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The diagnostic approach of monoclonal gammopathy of renal significance is based on the detection of a monoclonal immunoglobulin in the blood and urine, and the identification of the underlying clone through bone marrow and/or peripheral blood cytologic and flow cytometry analysis. However, the monoclonal component and its corresponding clone may be undetectable using these routine techniques. Since clone identification is the cornerstone for guiding therapy and assessing disease response, more sensitive methods are required. We recently developed a high-throughput sequencing assay from bone marrow mRNA encoding immunoglobulins (RACE-RepSeq). This technique provides both full-length V(D)J region (variable, diversity and joining genes that generate unique receptors as antigen receptors) of the monoclonal immunoglobulin and the dominant immunoglobulin repertoire. This allows analysis of mutational patterns, immunoglobulin variable gene frequencies and diversity due to somatic hypermutation. Here, we evaluated the diagnostic performance of RACE-RepSeq in 16 patients with monoclonal-associated kidney lesions, and low serum monoclonal immunoglobulin and free light chain levels at diagnosis. Bone marrow immunohistochemical analysis was negative in all 11 patients so tested and 7 of 12 patients had no detectable clone matching the kidney deposits using flow cytometry analysis. By contrast, RACE-RepSeq detected a dominant clonal light chain sequence of matched isotype with respect to kidney deposits in all patients. Thus, high throughput mRNA sequencing appears highly sensitive to detect subtle clonal disorders in monoclonal gammopathy of renal significance and suggest this novel approach could help improve the management of this kidney disease.
Assuntos
Nefropatias , Paraproteinemias , Humanos , Cadeias Leves de Imunoglobulina , Rim/patologia , Nefropatias/diagnóstico , Nefropatias/genética , Nefropatias/terapia , Paraproteinemias/diagnóstico , Paraproteinemias/genética , Paraproteinemias/terapia , RNARESUMO
Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear, but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid, and specific tool to detect low-abundance PC clones in BM and assign them to POEMS syndrome, with all the consequences for therapeutic options.
Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias lambda de Imunoglobulina/genética , Síndrome POEMS/genética , Biópsia , Medula Óssea/metabolismo , Medula Óssea/patologia , Mutação em Linhagem Germinativa , Humanos , Cadeias Leves de Imunoglobulina/análise , Cadeias Leves de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/análise , Linfonodos/metabolismo , Linfonodos/patologia , Técnicas de Diagnóstico Molecular/métodos , Síndrome POEMS/patologia , Análise de Sequência de ProteínaRESUMO
As a master regulator of functional Ig heavy chain (IgH) expression, the IgH 3' regulatory region (3'RR) controls multiple transcription events at various stages of B-cell ontogeny, from newly formed B cells until the ultimate plasma cell stage. The IgH 3'RR plays a pivotal role in early B-cell receptor expression, germ-line transcription preceding class switch recombination, interactions between targeted switch (S) regions, variable region transcription before somatic hypermutation, and antibody heavy chain production, but the functional ranking of its different elements is still inaccurate, especially that of its evolutionarily conserved quasi-palindromic structure. By comparing relevant previous knockout (KO) mouse models (3'RR KO and hs3b-4 KO) to a novel mutant devoid of the 3'RR quasi-palindromic region (3'PAL KO), we pinpointed common features and differences that specify two distinct regulatory entities acting sequentially during B-cell ontogeny. Independently of exogenous antigens, the 3'RR distal part, including hs4, fine-tuned B-cell receptor expression in newly formed and naïve B-cell subsets. At mature stages, the 3'RR portion including the quasi-palindrome dictated antigen-dependent locus remodeling (global somatic hypermutation and class switch recombination to major isotypes) in activated B cells and antibody production in plasma cells.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Formação de Anticorpos , Antígenos/metabolismo , Linfócitos B/metabolismo , Contagem de Células , Linhagem da Célula , Citometria de Fluxo , Marcação de Genes , Centro Germinativo/metabolismo , Heterozigoto , Switching de Imunoglobulina/genética , Imunoglobulina M/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Deleção de Sequência , Hipermutação Somática de Imunoglobulina/genética , Transcrição GênicaRESUMO
[This corrects the article DOI: 10.3389/fimmu.2024.1391404.].
