Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome-associated lymphoproliferation.

OBJECTIVE: Primary Sjögren's syndrome (SS) is an autoimmune disease associated with a high risk of developing non-Hodgkin's lymphoma. This study was undertaken to determine the nature of B cells driving lymphoproliferation in primary SS. METHODS: B cell subsets and function were analyzed in peripheral blood from 66 adult patients with primary SS (including 14 patients with B cell lymphoproliferative disease [LPD]) and 30 healthy donors, using flow cytometry, calcium mobilization, and gene array analysis. The reactivity of recombinant antibodies isolated from single B cells from patients with primary SS and LPD was tested using an enzyme-linked immunosorbent assay. RESULTS: We observed an expansion of an unusual CD21-/low B cell population that correlated with lymphoproliferation in patients with primary SS. A majority of CD21-/low B cells from patients with primary SS expressed autoreactive antibodies, which recognized nuclear and cytoplasmic structures. These B cells belonged to the memory compartment, since their Ig genes were mutated. They were unable to induce calcium flux, become activated, or proliferate in response to B cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. However, CD21-/low B cells from patients with primary SS remained responsive to Toll-like receptor (TLR) stimulation. Molecules specifically expressed in CD21-/low B cells that are likely to induce their unresponsive stage were detected in gene array analyses. CONCLUSION: Patients with primary SS who display high frequencies of autoreactive and unresponsive CD21-/low B cells are susceptible to developing lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.

Relapsing and remitting severe hypoglycemia due to a monoclonal anti-insulin antibody heralding a case of multiple myeloma.

CONTEXT: We report a novel case of insulin autoimmune syndrome (IAS) presenting with hypoglycemia due to production of a monoclonal anti-insulin antibody in a patient subsequently found to have multiple myeloma (MM). OBJECTIVE: The aim of the study was to describe the 5-yr clinical course of a patient with IAS and MM and to characterize the origin and function of the pathogenic antibody. METHODS: We conducted a longitudinal case history with laboratory investigations to characterize the anti-insulin antibody subtype, specificity, affinity, and origin. RESULTS: The patient presented with IAS, which worsened during treatment of hepatitis C. The patient was then discovered to have a monoclonal gammopathy that progressed to MM. Treatment of the MM induced remission of the neoplasia and IAS, which then followed a synchronized course of progression and response to therapy. An anti-insulin IgG(3)-λ that bound specifically but with low affinity to the insulin B chain (amino acids 9-30) and that was distinct from the primary MM IgG(3)-κ clone was recovered from the patient and cloned. The antibody bound insulin and showed mutations of normal affinity maturation. CONCLUSIONS: We describe a case of MM heralded by IAS, where full characterization of the pathogenic antibody revealed that the monoclonal anti-insulin antibody had originated from a self-reactive clone.

A rapid method for targeted modification and screening of recombinant bacterial artificial chromosome.

Modification of bacterial artificial chromosomes (BACs) has been a useful method to produce genomic DNA fragments for studying gene expression and function in vitro and in vivo. The original technique involved restrictions for BAC modification and required multiple cloning steps to target sequences into the shuttle vector. Selection and screening of BAC recombinants was accomplished by drug resistance and Southern blotting. We have developed a PCR-based method for producing the modified shuttle vectors and for screening for BACs carrying homologous integrants. The combination of these techniques allows for rapid and easy targeted BAC sequence deletion or insertion.

Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor gammadelta(+) gut intraepithelial lymphocytes.

CC chemokine receptor (CCR) 9, the receptor for the CC-chemokine CCL25/thymus-expressed chemokine (TECK), is mainly expressed by thymocytes and by intraepithelial (IEL) and lamina propria lymphocytes of the small intestine. To study the biologic role of CCR9, a mouse strain was generated in which the CCR9 gene was deleted. In spite of the high level of CCR9 found in double- and single-positive thymocytes and of the expression of its corresponding ligand on thymic stromal cells, CCR9 deletion had no major effect on intrathymic T-cell development. It was noted that there was only a one-day lag in the appearance of double-positive cells during fetal ontogeny in CCR9(-/-) thymi. When tested in chemotaxis assay, thymocytes isolated from CCR9(-/-) mice failed to respond to TECK/CCL25. Taken together, these results suggest that in thymocytes, CCR9 is the only physiologic receptor for TECK/CCL25, and that it is dispensable for proper T-cell development. Bone marrow pre-pro-B cells migrate in response to TECK/CCL25, but more mature B cells do not. Consistent with this observation, it was shown that there are fewer pre-pro-B cells in CCR9(-/-) mice than in wild-type mice. However, this diminution does not appear to have a detectable effect on the generation of a normal complement of mature B cells. Finally, it was shown that in the small intestine of CCR9-deficient mice, the intraepithelial T-cell-to-epithelial cell ratio is decreased, an observation that can be accounted for by a marked diminution of the T-cell receptor gammadelta(+) compartment.

