Germline IgH Research Articles

Evolution of immunogenetic components used to form ultralong VH CDR3 antibodies in Bovidae

Abstract Cattle has a restricted repository of immunoglobulin heavy chain gene segments with only one family of V, 4 J and 16 D segments functional. Among these segments are a V and D that, together, encode a heavy chain with an ultralong CDR3 region which manifests as a “stalk” supporting a “knob” domain of which only the knob has antigen binding capability. This antibody structure has boundless therapeutic potential, including for treatment of HIV and other antigens with veiled epitopes. Upon the discovery of this ultralong antibody in Bos taurus, speculation of its origin began. The components that should facilitate ultralong CDR3H antibodies appear in more species than just Bos taurus; they expand throughout the bovine subfamily as evidence by the presence of a V motif “TTVHQ” crested by an 8 base-pair (bp) duplication, and a comparatively long D segment. Several closely-related species encode the ultralong V segments gene in their germline IgH locus. Phylogenetically bookending the cluster that is genomically equipped for the ultralong cattlebody are the species Bos taurus and Bison bison with Bos mutus, indicus, and grunniens in between. The added length of the D segment is less consistent throughout the group as none of the species encode as long of segment as Bos taurus at 50 amino acids. Two species studied, Bos mutus and Bos grunniens, have 15 and 22 amino acids, respectively, encoded by their longest D segment, and Bison bison has 23. Though we do know the germline encodes the ultralong antibody, it is imperative to know if the gene rearrangement is actually expressed and employed in the mature repertoire.

Anti-insulin B cell receptors display skewed immunoglobulin gene usage and limited mutation in pre-symptomatic type 1 diabetes donors

Abstract Antibodies targeting insulin and other islet antigens predict type 1 diabetes (T1D) onset. Insulin autoantibody (IAA)-positive individuals progress to diabetes more rapidly, yet little is known about how IAA-precursor anti-insulin B cells (AIBCs) recognize insulin. Complementarity-determining regions (CDRs) of the B cell receptor (BCR) are structurally diverse loops that constitute the antigen-binding site, and amino acid variability in BCRs increases the breadth of repertoire antigen-specificities. To investigate anti-insulin BCRs during the earliest detectable stages of T1D, we enrolled eight insulin therapy-naïve Type 1 Diabetes TrialNet Pathway to Prevention participants who were positive for ≥2 islet autoantibodies and thus at high risk for diabetes. We stimulated donor peripheral blood mononuclear cells to drive BCR secretion as antibody, screened for AIBC-containing wells by ELISA, and immortalized these cells as hybridomas. We validated 25 monoclonal anti-insulin hybridoma lines and sequenced 16 heavy chain (IgH) and 14 light chain (IgL) immunoglobulin genes. 25% of VH and 50% of VL sequences contained at least one mutation within a CDR compared to germline, with 13% of VH and 29% of VL sequences having ≥6 mutations within the V-region. We surveyed previously and currently reported AIBC VH (n=25 BCRs), which revealed skewed VH and JH gene use compared to the total polyclonal IgH repertoire (n= 8790 BCRs, p < 0.001 and p < 0.05, respectively, chi-squared test). These data expose unique features of insulin recognition among AIBCs isolated from pre-symptomatic T1D donors, including V and J gene usage bias and potential for germline IgH insulin recognition, providing genetic and molecular insight into AIBC origins.

Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles

Definition of the specific germline immunoglobulin (Ig) alleles present in an individual is a critical first step to delineate the ontogeny and evolution of antigen-specific antibody responses. Rhesus and cynomolgus macaques are important animal models for pre-clinical studies, with four main sub-groups being used: Indian- and Chinese-origin rhesus macaques and Mauritian and Indonesian cynomolgus macaques. We applied the (Ig) gene inference tool IgDiscover and performed extensive Sanger sequencing-based genomic validation to define germline VDJ alleles in these 4 sub-groups, comprising 45 macaques in total. There was allelic overlap between Chinese- and Indian-origin rhesus macaques and also between the two macaque species, which is consistent with substantial admixture. The island-restricted Mauritian cynomolgus population displayed the lowest number of alleles of the sub-groups, yet maintained high individual allelic diversity. These comprehensive databases of germline IGH alleles for rhesus and cynomolgus macaques provide a resource toward the study of B cell responses in these important pre-clinical models.

