm and anti-DNA responses in MRL/Mp-lpr/lpr mice. The Ab produced by many anti-Sm hybridomas bind DNA and are encoded by Ig V genes used by anti-DNA hybridomas. In addition, some anti-Sm Ab that bind DNA have acquired mutations that improve DNA binding, indicating that DNA is a selecting Ag in the anti-Sm response. To gain insight into the basis for the dual binding ability of these Ab, we coexpressed the H chain from the anti-Sm hybridoma 2-12 with nine different L chains. Hybridoma 2-12 binds Sm but not DNA, yet expresses the same J558 VH gene as three anti-Sm hybridomas that bind ssDNA and at least one anti-DNA hybridoma that does not bind Sm. We found that most of the transfectoma Ab bind Sm, but their avidities vary over more than 3 orders of magnitude. Five of the nine transfectoma Ab bind ssDNA, and none bind dsDNA. In general, the ability to bind each Ag follows the binding ability of the hybridoma from which the L chain is derived. H Chain swapping experiments indicate that the H chain, VH CDR3 in particular, contributes to the binding of both Sm and DNA. We conclude that Sm and DNA select for distinct features of VH, V kappa, and VH CDR3, suggesting selection by both Ag in the anti-Sm response.

Amino Acid Sequence↗
PubMed1995

Differential susceptibility to tolerance induction in vitro of splenic B cells from several transgenic mouse lines. Role of B1 cells.

Spleen cells from transgenic mice, whose rearranged Ig receptors reflect the repertoires of B1 (CD5+ and "sister" B cells) or normal B2 cells, were examined for their ability to be rendered unresponsive. By using an anti-Ig tolerance protocol that is independent of receptor specificity, we previously reported that peritoneal B cells, containing primarily CD5+ and "sister" B cells, were not susceptible to unresponsiveness. Herein, we show that splenic B cells from two separate transgenic mouse lines, each expressing a B1-type receptor, are resistant to tolerance induction in vitro. In contrast, splenic B cells from two other transgenic mouse lines with a large representation of conventional B cells were sensitive to anti-lg-mediated unresponsiveness. This difference does not reside in the surface Ig density, cell cycle, or activation stage of these cells, but is reflected in the initial calcium mobilization and tyrosine phosphorylation after surface Ig cross-linking. Therefore, these results support the hypothesis that the antigenic specificity of B cell receptors may drive cells toward the B1 subset, as suggested by Cong et al. (Cong, Y-Z., E. Rabin, and H. H. Wortis. 1991. Int. Immunol. 3:467-476), and that B1 cell characteristics confer the ability of B cells to withstand in vitro tolerance induction, irrespective of their anatomical location. The possibility that this results from previous antigenic experience is discussed.

Animals↗
PubMed1995

Binding analysis of antibodies produced by precursor and branchpoint intermediates of an anti-influenza hemagglutinin B cell clone. Parallel replacement mutations do not confer increased avidity for hemagglutinin.

We have previously described a set of secondary (2 degrees) hybridomas that are specific for the Sb site of influenza hemagglutinin (HA(Sb)) and share the H37-68 ld (68ld). Cells producing 68ld Abs dominated the 2 degree response of one BALB/c mouse, but were virtually absent from the primary (1 degree) and 2 degree responses of other BALB/c mice. To understand the basis for the idiosyncratic nature of this response, we have assessed the functional importance of a conserved DJ junctional sequence. We find that substitutions of conserved residues within this sequence drastically reduced HA(Sb) binding, providing an explanation for the absence of this ld from 1 degree responses. We have also examined intraclonal affinity maturation by generating Abs inferred to have been produced by the unmutated precursors and branchpoint intermediates of the largest 68ld clone. Interestingly, unmutated Abs of this clone bound HA(Sb) well, comparable to that of some 2 degree 68ld hybridoma Abs. Abs produced by branchpoint intermediates of this clone have a lower affinity for HA(Sb) than their unmutated precursor, despite containing parallel amino acid replacement mutations. Thus, expansion of this clone did not follow the paradigm of stepwise increases in affinity. Together, these data underscore the interconnective nature of precursor frequency and somatic mutation in the formation of the 2 degree response to a given Ag.

Amino Acid Sequence↗
PubMed1995

The majority of murine VH12-expressing B cells are excluded from the peripheral repertoire in adults.

