[Role of the Nucleolus in Rearrangements of the IGH Locus].

The review summarizes the results from a series of studies focusing on the role that the nucleolus plays in maturation of the IGH locus and the choice of its partner genes in leukemia-associated translocations. The role of nuclear compartmentalization and nuclear localization of translocated oncogenes in ectopic activation of their transcription is discussed.

Single-Cell Analysis and Next-Generation Immuno-Sequencing Show That Multiple Clones Persist in Patients with Chronic Lymphocytic Leukemia.

The immunoglobulin heavy chain (IGH) gene rearrangement in chronic lymphocytic leukemia (CLL) provides a unique molecular signature; however, we demonstrate that 26/198 CLL patients (13%) had more than one IGH rearrangement, indicating the power of molecular technology over phenotypic analysis. Single-cell PCR analysis and next-generation immuno-sequencing identified IGH-defined clones. In 23% (18/79) of cases whose clones carried unmutated immunoglobulin heavy chain variable (IGHV) genes (U-CLL), IGH rearrangements were bialleic with one productive (P) and one non-productive (NP) allele. Two U-CLL were biclonal, each clone being monoallelic (P). In 119 IGHV-mutated (M-CLL) cases, one had biallelic rearrangements in their CLL (P/NP) and five had 2-4 distinct clones. Allelic exclusion was maintained in all B-clones analyzed. Based on single-cell PCR analysis, 5/11 partner clones (45%) reached levels of >5x10(9) cells/L, suggesting second CLL clones. Partner clones persisted over years. Conventional IGH characterization and next-generation sequencing of 13 CLL, 3 multiple myeloma, 2 Waldenstrom's macroglobulinemia and 3 age-matched healthy donors consistently identified the same rearranged IGH sequences. Most multiple clones occurred in M-CLL, perhaps indicative of weak clonal dominance, thereby associating with a good prognosis. In contrast, biallelic CLL occurred primarily in U-CLL thus being associated with poor prognosis. Extending beyond intra-clonal diversity, molecular analysis of clonal evolution and apparent subclones in CLL may also reflect inter-clonal diversity.

CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability.

The CCR4-NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre-B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4-NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA.

Primary Posttransplant Plasmablastic Lymphoma of the Tongue: Report of a Case With Immunohistochemical and Molecular Studies.

Plasmablastic lymphoma is a rare, highly aggressive lymphoma characterized by large lymphoid cells with immunoblastic or plasmablastic features, absent expression of CD45 and CD20, positivity for CD138, and monoclonal rearrangement of the immunoglobulin heavy chain gene. It was originally reported in oral cavity in the setting of underlying human immunodeficiency viral infection but may occur also in lymph nodes or extranodal sites after transplantation and, more rarely, immunocompetent patients. Herein, we report a case of PBL presenting as an ulcerated lesion of the tongue in an HIV-negative patient, 6 years after renal transplantation. To date, only rare cases of plasmablastic lymphoma presenting after solid organ transplantation have been reported. Although a reduction of immunosuppression and an aggressive chemotherapy were performed, the patient died after a few months because of septic and cardiovascular complications.

Not all IGHV3-21 chronic lymphocytic leukemias are equal: prognostic considerations.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Clonal B-cell lymphocytosis exhibiting immunophenotypic features consistent with a marginal-zone origin: is this a distinct entity?

