GLORIA

GEOMAR Library Ocean Research Information Access

X

Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
  • 1
    Online Resource
    Online Resource
    N -Glycosylation of the Ig Receptors Shapes the Antigen Reactivity in Chronic Lymphocytic Leukemia Subset #201
    The American Association of Immunologists ; 2023
    In:  The Journal of Immunology Vol. 211, No. 5 ( 2023-09-01), p. 743-754
    Details
    In: The Journal of Immunology, The American Association of Immunologists, Vol. 211, No. 5 ( 2023-09-01), p. 743-754
    Abstract: Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.
    Type of Medium: Online Resource
    ISSN: 0022-1767 , 1550-6606
    URL: Article
    DOI: 10.4049/jimmunol.2300330
    RVK:
    XA 54100
    RVK:
    XA 10000
    Language: English
    Publisher: The American Association of Immunologists
    Publication Date: 2023
    detail.hit.zdb_id: 1475085-5
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 2
    Online Resource
    Online Resource
    Higher Order Restrictions of the Immunoglobulin Repertoire in CLL: The Illustrative Case of Stereotyped Subsets #2 and #169
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 5453-5453
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 5453-5453
    Abstract: Stereotyped subset #2 (IGHV3-21/IGLV3-21) is the largest subset in CLL (~3% of all patients). Membership in subset #2 is clinically relevant since these patients experience an aggressive disease irrespective of the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene. Low-throughput evidence suggests that stereotyped subset #169, a minor CLL subset (~0.2% of all CLL), resembles subset #2 at the immunogenetic level. More specifically: (i) the clonotypic heavy chain (HC) of subset #169 is encoded by the IGHV3-48 gene which is closely related to the IGHV3-21 gene; (ii) both subsets carry VH CDR3s comprising 9-amino acids (aa) with a conserved aspartic acid (D) at VH CDR3 position 3; (iii) both subsets bear light chains (LC) encoded by the IGLV3-21 gene with a restricted VL CDR3; and, (iv) both subsets have borderline SHM status. Here we comprehensively assessed the ontogenetic relationship between CLL subsets #2 and #169 by analyzing their immunogenetic signatures. Utilizing next-generation sequencing (NGS) we studied the HC and LC gene rearrangements of 6 subset #169 patients and 20 subset #2 cases. In brief, IGHV-IGHD-IGHJ and IGLV-IGLJ gene rearrangements were RT-PCR amplified using subgroup-specific leader primers as well as IGHJ and IGLC primers, respectively. Libraries were sequenced on the MiSeq Illumina instrument. IG sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis conducted using an in-house, validated bioinformatics pipeline. Rearrangements with identical CDR3 aa sequences were herein defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. For the HC analysis of subset #169, we obtained 894,849 productive sequences (mean: 127,836, range: 87,509-208,019). On average, each analyzed sample carried 54 clonotypes (range: 44-68); the dominant clonotype had a mean frequency of 99.1% (range: 98.8-99.2%) and displayed considerable intraclonal heterogeneity with a mean of 2,641 subclones/sample (range: 1,566-6,533). For the LCs of subset #169, we obtained 2,096,728 productive sequences (mean: 299,533, range: 186,637-389,258). LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean: 148, range: 110-205); the dominant clonotype had a mean frequency of 98.1% (range: 97.2-98.6%). Intraclonal heterogeneity was also observed in the LCs, with a mean of 6,325 subclones/sample (range: 4,651-11,444), hence more pronounced than in their partner HCs. Viewing each of the cumulative VH and VL CDR3 sequence datasets as a single entity branching through diversification enabled the identification of common sequences. In particular, 2 VH clonotypes were present in 3/6 cases, while a single VL clonotype was present in all 6 cases, albeit at varying frequencies; interestingly, this VL CDR3 sequence was also detected in all subset #2 cases, underscoring the molecular similarities between the two subsets. Focusing on SHM, the following observations were made: (i) the frequent 3-nucleotide (AGT) deletion evidenced in the VH CDR2 of subset #2 (leading to the deletion of one of 5 consecutive serine residues) was also detected in all subset #169 cases at subclonal level (average: 6% per sample, range: 0.1-10.8%); of note, the 5-serine stretch