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Introduction: Follicular Lymphoma (FL) results from the malignant transformation of germinal center (GC) B cells. FL B cells display recurrent and diverse genetic alterations, some of them favoring their direct interaction with their cell microenvironment, including follicular helper T cells (Tfh). Although FL-Tfh key role is well-documented, the impact of their regulatory counterpart, the follicular regulatory T cell (Tfr) compartment, is still sparse. Methods: The aim of this study was to characterize FL-Tfr phenotype by cytometry, gene expression profile, FL-Tfr origin by transcriptomic analysis, and functionality by in vitro assays. Results: CD4+CXCR5+CD25hiICOS+ FL-Tfr displayed a regulatory program that is close to classical regulatory T cell (Treg) program, at the transcriptomic and methylome levels. Accordingly, Tfr imprinting stigmata were found on FL-Tfh and FL-B cells, compared to their physiological counterparts. In addition, FL-Tfr co-culture with autologous FL-Tfh or cytotoxic FL-CD8+ T cells inhibited their proliferation in vitro. Finally, although FL-Tfr shared many characteristics with Treg, TCR sequencing analyses demonstrated that part of them derived from precursors shared with FL-Tfh. Discussion: Altogether, these findings uncover the role and origin of a Tfr subset in FL niche and may be useful for lymphomagenesis knowledge and therapeutic management.
Assuntos
Linfoma Folicular , Linfócitos T Reguladores , Linfoma Folicular/imunologia , Linfoma Folicular/genética , Linfoma Folicular/patologia , Humanos , Linfócitos T Reguladores/imunologia , Perfilação da Expressão Gênica , Transcriptoma , Microambiente Tumoral/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células T Auxiliares Foliculares/imunologia , Células T Auxiliares Foliculares/metabolismo , Masculino , Feminino , Técnicas de Cocultura , Centro Germinativo/imunologiaRESUMO
Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3' cis-regulatory region (3'RR). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells.
Assuntos
Linfócitos B , Suicídio , Camundongos , Animais , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Switching de Imunoglobulina/genética , Antígenos/metabolismoRESUMO
Upregulated expression of the anti-apoptotic BCL2 oncogene is a common feature of various types of B-cell malignancies, from lymphoma to leukemia or myeloma. It is currently unclear how the various patterns of deregulation observed in pathology eventually impact the phenotype of malignant B cells and their microenvironment. Follicular lymphoma (FL) is the most common non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells, and its major hallmark is the t(14:18) translocation occurring in B cell progenitors and placing the BCL2 gene under the control of the immunoglobulin heavy chain locus regulatory region (IgH 3'RR), thus exposing it to constitutive expression and hypermutation. Translocation of BCL2 onto Ig light chain genes, BCL2 gene amplification, and other mechanisms yielding BCL2 over-expression are, in contrast, rare in FL and rather promote other types of B-cell lymphoma, leukemia, or multiple myeloma. In order to assess the impact of distinct BCL2 deregulation patterns on B-cell fate, two mouse models were designed that associated BCL2 and its full P1-P2 promoter region to either the IgH 3'RR, within a "3'RR-BCL2" transgene mimicking the situation seen in FL, or an Ig light chain locus context, through knock-in insertion at the Igκ locus ("Igκ-BCL2" model). While linkage to the IgH 3' RR mostly yielded expression in GC B-cells, the Igκ-driven up-regulation culminated in plasmablasts and plasma cells, boosting the plasma cell in-flow and the accumulation of long-lived plasma cells. These data demonstrate that the timing and level of BCL2 deregulation are crucial for the behavior of B cells inside GC, an observation that could strongly impact the lymphomagenesis process triggered by secondary genetic hits.
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Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.
Assuntos
Diferenciação Celular/imunologia , Imunoglobulina M/imunologia , Mutação de Sentido Incorreto , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas de Neoplasias/imunologia , Plasmócitos/imunologia , Substituição de Aminoácidos , Animais , Diferenciação Celular/genética , Humanos , Imunoglobulina M/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/genética , Plasmócitos/patologiaRESUMO
The immunoglobulin heavy chain (IgH) 3' regulatory region (3'RR) superenhancer controls B2 B-cell IgH transcription and cell fate at the mature stage but not early repertoire diversity. B1 B cells represent a small percentage of total B cells differing from B2 B cells by several points such as precursors, development, functions, and regulation. B1 B cells act at the steady state to maintain homeostasis in the organism and during the earliest phases of an immune response, setting them at the interface between innate and acquired immunity. We investigated the role of the 3'RR superenhancer on B1 B-cell fate. Similar to B2 B cells, the 3'RR controls µ transcription and cell fate in B1 B cells. In contrast to B2 B cells, 3'RR deletion affects B1 B-cell late repertoire diversity. Thus, differences exist for B1 and B2 B-cell 3'RR control during B-cell maturation. For the first time, these results highlight the contribution of the 3'RR superenhancer at this interface between innate and acquired immunity.