The biological and clinical significance of a clonal B-cell lymphocytosis with an immunophenotype consistent with marginal-zone origin (CBL-MZ) is poorly understood. We retrospectively evaluated 102 such cases with no clinical evidence to suggest a concurrent MZ lymphoma. Immunophenotyping revealed a clonal B-cell population with Matutes score ≤2 in all cases; 19/102 were weakly CD5 positive and all 35 cases tested expressed CD49d. Bone marrow biopsy exhibited mostly mixed patterns of small B-lymphocytic infiltration. A total of 48/66 (72.7%) cases had an abnormal karyotype. Immunogenetics revealed overusage of the IGHV4-34 gene and somatic hypermutation in 71/79 (89.8%) IGHV-IGHD-IGHJ gene rearrangements. With a median follow-up of 5 years, 85 cases remain stable (group A), whereas 17 cases (group B) progressed, of whom 15 developed splenomegaly. The clonal B-cell count, degree of marrow infiltration, immunophenotypic, or immunogenetic findings at diagnosis did not distinguish between the 2 groups. However, deletions of chromosome 7q were confined to group A and complex karyotypes were more frequent in group B. Although CBL-MZ may antedate SMZL/SLLU, most cases remain stable over time. These cases, not readily classifiable within the World Heath Organization classification, raise the possibility that CBL-MZ should be considered as a new provisional entity within the spectrum of clonal MZ disorders.

Expansion of CD27high plasmablasts in transverse myelitis patients that utilize VH4 and JH6 genes and undergo extensive somatic hypermutation.

Patients with the autoimmune disease multiple sclerosis (MS) typically present with the clinically isolated syndromes (CIS) transverse myelitis (TM) or optic neuritis (ON). B-cell disturbances have been well documented in patients with MS and CIS patients with ON, but not in CIS patients with TM, despite the fact that these patients have the worst clinical outcome of all CIS types. The goal of this study was to characterize the B-cell populations and immunoglobulin genetics in TM patients. We found a unique expansion of CD27(high) plasmablasts in both the cerebrospinal fluid and periphery of TM patients that is not present in ON patients. Additionally, plasmablasts from TM patients show evidence for positive selection with increased somatic hypermutation accumulation in VH4(+) B cells and receptor editing that is not observed in ON patients. These characteristics unique to TM patients may impact disease severity and progression.

Recirculating bone marrow B cells in C57BL/6 mice are more tolerant of highly hydrophobic and highly charged CDR-H3s than those in BALB/c mice.

To test whether mechanisms controlling the range of diversity of the developing antibody repertoire in C57BL/6 mice (IgH(b)) operate similarly to those identified in BALB/c mice (IgH(a)), we compared the sequences of VH 7183-containing H-chain transcripts from sorted adult bone marrow C57BL/6 B-cell subsets with those previously obtained from BALB/c mice. Patterns of VDJ gene segment utilization and CDR-H3 amino acid composition, charge, and average length in C57BL/6 pro-B cells were similar, although not identical, to BALB/c pro-B cells. However, C57BL/6 mature, recirculating B cells failed to demonstrate the reduction in the use of VH81X and the narrowing in the range of variance of CDR-H3 hydrophobicity that characterizes B-cell maturation in BALB/c mice. To further test the ability of the C57BL/6 strain to discard B cells expressing highly charged CDR-H3s, we introduced a mutant IgH(a) DH allele that forces use of arginine, asparagine, and histidine. Unlike BALB/c mice, C57BL/6 mice congenic for the charged DH maintained normal numbers of mature, recirculating B cells that were enriched for charged CDR-H3s. Together these findings indicate that the mature C57BL/6 B-cell pool permits expression of immunoglobulins with antigen-binding sites that are typically discarded during late-stage bone marrow B-cell development in BALB/c mice.

IL-7 functionally segregates the pro-B cell stage by regulating transcription of recombination mediators across cell cycle.

Ag receptor diversity involves the introduction of DNA double-stranded breaks during lymphocyte development. To ensure fidelity, cleavage is confined to the G(0)-G(1) phase of the cell cycle. One established mechanism of regulation is through periodic degradation of the RAG2 recombinase protein. However, there are additional levels of protection. In this paper, we show that cyclical changes in the IL-7R signaling pathway functionally segregate pro-B cells according to cell cycle status. In consequence, the level of a downstream effector of IL-7 signaling, phospho-STAT5, is inversely correlated with cell cycle expression of Rag, a key gene involved in recombination. Higher levels of phopho-STAT5 in S-G(2) correlate with decreased Rag expression and Rag relocalization to pericentromeric heterochromatin. These cyclical changes in transcription and locus repositioning are ablated upon transformation with v-Abl, which renders STAT5 constitutively active across the cell cycle. We propose that this activity of the IL-7R/STAT5 pathway plays a critical protective role in development, complementing regulation of RAG2 at the protein level, to ensure that recombination does not occur during replication. Our data, suggesting that pro-B cells are not a single homogeneous population, explain inconsistencies in the role of IL-7 signaling in regulating Igh recombination.

