Use Of Gene Segments Research Articles

#4851 MOLECULAR FEATURES OF CIRCULATORY B CELLS AND RENAL CELLS AT THE SINGLE-CELL LEVEL IN AUTOANTIBODY-NEGATIVE PRIMARY MEMBRANOUS NEPHROPATHY PATIENTS

Abstract Background and Aims Primary membranous nephropathy (PMN) is an organ-specific autoimmune disease that is the most common cause of idiopathic nephrotic syndrome in adults. Furthermore, the incidence of PMN is increasing, especially in young adults and children. The landmark discovery of nephritogenic autoantibodies against podocyte antigens such as M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing protein 7A (THSD7A) has provided a paradigm shift in diagnosis and treatment of PMN. However, there are still few patients with negative serum and renal tissue autoantibodies. This study aims to explore the molecular features of antibody-negative PMN. Method We sorted CD19+ cells from the peripheral blood of a pediatric patient with seven nephritogenic autoantibodies-negative PMN (NEG) by flow cytometry and performed single-cell transcriptome sequencing (scRNA-seq) and single-cell B cell receptor sequencing (scBCR-seq). Meanwhile, scRNA-seq was performed on renal biopsy tissues from the same patient. In addition, we included the scRNA-seq data of renal from anti-PLA2R antibody positive patients (POS) and healthy controls (CTRL), and the scBCR-seq data of B cell from CTRL for integrative analysis. Results The NEG patient showed typical nephrotic syndrome and was diagnosed as stage Ⅲ membranous nephropathy by renal biopsy. There was no evidence of anti-PLA2R or anti-THSD7A antibodies in peripheral blood and seven nephritogenic autoantibodies such as anti-PLA2R antibodies in renal tissue. After excluding secondary factors, the patient was diagnosed with PMN. Through scRNA-seq of CD19+ cells, we found that the number, characteristic gene, function and clonotype of naÏve B cells and memory B cells in NEG were significantly changed. Expanded CD38+ naÏve B cells in NEG had the molecular characteristics of CD19+CD24+CD27-CD38+, defined as transitional B cells. This group of cells had distinct function features of cell activation, and its up-regulated genes were involved in multiple aspects of the BCR signaling pathway. Pseudotime trajectory analysis suggested CD38+ naÏve B cells were highly enriched in the beginning of B cell differentiation. There was a preference in the use of VJ gene segments of B cells between NEG and CTRL, especially an increase of the IGHV3-23 and IGLV2-14 in NEG. The stronger pairing frequencies, IGLV2-14/IGLJ3 and IGKV2D-29/IGKJ2, were indicated in NEG. We identified 14 distinct kidney cell types by marker genes. Through re-clustering of glomerular parietal epithelial cells (PECs), the patients were clearly distinct from their control counterparts, indicating a major shift in gene expression for this cell type. PECs in NEG showed significant up-regulation of cellular communication network factor-related genes (CCN1, CCN2), phospholipase A and acyltransferase-related genes (PLAAT4, PLAAT3), and septin protein-related genes (SEPTIN2, SEPTIN7), accompanied by significant down-regulation of podocyte-related genes. In addition, there are clearly distinct cellular functions and pseudotime trajectory in PECs from NEG and POS, and genes such as CCN2, PLAAT4, SEPTIN2 might drive the special trajectory of PECs in NEG. For the podocytes, the genes related with extracellular matrix and cell adhesion were significantly enhanced in NEG, which consistented with the functional enrichment analysis of the differentially expressed genes and gene set-based scores. We calculated the gene set-based scores including genes encoding lumen-to-blood sodium transporters. Results indicated the enhanced expression of sodium transporters in distal nephrons of MN patients. More surprisingly, a group of proximal tubule epithelial cells showed significantly higher expression levels of sodium transporters. Among them, the expression of SLC5A12 encoding SMCT2 increased significantly. Conclusion We have systematic revealed the cell-type specific molecular features of PMN patients from circulation to renal tissue. Our research provides valuable evidence for the molecular diagnosis of PMN in children and insights into pathogenic mechanism of classical nephritogenic autoantibody-negative PMN.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Polygenic autoimmune disease risk alleles impacting B cell tolerance act in concert across shared molecular networks in mouse and in humans.