is also present in the germline VH CDR2 of the IGHV3-48 gene; (ii) the R-to-G substitution at the VL-CL linker, a ubiquitous SHM in subset #2 and previously reported as critical for IG self-association leading to cell autonomous signaling in this subset, was present in all subset #169 samples as a clonal event with a mean frequency of 98.3%; and, finally, (iii) the S-to-G substitution at position 6 of the VL CDR3, present in all subset #2 cases (mean : 44.2% ,range: 6.3-87%), was also found in all #169 samples, representing a clonal event in 1 case (97.2% of all clonotypes) and a subclonal event in the remaining 5 cases (mean: 0.6%, range: 0.4-1.1%). In conclusion, the present high-throughput sequencing data cements the immunogenetic relatedness of CLL stereotyped subsets #2 and #169, further highlighting the role of antigen selection throughout their natural history. These findings also argue for a similar pathophysiology for these subsets that could also be reflected in a similar clonal behavior, with implications for risk stratification. Disclosures Sutton: Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-128017
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 3
    Online Resource
    Online Resource
    VH CDR3-Focused Somatic Hypermutation in CLL IGHV-IGHD-IGHJ Gene Rearrangements with 100% IGHV Germline Identity
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4277-4277
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4277-4277
    Abstract: Classification of patients with chronic lymphocytic leukemia (CLL) based on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the sequence of the rearranged IGHV gene excluding the VH CDR3. This is mostly due to the difficulty in discriminating actual SHM from random nucleotides added between the recombined IGHV, IGHD and IGHJ genes. Hence, this approach may underestimate the true impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions i.e. the VH CDR3. Relevant to mention in this respect, studies from our group in CLL with mutated IGHV genes (M-CLL), particularly subset #4, have revealed considerable intra-VH CDR3 diversity attributed to ongoing SHM. Prompted by these findings, here we investigated whether SHM may also be present in cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3), focusing on two well characterized stereotyped subsets i.e. subset #1 (IGHV clan I/IGHD6-19/IGHJ4) and subset #6 (IGHV1-69/IGHD3-16/IGHJ3). These subsets carry germline-encoded amino acid (aa) motifs within the VH CDR3, namely QWL and YDYVWGSY, originating from the IGHD6-19 and IGHD3-16 gene, respectively. However, in both subsets, cases exist with variations in these motifs that could potentially represent SHM. The present study included 12 subset #1 and 5 subset #6 patients with clonotypic IGHV genes lacking any SHM (100% germline identity). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified by subgroup-specific leader primers and a high-fidelity polymerase in order to ensure high data quality. RT-PCR products were subjected to paired-end NGS on the MiSeq platform. Sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis was undertaken using an in-house purpose-built bioinformatics pipeline. Rearrangements with the same IGHV gene and identical VH CDR3 aa sequences were defined as clonotypes. Overall, we obtained 1,570,668 productive reads with V-region identity 99-100%; of these, 1,232,958 (mean: 102,746, range: 20,796-242,519) concerned subset #1 while 337,710 (mean: 67,542, range: 50,403-79,683) concerned subset #6. On average, 64.4% (range: 1.7-77.5%) of subset #1 reads and 49.2% (range: 0.7-70%) of subset #6 reads corresponded to rearrangements with IGHV genes lacking any SHM (100% germline identity). Clonotype computation revealed 1,831 and 1,048 unique clonotypes for subset #1 and #6, respectively. Subset #1 displayed a mean of 157 distinct clonotypes per sample (range: 74-267), with the dominant clonotype having a mean frequency of 96.9% (range: 96-98.2%). Of note, 44 clonotypes were shared between different patients (albeit at varying frequencies), including the dominant clonotype of 11/12 cases, which was present in 2-6 additional subset #1 patients. Subset #6 cases carried a higher number of distinct clonotypes per sample (mean: 219, range: 189-243) while the dominant clonotype had a mean frequency of 95.6% (range: 94.5-96.5%). Shared clonotypes (n=30) were identified also in subset #6 and the dominant clonotype