The normal IGHV1-69-derived B-cell repertoire contains stereotypic patterns characteristic of unmutated CLL.

The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought, and immunoglobulin gene analysis provides new clues. In the unmutated subset (U-CLL), there is increased usage of the 51p1-related alleles of the immunoglobulin heavy chain variable 1-69 gene, often combined with selected genes and with immunoglobulin heavy chain diversity IGHJ6. Stereotypic characteristics of the HCDR3 result and suggest antigen selection of the leukemic clones. We have now analyzed 51p1/IGHJ6 combinations in normal blood B cells from 3 healthy persons for parallel sequence patterns. A high proportion (33.3% of sequences) revealed stereotypic patterns, with several (15.0%) being similar to those described in U-CLL. Previously unreported CLL-associated stereotypes were detected in 4.8%. Stereotypes (13.6%) not detected in CLL also were found. The HCDR2-IGHJ6 sequences were essentially unmutated. Junctional amino acids in normal B cells were heterogeneous, as in cases of stereotyped CLL. Phenotypically, normal B cells expressing 51p1-derived immunoglobulin M were naive. This snapshot of the naive B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded immunoglobulin M of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL.

Replacement of Imu-Cmu intron by NeoR gene alters Imu germ-line expression but has no effect on V(D)J recombination.

The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3' regulatory region (3'RR). This polarized long-range effect of the 3'RR is disturbed upon insertion of NeoR gene in the IgH constant (C(H)) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Emu enhancer or Emu core enhancer (cEmu) by NeoR gene fully blocked V(D)J recombination and mu0 GL transcription which originates 5' of DQ52 and severely diminished Imu GL transcription derived from Emu/Imu promoter, suggesting a critical role for cEmu in the regulation of V(D)J recombination and of mu0 and Imu expression. Here we focus on the effect of NeoR gene on mu0 and Imu GL transcription in a mouse line in which the Imu-Cmu intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Emu. mu0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Imu transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Imu expression. Surprisingly, in stark contrast with the NeoR effect in the C(H) region, LPS-induced NeoR expression restored Imu transcript levels to normal. The data suggest that Emu enhancer may be the master control element that counteracts the down-regulatory "Neo effect" on Imu expression upon LPS stimulation. More importantly, they reveal a complex and developmentally regulated interplay between IgH enhancers in the control of Imu expression.

Sgamma3 switch sequences function in place of endogenous Sgamma1 to mediate antibody class switching.

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cmu exons with a set of downstream IgH constant region (C(H)) exons. Individual sets of C(H) exons are flanked upstream by long (1-10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Smu region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region-specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sgamma1 region was replaced with wild-type (WT) or synthetic 2-kb Sgamma3 sequences or a synthetic 2-kb Sgamma1 sequence. We found that both the inserted endogenous and synthetic Sgamma3 sequences functioned similarly to a size-matched synthetic Sgamma1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sgamma3 can function similarly to Sgamma1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype-specific functions of other endogenous S regions.

Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus.

Immunoglobulin variable region exons are assembled from discontinuous variable (V), diversity (D), and joining (J) segments by the process of V(D)J recombination. V(D)J rearrangements of the immunoglobulin heavy chain (IgH) locus are tightly controlled in a tissue-specific, ordered and allele-specific manner by regulating accessibility of V, D, and J segments to the recombination activating gene proteins which are the specific components of the V(D)J recombinase. In this review we discuss recent advances and established models brought forward to explain the mechanisms underlying accessibility control of V(D)J recombination, including research on germline transcripts, spatial organization, and chromatin modifications of the immunoglobulin heavy chain (IgH) locus. Furthermore, we review the functions of well-described and potential new cis-regulatory elements with regard to processes such as V(D)J recombination, allelic exclusion, and IgH class switch recombination.