Rhesus and Cynomolgus IGH Genotyping Yields Comprehensive Databases of Germline VDJ Alleles for In-Depth Immunological Studies

Rhesus and cynomolgus macaques are important animal models for the pre-clinical assessment of candidate drugs and vaccines. Four main sub-groups are used: Indian and Chinese origin rhesus macaques and Mauritian and Indonesian cynomolgus macaques. Here, we applied the IG gene inference tool IgDiscover and extensive Sanger sequencing-based genomic validation to define germline VDJ alleles in these 4 sub-groups, comprising 45 macaques in total. We observed a significant allelic overlap between Chinese and Indian origin rhesus macaques and, surprisingly, also between the two macaque species, consistent with significant levels of admixture. The island-restricted Mauritian cynomolgus population displayed the lowest number of alleles of the sub-groups, yet they maintained high individual allelic diversity. Our analysis resulted in comprehensive DBs of germline IGH alleles for rhesus and cynomolgus macaques, of which 64% and 79% of alleles respectively were not previously described, thereby facilitating high-quality B cell response studies in these species.

Detection of Immunoglobulin Heavy Chain Gene Clonality By High-Throughput Sequencing for Minimal Residual Disease Monitoring in Chronic Lymphocytic Leukaemia

Introduction: The negative minimal residual disease (MRD) after treatment has been recently accepted as endpoint for Chronic Lymphocytic Leukaemia (CLL) clinical trials. Conventionally, MRD can be detected by using multi-color Flow Cytometry (FC) with high sensitivity. Determination of the clonal immunoglobulin gene rearrangement can be a useful monitoring marker in a broad range of B-cell lymphoproliferative neoplasms. Moreover, the mutational status of immunoglobulin heavy chain variable (IgHV) rearrangement is considered one of the most important prognostic factors in CLL. Therefore, the identification of the IgHV rearrangement can be a useful marker both at diagnostic and as monitoring marker for MRD. Nowadays, high-throughput sequencing (HTS) technologies has enabled highly sensitive cancer genomic testing in clinical laboratories. There are same initiatives based on HTS to use IgHV rearrangement as marker for MRD monitoring in Acute lymphoblastic leukemia or multiple myeloma, but it remains unharmonized for application on CLL in the clinical laboratory. Objective: We evaluated the performance and clinical applicability of HTS assay for IgHV rearrangement in CLL MRD monitoring in 69 samples from 19 CLL patients treated. Methods: The libraries including IGH locus were performed using the Sequencing Multiplex Kit on IGH consensus primers. To simplify and make automatic the analysis of the data obtained, we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Assessment of IgHV mutational status by Sseq, genomic DNA (gDNA; 50-100 ng), were used for IgHV analysis. gDNA was amplified using locus-specific primer sets for IgHV designed to allow for the amplification of all known alleles of the germline IgH sequence, as described previously. Inmunophenotypic studies were performed on erythrocyte-lysed whole PB samples according to Euroflow procedures. PB white blood cells (WBC) was systematically stained with the eight color combination panel recently proposed by the ERIC group for MRD detection (Rawstron AC et al. 2016). Data acquisition was performed on a FACSCanto II flow cytometer Becton-Dickinson Biociences using the FACSDiva software (V8.0; BD). For data analysis, the Infinicyt softwareTM (Cytognos SL, Salamanca, Spain) was used. The MRD levels were reported as fraction of CLL cells of all nucleated cells. MRD negativity was define as a fraction <10 -4 However, for treatments that preferentially clear the blood (for example monoclonal antibodies) the MRD was confirm in the bone marrow as has been recommended. Results: Patient demographics and the results of IGH clonality tests are summarized in Table 1. Interpretable results were obtained with higher sensitivity compared with Sseq at diagnosis stage (19/19 samples), and we are able to determine the same clone at subsequent samples. Among 50 follow-up samples, 44 MRD were positive in both techniques (HTS and FC); 3 follow-up samples, were negative in both determinations (HTS and FC); in contrast, 3 follow-up samples, were negative by FC but positive by HTS (8.3, 17.2, 18.2 samples). This is the case of Patient 8, in which the last follow-up sample, the detection of the primary clone is positive by HTS, while by FC is undetected (Figure 1). In addition, it was possible to detect IgHV clone in all Patient 7 samples. It was under long-term monitoring by HTS and FC, and 17 months after initial diagnosis and first line of treatment (7.5 sample), it was detected a loss of response (Figure 2.). Conclusions: We evaluated an HTS IgHV assay using initial and follow-up samples of 19 CLL patients. Using our pre-designed primer set in library preparation, and developed our specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation, we were able to demonstrate that our method was more sensitive than FC in detecting positive follow-up samples and could be able to propose it for MRD monitoring in CLL. However, the prognostic impact of these low-level MRD detected by HTS should be validated with further investigations. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Ortiz: GILEAD SCIENCES: Research Funding. Terol:Abbvie: Consultancy; Gilead: Research Funding; Janssen: Consultancy, Research Funding; Astra Zeneca: Consultancy; Roche: Consultancy.

