Immunoglobulin Gene Repertoire in Ocular Adnexa Lymphomas (OAL): Hints on the Nature of the Antigenic Stimulation

Abstract OALs arising in the ocular adnexae account for 15% of all extranodal lymphomas and are mostly represented by mucosa-associated lymphoid tissue (MALT)-type lymphomas (OAML). OAML have been described to be significantly associated, although with a variable geographic distribution, with Chlamydiophila psittaci (CP) infection, the etiologic agent of human psittacosis. Though the role played by Chlamydiae in OAML pathogenesis is also strongly supported by the observation of clinical remissions after bacterial eradication alone, the actual mechanisms have not been yet elucidated. Previous analysis of the Immunoglobulin Heavy Chain Variable (IGHV) genes in OAML showed a restricted repertoire with mutated genes and ongoing somatic mutations, thereby alluding to an origin from an antigen–experienced B cell, which has undergone antigen-selection; however, data on the potential antigenic elements stimulating OAML B lymphocytes are not available. In order to get hints on the potential ligands, we aimed at investigating the Immunoglobulin structure and, in particular, the molecular features of the antigen binding site (i.e. the HCDR3) of IG genes expressed by OAML. We analyzed the monoclonal IGHV-D-J rearrangements in a series of 30 OAML cases. IGHV3 subgroup genes predominated (24/30, 80%), followed by IGHV4 (4/30, 13.3%) and IGHV1 (2/30, 6,7%) subgroup genes, in keeping with the normal B cell IG gene repertoire. According to the 98% identity cut-off value, 23/30 sequences (76,7%) were defined as “mutated”, with a percentage of identity ranging between 89.3% and 97.7%, whereas the remainder (7/30 sequences; 23.3%) had “unmutated” IGHV genes. At individual gene level, IGHV3-23 and IGHV3-43 were the most frequently used genes (6/30 sequences, 20%, and 4/30 sequences, 13,3%, respectively); interestingly, the latter is rarely used in the normal repertoire. No significant correlations were found between IGHV gene usage and CP infection. HCDR3 length ranged from 9 to 30 amino acids (median 16) and 20/28 HCDR3 sequences showed an isoelectric point (pI) value >6.0, (in 10/28 pI>8.0), a feature shared with antibodies with anti–DNA reactivity. Shared epitope recognition was excluded by cluster analysis of HCDR3 sequences, also among OAML cases using the same IGHV genes. When we aligned HCDR3 sequences to a panel of 8214 unique IGHVD- J sequences from various types of normal, autoreactive or malignant B cell clones, we observed significant similarity between 3 OAML cases and 2 auto-reactive antibodies, present in rheumatoid arthritis and Sjoegren’s syndrome, respectively, and 1 antibody with anti-thyroid peroxidase (TPO) reactivity. The latter case showed a HCDR3 similarity also with the IG gene of a Chronic Lymphocytic Leukemia case, a disease characterized by frequent expression of auto-reactive Igs. Interestingly, the same search performed using anti-Chlamydia antibodies did not show any significant similarity. In conclusion, these results suggest that OAML express a distinctive IG gene repertoire and may originate from B cells selected for their capacity to bind to auto-antigens in a similar way to what reported for gastric MALT lymphomas, where malignant B cells, instead of directly reacting against Helicobacter Pylori, are actually stimulated by tissue auto-antigens exposed during the inflammatory reaction, as confirmed by molecular analyses of antigen binding sites of the monoclonal IG. Our findings support the hypothesis that stimulation by auto-antigens, likely generated within an inflammatory background promoted by Chlamydial infection, may be the driving force also in OAML pathogenesis, rather than a direct action of the bacteria on B cells.

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The Immunoglobulin Gene Repertoire of Low-Count CLL-Like MBL Is Different from CLL: Diagnostic Considerations and Implications for Clinical Monitoring

Blood ◽

10.1182/blood.v112.11.779.779 ◽

2008 ◽

Vol 112 (11) ◽

pp. 779-779

Author(s):

Antonis Dagklis ◽

Claudia Fazi ◽

Cinzia Sala ◽

Valeria Cantarelli ◽

Cristina Scielzo ◽

...