Imunofenotipagem e rearranjo gênico em doenças pulmonares linfocíticas e linfoproliferativas / Immunophenotyping and gene rearrangement analysis in lymphoid/lymphoproliferative disorders of the lungs

OBJETIVO: Determinar a utilidade, na prática rotineira, da análise da clonalidade dos linfócitos T e B nos tecidos pulmonares por reação em cadeia da polimerase no diagnóstico das doenças linfoproliferativas pulmonares. MÉTODOS: Avaliaram-se, mediante análise imunohistoquímica e rearranjo molecular dos genes, 8 casos de pneumonia intersticial linfocítica (PIL) e 7 casos de doenças linfoproliferativas pulmonares. RESULTADOS: Todos os 8 casos de PIL expressaram imunocoloração moderada a forte para CD3, em contraste com apenas 2 casos de linfoma e 1 caso de pseudolinfoma. Rearranjo gênico foi detectado em 4 de 8 casos de PIL, o que mudou o diagnóstico de PIL para linfoma, indicando, assim, a importância da detecção de rearranjo gênico em casos de PIL. Nesta situação, rearranjo gênico usando-se os pares de primers VH/JH e Vgama11/Jgama12 foi detectado em 3 e 1 casos de PIL, respectivamente, e não foram detectadas anormalidades gênicas usando-se as pares Dbeta1/Jbeta2 e Vgama101/Jgama12. Uma associação positiva foi detectada entre a intensidade de imunoexpressão CD20 e CD68 e rearranjo gênico usando-se o par de primers VH/JH. Antes do rearranjo gênico, 4 pacientes com PIL morreram rapidamente, enquanto que, após o rearranjo gênico, apenas 1 paciente com PIL morreu. CONCLUSÕES: A detecção de células B e T monoclonais por imunofenotipagem e reação em cadeia da polimerase mostrou impacto no diagnóstico de linfomas pulmonares em pacientes previamente diagnosticados com PIL. Portanto, imunofenotipagem e reação em cadeia da polimerase devem ser incluídas como métodos de 'padrão ouro' na rotina diagnóstica.

OBJECTIVE: To determine the usefulness, in routine practice, of using polymerase chain reaction to analyze B and T lymphocyte clonality in pulmonary tissue as a tool for the diagnosis of pulmonary lymphoproliferative disorders. METHODS: Immunohistochemistry and molecular gene rearrangement analysis were performed in order to assess 8 cases of lymphoid interstitial pneumonia (LIP) and 7 cases of pulmonary lymphoproliferative disorders. RESULTS: All 8 cases of LIP presented moderate to strong immunostaining for CD3, compared with only 2 cases of lymphoma and 1 case of pseudolymphoma (p = 0.02). Gene rearrangement was detected in 4 of the 8 cases, which changed the diagnosis from LIP to lymphoma, showing the importance of gene rearrangement detection in cases of LIP. In this situation, gene rearrangement using the VH/JH and Vgamma11/Jgamma12 primer pairs was detected in 3 cases and 1 case, respectively, and no gene abnormalities were found using the Dbeta1/Jbeta2 and Vgamma101/Jgamma12 primer pairs in any of the cases. A significant positive association was found between the intensity of CD20 and CD68 expression and gene rearrangement using the VH/JH primer pair. Prior to the gene rearrangement, 4 patients with LIP died quickly, whereas only one patient with LIP died after the gene rearrangement. CONCLUSIONS: Detection of monoclonal B and T cells by immunophenotyping and polymerase chain reaction had an impact on the diagnosis of pulmonary lymphomas in patients previously diagnosed with LIP. Therefore, immunophenotyping and polymerase chain reaction should be used as 'gold standard' techniques in routine practice.

Models for antigen receptor gene rearrangement: CDR3 length.

