Igκ Gene Research Articles

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the DNA of the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of IGV gene subgroup-specific primers recognizing nearly all IGV genes together with primers for the J genes. By sequence analysis of IGV region genes from distinct cells, the clonal relationship of the B-lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D and J gene usage. Moreover, the presence and pattern of somatic IGV gene mutations give valuable insight into the differentiation stage of the B cells.

Expression of immunoglobulin G in human proximal tubular epithelial cells.

Proximal tubular epithelial cells (PTECs) have innate immune characteristics, and produce proinflammatory factors, chemokines and complement components that drive epithelial-mesenchymal transition (EMT). Our previous studies revealed that human mesangial cells and podocytes were able to synthesize and secrete immunoglobulin (Ig)A and IgG, respectively. The aim of the present study was to evaluate the expression of Igs in PTECs. Firstly, IgG was detected in the cytoplasm, the cell membrane and the lumen of PTECs in the normal renal cortex by immunohistochemistry. Secondly, Igγ gene transcription and V(D)J recombination were detected in single PTECs by nested PCR and Sanger sequencing. Thirdly, Igγ, Igκ and Igλ were clearly detected in an immortalized PTEC line (HK-2) by immunostaining and western blotting, in which RP215 (an antibody that predominantly binds to non-B cell-derived IgG) was used. In addition, Igγ, Igκ and Igλ gene transcripts, conservative V(D)J recombination in the Igγ variable region, recombination activating gene 1/2 and activation-induced cytidine deaminase were all detected in HK-2 cells. These data suggested that PTECs may express IgG in a similar manner to B cells. Furthermore, IgG expression was upregulated by TGF-β1 and may be involved in EMT.

Cytological analysis of lymphoblastic lymphoma/acute lymphoblastic leukemia in serous effusion

Objective: To study the diagnostic clues and significance in serous effusion cytology associated with lymphoblatic lymphoma/acute lymphoblastic leukemia (LBL/ALL). Methods: Forty-five serous effusion specimens with final diagnosis of LBL/ALL were collected from August 2011 to December 2019 at the First Affiliated Hospital of Zhengzhou University. All cases were reviewed for their clinical profiles, cytomorphologic features and ancillary studies. Cell blocks and immunocytochemistry were prepared in 22 cases; flow cytometric immunophenotyping was performed in three cases and gene rearrangement analysis (T-cell recepter, TCR and immunoglobulin, Ig) was performed in five cases. Results: Among the 45 cases, there were 35 males and 10 females with male to female ratio of 3.5∶1.0. The median age was 15 years. Mediastinal mass was the initial presentation in 39 patients (86.7%) and high LDL level were observed in 34 patients (75.6%). Microscopically, the majority of the specimens (86.7%) were hypercellular. The smears demonstrated dispersed lymphoblasts that were predominantly small to intermediate in size with scanty basophilic cytoplasm and irregular or convoluted nuclei with fine chromatin condensation and inconspicuous nucleoli. Mitoses were frequently observed. Karyorrhexis and apoptosis were seen in all cases. By immunophenotyping, TdT was expressed in 19 cases (86.4%) and CD99 in 20 cases (90.9%). Ki-67 expression varied from 65% to 95%. Flow cytometry in three cases demonstrated positivity for TdT, CD2, CD3 and CD7. Monoclonal TCR gene rearrangement was found in 4 of 5 cases, and both monoclonal TCR and Igκ gene were found in 1 case. Conclusions: In LBL/ALL, primary diagnosis could be made basing on clinical features (younger male patients with a mediastinum mass) and cytomorphology (monotonous, small to medium sized lymphoid cells with prominent irregular nuclei, fine chromatin and frequent mitoses, karyorrhexis and apoptosis). If immunocytochemistry and other ancillary studies are performed, the accuracy and reliability of the results could be improved.

JNK/AP1 Pathway Regulates MYC Expression and BCR Signaling through Ig Enhancers in Burkitt Lymphoma Cells.

