Multiregional sequencing and circulating tumour DNA analysis provide complementary approaches for comprehensive disease profiling of small lymphocytic lymphoma.

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.

The Yin and Yang of RNA surveillance in B lymphocytes and antibody-secreting plasma cells.

The random V(D)J recombination process ensures the diversity of the primary immunoglobulin (Ig) repertoire. In two thirds of cases, imprecise recombination between variable (V), diversity (D), and joining (J) segments induces a frameshift in the open reading frame that leads to the appearance of premature termination codons (PTCs). Thus, many B lineage cells harbour biallelic V(D)J-rearrangements of Ig heavy or light chain genes, with a productively-recombined allele encoding the functional Ig chain and a nonproductive allele potentially encoding truncated Ig polypeptides. Since the pattern of Ig gene expression is mostly biallelic, transcription initiated from nonproductive Ig alleles generates considerable amounts of primary transcripts with out-of-frame V(D)J junctions. How RNA surveillance pathways cooperate to control the noise from nonproductive Ig genes will be discussed in this review, focusing on the benefits of nonsense- mediated mRNA decay (NMD) activation during B-cell development and detrimental effects of nonsense-associated altered splicing (NAS) in terminally differentiated plasma cells. [BMB Reports 2019; 52(12): 671-678].

Massively Parallel Sequencing of Peritoneal and Splenic B Cell Repertoires Highlights Unique Properties of B-1 Cell Antibodies.

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained VH11 and VH12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Optimizing therapy for nodal marginal zone lymphoma.

Nodal marginal zone lymphoma (NMZL) is a rare form of indolent small B-cell lymphoma which has only been clearly identified in the last 2 decades and which to date remains incurable. Progress in therapeutic management has been slow, largely due to the very small number of patients treated and the heterogeneity of treatments administered; thus, standard-of-care treatment is currently nonspecific for this lymphoma entity. In this review, treatments routinely used to manage adult NMZL patients are presented, principally based on immunochemotherapy (when treatment is needed). Biological research behind the key axes of agents currently under development is described; development of novel agents is heavily based on data from gene profiling and genome-wide sequencing research, uncovering a number of critical deregulated pathways specific to NMZL tumors. These include B-cell receptor, JAK/STAT, NF-κB, NOTCH, and Toll-like receptor signaling pathways, as well as intracellular processes such as the cell cycle, chromatin remodeling, and transcriptional regulation in terms of epigenetic modifiers, histones, or transcriptional co-repressors, along with immune escape via T-cell-mediated tumor surveillance. These pathways are examined in detail and a projection of how the field may evolve in the near future for an efficient personalized treatment approach for NMZL patients is presented.

Age and vitamin E-induced changes in gene expression profiles of T cells.

T cells are vulnerable to age-associated changes. Vitamin E has been shown to improve T cell functions in the old. We studied gene expression profiles of T cells to better understand the underlying mechanisms of age and vitamin E-induced changes in T cell function. Young and old C57BL mice were fed diets containing 30 (control) or 500 (supplemented) ppm of vitamin E for 4 wks. Gene expression profiles of T cells were assessed using microarray analysis with/without anti-CD3/anti-CD28 stimulation. Genes associated with cytokines/chemokines, transcriptional regulation, signal transduction, cell cycle, and apoptosis were significantly up-regulated upon stimulation. Higher SOCS3 and lower growth factor independent 1 (Gfi-1) expression in old T cells may contribute to age-associated decline in proliferation. Higher Gadd45 and lower Bcl2 expression may contribute to increased apoptosis in old T cells. Vitamin E supplementation resulted in higher expression of genes involved in cell cycle regulation (Ccnb2, Cdc2, Cdc6) in old T cells. Vitamin E supplementation resulted in higher up-regulation of IL-2 expression in young and old T cells and lower up-regulation of IL-4 expression in old T cells following stimulation. These findings suggest that aging has significant effects on the expression of genes associated with signal transduction, transcriptional regulation, and apoptosis pathways in T cells, and vitamin E has a significant impact on the expression of genes associated with cell cycle and Th1/Th2 balance in old T cells. Further studies are needed to determine whether these changes are due to the effects of aging at a single-cell level or to the shift in the ratio of naïve:memory T cells with age.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

VH gene usage in immunoglobulin E responses of seasonal rhinitis patients allergic to grass pollen is oligoclonal and antigen driven.