Despite the various processing steps involved in V(D)J recombination, which could potentially introduce many biases in the length distribution of complementarity determining region 3 (CDR3) segments, the observed CDR3 length distributions for complete repertoires are very close to a normal-like distribution. This raises the question of whether this distribution is simply a result of the random steps included in the process of gene rearrangement, or has been optimized during evolution. We have addressed this issue by constructing a simulation of gene rearrangement, which takes into account the DNA modification steps included in the process, namely hairpin opening, nucleotide additions, and nucleotide deletions. We found that the near-Gaussian- shape of CDR3 length distribution can only be obtained under a relatively narrow set of parameter values, and thus our model suggests that specific biases govern the rearrangement process. In both B-cell receptor (BCR) heavy chain and T-cell receptor beta chain, we obtained a Gaussian distribution using identical parameters, despite the difference in the number and the lengths of the D segments. Hence our results suggest that these parameters most likely reflect the optimal conditions under which the rearrangement process occurs. We have subsequently used the insights gained in this study to estimate the probability of occurrence of two exactly identical BCRs over the course of a human lifetime. Whereas identical rearrangements of the heavy chain are highly unlikely to occur within one human lifetime, for the light chain we found that this probability is not negligible, and hence the light chain CDR3 alone cannot serve as an indicator of B-cell clonality.

[Immunophenotyping and gene rearrangement analysis in lymphoid/lymphoproliferative disorders of the lungs]. / Imunofenotipagem e rearranjo gênico em doenças pulmonares linfocíticas e linfoproliferativas.

OBJECTIVE: To determine the usefulness, in routine practice, of using polymerase chain reaction to analyze B and T lymphocyte clonality in pulmonary tissue as a tool for the diagnosis of pulmonary lymphoproliferative disorders. METHODS: Immunohistochemistry and molecular gene rearrangement analysis were performed in order to assess 8 cases of lymphoid interstitial pneumonia (LIP) and 7 cases of pulmonary lymphoproliferative disorders. RESULTS: All 8 cases of LIP presented moderate to strong immunostaining for CD3, compared with only 2 cases of lymphoma and 1 case of pseudolymphoma (p = 0.02). Gene rearrangement was detected in 4 of the 8 cases, which changed the diagnosis from LIP to lymphoma, showing the importance of gene rearrangement detection in cases of LIP. In this situation, gene rearrangement using the VH/JH and Vgamma11/Jgamma12 primer pairs was detected in 3 cases and 1 case, respectively, and no gene abnormalities were found using the Dbeta1/Jbeta2 and Vgamma101/Jgamma12 primer pairs in any of the cases. A significant positive association was found between the intensity of CD20 and CD68 expression and gene rearrangement using the VH/JH primer pair. Prior to the gene rearrangement, 4 patients with LIP died quickly, whereas only one patient with LIP died after the gene rearrangement. CONCLUSIONS: Detection of monoclonal B and T cells by immunophenotyping and polymerase chain reaction had an impact on the diagnosis of pulmonary lymphomas in patients previously diagnosed with LIP. Therefore, immunophenotyping and polymerase chain reaction should be used as 'gold standard' techniques in routine practice.

Spontaneous regression of intraoral mucosa-associated lymphoid tissue lymphoma: molecular study of a case.

Mucosa-associated lymphoid tissue (MALT) lymphoma presentation in the oral cavity is very rare. Reported herein is a case of intraoral MALT lymphoma of the minor salivary gland in a 70-year-old woman with Sjogren's syndrome. Unexpectedly, a spontaneous clinically and histologically confirmed regression occurred 1 month after the tumor biopsy for diagnosis. Considering that salivary MALT lymphoma is associated with Sjogren's syndrome and that the chronic inflammation caused by Sjogren's syndrome persisted, it is hypothesized that the tumor clone might be present in the regressed lesion. Minimal residual tumor clone identical with the primary lesion was detected using the polymerase chain reaction (PCR) clonality assay for immunoglobulin heavy chain gene (IgH) rearrangement. No recurrence was clinically evident 38 months after the diagnosis. Spontaneous regression of MALT lymphoma should be examined at the molecular level in addition to clinical and histological evaluations. When minimal residual disease is detected, close follow up is necessary for early detection of the tumor relapse.