In Burkitt lymphoma (BL), a chromosomal translocation by which the MYC gene is fused to an immunoglobulin (Ig) gene locus is frequently found. The translocated MYC gene is overexpressed, which is the major driver of BL tumorigenesis. Studies have shown that Ig enhancers are essential for MYC overexpression, but the involved mechanisms are not fully understood. In addition, the survival of BL cells relies on B-cell receptor (BCR) signaling, which is determined by the levels of Ig molecules expressed on the cell surface. However, whether MYC has any impact on Ig expression and its functional relevance in BL has not been investigated. Herein, we show that MYC upregulates Ig kappa (Igκ) expression in BL cells through two Igκ enhancers, the intronic enhancer (Ei) and the 3ʹ enhancer (E3ʹ). Mechanistically, by activating the JNK pathway, MYC induces the phosphorylation of c-Fos/c-Jun and their recruitment to AP1 binding sites in the Igκ enhancers, leading to the activation of the enhancers and subsequent Igκ upregulation. The AP1-mediated activation of the Igκ enhancers is also required for the expression of the translocated MYC gene, indicating positive feedback for the MYC overexpression in BL cells. Importantly, interrupting the JNK pathway inhibits both Igκ and MYC gene expression and suppresses BL cell proliferation. Our study not only reveals a novel mechanism underlying MYC overexpression in BL but also suggests that targeting the JNK pathway may provide a unique strategy to suppress BL tumorigenesis.

A linear-amplification VDJ-seq technique for quantification of immunoglobulin and T cell receptor diversity.

The V(D)J recombination is essential for generating a highly diverse repertoire of antigen receptors expressed on T and B lymphocytes. Here, we developed a linear-amplification VDJ-seq technique for quantifying V(D)J recombination of antigen receptor genes. This technique takes advantage of linear amplification using in vitro transcription and reverse transcription to avoid bias generated by the PCR amplification of low copy number of target DNA. The unrearranged alleles are removed by in vitro cleavage with the CRISPR-Cas9 system. The linear-amplification VDJ-seq assay was applied in quantification of the Vκ-Jκ recombination of the mouse Igκ gene with Jκ capture primers. The Jκ genes were detected in 95.86% of clean reads with more than half containing the Vκ gene, indicating high specificity of capturing and amplification. We also applied this approach to quantify the usage of Jα within the Trav12 gene family of the Tcra gene.

Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene subgroup-specific primers recognizing nearly all V genes together with primers for the J genes. By sequence analysis of V region genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

YY1 binds to the E3ʹ enhancer and inhibits the expression of the immunoglobulin κ gene via epigenetic modifications

The rearrangement and expression of immunoglobulin genes are regulated by enhancers and their binding transcriptional factors that activate or suppress the activities of the enhancers. The immunoglobulin κ (Igκ) gene locus has three important enhancers: the intrinsic enhancer (Ei), 3' enhancer (E3'), and distal enhancer (Ed). Ei and E3' are both required for Igκ gene rearrangement during early stages of B-cell development, whereas optimal expression of the rearranged Igκ gene relies on both E3' and Ed. The transcription factor YY1 affects the expression of many genes involved in B-cell development, probably by mediating interactions between their enhancers and promoters. Herein, we found that YY1 binds to the E3' enhancer and suppresses Igκ expression in B lymphoma cells by epigenetically modifying the enhancer. Knocking down YY1 enhanced Igκ expression, which was associated with increased levels of E2A (encoded by the TCF3 gene) and its binding to the E3' enhancer. Moreover, in germinal centre B cells and plasma cells, YY1 expression was reversely associated with Igκ levels, implying that YY1 might facilitate antibody affinity maturation in germinal centre B cells through the transient attenuation of Igκ expression.

Effects of spaceflight on the immunoglobulin repertoire of unimmunized C57BL/6 mice

Spaceflight has been shown to suppress the adaptive immune response, altering the distribution and function of lymphocyte populations. B lymphocytes express highly specific and highly diversified receptors, known as immunoglobulins (Ig), that directly bind and neutralize pathogens. Ig diversity is achieved through the enzymatic splicing of gene segments within the genomic DNA of each B cell in a host. The collection of Ig specificities within a host, or Ig repertoire, has been increasingly characterized in both basic research and clinical settings using high-throughput sequencing technology (HTS). We utilized HTS to test the hypothesis that spaceflight affects the B-cell repertoire. To test this hypothesis, we characterized the impact of spaceflight on the unimmunized Ig repertoire of C57BL/6 mice that were flown aboard the International Space Station (ISS) during the Rodent Research One validation flight in comparison to ground controls. Individual gene segment usage was similar between ground control and flight animals, however, gene segment combinations and the junctions in which gene segments combine was varied among animals within and between treatment groups. We also found that spontaneous somatic mutations in the IgH and Igκ gene loci were not increased. These data suggest that space flight did not affect the B cell repertoire of mice flown and housed on the ISS over a short period of time.

