Family Usage Research Articles

Quantitative Analysis of the B Cell Repertoire by Limiting Dilution Analysis and Fluorescent in Situ Hybridization

We have made a quantitative and concurrent analysis of B cell frequencies and V H gene family expression to study the influence of tissue type and age on the development and establishment of the primary B cell repertoire. Using LPS-mediated limiting dilution analysis and a panel of antigens we show that the newly generated B cell specificities from bone marrow get distributed without a bias to peripheral tissues such as spleen and Peyer's patches throughout the lifetime of the animal. Comparison of the B cell frequencies in animals of four different age groups (2-4 days old, 3, 12, and 18 months old) reveals that while the neonatal repertoire is comparable to that of adults, there was a selective twofold increase in the generation and distribution of B cells reactive with autologous mouse red blood cells in older mice compared to young ones. By means of a novel technique that employs fluorescent in situ hybridization and flow cytometry, we have also compared the V H gene family usage in large numbers of single B cells from these mice. Analysis of the same cell population (surface Ig +, functional B lineage cells) for expression of 7183, J558, and S107 V H families shows a preferential twofold increase in the use of V H 7183 in neonates compared to adults, while all three families show no significant difference in levels of expression during adult life or between primary and secondary lymphoid tissues. Taken together, our data indicate that specific and selective changes occur in both V H gene usage and antibody frequencies during murine ontogeny.

B precursor acute lymphoblastic leukemia third complementarity-determining regions predominantly represent an unbiased recombination repertoire: leukemic transformation frequently occurs in fetal life.

To determine whether the ALL (acute lymphoblastic leukemia) CDR3 (third complementarity-determining region) repertoire represents the recombination repertoire, or shows evidence of selectional processes inherent to normal B cell differentiation or malignant transformation, we analyzed 68 ALL CDR3 regions and included 127 previously published sequences in the analyses. We found no evidence of selection prior to malignant transformation as recombination was random with 1/3 "in frame" and 2/3 "out of frame" joinings and usage of all three D reading frames was observed. D and JH gene segments were predominantly unmutated which allowed a detailed analysis of gene usage and rearrangement characteristics. JH4 and JH6 usage (both 32.2%) was significantly different (p = 0.005) from that observed in peripheral B lymphocytes. D gene family usage roughly represented D gene family size with the exception of the DXP and DA/K family which were over- and underrepresented (p = < or = 0.05), respectively. D-D fusions were found in 26.2% of CDR3 regions. If less stringent criteria were applied DIR homology was found in 40/65 sequences, suggesting the frequent involvement of DIR gene segments in human CDR3 formation. The rearranged D genes were evenly distributed over the D locus, suggesting that D recombination is a predominantly random process, independent of physical location at the locus. Also, there was no correlation between JH gene usge and physical location of the rearranged D gene segment, which excludes a major contribution of the DJH replacement recombination mechanism. In 36.1% of CDR3 regions N-nucleotides at the DJH junction were absent. This frequency is higher than observed for peripheral B lymphocytes. It is suggested that for a number of ALL the initial transformational event took place in early fetal life. We conclude that ALL CDR3 sequences show no evidence of selection prior to malignant transformation, nor of extensive changes subsequent to malignant transformation.

Usage of TCRAV and TCRBV gene families in human fetal and adult TCR rearrangements.

We have investigated fetal and adult T-cell receptor (TCR) A and B V-gene repertoires both by fluorescence-activated cell sorter (FACS) analysis with the available TCR V region-specific mAbs and by the polymerase chain reaction (PCR) with TCR V gene family-specific oligonucleotides. Among the low number of CD3+ T cells, most of the TCR V regions tested for could be detected by FACS analysis in liver, bone marrow, and spleen derived from a 14-week-old fetus and two 15-week-old fetuses. Similarly, the PCR analysis showed that the majority of the TCRAV and TCRBV families were expressed in the peripheral organs of the 13-week-old fetus, although an apparent absence of particular TCR V families was found in liver and bone marrow. This was most probably the consequence of the low number of CD3+ T cells in these organs. In 17-week-old fetal thymi the level of expression of some TCRAV and TCRBV gene families, in particular those that contain a single member, was lower compared to post-partum thymi and adult peripheral blood mononuclear cells. The combined data of FACS and PCR analysis demonstrate that TCR V genes belonging to the majority of TCR V gene families can be used in TCR alpha and beta chain rearrangements during early human fetal life. Our data also suggest that the expression levels of some of the single member TCR V gene families may be influenced by the developmental stage.