of each subset #6 case was present in 3-5 additional subset #6 patients. Focusing on the VH CDR3, in particular the IGHD-encoded part, the following observations were made: (1) in both subsets, extensive intra-VH CDR3 variation was detected at certain positions within the IGHD gene; (2) in most cases, the observed aa substitutions were conservative i.e. concerned aa sharing similar physicochemical properties. Particularly noteworthy in this respect were the observations in subset #6 that: (i) the valine residue (V) in the D-derived YDYVWGSY motif was very frequently mutated to another aliphatic residue (A, I, L); (ii) in cases were the predominant clonotype carried I (also in the Sanger-derived sequence), several minor clonotypes carried the germline-encoded V, compelling evidence that the observed substitution concerned true SHM. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, very likely attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. While the prognostic/predictive relevance of this observation is beyond the scope of the present work, our findings highlight the possible need to reappraise definitions ('semantics') regarding SHM status in CLL. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-127979
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 4
    Online Resource
    Online Resource
    High-Throughput T Cell Receptor (TR) Repertoire Analysis of Virus-Specific T Cells: Implications for T Cell Immunotherapy and Viral Infection Risk Stratification
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 2057-2057
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 2057-2057
    Abstract: Viral infections, mainly by cytomegalovirus (CMV), Epstein Barr virus (EBV) and polyomavirus type I (BKV), are major causes of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As effective immune responses against human viruses rely on an armamentarium of T-cell receptor (TR) repertoire capable of recognizing a broad range of antigenic peptides of those pathogens, reconstitution of antiviral immunity, either by spontaneous generation of endogenous virus-specific T cells (VSTs) or by adoptive immunotherapy with VSTs, plays a critical role to fight infections. We here evaluated the diversity and clonality of TR repertoire of functional tri-virus-specific T cell products generated from immunocompetent donors (n=10) and compared their TR gene repertoire to that of peripheral blood mononuclear cells (PBMCs) from patients who had undergone allo-HSCT (n=5). To generate tri-VSTs, PBMCs derived from 15-20ml of peripheral blood of normal donors, were exposed to EBV, CMV and BKV overlapping peptides and cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors. Specificity of donor-derived VSTs and patient-derived PBMCs was measured by IFN-γElispot. TR diversity was investigated by next-generation sequencing on a MiSeq Sequencer, after amplification of TR beta chain gene rearrangements by RT-PCR with the BIOMED-2 protocol. Raw NGS reads were filtered based on their length and quality and the filtered-in sequences were submitted to IMGT/HighVQUEST. Metadata analysis and clonotype computation were performed using a validated in-house bioinformatics platform. As clonotype we defined sequences carrying the same TRBV gene and identical CDR3 amino acid sequence. Tri-VSTs provided 947,298 productive TRBV-TRBD-TRBJ rearrangements and a polyclonal and highly diverse TR gene repertoire, consisting of a total of 169,502 unique clonotypes (average: 16,950/sample, range 4,057-45,602), 64,971 (38.3%) of which were expanded (corresponding to more than one sequence). In terms of clonality, the mean relative frequency of the major clonotype in all tri-VSTs was 12.6% (range 3.3-29.2%). Interestingly, among tri-VST cell lines, 637 clonotypes were shared (present in 〉 2/10 samples), 80 were highly shared (present in 〉 3/10 samples) while 7 were present in 6-8 different VST lines and largely expanded, accounting for up to 29.2% of all sequences. Importantly, there were 65 of 96 major VST clonotypes shared, thus suggesting that they were potentially associated with recognition of the targeted viruses. Given that 4/10 VSTs cell lines were not specific for CMV, while being EBV-and BKV-specific, dominant TRs in those 4 cell lines can potentially be associated with EBV- or BKV-activity. By searching a public database of TR clonotypes with known reactivity against EBV and/or CMV (ShugayM, Nucleic