BACKGROUND: IgE is the pivotal-specific effector molecule of allergic reactions yet it remains unclear whether the elevated production of IgE in atopic individuals is due to superantigen activation of B cell populations, increased antibody class switching to IgE or oligoclonal allergen-driven IgE responses. OBJECTIVES: To increase our understanding of the mechanisms driving IgE responses in allergic disease we examined immunoglobulin variable regions of IgE heavy chain transcripts from three patients with seasonal rhinitis due to grass pollen allergy. METHODS: Variable domain of heavy chain-epsilon constant domain 1 cDNAs were amplified from peripheral blood using a two-step semi-nested PCR, cloned and sequenced. RESULTS: The VH gene family usage in subject A was broadly based, but there were two clusters of sequences using genes VH 3-9 and 3-11 with unusually low levels of somatic mutations, 0-3%. Subject B repeatedly used VH 1-69 and subject C repeatedly used VH 1-02, 1-46 and 5a genes. Most clones were highly mutated being only 86-95% homologous to their germline VH gene counterparts and somatic mutations were more abundant at the complementarity determining rather than framework regions. Multiple sequence alignment revealed both repeated use of particular VH genes as well as clonal relatedness among clusters of IgE transcripts. CONCLUSION: In contrast to previous studies we observed no preferred VH gene common to IgE transcripts of the three subjects allergic to grass pollen. Moreover, most of the VH gene characteristics of the IgE transcripts were consistent with oligoclonal antigen-driven IgE responses.

Characterization of childhood acute lymphoblastic leukemia xenograft models for the preclinical evaluation of new therapies.

Continuous xenografts from 10 children with acute lymphoblastic leukemia (ALL) were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Relative to primary engrafted cells, negligible changes in growth rates and immunophenotype were observed at second and third passage. Analysis of clonal antigen receptor gene rearrangements in 2 xenografts from patients at diagnosis showed that the pattern of clonal variation observed following tertiary transplantation in mice exactly reflected that in bone marrow samples at the time of clinical relapse. Patients experienced diverse treatment outcomes, including 5 who died of disease (median, 13 months; range, 11-76 months, from date of diagnosis), and 5 who remain alive (median, 103 months; range, 56-131 months, following diagnosis). When stratified according to patient outcome, the in vivo sensitivity of xenografts to vincristine and dexamethasone, but not methotrexate, differed significantly (P =.028, P =.029, and P =.56, respectively). The in vitro sensitivity of xenografts to dexamethasone, but not vincristine, correlated significantly with in vivo responses and patient outcome. This study shows, for the first time, that the biologic and genetic characteristics, and patterns of chemosensitivity, of childhood ALL xenografts accurately reflect the clinical disease. As such, they provide powerful experimental models to prioritize new therapeutic strategies for future clinical trials.

Susceptibility of B-cell lymphoma to human antibodies encoded by the V4-34 gene.

Our previous studies have shown that mAbs derived from the human V4-34 gene bind and kill human B-lymphocytes via membrane disruption. This study demonstrates the cytotoxicity of two V4-34 encoded mAbs, 216 and Z2D2, towards human B-cell lymphoma. In vitro, 216 and Z2D2 are cytotoxic to a variety of B-cell lymphomas obtained from patient biopsies. In vivo, increased survival was observed with both mAbs in a lymphoma model developed in scid mice with human B-cell line Nalm-6. Studies in mice show that these mAbs are well tolerated with minimum side effects. Since 216 and Z2D2 show increased toxicity towards cycling cells, V4-34 mAb-based therapy can be additive with drugs that block cell-cycle progression. Stem cells that are V4-34 mAb ligand negative would not be depleted. Together, these studies recommend an evaluation of the two completely human mAbs in a phase I trial for B-cell lymphoma.

Evaluation of the safety of recombinant P-selectin glycoprotein ligand-immunoglobulin G fusion protein in experimental models of localized and systemic infection.

P-selectin is a major component in the early interaction between platelets, endothelial cells, and inflammatory cells in the initial phases of the innate immune response. The major ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1) and this ligand is expressed on the surface of monocyte, lymphocyte, and neutrophil membranes. A truncated form of recombinant human P-selectin glycoprotein ligand-1 has been covalently linked to immunoglobulin G (rPSGL-Ig) and this fusion peptide functions as a competitive inhibitor of PSGL-1. As an inhibitor of neutrophil-endothelial cell adherence, rPSGL-Ig is in early clinical development for the treatment of ischemia reperfusion injury. To determine the potential for deleterious effects from inhibition in P-selectin-mediated neutrophil attachment in the presence of bacterial infection, the effects of therapeutic doses of rPSGL-Ig were tested in three standard laboratory sepsis models. The experimental models included: the murine systemic Listeria monocytogenes infection model, the Pseudomonas aeruginosa bacteremia model in neutropenic rats, and the cecal ligation and puncture (CLP)-induced peritonitis model in rats. Recombinant human PSGL-Ig had no adverse effects on mortality or immune clearance in systemic bacterial infection in any of the three infection models. The PSGL-1 inhibitor did significantly decrease local neutrophil infiltration and bacterial clearance in the peritoneum following CLP, but this did not increase the systemic levels of proinflammatory cytokines, the quantitative levels of bacteremia, or the overall mortality rate following CLP. The results indicate that rPSGL-Ig did not exacerbate infection in these experimental sepsis models.

Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage.

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.

Lymphoid hyperplasia of the large intestine: a case report with immunohistochemical and gene analysis.

A case of lymphoid hyperplasia arising in the large intestine of a 54-year-old woman is described. Barium enema X-ray and colonoscopic examination revealed multiple small polyps in the right side of the colon. Pathological findings from forceps biopsy revealed similar features to a mucosa-associated lymphoid tissue (MALT) lymphoma. A right hemicolectomy with mesenteric lymph node dissection was carried out. Histological sectioning demonstrated hypertrophic lymphoid follicles with well-formed germinal centers. Occasionally, lymphocytes infiltrated the crypts, in a way similar to that found in lymphoepithelial lesions, which was suggestive of a MALT lymphoma diagnosis. Cryptitis was also observed in the lamina propria. Immunohistochemically, proliferating lymphocytes were positive for CD20 (L26) and negative for CD45RO (UCHL-1). Analyses of immunoglobulin gene (IgHJH) rearrangement could not detect any monoclonality in these cells. These findings suggested that the present case should be categorized as lymphoid hyperplasia rather than lymphoma.

Mannosidase action, independent of glucose trimming, is essential for proteasome-mediated degradation of unassembled glycosylated Ig light chains.

In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acceptor sites into the variable domain of the murine Ig L chain kappaNS1, which is unfolded in unassembled molecules. We investigated the fate of kappaNS1 glycosylated at position 70 (K70) and of a double mutant (kappa18/70) in stably transfected HeLa cells. Degradation of both chains was impaired by lactacystin, a specific inhibitor of the proteasome. The mannosidase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromycin. In contrast, ER glucosidase inhibitors dramatically accelerated the degradation of the chains when added either pre- or posttranslationally. The accelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide, too. None of these drugs, except lactacystin, affected the degradation of unglycosylated kappaNS1 chains. We conclude that ER mannosidases and proteasome activities, but not glucose trimming (and therefore, most likely not the calnexin/calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glycoproteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.

Identification and characterization of BCL6 translocation partner genes in primary gastric high-grade B-cell lymphoma: heat shock protein 89 alpha is a novel fusion partner gene of BCL6.

Primary gastric high-grade B-cell lymphoma (HGBL) is a special type of non-Hodgkin's lymphoma. So far, the genetic features of this tumor have not been well characterized. Recently, a high incidence of BCL6 rearrangements has been detected in HGBL. However, no previous cytogenetic studies have found translocations involving the BCL6 locus (3q27) in HGBL, and the genetic basis underlying the BCL6 rearrangements in this tumor remains unclear. We therefore characterized the partner genes of BCL6 in five primary gastric HGBLs with a rearranged BCL6 gene by analyzing BCL6 transcripts using the 5' RACE (rapid amplification of cDNA end) strategy. BCL6 translocation partner genes were identified at the 5' end of the chimeric transcripts in all five cases, including the immunoglobulin heavy-chain (IGH) gene in three cases and the immunoglobulin lambda-light-chain gene and the heat shock protein 89 alpha (HSP89A) gene in the other two cases. The chimeric transcripts in all cases contained the intact BCL6 exon 2, but lacked exon 1, which was replaced by sequences from the partner genes, suggesting that BCL6 expression was under the control of regulatory sequences of the partner genes. These results, for the first time, indicate that immunoglobulin genes, especially IGH, are the most common BCL6 translocation partner genes in primary gastric HGBL and that HSP89A is a novel partner of BCL6. Because immunoglobulin genes are also the most frequent partners of BCL6 in nodal diffuse large B-cell lymphoma (DLBCL), these data suggest that primary gastric HGBL shares a common genetic basis with nodal DLBCL. Genes Chromosomes Cancer 27:69-75, 2000.

Expression of IgH chain genes in antibody response to ragweed and purified Amb a 1: evidence for differential regulation of isotype production.

A survey of the work with Ig response to allergens carried out previously reveals an allergen-specific response both by IgE and all of IgG subclasses. Response of non-sensitive people is characterized by the appearance of a variety of the IgG subclasses. We have reexamined ragweed and Amb a 1 specific Ig response in 54 nonsensitive and 147 atopic or atopic-allergic people using a new inverse sandwich immunoassay allowing discrimination based on antibody affinity. We show that non-sensitive people present no, 0 out of 54, Ig response with affinities higher than Ka 10(7) M(-1). The subpopulation of 66 atopics who never have experienced desensitization responds vigorously and solely (56 out of 66) with genes of the sequence gamma2-alpha2. Only ten showed an additional weak response from gamma1-alpha1. This suggests a possible association between the atopic state and selective activation of part of the gene sequence.