Most B cells produced in the bone marrow have some level of autoreactivity. Despite efforts of central tolerance to eliminate these cells, many escape to periphery, where in healthy individuals, they are rendered functionally non-responsive to restimulation through their antigen receptor via a process termed anergy. Broad repertoire autoreactivity may reflect the chances of generating autoreactivity by stochastic use of germline immunoglobulin gene segments or active mechanisms may select autoreactive cells during egress to the naïve peripheral B cell pool. Likewise, it is unclear why in some individuals autoreactive B cell clones become activated and drive pathophysiologic changes in autoimmune diseases. Both of these remain central questions in the study of the immune system(s). In most individuals, autoimmune diseases arise from complex interplay of genetic risk factors and environmental influences. Advances in genome sequencing and increased statistical power from large autoimmune disease cohorts has led to identification of more than 200 autoimmune disease risk loci. It has been observed that autoantibodies are detectable in the serum years to decades prior to the diagnosis of autoimmune disease. Thus, current models hold that genetic defects in the pathways that control autoreactive B cell tolerance set genetic liability thresholds across multiple autoimmune diseases. Despite the fact these seminal concepts were developed in animal (especially murine) models of autoimmune disease, some perceive a disconnect between human risk alleles and those identified in murine models of autoimmune disease. Here, we synthesize the current state of the art in our understanding of human risk alleles in two prototypical autoimmune diseases – systemic lupus erythematosus (SLE) and type 1 diabetes (T1D) along with spontaneous murine disease models. We compare these risk networks to those reported in murine models of these diseases, focusing on pathways relevant to anergy and central tolerance. We highlight some differences between murine and human environmental and genetic factors that may impact autoimmune disease development and expression and may, in turn, explain some of this discrepancy. Finally, we show that there is substantial overlap between the molecular networks that define these disease states across species. Our synthesis and analysis of the current state of the field are consistent with the idea that the same molecular networks are perturbed in murine and human autoimmune disease. Based on these analyses, we anticipate that murine autoimmune disease models will continue to yield novel insights into how best to diagnose, prognose, prevent and treat human autoimmune diseases.

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Perturbed CD8+ T cell immunity across universal influenza epitopes in the elderly.

Influenza epidemics lead to severe illness, life-threatening complications, and deaths, especially in the elderly. As CD8+ T cells are associated with rapid recovery from influenza, we investigated the effects of aging on antigen-specific CD8+ T cells across the universal influenza epitopes in humans. We show that aging is characterized by altered frequencies in T cell subsets, with naive T cells being partially replaced by activated effector/memory populations. Although we observed no striking differences in TCR signaling capacity, T cells in the elderly had increased expression of transcription factors Eomes and T-bet, and such changes were most apparent in CD8+ T cells. Strikingly, the numbers of antigen-specific CD8+ T cells across universal influenza epitopes were reduced in the elderly, although their effector/memory phenotypes remained stable. To understand whether diminished numbers of influenza-specific CD8+ T cells in the elderly resulted from alteration in TCR clonotypes, we dissected the TCRαβ repertoire specific for the prominent HLA-A*02:01-restricted-M158-66 (A2/M158 ) influenza epitope. We provide the first ex vivo data on paired antigen-specific TCRαβ clonotypes in the elderly, showing that influenza-specific A2/M158+ TCRαβ repertoires in the elderly adults varied from those in younger adults, with the main features being a reduction in the frequency of the public TRAV27-TRBV19 TCRαβ clonotype, increased proportion of private TCRαβ signatures, broader use of TRAV and TRBV gene segments, and large clonal expansion of private TCRαβ clonotypes with longer CDR3 loops. Our study supports the development of T cell-targeted influenza vaccines that would boost the T cell compartment during life and maintain the numbers and optimal TCRαβ signatures in the elderly.

Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/-Mb1-Cre+/- mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/- bone marrow. For Hdac3Δ/- B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/- progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells.