RAG2 involves the Igκ locus demethylation during B cell development

The genes encoding the immunoglobulin κ light chain are assembled during B cell development by V(D)J recombination. For efficient rearrangement, the Igκ locus must undergo a series of epigenetic changes. One such epigenetic mark is DNA methylation. The mechanism that the Igκ locus is selectively demethylated at the pre-B cell stage has not previously been characterized. Here, we employed bisulfite DNA-modification assays to analyze the methylation status of the Igκ locus in primary pre-B cells from RAG-deficient mice with pre-rearranged Igh knock-in allele. We observed that the Igκ locus was hypermethylated in RAG2-deficient pre-B cells but hypomethylated in RAG1-deficient pre-B cells, indicating that wild-type (WT) RAG2 involves the Igκ locus demethylation in a RAG1-independent manner prior to rearrangement. We generated a series of RAG2 mutants between residue 350 and 383. We showed that these mutants mediated the Igκ rearrangement but failed to regulate the Igκ gene demethylation. We further analyzed that these mutants could increase RAG recombinase activity in vivo. We conclude that residues 350–383 region are responsible for endogenous Igκ locus demethylation at pre-B cells. We propose that WT RAG2 has an intrinsic function to regulate the Igκ locus demethylation.

Plasmablastic lymphoma: a clinicopathologic analysis of 11 cases with review of literature

To investigate the clinicpathologic features and diagnosis of plasmablastic lymphoma (PBL). Eleven cases of PBL were collected and followed up, with review of the literature. HIV and EBV status and their relationships with the tumor were specially compared as well. In the current cohort, 10 patients were serologically HIV negative; the male to female ratio was 8 to 3, and the median age was 57 years. Ten cases showed extranodal involvement and one case was nodal based. At presentation, five patients had mid-facial involvement, including sinonasal area (3 cases) and oral cavity (2 cases). Histologically, six were PBL of oral mucosa type, and five were PBL with plasmacytic differentiation. In all cases, the neoplastic cells expressed CD138 and MUM-1, and were negative for CD20 and CD3ε; the median Ki-67 index was 80%. Five cases were EBER1/2 in situ hybridization positive. IgH or/and Igκ gene rearrangement was detected in all five cases examined. Most patients were no congenital or acquired immunodeficiency in the retrospective study. Of the died patients, EBER1/2 in situ hybridization were negative and their disease staging were Ⅳ, The neoplastic cells were immunoblastic or plasmablastic, sometimes the plasmacytoid cell can be seen and the neoplastic cell had mature plasma cell phenotype, the pathologic diagnosis of the lymphoma is still controversial now. Differentiate with plasma cell neoplasm is difficult, it is necessary to accumulate more cases for advanced study and observation in the future.

Reversing Tolerance in Isotype Switch-Competent Anti-Insulin B Lymphocytes.

Autoreactive B lymphocytes that escape central tolerance and mature in the periphery are a liability for developing autoimmunity. IgG insulin autoantibodies that predict type 1 diabetes and complicate insulin therapies indicate that mechanisms for tolerance to insulin are flawed. To examine peripheral tolerance in anti-insulin B cells, we generated C57BL/6 mice that harbor anti-insulin VDJH-125 site directed to the native IgH locus (VH125(SD)). Class switch-competent anti-insulin B cells fail to produce IgG Abs following T cell-dependent immunization of VH125(SD) mice with heterologous insulin, and they exhibit markedly impaired proliferation to anti-CD40 plus insulin in vitro. In contrast, costimulation with LPS plus insulin drives robust anti-insulin B cell proliferation. Furthermore, VH125(SD) mice produce both IgM and IgG2a anti-insulin Abs following immunization with insulin conjugated to type 1 T cell-independent Brucella abortus ring test Ag (BRT). Anti-insulin B cells undergo clonal expansion in vivo and emerge as IgM(+) and IgM(-) GL7(+)Fas(+) germinal center (GC) B cells following immunization with insulin-BRT, but not BRT alone. Analysis of Igκ genes in VH125(SD) mice immunized with insulin-BRT reveals that anti-insulin Vκ from the preimmune repertoire is selected into GCs. These data demonstrate that class switch-competent anti-insulin B cells remain functionally silent in T cell-dependent immune responses, yet these B cells are vulnerable to reversal of anergy following combined BCR/TLR engagement that promotes Ag-specific GC responses and Ab production. Environmental factors that lead to infection and inflammation could play a critical yet underappreciated role in driving loss of tolerance and promoting autoimmune disease.