The origin of B-cell chronic lymphocytic leukemia.

An immunobiologic approach has led to substantial changes in our current view of chronic lymphocytic leukemia (CLL). Several questions remain unsolved and the definition of the cell origin of CLL is still prominent. The presence of somatic mutations of IGHV genes indicates that, at least in a portion of cases, CLL cells had encountered an antigen during the natural history of the disease. Unmutated (UM) cases show a remarkable skewing in IGHV gene usage. In addition, all CLL cases, both mutated (M) and UM, show a common surface phenotype which is significantly activated and similar to the surface phenotype of antigen (Ag)-experienced B cells. The properties of CLL B-cell receptors (BCR) resemble those observed in normal B cells upon Ag interaction, and gene profiling analyses revealed that both subsets share striking similarities with the so-called memory B cells. The detailed analyses of the complementary determining region 3 (CDR3) sequences of the leukemic immunoglobulin (Ig) receptors showed that unrelated patients in different parts of the world express very similar if not identical BCR. Remarkably, similar V(H)DJ(H) rearrangements have been identified in both UM- and M-CLL, suggesting an antigenic selection in both subsets of the disease. From all this evidence, the concept has arisen that the cell of origin, regardless its mutational status, has to be "an Ag-experienced" B cell that gives rise to a malignant clone that appears to be more dynamic than previously appreciated and whose progression is favored by a number of molecular and cellular interactions that occur in tissues.

An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion.

The Ig heavy (H) chain plays a pivotal role in the regulation of primary B cell development through its association with a variety of other proteins including Igalpha and Igbeta, the surrogate light chain components and bona fide L chains, to form transmembrane signaling complexes. Little is known about how alterations in the structure of the H chain variable region influence association with these proteins, or the signaling capacity of the complexes that form. Here we describe a line of VH 'knockin' mice in which the transgene-encoded VH region differs by eight amino acid residues from the VH region in a VH knockin line we previously constructed and characterized. The transgenic H chain locus in the line of mice we characterized earlier efficiently promotes H chain allelic exclusion and all phases of primary B cell development, resulting in the generation of mature B1, marginal zone (MZ) and follicular (FO) B cell compartments. In contrast, the transgenic H chain locus in the new line fails to enforce allelic exclusion, as evidenced by the majority of peripheral B cells expressing two H chains on their surfaces. Moreover, this locus inefficiently drives bone marrow B lymphopoiesis and FO B cell development. However, this H chain locus does promote MZ B cell development, from precursors that appear to be generated during fetal and neonatal life. We discuss these data in the context of previous findings on the influence of Ig H chain structure on primary B cell development.

Absence of immunoglobulin class switch in primary lymphomas of the central nervous system.

Primary lymphomas of the central nervous system (PCNSLs) were investigated for their capacity to perform further maturation steps. We studied a series of 11 PCNSLs derived from immunocompetent patients for immunoglobulin (Ig) class switch recombination (CSR) by performing reverse transcriptase-polymerase chain reaction (RT-PCR) for transcripts of Ig constant region gene segments (IGHC). This analysis revealed exclusive transcription of IgM and IgD mRNA in the absence of IgG, IgA, or IgE transcription. This finding was corroborated at the protein level by the immunohistochemical demonstration of IgM on the surface of the tumor cells. The unexpected lack of CSR may be due to internal switch mu region deletions, which were detected in 7 of 11 cases. We also found that expression of activation-induced cytidine deaminase (AID), which is required for CSR and somatic hypermutation, was detectable by RT-PCR in 4 of 10 cases and by immunohistochemistry in one of three cases analyzed. This may indicate that ongoing somatic mutation, which is often observed in PCNSL, could be due to sustained AID expression in a fraction of cases and that intraclonal V gene diversity may occur in other cases at an earlier phase of tumor clone expansion, when AID may have been expressed.