Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal centerindependent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes.

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

Mantle cell lymphoma (MCL) is a heterogeneous disease with most patients following an aggressive clinical course while others have an indolent behavior. We performed an integrative and multidisciplinary analysis of 177 MCL to determine whether the immunogenetic features of the clonotypic B cell receptors may identify different subsets of tumors. ‘Truly unmutated’ (100% identity) IGHV genes were found in 24% cases, 40% were ‘minimally/borderline mutated’ (99.9-97%), 19% ‘significantly mutated’ (96.9-95%) and 17% ‘hypermutated’ (<95%). Tumors with high (≥97%) or low (<97%) mutational load used different IGHV genes and their gene expression profiles were also different for several gene pathways. A gene set enrichment analysis showed that MCL with high and low IGHV mutations were enriched in memory and naïve B-cell signatures, respectively. Furthermore, the highly mutated tumors displayed less genomic complexity, were preferentially SOX11 negative, and showed more frequently non-nodal disease. The best cut-off of germline identity of IGHV genes to predict survival was 3%. Patients with high and low mutational load had significant different outcome with 5-year overall survival of 59% and 40%, respectively (P=0.004). Nodal presentation and SOX11 expression also predicted for poor overall survival. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. In conclusion, these observations suggest the idea that MCL with mutated IGHV, SOX11 negativity, and non-nodal presentation correspond to a subtype of the disease with more indolent behavior.

We performed an immunogenetic analysis of 345 IGHV --IGHD --IGHJ rearrangements from 337 cases with primary splenic small B-cell lymphomas of marginal-zone origin. Three immunoglobulin (IG) heavy variable (IGHV) genes accounted for 45.8% of the cases (IGHV1-2, 24.9%; 12.8%; 8.1%). Particularly for the IGHV1-2 gene, strong biases were evident regarding utilization of different alleles, with 79/86 rearrangements (92%) using allele *04. Among cases more stringently classified as splenic marginal-zone lymphoma (SMZL) thanks to the availability of splenic histopathological specimens, the frequency of IGHV1-2*04 peaked at 31%. The IGHV1-2*04 rearrangements carried significantly longer complementaritydetermining region-3 (CDR3) than all other cases and showed biased IGHD gene usage, leading to CDR3s with common motifs. The great majority of analyzed rearrangements (299/345, 86.7%) carried IGHV genes with some impact of somatic hypermutation, from minimal to pronounced. Noticeably, 75/79 (95%) IGHV1-2*04 rearrangements were mutated; however, they mostly (56/75 cases; 74.6%) carried few mutations (97 --99.9% germline identity) of conservative nature and restricted distribution. These distinctive features of the IG receptors indicate selection by (super)antigenic element(s) in the pathogenesis of SMZL. Furthermore, they raise the possibility that certain SMZL subtypes could derive from progenitor populations adapted to particular antigenic challenges through selection of VH domain specificities, in particular the IGHV1-2*04 allele.

Immunoglobulin (IG) gene repertoire restrictions strongly support antigen selection in the pathogenesis of chronic lymphocytic leukemia (CLL). Given the emerging multifarious interactions between CLL and bystander T cells, we sought to determine whether antigen(s) are also selecting T cells in CLL. We performed a large-scale, next-generation sequencing (NGS) study of the T-cell repertoire, focusing on major stereotyped subsets representing CLL subgroups with undisputed antigenic drive, but also included patients carrying non-subset IG rearrangements to seek for T-cell immunogenetic signatures ubiquitous in CLL. Considering the inherent limitations of NGS, we deployed bioinformatics algorithms for qualitative curation of T-cell receptor rearrangements, and included multiple types of controls. Overall, we document the clonal architecture of the T-cell repertoire in CLL. These T-cell clones persist and further expand overtime, and can be shared by different patients, most especially patients belonging to the same stereotyped subset. Notably, these shared clonotypes appear to be disease-specific, as they are found in neither public databases nor healthy controls. Altogether, these findings indicate that antigen drive likely underlies T-cell expansions in CLL and may be acting in a CLL subset-specific context. Whether these are the same antigens interacting with the malignant clone or tumor-derived antigens remains to be elucidated.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV-or EBV-positive; 25/93); (3) double-positive (9/93). The doublenegative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.

Primary splenic small B-cell lymphomas mostly comprise the distinct entity of splenic marginal-zone lymphoma (SMZL) and the provisional category of splenic lymphoma/leukemia unclassifiable, mainly represented by the hairy cell leukemia variant and splenic diffuse red pulp small B-cell lymphoma (SDRL). Until recently, histopathologic examination of splenectomy specimens was considered mandatory for the diagnosis of SMZL. However, nowadays, mainly because of advances in chemoimmunotherapy, splenectomy is performed much less frequently. We evaluated the diagnostic efficacy of bone marrow biopsy (BMB) histopathology in the diagnostic approach toward SMZL and SDRL and tested whether it may serve as a substitute for spleen histopathology in the differential diagnosis between these 2 entities. To this end, we conducted a paired assessment of BMB and spleen diagnostic samples from 46 cases with a diagnosis of SMZL (n=32) or SDRL (n=14) based on spleen histopathology. We demonstrate that detailed immunohistopathologic BMB evaluation offers adequate evidence for the confirmation of these entities and their differential diagnosis from other small B-cell lymphoma histotypes. Notably, the immunophenotypical profile of SMZL and SDRL was identical in both BMB and spleen specimens for 21 evaluated markers. Paired assessment of BMB and spleen specimens did not identify discriminating patterns of BMB infiltration, cytology, and/or immunohistology between SMZL and SDRL. Accordingly, bone marrow histopathology contributes significantly in confirming the diagnosis of SMZL and SDRL. However, presently it is not possible to distinguish SMZL from SDRL on the basis of BMB evaluation alone; hence, histopathologic examination of the spleen remains the "gold standard" approach.

Supplementary data are available at Bioinformatics online.