Dynamic Control of Long-Range Genomic Interactions at the Immunoglobulin κ Light-Chain Locus.

The Igκ locus, which is spread over 3Mb of genomic DNA and contains >100 variable (V) genes, serves as an important model system to study long-range chromatin interactions. Here, we will discuss how in developing B cells in the bone marrow the accessibility of individual Vκ segments is controlled by many lineage-specific and ubiquitously expressed transcription factors that act on various cis-regulatory elements, including promoters, enhancers, and insulators. This dynamic control furthermore involves changes in subnuclear localization, histone modification, DNA demethylation, and three-dimensional locus compaction. In pro-B cells, the Igκ locus adopts a poised conformation as full contraction has been achieved and many key transcription factors already occupy the locus. Subsequently, the combined activation of pre-B cell antigen receptor signaling pathways and attenuation of IL-7R signaling in small resting pre-B cells dramatically modifies the transcription factor landscape, supporting the induction of monoallelic Igκ gene rearrangements. Hereby, the intronic and 3' Igκ enhancer elements coordinately focus their activities in the Vκ region toward frequently used Vκ genes. Recent work has drawn attention to the intriguing role of the CTCF-associated regulatory elements Cer and Sis, which are located in the Vκ-Jκ intervening region and control Igκ locus contraction and Vκ repertoire diversity. This involves CTCF-mediated locus insulation, restricting enhancer activity to the Vκ region and suppressing the preferential recombination to proximal Vκ genes. A picture emerges in which the dynamic control of long-range genomic interactions ensures correct timing of Igκ locus recombination and provides appropriate opportunities for individual Vκ gene segments to engage in Vκ-Jκ rearrangement.

Vκ Gene Repertoire and Locus Contraction Are Specified by Critical DNase I Hypersensitive Sites within the Vκ-Jκ Intervening Region

The processes of Ig gene locus contraction and looping during V(D)J-recombination are essential for creating a diverse Ab repertoire. However, no cis-acting sequence that plays a major role in specifying locus contraction has been uncovered within the Igκ gene locus. In this article, we demonstrate that a 650-bp sequence corresponding to DNase I hypersensitive sites HS1-2 within the mouse Igκ gene V-J intervening region binds CCCTC-binding factor and specifies locus contraction and long-range Vκ gene usage spanning 3.2 Mb in pre-B cells. We call this novel element Cer (for "contracting element for recombination"). Targeted deletion of Cer caused markedly increased proximal and greatly diminished upstream Vκ gene usage, higher allele usage, more splenic Igκ(+) B cells, and nonlineage-specific Igκ rearrangement in T cells. Relative to wild-type mice, Cer-deletion mice exhibited similar levels of Vκ gene germline transcription and H3K4me3 epigenetic marks but displayed a dramatic decrease in locus contraction in pre-B cells. Thus, our studies demonstrate that DNase I hypersensitive sites HS1-2 within the Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire.

CTCF mediates long-range interaction between silencer Sis and enhancer Ei and inhibits VJ rearrangement in pre-B cells

Igκ VJ rearrangement occurs only in one allele. It is not fully understood how this allelic exclusion is regulated. We previously identified a recombination silencer and heterochromatin targeting element in the mouse Igκ gene locus, which is termed Sis. Using chromosome conformation capture (3C) technology, here, we demonstrate that Sis and Igκ gene intronic enhancer (Ei) exhibits physical interaction with the intervening DNA looping out. Knock-down of CCCTC-binding factor, CTCF, Ei-Sis interaction is lost and enhancement of VJ rearrangement is detected. Therefore, we propose that the interaction between Sis and Ei mediated by CTCF inhibits the VJ rearrangement and participates in the monoallelic silencing aspect of allelic exclusion regulation.