B-cell repertoire responses to varicella-zoster vaccination in human identical twins

Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus.

Hematopoiesis in the equine fetal liver suggests immune preparedness.

We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100days of gestation (DG) are active sites of hematopoiesis based on the expression of signature messenger RNA (mRNA) (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage, and surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth.

DH reading frame usage influences HIV-1 epitope recognition (MPF2P.804)

Abstract Background: Some HIV-1-specific neutralizing antibodies, such as 2F5 and 4E10, contain a long H chain complementarity determining region 3 (CDR-H3) includes patches of hydrophobic and charged amino acids, but B cells bearing Igs with charged or hydrophobic CDR-H3s are normally culled from mature B cell subsets. Thus, DH influence on neutralizing antibody generation is very worth to explore. Methods: Mice-BALB/c mice cohort limited to the use of single DH gene segments were generated. ΔD-DFL is a human-like repertoire control. ΔD-DµFS promotes the use of hydrophobic amino acid repertoire. The ΔD-iD is charged amino acid enriched repertoire. Immunization-each strain of 10 mice was immunized with HIV-1 JR-FL gp140 protein. Epitope Identification, serum Assay were conducted. Results: As a general rule, the heterogeneity of the anti-JR-FL gp140 response varied by DH genotype with ΔD-DFL>Wild Type (WT)>>ΔD-DμFS, ΔD-iD. Conversely, the intensity of the response was greatest in the ΔD-iD. However, linear response was not identified with the response to natural HIV-1 epitope. Anti-JR-FL gp140 antibodies present in the sera of the DH altered did not block HIV-1 envelope CD4, 2F5, 2G12 binding site. Conclusion: The pattern of epitope recognition and antigen binding in the response to T dependent antigen, in part, on DH gene segment sequence. Restrictions imposed by natural selection of DH sequence may underlie some of the difficulty that patients generate neutralizing antibody.

Chromatin conformation governs T-cell receptor Jβ gene segment usage.

T cells play fundamental roles in adaptive immunity, relying on a diverse repertoire of T-cell receptor (TCR) α and β chains. Diversity of the TCR β chain is generated in part by a random yet intrinsically biased combinatorial rearrangement of variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments. The mechanisms that determine biases in gene segment use remain unclear. Here we show, using a high-throughput TCR sequencing approach, that a physical model of chromatin conformation at the DJβ genomic locus explains more than 80% of the biases in Jβ use that we measured in murine T cells. This model also predicts correctly how differences in intersegment genomic distances between humans and mice translate into differences in Jβ bias between TCR repertoires of these two species. As a consequence of these structural and other biases, TCR sequences are produced with different a priori frequencies, thus affecting their probability of becoming public TCRs that are shared among individuals. Surprisingly, we find that many more TCR sequences are shared among all five mice we studied than among only subgroups of three or four mice. We derive a necessary mathematical condition explaining this finding, which indicates that the TCR repertoire contains a core set of receptor sequences that are highly abundant among individuals, if their a priori probability of being produced by the recombination process is higher than a defined threshold. Our results provide evidence for an expanded role of chromatin conformation in VDJ rearrangement, from control of gene accessibility to precise determination of gene segment use.

Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.

Antibody Vh Repertoire Differences between Resolving and Chronically Evolving Hepatitis C Virus Infections

Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of naïve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both naïve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in naïve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in naïve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion.

Use of similar immunoglobulin VH gene segments by MALT lymphomas of the ocular adnexa