We have previously demonstrated that at birth most productive (P) VH12 rearrangements in B10.H-2aH-4bp/Wts (2a4b) mice encode a ten-amino acid CDR3, and that a significant fraction of the expected repertoire is absent. We have now examined the adult VH12 CDR3 repertoire involving all four JH gene segments in both peritoneum and spleen. Of the 74 P VH12 rearrangements from these tissues 67 encode a CDR3 of ten amino acids and include a Gly in the fourth position (designated 10/G4). Most of these rearrangements appear to derive from phosphatidylcholine (PtC)-specific B cells, which also have a 10/G4 VHCDR3, since few 10/G4 P rearrangements were present in spleen cells depleted of PtC-specific B cells. Thus, the VH12 B cell repertoire in adult mice is largely restricted to the use of a single CDR3 motif and to a single antigen specificity. This bias results from two selection events: (1) selective exclusion of most VH12 B cells from the peripheral repertoire, and (2) clonal expansion in the periphery of VH12 B cells that have a 10/G4 VHCDR3 and bind PtC. Analysis of VH12-JH1 rearrangements in viable motheaten (mev/mev) mice, which have an abnormal B cell repertoire due to a defective phosphatase (Hcph) and have barely detectable numbers of PtC-specific B cells, indicates that selective exclusion of VH12 B cells from the peripheral repertoire occurs normally, but that clonal expansion of 10/G4 VH12 B cells is minimal. This is evidence that the selective exclusion of VH12 B cells from the peripheral repertoire and the clonal expansion of VH12 B cells with a 10/G4 CDR3 are due to independent signaling events.

Age Factors↗
PubMed1995

The influence of variable-region somatic mutations on the specificity and pathogenicity of murine monoclonal anti-DNA antibodies.

Antibodies to DNA (anti-DNA) occur prominently in systemic lupus erythematosus and provoke inflammatory damage in the kidneys. To determine the factors that confer pathogenicity on antibodies of this specificity, we investigated the in vitro and in vivo glomerular binding by members of four clonally related sets of monoclonal anti-DNA antibodies from lupus mice. Somatic mutations within the clonal sets enhanced binding to double-stranded DNA (dsDNA). Binding to permeabilized glomeruli in vitro was observed among affinity-purified preparations of these antibodies independent of specificity for dsDNA. In normal mice injected with hybridoma cell lines, nephritis as assessed by histology and immunofluorescence did not correlate with antibody affinity for DNA. By multivariate analysis, in vitro glomerular binding was the most predictive parameter of histologic outcome. These findings indicate that somatic mutations occurring during maturation of the autoimmune response do not necessarily enhance pathogenicity.

Amino Acid Sequence↗
PubMed1994

Development of B-1 cells: segregation of phosphatidyl choline-specific B cells to the B-1 population occurs after immunoglobulin gene expression.

Adult mice have two easily recognizable subsets of B cells: the predominant resting population of the spleen, called B-2, and those called B-1, which predominate in coelomic cavities and can express CD5. Some antibody specificities appear to be unique to the B-1 population. Cells expressing antibody specific for phosphatidyl choline (PtC) are the most frequent, comprising 2-10% of peritoneal B cells in normal mice. To understand the basis for the segregation of the anti-PtC specificity to this population, we have produced transgenic (Tg) mice expressing the rearranged VH12 and V kappa 4 genes of a PtC-specific B-1 cell lymphoma. We find that VH12-Tg and VH12/V kappa 4 double-Tg mice develop very high numbers of PtC-specific peritoneal and splenic B cells. These cells have the characteristics of B-1 cells; most are CD5+, and are all IgMhi, B220lo, and CD23-. In the peritoneum these cells are also CD11b+. In addition, adult mice have many splenic B cells (up to one third of Tg+ cells) that express the VH12 Tg but do not bind PtC, presumably because they express a V kappa gene other than V kappa 4. These cells appear to be B-2 cells; they are CD23+, CD11b-, IgMlo, B220hi, and CD5-. Thus, mice given either the VH12 Tg alone or together with the V kappa 4 Tg develop a large population of PtC-specific B cells which belong exclusively to the B-1 population. Since B-2 cells can express the VH12 and V kappa 4 gene separately, we interpret these data to indicate that the events leading to the segregation of PtC-specific B cells to the B-1 population in normal mice are initiated after Ig gene rearrangement and expression. These data are discussed with regard to hypotheses of the origin of B-1 cells. We also find that VH12-Tg mice have a marked decrease in the generation of Tg-expressing B cells in adult bone marrow, but not newborn liver. We speculate that this may be related to positive selection of VH12-expressing B cells during differentiation.

Animals↗
PubMed1994

The anti-La response of a single MRL/Mp-lpr/lpr mouse: specificity for DNA and VH gene usage.