Immunoglobulin heavy chain expression shapes the B cell receptor repertoire in human B cell development.

Developing B cells must pass a series of checkpoints that are regulated by membrane-bound Ig(mu) through the Igalpha-Igbeta signal transducers. To determine how Ig(mu) expression affects B cell development and Ab selection in humans we analyzed Ig gene rearrangements in pro-B cells from two patients who are unable to produce Ig(mu) proteins. We find that Ig(mu) expression does not affect V(H), D, or J(H) segment usage and is not required for human Igkappa and Iglambda recombination or expression. However, the heavy and light chains found in pro-B cells differed from those in peripheral B cells in that they showed unusually long CDR3s. In addition, the Igkappa repertoire in Ig(mu)-deficient pro-B cells was skewed to downstream Jkappas and upstream Vkappas, consistent with persistent secondary V(D)J rearrangements. Thus, Ig(mu) expression is not required for secondary V(D)J recombination in pro-B cells. However, B cell receptor expression shapes the Ab repertoire in humans and is essential for selection against Ab's with long CDR3s.

Somatic hypermutation shapes the antibody repertoire of memory B cells in humans.

High-affinity antibodies produced by memory B cells differ from antibodies produced in naive B cells in two respects. First, many of these antibodies show somatic hypermutation, and second, the repertoire of antibodies expressed in memory responses is highly selected. To determine whether somatic hypermutation is responsible for the shift in the antibody repertoire during affinity maturation, we analyzed the immunoglobulin lambda light chain (Iglambda) repertoire expressed by naive and antigen-selected memory B cells in humans. We found that the Iglambda repertoire differs between naive and memory B cells and that this shift in the repertoire does not occur in the absence of somatic hypermutation in patients lacking activation-induced cytidine deaminase (AID). Our work suggests that somatic hypermutation makes a significant contribution to shaping the antigen-selected antibody repertoire in humans.

Circulating human B cells that express surrogate light chains display a unique antibody repertoire.

Circulating human B cells that coexpress V-preB and conventional L chains (V-preB+L+ B cells) are a recently described subset of B cells that express Abs with features of self-reactivity. Initial analysis of V-preB+L+ B cells was limited to Ig-kappa and to the small, underused VH5 family. To determine whether Abs commonly expressed by V-preB+L+ B cells show similar features, we analyzed Ig H chains from three highly expressed VH families, VH1, VH3, and VH4, and Ig-lambda. We find that VH1 and VH3 Abs expressed by V-preB+L+ B cells resemble VH5 in that they display increased JH6 use, long CDR3s, and an increased frequency of D-D fusions. Abs in all three of these VH families also show skewed D reading frame use resulting in predominance of hydrophobic amino acids, which are counterselected in conventional B cells. Like Ig-kappa genes, the Ig-lambda genes in V-preB+L+ B cells show long CDR3s, but they differ from Ig-kappa genes in that they display no evidence of receptor editing. We conclude that a large number of H and L chain Abs expressed by V-preB+L+ B cells display features associated with self-reactive Abs.

B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

Immunoglobulin heavy chain variable region gene replacement As a mechanism for receptor revision in rheumatoid arthritis synovial tissue B lymphocytes.