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity.

In this study we have analyzed the TCR V alpha and V beta regions at the DNA level in the CD4+CD45RO+ memory T cell population of synovial tissue infiltrating T lymphocytes of three rheumatoid arthritis (RA) patients and one patient with chronic arthritis. Cell lines of CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD8+CD45RO- T lymphocyte populations were generated following FACS cell sorting of freshly isolated synovial tissue mononuclear cell infiltrates (STMC) and of freshly isolated peripheral blood mononuclear cells (PBMC) of these patients. The phenotypic and molecular analyses have revealed the following. (i) The TCR repertoires of tissue infiltrating T lymphocytes in the various subsets were extensive on the basis of TCR V gene family usage. (ii) Furthermore, each patient displayed individual specific TCR V gene expression patterns in the various STMC and PBMC derived T cell subsets. However, the majority of these arthritis patients manifested increased expression of multiple TCR V gene families in the synovial tissue derived CD4+CD45RO+ T cell population when compared with the peripheral blood derived CD4+CD45RO+ subset. Of these gene families, we found enhanced expression of the TCR V alpha 7 and V beta 11 gene segments in synovial tissue to be shared by all four patients analyzed. (iii) Nucleotide sequence analysis of the CDR3 regions of a number of TCR V regions in the CD4+CD45RO+ T cell subsets has revealed that the CDR3 regions comprised within synovial tissue derived TCR V regions differed from those found in peripheral blood derived TCR V regions. These differences in CDR3 diversity might be the consequence of a specific interaction with particular MHC-peptide complexes expressed at the site of inflammation. (iv) The CDR3 region analysis also showed individual specific amino acid motifs within the N-D-N regions of all analyzed TCR V beta genes derived from PBMC as well as STMC.

Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries.

Thyroid lymphocyte RNA from a Hashimoto patient with high serum levels of autoantibodies to thyroid peroxidase (TPO) was used to construct a phage display antibody library in the phagemid vector pComb3. The library (100,000cfu) encoded IgG1 heavy chains together with kappa light chains. Selection of the phages displaying TPO antibody on TPO-coated ELISA plates yielded a phage population enriched for surface expression of TPO antibody Fabs. 3 different Fabs specific for TPO were subsequently isolated with affinities in the region of 10(9) molar-1. 2 of the Fabs recognised the same, or closely related, epitopes on TPO whereas the third Fab recognised a different epitope. These 2 epitopes were recognised by TPO autoantibodies in the serum of the lymphocyte donor and a series of 10 patient sera. Available sequence data showed that several non-self antibodies and non-thyroid autoantibodies use the same V kappa and VH germline genes as TPO autoantibodies. There appeared to be no clear relationship between gene sequence or gene family usage by TPO autoantibodies of the same or similar epitope specificity.

IL-2 receptor-positive intrathyroidal lymphocytes in Graves' disease. Analysis of V alpha transcript microheterogeneity.