Autoantibodies to ribonucleoproteins (RNP) occur prominently in human systemic lupus erythematosus and murine lupus models. In previous studies we demonstrated a relationship in MRL/Mp-lpr/lpr (MRL/lpr) mice between antibodies to Sm, an RNP autoantigen, and antibodies to DNA. Thus, many anti-Sm monoclonals bound DNA and expressed the same V region genes as anti-DNA. In addition, many had multiple VHCDR3 Arg residues suggestive of selection by DNA, and some had somatic mutations suggesting selection for mutant B cells by DNA. To determine whether autoantibodies to other RNP antigens are also associated with the anti-DNA response, we have analyzed the response to the La RNP. Six anti-La B cell hybridomas were generated from a single MRL/lpr mouse. Southern blot analysis of Ig V gene rearrangements and V gene sequences indicated two clonally related pairs, suggesting an oligoclonal response. Antibodies from all six hybridomas bound single-stranded DNA, while antibodies from five hybridomas bound double-stranded DNA. Two hybridomas expressed a VH7183 gene which is used by members of two previously reported anti-DNA clones and two anti-Sm/DNA clones of MRL/lpr origin. These data demonstrate an association between the anti-La and anti-DNA responses in MRL/lpr mice, suggesting that cross-reactive anti-RNP and anti-DNA responses are a general feature of autoimmunity in this lupus model.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. Differences from spontaneous anti-DNA in the content and location of VH CDR3 arginines.

Immunization of normal mice with bacterial DNA induces a significant anti-DNA response that includes antibodies resembling some lupus anti-DNA in their binding properties, although lacking specificity for mammalian dsDNA. To determine the structure of these induced antibodies and their relationship to anti-DNA from lupus mice, we have characterized the clonality and selected V-region sequences of a panel of 20 anti-DNA antibodies from 3 BALB/c mice immunized with ssDNA from Escherichia coli. Southern blot analysis of H and L chain rearrangements indicated that two of the animals expressed pairs of clonally related antibodies. Amino acid sequences of 10 of the induced antibodies demonstrated predominant utilization of J558 family VH genes and JH4 in association with various DH, J kappa and V kappa genes. Among the VH CDR3 of these 10 antibodies, 4 displayed arginine residues as a result of N region additions. None of these antibodies, however, had more than one arginine residue in VH CDR3 nor arginines at positions 100 or 100a, characteristic features of lupus antibodies to dsDNA. These results suggest that normal mice immunized with bacterial DNA display certain facets of DNA Ag drive, although lacking the mechanisms for the production of antibodies to mammalian dsDNA.

Amino Acid Sequence↗
PubMed1993

Overlap of the anti-Sm and anti-DNA responses of MRL/Mp-lpr/lpr mice.

Autoantibodies specific for the Sm ribonucleoprotein are spontaneously produced in patients with SLE and in mice of the MRL mouse strains. We have previously reported the characterization of the clonality and V region gene use of 41 MRL/Mp-lpr/lpr (MRL/lpr)-derived B cell hybridomas selected for Sm binding. In this report, we show that many of the expressed V genes of these hybridomas are also expressed by anti-DNA hybridomas of MRL/lpr mice. Moreover, the anti-Sm hybridomas from nine clonal groups produce antibodies that bind ssDNA, and those of five clones produce antibodies that also bind dsDNA. Sm/DNA-specific hybridomas, but not Sm-only-specific hybridomas, have a higher than expected content of arginine residues in CDR3 of the H chain, similar to MRL/lpr hybridomas selected on the basis of DNA binding. One clone displays intraclonal differences in DNA binding, inasmuch as the most extensively mutated members produce antibodies that are able to bind dsDNA and have a higher affinity for ssDNA than the least mutated members of this clone. Thus, DNA appears to be a selecting Ag in this response. These data indicate an overlap in the anti-Sm and anti-DNA autoimmune responses in MRL mice that may have implications for the activation of anti-Sm B cells, and for defining the spectrum of Ag targeted in SLE.

Amino Acid Sequence↗
PubMed1993

V region gene analysis of anti-Sm hybridomas from MRL/Mp-lpr/lpr mice.

Anti-Sm autoantibodies are unique to SLE, but are present in only 25% of patients with this disease. This response also occurs at a similar frequency in mice of the autoimmune MRL strains. Previous analyses of the anti-Sm response in these mice indicate that its occurrence is controlled by stochastic events, and suggest that Sm is the driving Ag. To further elucidate the role of Ag in this response, and to test the hypothesis that the 25% incidence is due to a requirement for particular Ig gene rearrangements or somatic mutations, we have analyzed the specificity and V-region gene sequences of 41 anti-Sm B cell hybridomas derived from nine anti-Sm-positive MRL/Mp-lpr/lpr mice. The majority of hybridomas are specific for the D peptide of the Sm particle. Hybridomas of independent origin express unique VH/V kappa combinations with diverse junctional sequences and are variable in the extent of somatic mutation. Thus, the response does not appear to be dependent upon the occurrence of a rare Ig gene rearrangement or specific somatic mutation. The response exhibits restriction in JH and VH gene use, and in individual mice is oligoclonal, suggestive of Ag selection. In the few B cells for which mutations can be identified, the evidence for selection of mutant B lymphocytes, based on patterns of mutation, is ambiguous. However, there is remarkably little intraclonal diversity, suggesting that the overall mutation rates in these clones are low.