A new hypersensitive site, HS10, and the enhancers, E3' and Ed, differentially regulate Igκ gene expression.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). We previously discovered, using the chromosome conformation-capture technique, that Ei and E3' interact with a novel DNA sequence near the 3' end of the Igκ locus, specifically in B cells. In the present investigation, we examined the function of this far downstream element. The sequence is evolutionarily conserved and exhibits a plasmacytoma cell-specific DNase I-hypersensitive site in chromatin, henceforth termed HS10 in the locus. HS10 acts as a coactivator of E3' in transient transfection assays. Although HS10(-/-) mice exhibited normal patterns of B cell development, they were tested further along with E3'(-/-) and Ed(-/-) mice for their Igκ expression levels in plasma cells, as well as for both allelic and isotype exclusion in splenic B cells. HS10(-/-) and Ed(-/-), but not E3'(-/-), mice exhibited 2.5-fold lower levels of Igκ expression in antigenically challenged plasma cells. E3'(-/-) mice, but not HS10(-/-) mice, exhibited impaired IgL isotype and allelic exclusion in splenic B cells. We have suggestive results that Ed may also weakly participate in these processes. In addition, HS10(-/-) mice no longer exhibited regional chromosome interactions with E3', and they exhibited modestly reduced somatic hypermutation in the Jκ-Cκ intronic region in germinal center B cells from Peyer's patches. We conclude that the HS10, E3', and Ed differentially regulate Igκ gene dynamics.

Cloning and sequencing of the light chain variable region from NS-1 myeloma

The aim of this study was to clone the gene encoding the light chain variable region (V(L)) of the murine myeloma cell line P3/NS1/1-Ag4-1 (NS-1). Total RNA was prepared from the NS-1 cell line and its fusion hybridoma cell line 2F9. The V(L) gene was amplified using reverse transcription PCR (RT-PCR) with family-specific primer pairs. The PCR products were cloned into the pGEM(®)-T easy vector, transfected into E. coli DH5α cells and the positive recombinants were identified and purified with EcoRI digestion and agarose gel electrophoresis. The sequence was identified using an automatic DNA sequencer and analyzed online using the IMGT/V-QUEST program (version 3.2.21). The PCR product that was amplified with the P9 and P14 primers was approximately 392 bp in size. Following digestion with EcoRI, the band of interest was also detected in positive recombinants using agarose gel electrophoresis. The sequences from the NS-1 and 2F9 cells were identical. The whole sequence was 387 bp long, encoding 128 amino acids (AA), including a 60 bp leader sequence. There was a TAA stop codon at 385-387 bp and only one cysteine was found, at 112AA/128AA. Analysis using IMGT/V-QUEST determined the cloned V- and J-segments as murine IG(κ)KV3-12*01 and IG(κ)KJ2*01, respectively. Accordingly, the NS-1 V(L) gene belongs to the Ig(κ) gene family V3 subgroup. The NS-1 V(L) gene was successfully cloned and it is a pseudo-Igκ chain gene in NS-1 cells. This study may aid the sequencing of genes encoding monoclonal antibodies produced by mouse hybridomas raised with NS-1 myeloma.

The Igκ gene enhancers, E3' and Ed, are essential for triggering transcription.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3' significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3' eliminates such recombination. Furthermore, deletion of either E3' or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3' and Ed are essential for Igκ gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ(+) B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Igλ-chains, despite the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer-deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3' knockout mice in triggering germline transcription and Vκ gene rearrangements to both Jκ and RS elements.

Divergent Roles of RelA and c-Rel in Establishing Chromosomal Loops upon Activation of the Igκ Gene

Precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening DNA, but how trans-acting regulatory proteins work to establish and maintain DNA loops during gene activation remains largely unexplored. LPS-induced transcription of the mouse Igkappa gene in B lymphocytes utilizes three distal enhancers and requires the transcription factor NF-kappaB, whose family members include RelA and c-Rel. Using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that LPS-induced Igkappa gene activation creates chromosomal loops by bridging together all three pairwise interactions between the distal enhancers and RNA polymerase II, the apparent molecular tie for the bases of these loops. RelA and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis, or c-Rel. We have thus identified both essential and nonessential events that establish higher order chromatin reorganization during Igkappa gene activation.