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue type (MALT lymphomas) develop from acquired reactive infiltrates directed against external or autoantigens. Although some European cases of ocular adnexal MALT lymphoma have been associated with Chlamydia psittaci infections, C. psittaci has not been detected in large studies of US-based cases. To evaluate whether the growth of US-based ocular adnexal MALT lymphomas may be promoted by a similar antigen, we identified and analyzed the expressed immunoglobulin VH genes in 10 cases. Interestingly, the VH genes in two cases used the same VH1 family V1-2 gene segment, and three cases used the same VH4 family V4-34 gene segment. The other five cases all used different gene segments V4-31, V5-51, V3-23, V3-30, and V3-7. All of the VH genes were mutated from germ line, with percent homologies ranging between 96.9 and 89.0%. The distribution of replacement and silent mutations within the VH genes was nonrandom consistent with the maintenance of immunoglobulin function and also strongly suggestive of antigen selection in the six VH genes with highest mutation loads. The CDR3 sequences in two of three VH-34 cases were the same size (15 amino acids) and had similar sizes in the two VH1-2 cases (18 and 16 amino acids). In conclusion, US-based MALT lymphomas of the ocular adnexa preferentially express a limited set of VH gene segments not frequently used by other MALT lymphomas and consistent with some recognizing similar antigens. Analysis of somatic mutations present within the VH genes is also consistent with antigen binding stimulating the growth of these lymphomas.

Relapsed MALT Lymphomas Are Associated with a Restricted VH3-30.3 Immunoglobulin VH Gene Repertoire and Are Targeted by Aberrant Somatic Hypermutation

Mucosa associated lymphoid tissue (MALT) type lymphomas are B-cell neoplasms that develop out of a reactive infiltrate; Sjoegren syndrome, Hashimoto thyroiditis, and Helicobacter pylori gastritis provide the pathogenetic background. Direct antigen stimulation through surface immunoglobulin (Ig) molecules may be playing an important role in the development of MALT lymphomas. In rare cases MALT lymphomas may undergo transformation into extranodal diffuse large B-cell lymphomas (eDLBCL). Previous reports suggested salivary gland MALT lymphomas expressed a restricted Ig VH gene repertoire with over use of VH1-69 gene segments. Because knowledge about the VH gene used by MALT lymphoma of gastric and extragastric origin and by eDLBCL is limited, we sequenced the VH genes from 11 MALT lymphomas [5 of gastric and 6 of extragastric origin (3 salivary gland, 3 thyroid)] and from 10 eDLBCL, all arisen from MALT lymphoma and still exhibiting a low grade component [= so called “transformed MALT lymphoma”: 5 of gastric and 5 of extragastric origin (3 salivary gland and 2 thyroid)]. MALT lymphomas used gene segments of the VH1 (1–69: 2 salivary gland and 1 thyroid), VH3 (3–30.3: 1 thyroid), VH4 (4–34: 1 thyroid, 1 salivary gland and 2 gastric), VH5 (5–51: 2 gastric) and VH6 (6-1: 1 gastric) families. Extranodal DLBCLs used segments derived from VH1 (1–69: 1 gastric and 1 salivary gland), VH2 (2–70: 1 thyroid), VH3 (3–23: 1 gastric; 3–30: 1 gastric; 3–30.3: 1 gastric and 2 salivary glands) and VH4 (4–34: 1 gastric) families as shown in table 1. The VH1-69 gene segment was found in 3 of 6 extragastric MALT lymphomas (2 salivary glands and 1 thyroid), in one gastric and one salivary gland eDLBCL but in no gastric MALT lymphoma. Further, 4 of the 21 lymphomas relapsed, 3 eDLBCL and one MALT lymphoma – and remarkably, all of them used the VH3-30.3 gene segment. Comparing the frequency of somatic hypermutation (SHM) of the immunoglobuline locus and aberrant somatic hypermutation (ASHM) of the four proto-oncogenes PAX-5, PIM-1, Rho/TTF and c-MYC between the relapsed (n=4) and non relapsed lymphomas (n=17) a ~2.9 fold (5.8 vs 2.0 × 10−2/bp, p=0.017) and 2.1 fold (0.067 vs 0.032 × 10−2/bp, p=0.027) higher mutation rate for SHM and ASHM in relapsed lymphomas was observed. Also, the AID mRNA relative expression number was 1.7 fold higher in the group of relapsed lymphomas (4.66 vs 2.71, p=0.049). Performing immunohistochemical analysis for AID a significant positive correlation (p=0.01; correlation coefficient (Spearman rho) = 0.794) was observed when comparing protein expression with mRNA levels. These results are consistent with the model in which only certain B cells displaying specific patterns of VH immunoglobuline molecules binding to an as yet undefined antigen together with a highly aberrant somatic hypermutation process are preferentially selected for malignant transformation and determine the natural course of disease.Table1: VH gene sement used by MALT lymphomas and eDLBCLCaseTypeOriginVH geneRelapseGHM2eDLBCLstomach1–69noGHM3eDLBCLstomach3–23noGHM5eDLBCLstomach3-30-3yesGHM7eDLBCLstomach4–34noGHM8eDLBCLstomach3–30noEHM12eDLBCLthyroid6–1noEHM15eDLBCLsalivary gland3-30-3yesEHM16eDLBCLsalivary gland3-30-3yesEHM18eDLBCLthyroid2–70noEHM19eDLBCLsalivary gland1–69noENM14MALTthyroid3-30-3yesENM21MALTsalivary gland1–69noENM29MALTsalivary gland1–69noENM30MALTsalivary gland4–34noENM33MALTthyroid1–69noENM35MALTthyroid4–34noGNM36MALTstomach5–51noGNM37MALTstomach4–34noGNM38MALTstomach6–1noGNM39MALTstomach5–51noGNM41MALTstomach4–34no