Keyword(s):

B Cells ◽

General Population ◽

B Cell ◽

B Lymphocytes ◽

Low Frequency ◽

Clinical Monitoring ◽

Gene Repertoire ◽

Healthy Individuals ◽

Molecular Features ◽

Ighv Gene

Abstract The term Monoclonal B Lymphocytosis (MBL) defines the presence of Monoclonal B Lymphocytes in the blood of otherwise healthy individuals. Though phenotypically heterogeneous, most MBL cases resemble CLL cells (CD5+, CD20dim, CD79b dim, sIgdim). The interest in MBL increased after this entity was included in the revised NCI-WG/IWCLL guidelines for the diagnosis and management of CLL (Hallek et al, 2008) and defined as “the presence of fewer than 5×109/L B lymphocytes” in the peripheral blood. However, the concentration of MBL in the blood of any given individual is extremely variable accounting in some cases for the vast majority of circulating B cells while being a negligible portion of them in others. It is then plausible that molecular differences could exist between low- vs. high-count MBL, being the latter likely more advanced on the way to become CLL. In this context, it was recently reported that subjects with <5×109/L CLL-like MBL but with lymphocytosis will require treatment at a rate of 1.1% per year. The absolute B-cell count turned out to be the only independent prognostic factor associated with progressive lymphocytosis, as all MBL cases studied had immunoglobulin (IG) gene features and cytogenetic abnormalities similar to good prognosis CLL. In contrast, very little is known about low-count MBL cases accidentally found in the general population. By cytofluorograph analysis, we identified 89 CLL-like MBL in the blood of 1725 healthy individuals >18 years old (5.1%) and analyzed the IGHV-D-J rearrangements expressed by 51 of them, the majority being characterized by few clonal B cells (mean 6.9% of circulating B lymphocytes, with only 13 cases >10%). CLL-like MBL cells showed a predominance of IGHV3 genes, followed by IGHV4 genes, resembling the normal repertoire. The most frequent IGHV gene in MBL was IGHV4- 59/61, rarely used in CLL. The MBL repertoire was also conspicuous for the lack of the IGHV1-69 gene (the predominant gene in unmutated CLL, ~25%) and the low frequency of the IGHV4-34 gene (2/51 sequences, 3.9%), the most frequent gene in mutated CLL (~12%). Following the 98% identity cut-off value, 36/51 sequences (70.5%) were defined as “mutated”, whereas the remainder had “unmutated” IGHV genes. Alignment of MBL HCDR3 sequences to a comprehensive panel of CLL HCDR3 sequences identified 2/51 (3.9%) MBL cases with a sequence similar to previously described CLL cases (“stereotyped receptors”). These results show that the IG gene repertoire in low-count MBL, accidentally found in the general population, does not show the typical CLL-related biases in terms of IGHV gene usage. This cannot be simply explained by the higher number of mutated cases among MBL, as unmutated cases account for almost a third of CLL-like MBL, indicating a molecular heterogeneity. In addition, HCDR3 stereotypy in MBL is significantly less frequent than in CLL (>25% of cases). Occasional MBL may indeed express a CLL “stereotyped receptor”, implying that the potential to evolve into a leukemia exists within MBL, though at low frequency, and may depend on precise selection mechanisms based on the molecular features of the B cell receptor. Taken together, our results strongly suggest that the detection of MBL in an otherwise healthy subject is not always equivalent to a pre-leukemic state. Differential IG molecular features might provide a better tool to discriminate individuals at risk of progression than an arbitrary mathematical threshold. Detailed IG molecular analysis of individual MBL may help to identify those few cases that necessitate continuous clinical monitoring to anticipate disease progression and, on the other hand, to avoid the burden of lengthy and expensive follow-ups in the vast number of persons who are extremely unlikely to develop CLL, though carrying MBL in their blood.