There has been considerable interest recently in the possible restriction of the TCR repertoire in autoimmune disorders, because such restriction would have important therapeutic implications. Reports of restriction of the TCR V alpha but not V beta repertoire in the thyroid in Graves' disease could not be repeated in an earlier study. Using RNA derived from matched peripheral blood, thyroid tissue, intrathyroidal lymphocytes (ITL), and IL-2R+ and IL-2R- subpopulations of ITL from Graves' patients, we conducted reverse transcription polymerase chain reaction/Southern blot analysis of TCR V alpha family usage. No evidence was found for V alpha restriction in the IL-2R+ subpopulation of ITL from eight patients, one of whom was operated on within 1 mo of diagnosis. We have further analyzed samples from seven of these patients by resolution on denaturing polyacrylamide gels. Typically, a single dominant band was amplified with surrounding minor bands in a normal distribution. Dominant and minor species were both the result of amplification from in frame V alpha transcripts, and the minor bands were separated in size by increments of 3 bp. We found no evidence for reduced heterogeneity of the V alpha transcripts in ITL or IL-2R+ ITL populations relative to peripheral blood in the vast majority of samples. This therefore suggests that there is little difference between the blood lymphocyte population and the activated T cell population in the thyroid in patients with Graves' disease, and indicates that in autoimmune thyroid disease, this method of analysis is not sufficient to distinguish between autoreactive and bystander T cell populations.

B‐Cell Abnormalities in AIDS: Stable and Clonally‐Restricted Antibody Response in HIV‐1 Infection

Antibodies in sera of HIV-1 infected individuals against the HIV-1 core protein (p24), HIV-1 envelope glycoprotein (gp120) and reverse transcriptase (RT) are characterized by a skewed light chain isotype expression. The kappa/lambda ratios of antibodies to p24 and gp120 in infected individuals were found to be unique in each individual, but constant over several years independently from disease progression. The oligoclonal nature of the anti-HIV-1 antibodies suggested by skewed kappa/lambda expression was confirmed with isoelectric focusing of affinity purified antibodies to p24 and gp120. Analysis of the utilization of V gene families in purified anti-p24 and anti-gp120 antibodies revealed a restricted and biased VH gene family usage. In contrast, the utilization of VK gene families appeared to be random. The finding of stable and restricted antibody responses in infected individuals could be one of the causes for the failure to produce antibodies to HIV-1 that are effective against escape virus variants.

V H gene family utilization of anti-acetylcholine receptor antibodies in experimental autoimmune myasthenia gravis

The immunoglobulin heavy chain (V H) gene family usage in experimental autoimmune myasthenia gravis (EAMG) model was investigated by RNA slot blot hybridization using V H gene family specific probes. Anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) isolated from susceptible C57BL/6 and resistant BALB/c mice were found to be encoded by V H genes from at least six different families. The Vgam3.8 family was overrepresented in α-bungarotoxin blocking mAbs. Expression of cross-reactive idiotopes by anti-AChR mAbs was irrespective of the V H gene family usage. Strain dependent differences in susceptibility for EAMG were not reflected in an aberrant V H gene family usage of anti-AChR mAbs.

The Variable-Region Specificity of Bacterial Fab-Binding Proteins: The Search for B-Cell Superantigens

Due to the biologic implications for clinical infection, defining the Fab-binding specificities of certain bacterial Ig-binding proteins has been an area of considerable interest. To elucidate the structural and genetic correlates of the Fab-binding specificity of a prototypic Fab-binding bacterial protein, staphylococcal protein A (SpA), we have developed several new approaches. In earlier studies, we used a panel of peptide-induced serologic reagents to identify the V-region family usage in monoclonal Ig and then assessed their binding interaction with the Fab-specific binding site of SpA. To identify the conserved V-region sequences that correlate with SpA binding, the SpA-binding abilities of a group of purified, B-cell-line derived, monoclonal Ig of known sequence were also assessed. More recently, we have studied the binding of SpA with a combinatorial Ig expression library made with a phage surface-display vector. These studies have rigorously demonstrated that SpA binding is restricted to VH3 Fab: The vast majority of VH3 Fab bind SpA, and diverse VH3 genes can encode for SpA binding. We then used the labeled Fab-specific SpA as a VH3-specific phenotypic marker in multiparameter flow cytometric analyses to study human B-cell repertoire expression. These studies indicate that SpA possesses the Fab-binding specificity predicted for a B-cell superantigen, and we speculate that this type of unconventional antigen may have potent capabilities of influencing the formation of human immune repertoires.