Amino Acid Sequence↗
PubMed1993

VH CDR3-dependent positive selection of murine VH12-expressing B cells in the neonate.

Five to fifteen percent of peritoneal B1 (CD5+) cells from unmanipulated mice produce antibodies that bind bromelain-treated mouse red blood cells and the hapten phosphatidylcholine (PtC). The majority of these B cells express either of two VH/V kappa gene combinations, VH12/V kappa 4 or VH11/V kappa 9. Both the VH11 and VH12 genes are rearranged to JH1 and encode third complementarity determining regions (CDR3) of restricted length and sequence. These and other observations argue strongly that PtC-specific B1 cells are antigen selected. To determine when selection of PtC-specific B1 cells begins in mice we have used the polymerase chain reaction to amplify VH12-D-JH1 rearrangements from livers of fetal and neonatal mice, and determined the CDR3 encoding sequences of individual clones. We find an unusually low ratio of productive (P) to non-productive (NP) rearrangements (0.4-1.0) at both developmental stages. P rearrangements in day 1 neonates are biased in D gene use and in the sequence and length of their deduced VHCDR3. These biases are similar to those of PtC-specific B1 cells in the adult peritoneum. D gene use and CDR3 length and sequence are significantly less biased among VH12 P rearrangements 2 to 3 days earlier in the day 18 fetal liver. We suggest that this rapid change in repertoire is due to positive ligand selection that is dependent on the sequence of VHCDR3. We suggest further that the majority of VH12-expressing cells are not ligand selected and consequently undergo programmed cell death. The evidence of restriction in day 1 neonatal livers and the low P/NP ratio in the fetus suggests that selection of VH12-expressing cells begins before birth.

Amino Acid Sequence↗
PubMed1993

Mechanisms that limit the diversity of antibody: three sequentially acting mechanisms that favor the spontaneous production of germline encoded anti-phosphatidyl choline.

Antibody to phosphatidyl choline (PtC) is produced spontaneously in mice, by approximately 2-10% of naturally occurring CD5+ (B1) B cells in the peritoneum. Much of this antibody is encoded by the VH11 gene associated with a specific V kappa 9 gene. Constraints on the size and structure of the H chain CDR3 have been defined from nucleotide sequences of genes expressed by hybridomas and lymphomas derived from adult mice. All employ JH1 and all encode tyrosine as the first amino acid in CDR3, which is either nine or 10 amino acids long; the last six are always the same, start with tyrosine, and are rich in aromatic amino acids. Those with nine amino acids in CDR3 have glycine or serine in the second position and asparagine, serine or proline in the third; those with 10 have an additional aspartate or glycine inserted after the first tyrosine. DSP2 genes are used by 80% and DFL16 by 20%. Productively rearranged VH11 genes in neonates and in 18 day fetal liver display a greater, but still limited degree of diversity. All four JH genes are used and the length of CDR3 varies from three to 12 amino acids, but the first is tyrosine in 58 of 61 and DSP2 genes are used by 80% of these productive VDJ assemblies. Non-productive rearrangements of VH11 in fetal liver show a different pattern; 41% use DFL16 genes and only 40% have a TAx codon in the first position of CDR3. All rearrangements show evidence of a bias in favor of joining the VH11 gene to D genes at positions of matching nucleotide overlap.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

High frequency expression of S107 VH genes by peritoneal B cells of B10.H-2aH-4bP/WTS mice.

Our previous analyses of peritoneal Ly-1 B cells indicate that a high percentage express VH genes of the VH11 and VH12 families, and that this bias is due to clonal selection. The antibodies encoded by these genes bind the same hapten, phosphatidyl choline (PtC). Twenty-one of 73 hybridomas generated from fusions with peritoneal Ly-1 and Ly-1 sister population B cells of B10.H-2aH-4bp/Wts mice produce anti-PtC specific antibodies. We show here that 19 of these express VH11 and VH12 family genes and two express VH36-60 family genes. To assess whether there is a bias in VH gene use among non-PtC-specific hybridomas we analyzed the remaining 52 hybridomas for VH family expression by using VH family-specific probes in an RNA dot blot assay and by Ig mRNA sequencing. We find a seven-fold increase in the expression of the VHS107 family genes, and only slight differences in the expression of VH genes of other families relative to splenic B cells. We attribute the increase in VHS107 gene expression to clonal selection inasmuch as five of the seven VHS107+ hybridomas express the same VH gene (V11) and VL association is nonrandom. The bias in VH gene use among the entire panel of 73 peritoneal hybridomas is to the extent that approximately one-third express one of three genes: the V11 gene of the S107 family, the CH34 gene of the VH11 family, and the VH12 family gene.

Animals↗