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Acquisition of Mannoses on the Surface Immunoglobulin Binding Site Reveals Functional Status and Cell of Origin in Diffuse Large B Cell Lymphomas

Blood ◽

10.1182/blood-2018-99-118318 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 677-677

Author(s):

Giorgia Chiodin ◽

Philip Rock ◽

Enrica Antonia Martino ◽

Beatriz Valle Argos ◽

Graham Packham ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Binding Site ◽

Tumor Cells ◽

Antigen Binding ◽

Cell Of Origin ◽

B Cell Lymphomas ◽

Antigen Binding Site ◽

Surface Immunoglobulin ◽

Large B Cell

Abstract Acquisition of mannosylated glycans in the surface immunoglobulin (sIg) variable region (sIgV) antigen-binding site is a unique tumor-specific structural change of certain lymphomas, including all follicular lymphomas (FL) and ~40% diffuse large B-cell lymphomas (DLBCL). Mannosylation of the sIgV allows binding to environmental lectins including DC-SIGN (Coelho V et al, PNAS 2010). SIgV engagement is generally required for survival of DLBCL cells (Young RM et al, PNAS 2015), but how sIgV mannosylation distributes and affects behavior in the two germinal center B-cell-like (GCB-like) or activated B-cell-like (ABC-like) DLBCL subsets is unknown. While the mannosylation of the sIgV is tumor specific and irreversible, there are other natural N-glycosylation sites in the sIg constant region (sIgC). In secreted IgM these are mainly fully glycosylated and that is seen in sIgM of normal B cells (Krysov S et al, Blood 2010). However, engagement of sIgM by anti-IgM leads to expression ofan immature (mannosylated) form in both tumor and normal B cells. This conversion is dynamic, and tumor B cells restore expression of sIgC with mature glycans following BCR disengagement in vitro(Krysov S et al, Blood 2010). In this study, the glycosylation patterns of sIgV and sIgC were analyzed in GCB-like (n=6) vs ABC-like DLBCL lines (n=2) and primary samples (n=8) by IGHV-D-J sequencing, DC-SIGN binding and immunoblot of the biotinylated sIg following digestion by EndoH (specific for the mannosylated sugars) or by PNGase (removes all sugars). We found acquisition of N-mannosylation sequence motifs in the IGHV-D-J transcripts of all GCB-DLBCL lines with t(14;18), indicating a likely relationship with FL. In contrast, neither of the ABC-DLBCL lines had acquired sites, confirming a separate origin. DC-SIGN binding, which is specific for mannosylated IgV structures on the tumor cells, was observed in all GCB-DLBCL and not in the ABC-DLBCL, confirming that the acquired sites were glycosylated. These results allowed us to discriminate DLBCL cases into "DC-SIGN binders" (DB-DLBCL) vs "DC-SIGN non-binders" DLBCL (NB-DLBCL). Analysis of the carbohydrate structures on the sIgC revealed that the immature form was confined to the NB-DLBCL lines (2/2), while the DB-DLBCL expressed a mature fully glycosylated form (6/6). Consistent with the nature of ABC-DLBCL, these results revealed an activated BCR status of the NB-DLBCL. This was confirmed in the 8 primary samples (5/8 DB, 3/8 NB), which expressed an immature (activated) sIgC in 3/3 NB-DLBCL and a mature sIgC in 5/5 DB-DLBCL. However, engagement of anti-IgM F(ab')2 polyclonal antibody converted the inactive sIg form of DB-DLBCL into an activated sIg with relative increase of the immature sugars. It was evident that the mannosylated sites on the sIgC were not available for DC-SIGN binding, which is confined to the sIgV sites. We verified BCR activation status by investigating constitutive phosphorylation of SYK, BTK and PLCγ2, which are recruited to the membrane upon BCR activation, prior to endosome formation (Phelan JD et al, Nature 2018), in 2 DB-DLBCL lines (NU-DHL1 and SU-DHL6) and 2 NB-DLBCL (HBL-1 and TMD8). Basal phosphorylation of SYK, BTK and PLCγ2 was higher in the NB-DLBCL, consistent with the activated status associated with an immature sIgC. Our results reveal a functional dichotomy in DLBCL, which indicates: first, the cell of origin dictates whether sIgV carries mannoses in the antigen-binding site; second, reversible sIgC mannosylation associates with activation via sIg. Interestingly, this feature of activation is in ABC-DLBCL, which lacks IgV mannosylation. It is consistent with the suggestion that occupation of the antigen-binding sites with mannoses blocks further engagement of the receptor by 'antigen'. However, acquisition of mannoses in the sIgV sites appears to confer an ability to interact with environmental lectins such as DC-SIGN, whereas the sIgC sites fail to do this, suggesting an alternative function. Clearly, the post-translational modification targets several sites in sIg. Sites in the sIgC have a similar, possibly maturational, function in normal B cells, but in tumor cells the irreversible addition of mannoses to the sIgV adds a tumor-specific function. Disclosures Packham: Aquinox: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