Mature B cells can alter their antibody repertoires by several mechanisms, including immunoglobulin heavy chain variable region (V(H)) replacement. This process changes the antigen combining site by replacing a portion of the original V(H)/diversity/heavy chain joining region (V(H)DJ(H)) rearrangement with a corresponding portion of a new V(H) segment. This exchange can involve cryptic heptamer-like sequences embedded in the coding regions of V(H) genes. While studying the B lymphocytes that expand in the synovial tissues of patients with rheumatoid arthritis (RA), clones with V(H)DJ(H) variants that were apparently generated by V(H) replacement were identified with surprising frequency (approximately 8%). Examples of multiple independent V(H) replacement events occurring in distinct progeny clones were also identified. These secondary V(H) rearrangements were documented at both the cDNA and genomic DNA levels and involved several heptamer-like sequences at four distinct locations within V(H) (three sites in framework region 3 and one in complementarity determining region 2). The identification of blunt-ended double-stranded DNA breaks at the embedded heptamers and the demonstration of recombinase activating gene (RAG) expression suggested that these rearrangements could occur in the synovial tissues, presumably in pseudo-germinal centers, and that they could be mediated by RAG in a recognition signal sequence-specific manner. The presence of V(H) mutations in the clones that had undergone replacement indicated that these B cells were immunocompetent and could receive and respond to diversification signals. A relationship between these secondary V(H) gene rearrangements and the autoimmunity characteristic of RA should be considered.

DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation.

Cancer susceptibility genes have been classified into two groups: gatekeepers and caretakers. Gatekeepers are genes that control cell proliferation and death, whereas caretakers are DNA repair genes whose inactivation leads to genetic instability. Abrogation of both caretaker and gatekeeper function markedly increases cancer susceptibility. Although the importance of Ku80 in DNA double-strand break repair is well established, neither Ku80 nor other components of the non-homologous end-joining pathway are known to have a caretaker role in maintaining genomic stability. Here we show that mouse cells deficient for Ku80 display a marked increase in chromosomal aberrations, including breakage, translocations and aneuploidy. Despite the observed chromosome instabilities, Ku80-/- mice have only a slightly earlier onset of cancer. Loss of p53 synergizes with Ku80 to promote tumorigenesis such that all Ku80-/- p53-/- mice succumb to disseminated pro-B-cell lymphoma before three months of age. Tumours result from a specific set of chromosomal translocations and gene amplifications involving IgH and c-Myc, reminiscent of Burkitt's lymphoma. We conclude that Ku80 is a caretaker gene that maintains the integrity of the genome by a mechanism involving the suppression of chromosomal rearrangements.

Circulating human B cells that express surrogate light chains and edited receptors.

Immunoglobulin gene recombination can result in the assembly of self-reactive antibodies. Deletion, anergy or receptor editing normally silence B cells that produce these autoantibodies. Receptor editing is highly efficient in mouse B cells that carry pre-recombined autoantibody transgenes or gene "knock-ins". However, it has been difficult to identify cells that have edited receptors in unmanipulated mice and humans. To try to identify such cells we isolated and characterized B cells that coexpress surrogate and conventional light chains (V-preB+L+) from the blood of normal human donors. V-preB+L+ B cells express RAG mRNA, display an unusual heavy and light chain antibody repertoire consistent with antiself reactivity, and show evidence of receptor editing. These cells accumulate in the joints of patients with rheumatoid arthritis, consistent with a role for V-preB+L+ B cells and receptor editing in autoimmune disease.

Autosomal primary immunodeficiencies affecting human bone marrow B-cell differentiation.

Since the initial report of X-linked agammaglobulinemia by Bruton, numerous autosomal primary immune deficiencies affecting early B-cell differentiation have been described in humans. The identification of these autosomal mutations has been facilitated by phenotype comparison with knockout mice. In mice, defects in B-cell development have been observed after disruption of genes encoding transcription factors, the interleukin-7 pathways as well as structural or signaling components of the pre-B-cell receptor. In general, the phenotypes of primary immune deficiencies in humans correlate with those observed in mutant mice, validating the use of the mouse model approach. In addition, we report a follow-up analysis of an autosomal primary deficiency in a young female patient born from consanguinous parents and characterized by the absence of pre-B and B-cell compartments. The patient's gene defect was identified as a cytosine insertion at the beginning of the CH1 exon of the Ig(mu) gene, resulting in a stop codon at position 48 and the absence of Ig(mu) chain expression. The precise phenotype of this patient is compared to other autosomal primary immunodeficiencies affecting humans and mice.

Antibody regulation of B cell development.

Antibodies on the surface of B lymphocytes trigger adaptive immune responses and control a series of antigen-independent checkpoints during B cell development. These physiologic processes are regulated by a complex of membrane immunoglobulin and two signal transducing proteins known as Ig alpha and Ig beta. Here we focus on the role of antibodies in governing the maturation of B cells from early antigen-independent through the final antigen-dependent stages.

Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V(D)J-recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2. We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.

Antigen receptor engagement turns off the V(D)J recombination machinery in human tonsil B cells.