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Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Sequências Reguladoras de Ácido Nucleico/fisiologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linhagem Celular , Genes de Cadeia Pesada de Imunoglobulina/genética , Imunidade Inata , Camundongos , Análise de Sequência de DNA , Transcrição Gênica , Recombinação V(D)JRESUMO
The four transcriptional enhancers located in the 3' regulatory region (3'RR) of the IgH locus control the late phases of B-cell maturation, namely IgH locus transcription, somatic hypermutation and class switch recombination. Doctor Jekyll by nature, the 3'RR acts as Mister Hyde in case of oncogenic translocation at the IgH locus taking under its transcriptional control the translocated oncogene. The aim of this review is to show this duality on the basis of the latest scientific advances in the structure and function of the 3'RR and to hIghlight the targeting of the 3'RR as a potential therapeutic approach in mature B-cell lymphomas.
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Linfócitos B/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/fisiologia , Linfoma/patologia , Sequências Reguladoras de Ácido Nucleico/fisiologia , Animais , Linfócitos B/patologia , Carcinogênese/genética , Humanos , Linfoma/tratamento farmacológico , Linfopoese/genética , Fatores de Transcrição/fisiologiaRESUMO
Deregulation and mutations of c-myc have been reported in multiple mature B-cell malignancies such as Burkitt lymphoma, myeloma and plasma cell lymphoma. After translocation into the immunoglobulin heavy chain (IgH) locus, c-myc is constitutively expressed under the control of active IgH cis-regulatory enhancers. Those located in the IgH 3' regulatory region (3'RR) are master control elements of transcription. Over the past decade numerous convincing demonstrations of 3'RR's contribution to mature c-myc-induced lymphomagenesis have been made using transgenic models with various types of IgH-c-myc translocations and transgenes. This review highlights how IgH 3'RR physiological functions play a critical role in c-myc deregulation during lymphomagenesis.
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Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-myc/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Transcrição Gênica , Translocação GenéticaRESUMO
Functional B-cells are essential for the formation of oil granulomas. The IgH 3' regulatory region (3'RR) activates important check-points during B-cell maturation. We investigated if 3'RR-deficient B-cells remain efficient to develop oil granulomas in response to pristine. B-cells expressing an IgH 3'RR-deficient allele were similarly recruited to wild type allele expressing B-cells in the granuloma. No differences were observed between 3'RR-deficient mice and control mice for granuloma numbers, cellular composition and ability to express mRNA transcripts for several pro- and anti-inflammatory cytokines. Altogether these results suggest a normal role for 3'RR-deficient B-cells in the development of an acute B-cell-mediated inflammatory response.
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Linfócitos B/citologia , Granuloma/patologia , Cadeias Pesadas de Imunoglobulinas/genética , Alelos , Animais , Anti-Inflamatórios/química , Linfócitos B/imunologia , Feminino , Heterozigoto , Humanos , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Baço/metabolismoRESUMO
The IgH 3' regulatory region (3'RR) controls class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The mouse 3'RR contains four enhancer elements with hs1,2 flanked by inverted repeated sequences and the centre of a 25-kb palindrome bounded by two hs3 enhancer inverted copies (hs3a and hs3b). hs4 lies downstream of the palindrome. In mammals, evolution maintained this unique palindromic arrangement, suggesting that it is functionally significant. Here we report that deconstructing the palindromic IgH 3'RR strongly affects its function even when enhancers are preserved. CSR and IgH transcription appear to be poorly dependent on the 3'RR architecture and it is more or less preserved, provided 3'RR enhancers are present. By contrast, a 'palindromic effect' significantly lowers VH germline transcription, AID recruitment and SHM. In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance.