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Distinct clonal evolution of B-cells in HIV controllers with neutralizing antibody breadth

eLife ◽

10.7554/elife.62648 ◽

2021 ◽

Vol 10 ◽

Author(s):

Deniz Cizmeci ◽

Giuseppe Lofano ◽

Evan Rossignol ◽

Anne-Sophie Dugast ◽

Dongkyoon Kim ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Somatic Hypermutation ◽

Clonal Evolution ◽

Neutralizing Antibody ◽

Affinity Maturation ◽

Gene Repertoire ◽

Immunoglobulin Gene ◽

Hiv Controllers ◽

Hiv 1

A minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high-level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor (BCR) repertoires in 12,591 HIV-1 envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

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Sequence-Based Evidence for Antigen Selection in Mantle Cell Lymphoma: Remarkable Immunoglobulin Gene Repertoire Biases, Stereotyped Antigen-Binding Sites and Recurrent Hypermutations in Certain Subsets.

Blood ◽

10.1182/blood.v114.22.1933.1933 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1933-1933 ◽

Cited By ~ 1

Author(s):

Anastasia Hadzidimitriou ◽

Andreas Agathagelidis ◽

Fiona Murray ◽

Marie-Helene Delfau-Larue ◽

Nikos Darzentas ◽

...

Keyword(s):

Mantle Cell Lymphoma ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Somatic Hypermutation ◽

Antigen Binding ◽

Gene Repertoire ◽

Immunoglobulin Gene ◽

Antigen Binding Site ◽

Ighv Gene ◽

Mantle Cell

Abstract Abstract 1933 Poster Board I-956 According to the 2008 WHO Classification, mantle cell lymphoma (MCL) most likely derives from neoplastic transformation of a peripheral B cell of the inner mantle zone, mostly of naïve pre-germinal center type. Analysis of the immunoglobulin (IG) repertoire in MCL has revealed a bias in IG heavy variable (IGHV) gene usage and a predominance of unmutated rearrangements, with a variable minor proportion of cases carrying mutated IGHV genes (according to the 98% identity cutoff). However, limited knowledge exists regarding antigen-binding site sequence restrictions or specific somatic hypermutation (SHM) patterns in MCL. We examined the productive IGHV-D-J rearrangements of 651 MCL cases (including 398 cases from the European MCL Network), the largest series to date. We confirm and significantly extend previous observations that the IGHV gene repertoire is remarkably biased, with only three genes accounting for 40.3% of cases (IGHV3-21, 16.7%; IGHV4-34, 15.3%; IGHV1-8: 8.3%). Using bioinformatics approaches previously applied to CLL, biased associations of certain IGHV, IGHD and IGHJ genes with restricted (stereotyped) heavy complementarity-determining regions (HCDR3s) were identified in 68/651 cases (10.4%). Overall, 24 subsets of cases with stereotyped HCDR3s were recognized. Eight of 24 subsets included 3-7 cases each (“confirmed”), while the remaining 16 subsets included two cases each and were considered “provisional”. Stereotyped HCDR3s were found predominantly amongst rearrangements utilizing the IGHV3-21 and IGHV4-34 genes and the IGHJ6 gene. Notably, the MCL HCDR3 stereotypes identified here were distinct from those previously reported in CLL. Based on SHM analysis, the sequences were divided into three groups: (i) truly unmutated (100% germline identity, GI): 189/651 sequences (29.2%); (ii) minimally/borderline mutated (98-99.9% GI): 306/651 sequences (47%); and (iii) mutated (<98% GI): 155/651 sequences (23.8%), of which 93 had a GI<97%. In keeping with previous reports, the IGHV gene repertoire of the three identity groups differed considerably. For instance, the IGHV3-21 gene was used by 7% of rearrangements with <98% identity vs. 22.8% of rearrangements with 100% identity. In contrast, the IGHV3-23 gene was over-represented among mutated rearrangements (26.4%). Shared (“stereotyped”) amino acid (AA) changes across the entire IGHV gene sequence were identified for certain groups of sequences, especially those utilizing the IGHV1-8, IGHV3-21 and IGHV3-23 genes. A comparison to published series from other entities, in particular CLL, revealed that several stereotyped mutations identified in MCL were “disease-biased”. Importantly, stereotyped AA changes were also observed in the borderline or minimally mutated groups, indicating that even a low level of mutations may be functionally relevant. In conclusion, MCL is characterized by a highly distinctive IG gene repertoire with restricted HCDR3s and very precisely targeted and, probably, functionally driven SHM, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Based on the evidence presented here, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases. Disclosures: No relevant conflicts of interest to declare.