The Lysine Histone Methyltransferase SETD2 Is Required for Appropriate Immunoglobulin VDJ Recombination

Rearrangement of the immunoglobulin heavy chain locus (IgH) through non-homologous end-joining (NHEJ)-mediated VDJ recombination is a requisite step in normal lymphocyte development. This process is initiated through cleavage of DNA by RAG1/2 recombinases and repair by classical NHEJ pathway factors. Loss or defects of these factors can impair end-processing (DNA-PKcs, ARTEMIS) or end-ligation (KU70/80, XRCC4, XLF, LIGIV) of rearrangement and are drivers of disease, notably, severe combined immunodeficiency (SCID). In addition to these core NHEJ factors, other proteins, such as ATM kinase, have been shown to be critical for the stabilization of the post-cleavage VDJ intermediates to ensure proper signal joint formation. While epigenetic regulation of this process has been described through the maintenance of accessibility for transcription factors and RAG1/2, very little is known about the role of chromatin modifications on NHEJ-mediated VDJ recombination.Histone H3 lysine-36 tri-methylation (H3K36me3) is a histone modification associated with actively transcribed genes and has been shown to be critical for many biological processes, including transcription, mismatch repair and homologous recombination. Tri-methylation is catalyzed by the non-redundant histone methyltransferase, SETD2. Loss of function mutations in SETD2 or dominant negative onco-histone mutations in the H3K36 residue have been described in a broad array of tumors, including several hematopoietic malignancies. To study the role of Setd2 in hematopoiesis, we generated conditional knockout mice, where loss of Setd2 results in the ablation of the H3K36me3 mark. While heterozygous mice had no overt phenotype, mice with homozygous loss of Setd2(Setd2KO) displayed aberrant hematopoiesis, with a significant depletion of lymphoid primed progenitor population and B/T-cells in hematopoietic tissues. Setd2 loss early in hematopoiesis lead to a profound block of B-cell development at the proB cell stage and an early block in T-cell development, in a compartment where TCRβ rearrangements initiate. In Setd2KO mice, V to DJ rearrangements of the IgH locus were impaired, suggesting that the block at the proB stage of development was due to defective VDJ recombination. To assess this, we crossed our knockout mice with an IgHelMD4 mouse strain, and found that the block at the proB stage could be bypassed by the expression of a transgenic, fully rearranged IgH locus.We confirmed global loss of H3K36me3 in proB cells from our Setd2KO mice by ChIP-Seq and found that the ablation of this mark did not affect germline IgH locus accessibility (by ATAC-seq) or local chromatin architecture (H3K4me3 or H3K9ac) at a well-studied critical regulatory region near the Eμ enhancer. Loss of Setd2 and H3K36me3 only mildly effected sterile transcription of IgH genes and did not effect the overall cellular proliferative rate or the recruitment of NHEJ factors. Furthermore, loss of Setd2 did not cooperate to generate B-cell lymphomas in a p53-/- background. However, sequencing of VDJ recombination products from knockout mice indicated not only decreased clonality of products and variable gene usage but also resulted in alterations of complementarity defining region-3 (CDR3) hypervariable sequences. These results phenocopy defects or loss of NHEJ end-ligation factors and suggest that Setd2 and H3K36me3 could play a pivotal role in the end-ligation steps of NHEJ.Defects in end-ligation and resolution of VDJ intermediates have previously been shown to result in the formation of aberrant coding-signal hybrid joints. To see if this was occurring with loss of Setd2, we generated knockout murine proB v-Abl transformed lines where we could induce κ light chain rearrangement upon addition of an Abl kinase inhibitor. Remarkably, upon induction in Setd2KO v-Abl lines, we detected the formation of hybrid joints by both PCR and by the loss of a GFP-based coding-joint formation signal from a reporter expressed in these cells. We additionally could detect hybrid joint formation in the endogenous κ light chain locus of Setd2KO splenocytes, further indicating a role for Setd2/H3K36me3 in the end-ligation process. These studies demonstrate a novel and critical role for SETD2 and H3K36me3 in preventing aberrant, non-functional VDJ end joining of NHEJ-induced DNA breaks during lymphocyte development. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic modifications of the VH region after DJH recombination in Pro-B cells.