Monoclonality and oligoclonality of T cell receptor β gene in angioimmunoblastic T cell lymphoma

Angioimmunoblastic T cell lymphoma (AILT) is an aggressive T cell lymphoma with an incidence of approximately 1–2% of all non-Hodgkin lymphoma. The detection of clonal T cell receptor (TCR) gene...

The kappa immunoglobulin light chain repertoire of peripheral blood B cells in patients with juvenile rheumatoid arthritis

The frequent appearance of antinuclear antibodies in patients with juvenile rheumatoid arthritis (JRA) indicates a loss of tolerance in B cell differentiation and/or activation. In this analysis, we were interested whether particular changes in the immunoglobulin light chain repertoire might exist in early-onset pauciarticular arthritis (EOPA) patients thereby potentially revealing distinct molecular patterns, which characterize defects in central tolerance mechanisms as well as an autoreactive peripheral B cell repertoire. Using single cell sorting and single cell PCR the distribution of Vκ Jκ rearrangements has been analyzed in individual naïve B cells of patients with EOPA–JRA and healthy individuals. The immunoglobulin kappa light chain repertoire of peripheral blood B cells in EOPA patients seems to be skewed to a decreased use of downstream Vκ gene segments indicating increased events of secondary V(D)J-recombination. Another prominent molecular pattern in JRA B cells seem to be a restricted combination of Vκ Jκ rearrangements based on the predominant utilization of the Jκ 1 and 2 gene segment. The current study indicates disturbances in the peripheral B cell pool in juvenile rheumatoid arthritis. The peripheral blood B cell pool of JRA patients did show molecular changes in the kappa light chain repertoire which, in part, could be a sequel of secondary V(D)J-recombination and of a molecular bias during immunoglobulin rearrangement in the bone marrow. Thus, B cell tolerance might be broken by more than one pathogenic mechanism.

Molecular Characterization of Complete and Incomplete Immunoglobulin Heavy Chain Gene Rearrangements in Hairy Cell Leukemia

PurposeWe analyzed patients with hairy cell leukemia (HCL) to achieve a better understanding of the differentiation stage reached by HCL cells and to define the key role of the diversification of cell surface makers, especially CD25 expression. Patients and MethodsWe analyzed 38 previously untreated patients with HCL to characterize their complete (VDJH) and incomplete (DJH) immunoglobulin (Ig) heavy chain (IgH) rearrangements, including somatic hypermutation pattern and gene segment use. ResultsA correlation between immunophenotypic profile and molecular data was seen. All 38 cases showed monoclonal amplifications: VDJH in 97%, DJH in 42%, and both in 39%. Segments from the DH3 family were used more in complete compared with incomplete rearrangements (45% vs. 12%; P < .005). Furthermore, comparison between molecular and immunophenotypic characteristics disclosed differences in the expression of CD25 antigen; CD25– cases, a phenotype associated with HCL variant, showed complete homology to the germline in 3 of 5 cases (60%), whereas this characteristic was never observed in CD25+ cases (P < .005). Moreover, VH4-34, VH1-08, and JH3 segments appeared in 2, 1, and 2 CD25– cases, respectively, whereas they were absent in all CD25+ cases. ConclusionThese results support that HCL is a heterogeneous entity including subgroups with different molecular characteristics, which reinforces the need for additional studies with a larger number of patients to clarify the real role of gene rearrangements in HCL.