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Rituximab-resistant splenic memory B cells and newly engaged naive B cells fuel relapses in patients with immune thrombocytopenia

Science Translational Medicine ◽

10.1126/scitranslmed.abc3961 ◽

2021 ◽

Vol 13 (589) ◽

pp. eabc3961

Author(s):

Etienne Crickx ◽

Pascal Chappert ◽

Aurélien Sokal ◽

Sandra Weller ◽

Imane Azzaoui ◽

...

Keyword(s):

B Cells ◽

Autoimmune Diseases ◽

B Cell ◽

Single Cell ◽

Immune Thrombocytopenia ◽

Plasma Cells ◽

Surface Expression ◽

Memory B Cells ◽

Gene Repertoire ◽

Immunoglobulin Gene

Rituximab (RTX), an antibody targeting CD20, is widely used as a first-line therapeutic strategy in B cell–mediated autoimmune diseases. However, a large proportion of patients either do not respond to the treatment or relapse during B cell reconstitution. Here, we characterize the cellular basis responsible for disease relapse in secondary lymphoid organs in humans, taking advantage of the opportunity offered by therapeutic splenectomy in patients with relapsing immune thrombocytopenia. By analyzing the B and plasma cell immunoglobulin gene repertoire at bulk and antigen-specific single-cell level, we demonstrate that relapses are associated with two responses coexisting in germinal centers and involving preexisting mutated memory B cells that survived RTX treatment and naive B cells generated upon reconstitution of the B cell compartment. To identify distinctive characteristics of the memory B cells that escaped RTX-mediated depletion, we analyzed RTX refractory patients who did not respond to treatment at the time of B cell depletion. We identified, by single-cell RNA sequencing (scRNA-seq) analysis, a population of quiescent splenic memory B cells that present a unique, yet reversible, RTX-shaped phenotype characterized by down-modulation of B cell–specific factors and expression of prosurvival genes. Our results clearly demonstrate that these RTX-resistant autoreactive memory B cells reactivate as RTX is cleared and give rise to plasma cells and further germinal center reactions. Their continued surface expression of CD19 makes them efficient targets for current anti-CD19 therapies. This study thus identifies a pathogenic contributor to autoimmune diseases that can be targeted by available therapeutic agents.

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High Level Phosphatase Activity Revealed in Chronic Lymphocytic Leukemia Cells That Use Mutated Immunoglobulin Heavy Chain Variable Region Genes and Lack High-Level Expression of the Zeta-Associated Protein 70 (ZAP-70).

Blood ◽

10.1182/blood.v110.11.743.743 ◽

2007 ◽

Vol 110 (11) ◽

pp. 743-743

Author(s):

Wendy J. Fantl ◽

Rolla L. Yafawi ◽

Santosh K. Putta ◽

Ying-Wen Huang ◽

Helen L. Francis-Lang ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Phosphatase Activity ◽