The Polymorphisms on Igκ Gene Are Related to Susceptibility of Breast Cancer and Gastric Cancer

The phenomenon of immunoglobulin (Ig) expression in cancer cells has been discovered recently; the Ig protein expressed and secreted in cancer cells was found to be in favor of tumor growth. Two single-nucleotide polymorphism (SNP) loci rs232230 (5658C/G) and rs232228 (3635T/C) were identified on Igkappa gene previously, and we have demonstrated that they were associated with nasopharyngeal carcinoma susceptibility. In the present study, we further performed the study focused on these two SNPs in gastric and breast cancer, trying to demonstrate the association between the genotypes of the two SNPs and the susceptibility of gastric cancer and breast cancer. Our results suggested that the 5658-G allele and 3635-C allele were risk factors for both gastric cancer (odds ratio [OR]: 1.64 and 1.67, respectively) and breast cancer (OR: 1.94 and 1.56, respectively). Further, we also identified that they were related to other risk factors, including Helicobacter pylori (Hp) infection in gastric cancer and age in breast cancer.

Early Human B-Lineage Cells Can Make Functional Igκ Gene Rearrangements and Express Igκ Protein in the Absence of Functional VDJH Rearrangements.

One of the tools available to study human B cell development are xenogeneic murine stromal cells that serve as a microenvironment to support development of B-lineage cells from human hematopoietic stem cells. Efforts to clarify the role of IL-7 signaling in human B cell development have been rejuvenated by the observation that cord blood CD34+ stem cells cultured on murine MS-5 stromal cells develop into CD19+ B-lineage cells expressing the IL-7R (i.e., IL-7Rα chain, CD127 and gamma common chain, CD132), and that signaling through IL-7R is essential for development of CD19+ B-lineage cells in this model. CD19+/CD127+ cells have survival attributes and patterns of Bcl-2 and p27KIP1 expression that are distinct from CD19+/CD127− cells. CD127+ cells are Bcl-2hi, p27KIP1lo and CD127− cells are Bcl-2lo, p27KIP1hi. However, the relationship between IL-7R expression/signaling and the onset/progression of IgH and IgL rearrangement patterns in early human B-lineage cells is unknown. We hypothesized that IL-7R expression would signify the potential for IgH gene rearrangement and eventually IgL gene rearrangement, whereas absence of expression would signify failure to make in-frame rearrangements. To test this hypothesis, CD19+ cells from three independent 4-wk cord blood/MS-5 xenogeneic cultures were FACS-purified into CD19+/CD127+ and CD19+/CD127− fractions, and a quantitative real time PCR assay was employed to characterize IgH and IgL rearrangements at the level of the sorted populations. The majority of cells in both fractions had incomplete DJH rearrangements. However, < 10% of CD127+ and CD127− cells had complete VDJH rearrangements and those that did primarily used VH1, 2 and 3 families. This data was in agreement with immunofluorescent staining which showed that ~ 5% of CD127+ and CD127− cells expressed cytoplasmic Igμ heavy chains. Neither elimination of IL-7 signaling nor addition of exogenous IGF-1 promoted the appearance of cells expressing Igμ heavy chains. Thus, many of the early B-lineage cells that developed in this model had an IgH rearrangement profile that resembled pre-B-I cells isolated from pediatric marrow. Unexpectedly, analysis of IgL gene rearrangements in the CD19+/CD127+ and CD19+/CD127− fractions showed profound differences. CD127+ cells harbored very few rearrangements at the Igκ and Igλ light chain loci (estimated to be < 5% of cells). However, CD127− cells showed extensive VJκ and VJλ rearrangements, as well as evidence that the kappa deleting element had undergone rearrangement with Vκ genes and the intron recombination signal sequence. Thus, despite having only a small percentage of cells with complete VDJH rearrangements, the CD127− fraction showed a high level of recombinatorial activity in the light chain loci. Many of the VJκ rearrangements were in-frame, since Igκ protein was detected in the majority of CD127− cells by flow cytometry, immunohistochemistry, and Western blotting. These results show that in-frame rearrangement at the IgH locus is not a requirement for developing human B-lineage cells to initiate rearrangement at the IgL loci. They further suggest that the low frequency of cells with complete VDJH rearrangements reflects epigenetic silencing of VH gene clusters, or the absence of a stimulus in the xenogeneic culture that induces VDJH rearrangements. The existence of cells expressing Igκ light chain protein in the absence of Igμ heavy chain protein may represent a heretofore unrecognized population of human B-lineage cells with heightened recombinatorial flexibility and/or a population destined to die in the marrow.