The variable region of murine immunoglobulin heavy chain (Igh) is assembled by sequential DH -JH and VH -DJH recombination. The accessibility of the Igh locus determines the order of rearrangement. Because of the large number of VH genes and the lack of a suitable model, the epigenetic modifications of VH genes after DJH recombination have not previously been characterized. Here, we employed two v-Abl pro-B cell lines, in which the Igh locus is in germline and DJH -recombined configurations, respectively. The DJH junction displays the characteristics of a recombination centre, such as high levels of activation-associated histone modifications and recombination-activating gene protein (RAG) binding in DJH -rearranged pro-B cells, which extend the recombination centre model proposed for the germline Igh locus. The different domains of the VH region have distinct epigenetic characteristics after DJH recombination. Distal VH genes have higher levels of active histone modifications, germline transcription and Pax5 binding, and good quality recombination signal sequences. Proximal VH genes are relatively close to the DJH recombination centre, which partially compensates for the low levels of the above active epigenetic modifications. DJH recombination centre might serve as a cis-acting element to regulate the accessibility of the VH region. Furthermore, we demonstrate that RAG weakly binds to functional VH genes, which is the first detailed assessment of RAG dynamic binding to VH genes. We provide a way for VH -DJH recombination in which the VH gene is brought into close proximity with the DJH recombination centre for RAG binding by a Pax5-dependent chromosomal compaction event, and held in this position for subsequent cleavage and VH -DJH joining.

Correction: Shaping of Human Germline IgH Repertoires Revealed by Deep Sequencing

Larimore, K., M. W. McCormick, H. S. Robins, and P. D. Greenberg. 2012. Shaping of human germline IgH repertoires revealed by deep sequencing. J. Immunol. 189: [3221–3230][1]. It has been brought to our attention that the published sequence of the HCDR3 for 4E10 in [Fig. 6][2] was copied

Shaping of Human Germline IgH Repertoires Revealed by Deep Sequencing

To understand better how selection processes balance the benefits of Ig repertoire diversity with the risks of autoreactivity and nonfunctionality of highly variable IgH CDR3s, we collected millions of rearranged germline IgH CDR3 sequences by deep sequencing of DNA from mature human naive B cells purified from four individuals and analyzed the data with computational methods. Long HCDR3 regions, often components of HIV-neutralizing Abs, appear to derive not only from incorporation of long D genes and insertion of large N regions but also by usage of multiple D gene segments in tandem. However, comparison of productive and out-of-frame IgH rearrangements revealed a selection bias against long HCDR3 loops, suggesting these may be disproportionately either poorly functional or autoreactive. Our data suggest that developmental selection removes HCDR3 loops containing patches of hydrophobicity, which are commonly found in some auto-antibodies, and at least 69% of the initial productive IgH rearrangements are removed from the repertoire during B cell development. Additionally, we have demonstrated the potential utility of this new technology for vaccine development with the identification in all four individuals of related candidate germline IgH precursors of the HIV-neutralizing Ab 4E10.

Feasiblity and Implication of Bidirectional Sequencing Using a Multiplex Framework 2 Region Primer for Somatic Mutation Analysis of the Immunoglobulin (IgH) Heavy Chain in Chronic Lymphocytic Leukemia (CLL).