Ig VH gene family usage in spleen cells of CBA/J mice immunized with experimental autoimmune thyroiditis (EAT) inducer antigens.

Experimental autoimmune thyroiditis (EAT) is an autoimmune disorder of the thyroid gland induced in susceptible strains of mice by thyroglobulin (Tg). We recently showed that low Mr (< 10 kDa) Tg tryptic fragments and a 40 amino-acid peptide (F40D) from Tg could induce EAT as well as native Tg. Because it has been reported that autoantibodies (A-Abs) express VH families preferentially located in the D-proximal VH gene segment, we investigated whether A-Abs specific for one pathogenic peptide from Tg were also skewed towards D proximal VH gene segment. In that respect, we immunized CBA/J mice with EAT inducer antigens of decreasing sizes: Tg (660 M(r)), < 10 kDa Tg trypic fragments or F40D peptide (4.9 kDa M(r)) from Tg. The VH gene segments utilized by immune spleen cells were determined by hybridization to total spleen cell RNA previously deposited onto nylon membranes and densitometric scans. This study was conducted on days 7 and 9 after determination of the maximum amounts of mRNA coding for immunoglobulins and on day 28 when A-Ab levels are the highest. Results were compared to VH gene segment expression both in normal and adjuvant-injected mice. We found that immunization of CBA/J mice with EAT inducer antigens stimulate B cells the restriction of which, in terms of VH family usage, depends on the size of the immunizing antigen: the larger the antigen, the higher the numbers of VH families used. Moreover, we found that B cell stimulation consecutive to immunization with the peptidic antigen inducing EAT occurs in VH Q52 family, a VH encoded by D-proximal gene segment.

CD5 and immunoglobulin VH gene expression in B-cell lines from patients with autoimmune diseases.

We studied the CD5 mRNA expression and VH gene family usage in Epstein-Barr virus (EBV)-immortalized B-cell lines derived from the blood of patients with type 1 diabetes (IDDM) of recent onset and of patients with polyneuritis cranialis multiplex (cranial neuritis; CN). After immortalization with EBV, at least 10 cell lines from each subject were tested for surface CD5 and CD20. mRNA expression was studied using cDNA probes for the six VH families as well as for CD5. The EBV lines from the IDDM patients used the VHIV family more frequently and VHI and VHII families less frequently than lines from controls. EBV lines from CN patients expressed the VHI and VHII families more often than those of the controls. When the IDDM and CN lines were compared, the lines derived from IDDM patients were found to use VH families I and II less frequently and VH families IV and V more frequently than lines from CN patients. There were no significant differences in the mean numbers of CD5+ B cells in the cell lines tested. More than half of the lines from each patient expressed CD5 at the mRNA level. No correlation was seen between the expression of surface CD5 and the level of CD5 mRNA expression. There was, however, a positive correlation between the usage of VH families III, V and VI, and the CD5 mRNA expression. In conclusion, the usage of VH families I to VI seemed to differ in patients with IDDM and CN. No differences were seen in the surface CD5 expression, but the lines expressing CD5 mRNA preferentially used the VH families III, V and VI.

Strain-dependent restricted VH and VL usage by anti-bacterial levan monoclonal antibodies.

The immune response to polysaccharides is highly regulated and has several distinguishing features, including restricted clonotype and isotype expression. The basis for this highly restricted response is not fully understood. To address these questions in a systematic manner, we have generated a panel of 102 mAb from CBA/CaHN (CBA/Ca) and BALB/cAnN (BALB/c) mice after one and two injections of bacterial levan (BL), a beta(2----6)-linked polyfructosan with beta(2----1)-linked fructose branch points (inulin determinant, In). This panel of mAb was examined for isotype, fine specificity, VH and VL region gene family usage and relationships between these parameters. After one or two injections of BL in both strains, mAb were IgM and IgG3. Fine specificity and VH/VL gene family usage differed markedly, however, between the two strains. Only 4% (2/51) of CBA/Ca mAb recognized the In determinant, whereas 77% (40/51) of BALB/c mAb recognized this epitope. In both strains, VH usage was restricted and certain families were overrepresented. In CBA/Ca mice, the overall response to BL was dominated by VHJ558 (45%, 23/51), the largest VH family, but VH36-60 (27%, 14/51) and VHJ606 (25%, 13/51) were also highly utilized and overrepresented. In BALB/c mice, the overall response to BL was dominated by VHJ606 (79%, 39/49 designated), a relatively small VH family. More importantly, after a single immunization with BL one particular VH/VL pair (VHJ606/ kappa 11) was used by 88% (36/41) of BALB/c mAb and was associated with In reactivity. In summary, BALB/c and CBA/Ca responses to BL differ in fine specificity and VH/VL usage.