The germinal center (GC) is an anatomic compartment found in peripheral lymphoid organs, wherein B cells undergo clonal expansion, somatic mutation, switch recombination, and reactivate immunoglobulin gene V(D)J recombination. As a result of somatic mutation, some GC B cells develop higher affinity antibodies, whereas others suffer mutations that decrease affinity, and still others may become self-reactive. It has been proposed that secondary V(D)J rearrangements in GCs might rescue B cells whose receptors are damaged by somatic mutations. Here we present evidence that mature human tonsil B cells coexpress conventional light chains and recombination associated genes, and that they extinguish recombination activating gene and terminal deoxynucleotidyl transferase expression when their receptors are cross-linked. Thus, the response of the recombinase to receptor engagement in peripheral B cells is the opposite of the response in developing B cells to the same stimulus. These observations suggest that receptor revision is a mechanism for receptor diversification that is turned off when antigen receptors are cross-linked by the cognate antigen.

A non-XLA primary deficiency causes the earliest known defect of B cell differentiation in humans: a comparison with an XLA case.

We report a detailed comparison of B cell defects in two patients, one XLA and one non-XLA. Both had severe agammaglobulinemia with a total absence of CD19+ cells in the periphery. In the non-XLA case, CD19 expression was also highly impaired in the bone marrow, resulting in the absence of both B and preB compartments. Early proB cells were present since CD34+CD10+ and some CD19+CD10+ mostly CD34+ were identified, although diminished. By contrast, in the XLA patient the CD34+CD19+ proB cells were increased whereas the CD34-CD19+ preB cell population was low. Semi-quantitative RT-PCR analysis performed on mononuclear bone marrow cells from the non-XLA patient indicated that lambda-like, VpreB, Rag-1, Rag-2 and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Ig alpha, Ig beta, VH-C mu and V kappa-C kappa transcripts characteristic of later stages were severely depressed. By contrast in the XLA patient most of these transcripts were observed in normal amounts. The phenotype of the non-XLA patient resembles that of Pax-5 or Ig beta knock-out mice, but since the coding sequence of both cDNAs were shown to be normal, the blockage might rather result from an altered regulation of one of these genes or from defect of other genes. All these data indicate that the non-XLA patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, before the onset of Ig gene rearrangements. From all agammaglobulinemias reported so far, including XLA cases and those resulting from C mu gene defects, the non-XLA patient exhibits the earliest blockage in the B cell differentiation pathway.

Individual CD34+CD38lowCD19-CD10- progenitor cells from human cord blood generate B lymphocytes and granulocytes.

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19-/CD10- primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38low-CD19-CD10- cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34-CD10+- CD19+cIgM- phenotype of late pro-B cells and transcripts of Pax-5, lambda-like, and mu chain were detected. We further show that 7% of CD34+CD38lowCD19- cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10- cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

A human non-XLA immunodeficiency disease characterized by blockage of B cell development at an early proB cell stage.

We report a detailed analysis of a B cell defect affecting a patient girl born from first cousin parents, characterized by a severe non-X-linked agammaglobulinemia with a total absence of CD19- cells in the periphery. In the bone marrow, CD19 expression was also highly impaired, resulting in the absence of both B and preB compartments. By contrast, CD34+CD10+, CD34psiL+, and some CD19+CD10+ mostly CD34+ early proB cells were present, although diminished. Semiquantitative RT-PCR analysis performed on mononuclear bone marrow cells indicated that lambda-like, VpreB, Rag-1, Rag-2, and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Igalpha, Igbeta, VH-Cmu, and Vkappa-Ckappa transcripts characteristic of later stages were severely depressed. This phenotype resembles that of Pax-5 knock-out mice, but since the coding sequence of the patient Pax-5 cDNA was shown to be normal, the defect might rather result from an altered regulation of this gene. All these data indicate that the patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, i.e., earlier than X-linked agammaglobulinemia, before the onset of Ig gene rearrangements.

Cell surface expression of surrogate light chain (psi L) in the absence of mu on human pro-B cell lines and normal pro-B cells.

Surrogate light chains (psi L) encoded by lambda-like (lambda 5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the mu chain. These mAb provide the first characterization of human pro-B cell lines expressing surface psi L. We demonstrate that surface psi L expression is considerably enhanced upon interleukin-7 stimulation and that the psi L complex is formed independently of the Ig alpha/Ig beta heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34+ CD38+ psi L+, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.