Background: A somatic mutation rate of >2% of the immunoglobulin (IgH) heavy chain gene, in comparison to germline DNA, is a positive prognostic indicator in chronic lymphocytic leukemia (CLL). Genomic DNA is amplified using the Biomed-2 multiplex PCR primer set, sequenced in the reverse direction only using a 3′ JH primer, and compared to the germline sequence in VBase, a comprehensive directory of all human germline variable region sequences compiled from over a thousand published sequences. It is unknown whether forward sequencing using the multiplex 5′ Biomed-2 primers in a single reaction is feasible or whether doing so improves the accuracy of the mutational analysis.Methods: DNA was extracted from peripheral blood or bone marrow mononuclear cells from patients with CLL using the QIAGEN QIAspin DNA mini-kit. The FR2 region of the IgH gene was amplified using tube B of the Biomed-2 multiplex PCR primer set, separated in 2% agarose gel, excised, and prepared for sequencing using the QIAGEN QIAquick gel extraction kit. Bidirectional DNA sequencing was performed with the full set of multiplex 5′ FR2 primers combined in one reaction for the forward sequence and in a second reaction with the Biomed-2 3′ JH primer for the reverse sequence. A consensus sequence was obtained and mutation rates were calculated based on the consensus sequence and the reverse sequence separately.Results: The FR2 region was amplified from 30 CLL patients. 20 samples produced bidirectional consensus DNA sequences, 2 sequenced only with multiplex FR2 primers, 6 sequenced only with a JH primer, and 2 produced no sequences. Compared with unidirectional JH sequencing, bidirectional sequencing did not reassign any patient in the germline IgH group to the mutated category. In 15 patients, the uni- and bi-directional sequencing were identical. In 4 patients, a single basepair difference was noted. A random permutation test yields a two-sided p-value as 12.5%, indicating that no significant difference was detected between the uni- and bi-directional sequencing. Multiplex bidirectional sequencing did provide sequence information within the CDR3 region at the 3′ terminus of the amplimer which is partially truncated when using the JH primer alone.Conclusion: Bidirectional sequencing does not provide a somatic IgH mutation rate that differs significantly from that obtained from the reverse sequence alone and does not likely influence the IgH mutational prognostic assignment. Nonetheless, the bidirectional consensus sequence adds bases in the CDR3 region which can be used for research applications such as the generation of patient-specific primers for PCR. It is also noteworthy that multiplex PCR using the Biomed-2 primers is feasible.

Telomere Length Identifies Two Distinct Populations with Different Outcome among VH-Unmutated CLL Patients.

Introduction Telomere restriction fragments (TRF) length is a well-known prognostic indicator in CLL. Long telomeres have been associated with VH-mutated status, while VH-unmutated patients are regarded as having uniformly short telomeres. However, little is known on cases in which discordance exists between these two parameters. Patients and methods 138 B-CLL patients were analyzed for TRF length and VH mutational status. All samples were taken before the start of anti-CLL treatment. M/F ratio was 93/45. Median age was 60 years. According to Binet staging, 82 patients were A, 30 B and 26 C. CD38 and ZAP70 expression and FISH for Ch 11, 12, 13, 17, were available in 72% of patients. Median follow-up was 33 months (range 6–290). TRF length was evaluated by Southern blot as previously described (Ladetto M et al, Blood 2004). VH mutational status was evaluated by direct sequencing according to standard methods. Results VH sequencing was successful in 127 patients: 82 were VH-mutated and 45 VH-unmutated. Median TRF length was 6097bp (1465–14837). As expected VH-unmutated patients had shorter telomeres (median 4000bp) compared to VH-mutated patients (median 7399bp) (p<0.0001). A mathematical model has been employed to analyze the distribution of TRF length in VH-mutated and unmutated patients. While mutated patients had a homogeneous distribution, unmutated patients showed a bimodal distribution with no patients falling between 4137 and 4710bp (fig1a). This allowed to discriminate a larger population (26 patients) with very short TRF length (concordant patients), and a smaller (19 pts) with a TRF length superimposable to that of VH-unmutated patients (p=0.21) (discordant patients). Concordant and discordant patients had similar patterns of VH usage and similar levels of homology (H) to the germline IgH sequence (i.e. H=100% vs H<100% and >99% vs H<99% and >98%). In addition they showed no difference for all the available (previously mentioned) biological and clinical parameters. However the two population had significant differences in terms of clinical outcome: 5-year PFS was 22% in concordant patients and 55% in discordant (p<0.05) (fig1b). Moreover, only 1 patient has already died among discordant patients as opposed to 9 among concordant (fig1c). Of note, the outcome of discordant patients was not significantly different from that of VH-mutated patients. The role of telomere length as survival predictor was further supported by performing univariate and multivariate analysis indicating that TRF length is the most powerful prognostic indicator among those considered (age, sex, Binet, CD38, ZAP70, FISH and mutational status). Conclusion VH-unmutated patients are highly heterogeneous and this heterogeneity can be revealed through evaluation of TRF length. [Display omitted]

Mutator Genes UDG and AID Appear To Be Normal in Class-Switch Deficient and Clonally Homogeneous Waldenstrom's Macroglobulinemias Having Either Germline or Hypermutated Clonotypic IgH VDJ.