Differential expression of VH gene families in peripheral B cell repertoires of newborn or adult immunoglobulin H chain congenic mice.

The pattern of VH gene family expression in the primary B cell repertoire of the mouse is strain dependent. In C57Bl/6 mice, the VH J558 family is expressed by more than 45% of the cells, while the expression of VH 7183, VH Q52, and VH 36-60 families together does not exceed 20%. In BALB/c mice, relative expression of VH J558 is lower than 35%, while the sum of the other three families reaches 25%. To assess which genetic loci control strain-specific VH gene family expression, we studied VH gene family usage in splenic B cell repertoires of different congenic strains of mice. Changes in major histocompatibility complex or immunoglobulin (Ig) K light chain genes did not modify VH gene family expression in adult mice. Differences at the IgH locus, however, modified VH gene family usage. In 1-d-old mice, the strain-specific VH gene family expression pattern is determined by the IgH haplotype. In adult mice, the VH gene family expression pattern of resting B cells is independent of the IgH locus and follows the genetic background of the congenic strain, while it is determined by the IgH haplotype among Ig-secreting spleen cells. In F1(B6 x BALB/c) mice, each of the two spleen B cell populations, sorted on the basis of mu heavy chain allotype expression, shows an independent VH gene family expression pattern, determined by the IgH locus. The implications of these results in the control of VH gene family expression, and in the selection of peripheral B cell repertoires are discussed.

Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.

Evidence for selective accumulation of intrathyroidal T lymphocytes in human autoimmune thyroid disease based on T cell receptor V gene usage.

We have investigated the T cell receptor V alpha and V beta gene family usage by T lymphocytes infiltrating affected thyroids in patients with autoimmune thyroid disease. We show that the intrathyroidal T lymphocytes from patients (n = 6) with autoimmune thyroid disease display a widespread usage of V beta gene families with an average of 14.4/19 V beta gene families similar to the peripheral T lymphocytes of the same patients. Because we recently reported that the utilization of V alpha gene families is markedly reduced within these mitogen-stimulated intrathyroidal T cell populations, as well as within intact tissue from similar patients (n = 4) (overall mean of 4.0/18 families detected), these results indicate that in thyroids of patients with autoimmune thyroid disease the lymphocytes are selectively accumulating based on their V alpha rather than V beta elements. This preferential hTcR V alpha and widespread V beta gene usage was not mimicked in most 7-d autologous mixed lymphocyte reactions using non-T cell stimulators (n = 6) or EB-virus immortalized autologous B cell lines (n = 3). Hence, the selective V gene utilization by intrathyroidal T cells is likely to be secondary to multiepitopic thyroidal autoantigens activating thyroid infiltrating T cells or to the presence of a superantigenlike thyroidal self-antigen, capable of determining a selective infiltration or activation of a variety of T lymphocytes on the basis of their V alpha gene usage.

V beta gene family usage in spontaneous lymphomas of AKR mice: evidence for defective clonal deletion.

T-cell receptor (TCR) ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4+8+) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V ß gene family usage, 2 out of 14 lymphomas expressed V ß6. Normally, V ß6+ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V ß6+ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.

Ig V kappa family repertoire of plasma cells derived from autoimmune MRL mice.