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Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Sequências Repetidas Invertidas , Sequências Reguladoras de Ácido Nucleico , Animais , Feminino , Switching de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The IgH 3'regulatory region (3'RR), encompassing the four transcriptional enhancers hs3a-hs1,2-hs3b-hs4, has a key role on class switch recombination, somatic hypermutation, IgH transcription and B-cell fate. In plasma cells, transcribed IgH and IgL loci often colocalized in transcription factories and an IgL transcription defect might translate into lowered IgH transcription. We explored whether the 3'RR would affect lymphomagenesis in Igλ-Myc transgenic mice prone to lymphoproliferations. Breeding Igλ-Myc transgenics in a background deficient for the 3'RR influences lymphomagenesis toward less mature lymphomas (16% vs 54%, p = 0.01, Z test for two population proportions). In a 3'RR-deficient background mature tumors less often expressed the CD43 antigen (54% vs 0%, p = 0.02), a membrane glycoprotein expressed on activated mature B-cells. In contrast, in a 3'RR-deficient background tumors more often expressed the CD5 antigen (32% vs 12%, p = 0.05) that may serve to control autoimmunity and that is suspected to play a role in leukemic transformation. Lymphoma myc transcript levels, the Ki67 index of proliferation, the clonality, the usage of V(D)J segments, and their somatic hypermutation status were not affected in the 3'RR-deficient background. In conclusion, most probably through its action during the maturation process, the 3'RR can influence lymphomagenesis even when not linked with an oncogene.
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Linfócitos B/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Antígeno Ki-67/metabolismo , Linfoma/imunologia , Animais , Cadeias Pesadas de Imunoglobulinas/metabolismo , Antígeno Ki-67/análise , Camundongos , Camundongos TransgênicosRESUMO
In mature B cells, class switch recombination (CSR) replaces the expressed constant Cµ gene with a downstream C(H) gene. How the four transcriptional enhancers of the IgH 3' regulatory region (3'RR) control CSR remains an open question. We have investigated IgG1 CSR in 3'RR-deficient mice. Here we show that the 3'RR enhancers target the S(γ1) acceptor region (and poorly the S(µ) donor region) by acting on epigenetic marks, germline transcription, paused RNA Pol II recruitment, R loop formation, AID targeting and double-strand break generation. In contrast, location and diversity of S(µ)-S(γ1) junctions are not affected by deletion of the 3'RR enhancers. Thus, the 3'RR controls the first steps of CSR by priming the S acceptor region but is not implicated in the choice of the end-joining pathway.
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Cadeias Pesadas de Imunoglobulinas/genética , Sequências Reguladoras de Ácido Nucleico/fisiologia , Baço/citologia , Animais , Clonagem Molecular , Epigênese Genética , Feminino , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Região de Troca de Imunoglobulinas , Masculino , Camundongos , Proteínas Recombinantes , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
We report that the IgH 3' regulatory region (3'RR) has no role on µ chain transcription and pre-BCR expression in B cell progenitors. In contrast, analysis of heterozygous IgH aΔ3'RR/bwt mice indicated that the 3'RR controls µ chain transcripts in mature splenocytes and impacts membrane IgM density without obvious effect on BCR signals (colocalisation with lipid rafts and phosphorylation of Erk and Akt after BCR crosslinking). Deletion of the 3'RR modulates the B cell fate to less marginal zone B cells. In conclusion, the 3'RR is dispensable for pre-BCR expression and necessary for optimal commitments toward the marginal zone B cell fate. These results reinforce the concept of a dual regulation of the IgH locus transcription and accessibility by 5' elements at immature B cell stages, and by the 3'RR as early as the resting mature B cell stage and then along further activation and differentiation.
Assuntos
Linfócitos B/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias mu de Imunoglobulina/genética , Animais , Linfócitos B/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias mu de Imunoglobulina/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sequências Reguladoras de Ácido NucleicoRESUMO
The IgH 3' regulatory region (3'RR), encompassing the four transcriptional enhancers hs3a-hs1,2-hs3b-hs4, is a potent lymphoma oncogene deregulator but its role in B cell-mediated inflammatory responses is unknown. We investigated the 3'RR involvement in the in vivo pristane-induced inflammatory response in BALB/c mice. The lack of the 3'RR in BALB/c mice had no wide effect on the incidence, the kinetic of development and the cellular composition of peritoneal ascites. Ascite pro-inflammatory cytokines levels (IL-6, IL-21, IL-12/23, TNF-α) were unchanged while anti-inflammatory cytokines levels (IL-10, interferon-γ) were slightly increased in 3'RR-deficient BALB/c mice as compared to wt BALB/c mice. In conclusion, the 3'RR is dispensable for the efficient recruitment of immune cells and the normal development of an inflammatory response in the in vivo pristane-induced inflammatory model. The 3'RR might be considered as a potential suitable target for anti-lymphoma pharmacological therapy without potent adverse effect on normal immune and inflammatory responses.