Monoclonal TCR-Vβ13.1+/CD4+/NKa+/CD8−/+dim T-LGL lymphocytosis: evidence for an antigen-driven chronic T-cell stimulation origin

AbstractMonoclonal TCRαβ+/CD4+ T-large granular lymphocyte (T-LGL) lymphocytosis is a T-cell disorder with a restricted TCR-Vβ repertoire. In the present study we explored the potential association between the expanded TCR-Vβ families, the CDR3 sequences of the TCR-Vβ gene, and the HLA genotype of patients with monoclonal TCRαβ+/CD4+ T-LGL lymphocytosis. For that purpose, 36 patients with monoclonal TCRαβ+/CD4+ T-LGL lymphocytosis (15 TCR-Vβ13.1 versus 21 non–TCR-Vβ13.1) were selected. For each patient, both the HLA (class I and II) genotype and the DNA sequences of the VDJ-rearranged TCR-Vβ were analyzed. Our results show a clear association between the TCR-Vβ repertoire and the HLA genotype, all TCR-Vβ13.1+ cases being HLA-DRB1*0701 (P = .004). Interestingly, the HLA-DR7/TCR-Vβ13.1–restricted T-cell expansions displayed a highly homogeneous and strikingly similar TCR arising from the use of common TCR-Vβ gene segments, which shared (1) unique CDR3 structural features with a constantly short length, (2) similar combinatorial gene rearrangements with frequent usage of the Jβ1.1 gene, and (3) a homolog consensus protein sequence at recombination junctions. Overall, these findings strongly support the existence of a common antigen-driven origin for monoclonal CD4+ T-LGL lymphocytosis, with the identification of the exact peptides presented to the expanded T cells deserving further investigations.

Dissecting the Anti-Desmoglein Autoreactive B Cell Repertoire in Pemphigus Vulgaris Patients

Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. V(H) gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two V(H) genes. V(L) gene segment use was diverse even among IgG hybridomas suggesting that the V(L) is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in V(H) use, is somatically mutated, and is Ag selected.

Discrete T Cell Populations with Specificity for a Neo-Self-Antigen Bear Distinct Imprints of Tolerance

Mice expressing the Torpedo acetylcholine receptor alpha-chain as a neo-self-Ag exhibit a reduced frequency of T cells responding to the immunodominant epitope Talpha146-162 indicating a degree of tolerance. We characterized tolerance induction in these animals by analyzing the residual Talpha146-162-responsive T cell population and comparing it to that of nontransgenic littermates. Using CD4(high) sorting, we isolated the vast majority of Ag-reactive T cells from both strains of mice. Quantitative studies of the CD4(high) populations in transgenic mice following immunization with Talpha146-162 revealed a diminished expansion of cells expressing the canonical TCRBV6 but not other TCRBV gene segments when compared with nontransgenic littermates. In addition, CD4(high) cells from transgenic mice were functionally hyporesponsive to Talpha146-162 in terms of proliferation and cytokine secretion regardless of TCRBV gene segment use. TCR sequence analysis of transgenic Vbeta6(+)CD4(high) cells revealed a reduced frequency of cells expressing a conserved motif within the TCRbeta CDR3. Thus, the canonical Talpha146-162 responsive, Vbeta6(+) population demonstrates both quantitative and qualitative deficits that correlate with an altered TCR repertoire whereas the non-Vbeta6 population in transgenic mice exhibits only a reduction in peptide responsiveness, a qualitative defect. These data demonstrate that discrete autoreactive T cell populations with identical peptide/MHC specificity in Torpedo acetylcholine receptor-alpha-transgenic animals bear distinct tolerance imprints.