Clonotypic B cells of Waldenstrom's macroglobulinemia (WM) are characterized as CD20+IgM+IgD+ cells that are usually somatically mutated in IgH VDJ but for some patients, the clonotypic IgH VDJ is germline (unmutated).For both mutated and unmutated clones, WM lack ongoing somatic hypermutation (SHM) and class switch recombination (CSR). This may be due to abnormalities in switching and/or mutator genes. To understand the nature of unswitched tumor B cells, uracil DNA glycosylase (UDG) and activation-induced cytidine deaminase (AID), the two essential elements for CSR, were analysed in WM. Analysis of 12 WM clones characterized by somatic hypermutation showed that the mutation profile of VH genes had normal transition/transversion ratios at C or G, and thus did not suggest UDG abnormalities. Expression of AID was determined by single stage RT-PCR. Out of 14 patients studied (2 unmutated and 12 mutated VH clones), two of them (WM1-01 and WM1-08,with mutation rates of 0% and 6.2% respectively) gave positive bands. In WM1-01, despite having a germline IgH VDJ, AID is consistently expressed in two bone marrow samples collected three years apart and from which the identical unmutated clonotypic VDJ sequence was isolated. Full-length (FL) AID transcripts of WM have a conserved sequence, thus ruling out the possibility of functional defects due to point mutation. In addition, detection of AID in an unmutated VH clone suggested that lack of SHM does not result from an inability to produce AID. In addition to FL transcripts, three other splice variants were identified in both patients. Single cell analysis indicated that only a small compartment (10% or less), not all, of clonotypic B cells expressed AID, and multiple isoforms may be detectable in individual cells. Whether these splice variants that contain truncated C-terminal ends play a role in the regulation of CSR in WM remains to be investigated. Splice variants, nevertheless, may not characterize tumor B cells since up to 10% of AID-expressing normal activated B cells (n=3) also carried them. In vitro activation of clonotypic WM B cells by CD40L and IL4, using conditions that induced CSR in normal B cells, did not yield detectable class switching in WM B cells. In cultures of B cells from WM, the number of non-clonal B cells increased but the clonotypic B cells did not appear to expand, as indicated by the reduction of clonotypic IgM transcript at 5-days of culture. Thus, as well as failing to undergo somatic mutation or class switching, WM tumor B cells appear unresponsive to CD40L+IL4. They may be fundamentally unresponsive to signals for class switching and their clonal expansion may depend upon alternate signaling pathways.

Identification of the Earliest B Lineage Stage in Mouse Bone Marrow

We have identified a very early stage of B lineage cells in the CD45R (B220) +CD24 (HSA) − pre-pro-B fraction of mouse bone marrow delineated by expression of AA4.1, a molecule found on stem cells and early B lineage cells. These cells are B lineage precursors based on their capacity to generate B lineage cells rapidly in stromal-dependent culture and their expression of high levels of germline IgH transcripts in the absence of Rag-1/2. Half of these AA4.1 + precursors express low levels of CD4, characteristic of lymphoid progenitors, but few if any have up-regulated CD19, a molecule expressed very early in the B lineage. Furthermore, expression of genes encoding pre-B and B cell receptor components (mb-1, B29, and λ5) and transcription factors necessary for B lineage differentiation (BSAP, E12, E47, and Id) provide further support for designating these cells as the earliest B cell precursors.

Non-radioactive detection of immunoglobulin and T cell receptor gene rearrangement in acute lymphoblastic leukaemia.

Rearrangements of the heavy chain immunoglobulin gene and T cell receptor beta gene were investigated in 25 patients suffering from precursor B cell acute leukaemia and six patients suffering from T cell acute leukaemia using biotinylated DNA probes. All precursor B acute leukaemia patients had IgH gene rearrangements and 63% of those studied also had TCR beta gene rearrangements. All T cell acute leukaemia patients had TCR beta gene rearrangements and germline IgH configuration. Dilution experiments indicated that DNA from leukaemic cells representing 1-2% of a tested sample could be detected using this technique which compares favourably to radioactive DNA probes.

Isolation of an IgH gene circular DNA clone from human bone marrow

Circular DNA was obtained from human bone marrow. Then a phage library was prepared and screened by use of two probes of the IgH gene; 5'-DHQ52, containing the 5' flanking region of DHQ52, and JH4.3, containing the sequence from JH3 to the 3' flanking region of JH6. One clone, HBMC-1, that was DHQ52+JH4.3- was obtained. HBMC-1 had the germline IgH region upstream of JH1 and the 3' flanking region of DXP1. A recombination signal sequence flanking the 5' side of the JH1 segment was attached to the recombination signal sequence flanking the 3' side of DXP1 forming a head-to-head structure of two 7mers with 10 nucleotides in-between. HBMC-1 is thus considered to be a circular DNA deleted as a consequence of DXP1-JH1 joining of the IgH gene.