Intracellular Signaling ◽

Variable Region ◽

Lymphocytic Leukemia ◽

High Level Expression ◽

Molecular Features ◽

Bcr Signaling ◽

High Level

Abstract Signals propagated through the B cell receptor (BCR) guide the maturation and survival of B cells and might factor in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). BCR signaling in CLL cells was investigated at the single-cell level using multiparametric flow cytometry. Concurrent analysis was performed using fluorochrome-conjugated antibodies specific for B-cell surface antigens and a panel of antibodies recognizing specific phospho-peptide epitopes within a selected group of intracellular signaling proteins. CLL samples from patients (N=6) showed weak or minimal signaling activity at p72SYK/p70ZAP, Erk1/2, B-Cell linker protein (BLNK) and phospholipase-Cγ-2, (PLCγ2) when stimulated only at the BCR with anti-μ crosslinking, whereas a robust signal was observed in a control Ramos B cell line. The low-level signaling in CLL cells could be accounted for by either a defect in activation of a key protein required for signaling, or by enhanced inhibition mediated by phosphatases such as SHIP-1, SHIP-2, SHP-1, or SHP-2. To determine whether phosphatases were preventing or dampening BCR activation in CLL samples, CLL cells were treated with hydrogen peroxide (H2O2), a physiologic phosphatase inhibitor generated during BCR signaling that has been used previously to reveal dysregulated BCR signaling in follicular lymphoma (Sing et al., Cell 2005, Reth., Nat. Immunol. 2002, Irish et al., Blood 2006). H2O2 treatment of CLL cells that had molecular features associated with indolent disease (e.g. use of mutated immunoglobulin heavy chain variable region genes (IgVH) and a low level expression of ZAP70) induced high-levels of phosphorylated p72SYK/p70ZAP, ERK1/2, BLNK, and PLCγ2, independent of surface F(ab)2anti-μ ligation. In contrast, CLL-B cells that had molecular features associated with more aggressive disease (expression of unmutated IgVH and high-level expression of ZAP-70) were significantly less responsive to this treatment, even in the presence of F(ab)2anti-μ. Exposure of blood B cells from healthy donors to H2O2 failed to elicit a substantial increase in phosphorylation of these same intracellular signaling proteins. These studies reveal a previously unrecognized, constitutive high-level phosphatase activity in CLL cells that express mutated IgVH and low levels of ZAP-70, possibly contributing to the attenuated signaling observed in these cells following surface IgM ligation. Because this high level of constitutive phosphatase activity was not observed in CLL cells that express unmutated IgVH with high-level expression of ZAP70, flow cytometric measurement of the increased phosphorylation triggered by H2O2 could provide for a useful means with which to distinguish leukemia cells from patients with indolent versus aggressive CLL. Evaluation of additional samples is ongoing to test this hypothesis.

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The Normal IGHV1-69-derived B Cell Repertoire Contains “Stereotypic” Patterns Characteristic of Unmutated CLL.

Blood ◽

10.1182/blood.v114.22.4370.4370 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4370-4370

Author(s):

Francesco Forconi ◽

Kathleen N Potter ◽

Isla Wheatley ◽

Nikos Darzentas ◽

Elisa Sozzi ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Subset ◽

Lymphocytic Leukemia ◽

Immunoglobulin Gene ◽

Cell Of Origin ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Germinal Center B Cell

Abstract Abstract 4370 The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought and immunoglobulin gene analysis provides new clues. The immunoglobulin heavy variable gene (IGHV) status has clinical relevance in CLL, where two subsets, delineated by the absence or presence of somatic mutation, have markedly different prognoses. The unmutated subset (U-CLL), of inferior prognosis, appears to derive from a pre-germinal center B cell. In U-CLL, there is strikingly increased usage of the 51p1-related alleles of the IGHV1-69 gene, often combined with selected IGHD genes and with IGHJ6. Shared sequence “stereotypic” characteristics of the HCDR3 result, and suggest antigen selection of the leukemic clones. In this study, we have analyzed 147 51p1/IGHJ6 rearrangements from 3 healthy individuals (>51yr) and sought sequence patterns parallel to those of U-CLL. A pre-established dataset of 313 51p1/IGHJ6 rearrangements from patients with U-CLL was used as a reference. A high proportion (49/147, 33.3%) of normal sequences revealed stereotypic patterns, several (22/147, 15%) being similar to those described in U-CLL. Additional CLL-associated stereotypes, not yet reported, were detected in 7/147 sequences (4.8%). Stereotypes (13.6%) not detected in CLL were also found in 20/147 (13.6%) 51p1/IGHJ6 combinations. The HCDR2-IGHJ6 sequences were almost exclusively unmutated (143/147, 97,3% sequences had ≥98% homology to germline). Junctional amino acids in normal B cells were heterogeneous, as in the cases of CLL with stereotyped 51p1/IGHJ6 B-cell receptors. Normal B cells expressing 51p1-derived IgM (4.8% of all B-cells) had a phenotype of naïve B-cells, similar to 51p1-negative (CD27-) B cells, i.e. IgM+ IgD+ CD23+ CD38+, with a small percentage of CD5+ B cells, not found in the memory B-cell subset. This snapshot of the naïve B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded IgM 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. Disclosures: No relevant conflicts of interest to declare.