V kappa gene family usage was determined in the resident in vivo-activated plasma cells of individual diseased MRL mice by using in situ hybridization. In this way, the entire autoimmune repertoire could be analyzed. Autoantibody levels and extent of glomerulonephritis were also measured, so that the severity of disease could be assessed. It was found that V kappa expression was highly variable from mouse to mouse. Some animals displayed a V kappa family repertoire similar to mitogen-stimulated cells and consistent with the size of the families. These animals tended to have lower disease indices. Other animals, which had higher disease indices, displayed considerable over- or underutilization of individual V kappa families. However, no particular V kappa families were repeatedly biased in their expression, as was found at the VH level with J558. Importantly, in the 10% of animals that expressed VH J558 exclusively, four or more V kappa families were expressed and multiple antiself specificities were produced. The data are most consistent with a number of J558 genes being expanded in a variety of self-specificities. However, because only VH J558 is expressed in these sicker animals, nonspecific polyclonal activation is highly unlikely. These results underscore the continuing evolution of the autoimmune repertoire, with considerable diversity at early stages followed by a highly selected repertoire in which a potential role for nonspecific polyclonal activation is virtually excluded.

X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) patients manifest a very low production of immunoglobulins (Ig) of all classes and plasma cells are virtually absent. The XLA gene plays a crucial role in the transition of pre-B cells to later B cell stages, as hardly any slg-positive B lymphocytes can be detected. In the bone marrow almost normal numbers of pre-B lymphocytes are present. These cytoplasmatic C mu+ pre-B lymphocytes appear to express truncated M heavy chain molecules lacking the variable region segment. The T lymphocyte compartment is intact: the numbers of mature T cell receptor (TcR) alpha beta expressing T lymphocyte populations and their proliferative responses to antigens are normal. That the B cells are primary and exclusively affected was proven by X-chromosome inactivation studies. There is no evidence that the XLA gene is directly involved in the Ig gene rearrangements since B lymphoblastoid cell lines (BLCLs) established from peripheral blood of XLA patients were found to produce IgM molecules composed of complete Ig heavy and light chains and were shown to contain normal VHDJH recombinations. The data do not exclude the involvement of the XLA gene in a B cell specific process that makes the Ig loci accessible for recombination. Investigations on the degree of diversity of immunoglobulins generated by XLA patients exposed no limitations in the VH family usage. Sequence analysis of expressed VH3 and VH4 rearrangements however revealed that some genetic elements of the Ig locus might be over-represented and that a high portion of rearrangements was generated by unconventional mechanisms. By restriction length polymorphism (RFLP) and pulsed field gel electrophoreses analyses the XLA gene was mapped to an 8- to 12-Mb DNA fragment located in the Xq22 region. The known location of the XLA gene on the X-chromosome with closely linked RFLP markers and the availability of X-chromosome inactivation assays provides methods for carrier detection and prenatal diagnosis.

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.

Molecular analysis of VxIII variable regions of polyclonal rheumatoid factors during rheumatoid arthritis

We report the first molecular characterization of V kappa regions of the main human autoantibodies occurring during rheumatoid arthritis, the polyclonal rheumatoid factors. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from both peripheral blood and synovial fluid rheumatoid factor-secreting cells, nucleotide analysis of the V kappa III family usage shows the following: (a) at least three different V kappa III genes are used to encode polyclonal rheumatoid factors in a single patient, (b) each one of these genes seems more or less somatically mutated (from 1 to 14 mutations), (c) the mutation process preferentially affects the complementarity determining regions suggesting a selective pressure of antigen and (d) there is no clear difference between the mutation rates affecting the synovial fluid and peripheral blood rheumatoid factor-secreting cells. These results are able to explain some of the known idiotypic differences between monoclonal and polyclonal rheumatoid factors in humans. They also provide evidence that polyclonal autoantibodies arising during an autoimmune disease can be the products of multiple somatically mutated genes and suggest that this process is antigen driven, whether this antigen is the Fc piece of IgG or another unknown antigen.