Expression of a human IgG4 antibody, BAB2, with specificity for the major Birch pollen allergen, Bet v 1 in Escherichia coli: recombinant BAB2 Fabs enhance the allergic reaction.

BACKGROUND: Antigen recognition by antibodies of different isotypes can result in completely different effects as exemplified by Type I allergy. While the IgE-antibody-mediated release of biological mediators constitutes the immunopathological basis for the immediate symptoms observed in allergic patients, allergen-specific IgG antibodies are thought to have protective effects. METHODS: Cell lines secreting five human monoclonal IgG antibodies (BAB1-BAB5) with specificity for the major birch pollen allergen Bet v 1 were established from a birch-pollen-allergic patient who had received birch- pollen-specific immunotherapy. The influence of the Bet v 1-specific IgG antibodies on IgE binding to Bet v 1 was investigated. BAB2 was expressed in Escherichia coli as recombinant Fab, purified and tested for its ability to modulate Bet v 1-induced immediate-type skin reactions. RESULTS: The BAB antibodies belonged to different IgG subclasses (BAB1: IgG1; BAB2, BAB3, BAB5: IgG4; and BAB4: IgG2) reflecting a tendency towards Th2. BAB1 represented the only antibody which strongly blocked IgE binding to Bet v 1, whereas BAB 3-BAB5 had little effect on IgE binding. Surprisingly, natural BAB2 antibodies as well as recombinant BAB2 Fabs strongly enhanced IgE binding to Bet v 1 and Bet v 1-induced immediate-type skin reactions and thus represent 'enhancing antibodies'. CONCLUSION: The demonstration that anti-allergen IgG antibodies can also enhance IgE binding to a given allergen explains the unpredictability of specific immunotherapy as well as the controversy on the role of IgG in atopy.

Strain specific recombinant antibodies to potato virus Y potyvirus.

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY(O)). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY(O) whilst failing to detect three isolates of PVY(N) and 12 isolates of PVY(NTN). Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vlambda1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.

Overexpanded B cell clone mediating leukemic arthritis by abundant secretion of interleukin-1beta: a case report.

The role of cytokines in leukemic arthritis is unknown. The presentation of a patient with B cell chronic lymphocytic leukemia and destructive arthritis of the wrist joints prompted us to study the synovial cytokine pattern by immunohistologic analysis. In addition, rearranged V(H) and V(L) immunoglobulin genes were sequenced to assess B cell clonality. Heavy infiltrations of CD20+ cells with lambda light chain restriction were found in the synovial tissue. Sequencing demonstrated overexpansion of a single B cell clone (DP58/D/J(H)4b and IGLV3S2/Jlambda2-Jlambda3 for V(H) and V(L), respectively) in the peripheral blood. Identical V(H) and V(L) rearrangements were found in the synovial infiltrates. Somatic mutations were found in both the peripheral blood and the synovial clone. Immunohistologic study revealed the presence of abundant interleukin-1beta (IL-1beta) and, to a lesser degree, tumor necrosis factor beta (TNFbeta) (lymphotoxin). In contrast, TNFalpha, interferon-gamma, IL-4, IL-6, and IL-10 were rarely found in the synovial infiltrates. Therefore, IL-1beta secreted in great amounts by leukemic B cells appears to be the major cytokine that mediates joint destruction in leukemic arthritis.

Base-specific sequences that bias somatic hypermutation deduced by analysis of out-of-frame human IgVH genes.

Somatic hypermutation introduces mutations into IgV genes during affinity maturation of the B cell response. Mutations are introduced nonrandomly, and are generally targeted to the complementarity determining regions (CDRs). Subsequent selection against mutations that result in lower affinity or nonfunctional Ig increases the relative number of mutations in the CDRs. Investigation of somatic hypermutation is hampered by the effects of selection. We have avoided this by studying out-of-frame human IgVH4.21 and 251 genes, which, being unused alleles, are unselected. By comparison of the frequency of A, C, G, and T nucleotides at positions -3 to +3 around mutated or unmutated A, C, and G nucleotides, we have identified flanking sequences that most commonly surround mutated bases. Distinct trends in flanking sequences that were unique for each base were observed. Statistically significant trends that were common to both IgVH4.21 and 251 were used to deduce motifs that bias somatic hypermutation. The motifs deduced from this data, with targeted bases in regular type, are AANB, WDCH, and DGHD (where W = A/T, B = C/G/T, D = A/G/T, H = A/C/T, and N = any base). Mutations from C and G in two further groups of out-of-frame human IgVH genes, not used in the deduction of the motifs, occurred significantly within the motifs for C and G. The proposed target sequence for G is within the reverse complement of the target sequence for C, suggesting that the hypermutation mechanism may target only G or C. The mutation in the complementary base would appear on the other strand following replication.