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IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics

Blood ◽

10.1182/blood.2020008311 ◽

2020 ◽

Author(s):

Ferran Nadeu ◽

Romina Royo ◽

Guillem Clot ◽

Martí Duran-Ferrer ◽

Alba Navarro ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Immunoglobulin Gene ◽

Biological Features ◽

Mutational Status ◽

Bcr Signaling

B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL) and memory-like B-cells (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes we have characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases, being 30/79 (38%) i-CLL, 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL. All stereotype subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped B-cell receptor immunoglobulins. IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. Contrarily, i-CLL lacking the IGLV3-21R110 mirrored memory-like/mutated IGHV cases. IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Altogether, IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes.

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

Blood ◽

10.1182/blood-2009-06-225813 ◽

2010 ◽

Vol 115 (1) ◽

pp. 71-77 ◽

Cited By ~ 60

Author(s):

Francesco Forconi ◽

Kathleen N. Potter ◽

Isla Wheatley ◽

Nikos Darzentas ◽

Elisa Sozzi ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Heavy Chain ◽

Immunoglobulin Heavy Chain ◽

Immunoglobulin M ◽

Lymphocytic Leukemia ◽

Immunoglobulin Gene ◽

Cell Of Origin ◽

Cell Repertoire ◽

B Cell Repertoire

Abstract 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.

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Distinct clonal evolution of B-cells in HIV controllers with neutralizing antibody breadth

10.1101/2020.09.02.277566 ◽

2020 ◽

Author(s):

Deniz Cizmeci ◽

Giuseppe Lofano ◽

Anne-Sophie Dugast ◽

Dongkyoon Kim ◽

Guy Cavet ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Somatic Hypermutation ◽

Clonal Evolution ◽

Neutralizing Antibody ◽

Affinity Maturation ◽

Gene Repertoire ◽

Immunoglobulin Gene ◽

Hiv Controllers ◽

Hiv 1

AbstractA minor subset of individuals infected with HIV-1 develop antibody neutralization breadth during the natural course of the infection, often linked to chronic, high level viremia. Despite significant efforts, vaccination strategies have been unable to induce similar neutralization breadth and the mechanisms underlying neutralizing antibody induction remain largely elusive. Broadly neutralizing antibody responses can also be found in individuals who control HIV to low and even undetectable plasma levels in the absence of antiretroviral therapy, suggesting that high antigen exposure is not a strict requirement for neutralization breadth. We therefore performed an analysis of paired heavy and light chain B-cell receptor repertoires in 12,591 HIV-1 Envelope-specific single memory B-cells to determine alterations in the BCR immunoglobulin gene repertoire and B-cell clonal expansions that associate with neutralizing antibody breadth in 22 HIV controllers. We found that the frequency of genomic mutations in IGHV and IGLV was directly correlated with serum neutralization breadth. The repertoire of the most mutated antibodies was dominated by a small number of large clones with evolutionary signatures suggesting that these clones had reached peak affinity maturation. These data demonstrate that even in the setting of low plasma HIV antigenemia, similar to what a vaccine can potentially achieve, BCR selection for extended somatic hypermutation and clonal evolution can occur in some individuals suggesting that host-specific factors might be involved that could be targeted with future vaccine strategies.

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