Acids Research, 2018), we found 8 shared EBV-specific and 4 shared CMV-specific clonotypes among our VSTs and the 499 public clonotypes. When we compared the produced VSTs with PBMCs from 3 allo-grafted patients with circulating CMV-, BKV- and EBV-specific T cells and previous viral reactivation, we detected 163 shared clonotypes. Likewise, we observed 21 and 23 shared clonotypes in similar frequencies, between VSTs and PBMCs from 2 patients with CMV- or BKV-specific T cell immunity. These data identify clones that potentially expand in vivo and protect patients from viral infections. Overall, our findings reveal high levels of TR clonality in cell lines enriched for T cells reactive against EBV and/or CMV and/or BKV and provide insights into the TR repertoire of ex vivo- or endogenously-generated VSTs. Our approach may help to identify optimal TRs for immunotherapy as well as TRs which can be used as a tool for risk stratification of viral infections. Disclosures Agathangelidis: Gilead: Research Funding. Gemenetzi:Gilead: Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Abbvie: Research Funding; Janssen: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-118851
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 5
    Online Resource
    Online Resource
    Chystallographic Evidence of Autologous Recognition By a Clonotypic B Cell Receptor in Chronic Lymphocytic Leukemia
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 4129-4129
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 4129-4129
    Abstract: Chronic lymphocytic leukemia (CLL) leukemic cells express B-cell receptor immunoglobulin (BcR IG) whose signaling is of paramount importance throughout the natural history of the disease. Indeed, signaling pathways downstream of the BcR are constitutively active in all cases of CLL and inhibitors of the Bruton's tyrosine kinase BTK (Ibrutinib) or PI3Kδ (Idelalisib), two downstream signaling effectors, are clinically effective. This functional evidence complements earlier molecular observations supporting antigen drive in CLL ontogeny, including the distinction of CLL into cases with somatically hypermutated BcR IG (M-CLL) that have a significantly better outcome compared to those with unmutated, germline-like receptors (U-CLL). CLL also displays a remarkably skewed BcR IG gene repertoire, culminating in the existence of highly homologous, stereotyped BcR IG in 〉 30% of cases, indicating selection by a limited set of antigenis. A number of potential antigenic elements have been described, being recognized by the monoclonal receptors and able to deliver intracellular signals. More recently, it has been reported that CLL cells are endowed with the apparently unique property of autonomous signaling, since individual CLL-derived BcR IG can promote Ca2+ influx and NF-κB target gene transcription in a reconstituted B cell system upon self-recognition of common BcR-intrinsic epitopes. However, the precise molecular details of such process are unknown. In order to gain insight into the molecular interactions, particularly to further understand the role played by autonomous signaling, we determined the crystal structures of two BcR IG of CLL cases assigned to subset #4. This is a CLL subset expressing stereotyped, G(κ)-switched BcR IG encoded by the IGHV4-34/IGKV2-30 gene combination. Subset #4 accounts for ~1% of all CLL and is the largest within M-CLL, distinctive for a particularly indolent clinical course. BcR IG derived from two subset #4 cases were found to bind autologously via their VH CDR3 loops to a composite surface spanning the variable and constant regions of the heavy chain; the relevant epitope is conserved in all cases belonging to subset #4 and differs from other non-subset #4 BcR IG. This specific self-recognition was identified as dependent on the individual IG gene usage in the BcR, and is functionally relevant as it occurs in solution and leads to intracellular signalling in B cells. Analysis of epitope and paratope mutants revealed that the interactions observed in the crystal structures are mediated by a few critical amino acid residues. Indeed, the distinctively conserved amino acid residues in the VH CDR3 loop of the BcR IG both dictate a specific VH-VK pairing and shape the combining site for autologous recognition. Moreover, the epitope comprises specific amino acids from the CH1 domain that restrict the autologous recognition to IgG molecules. Finally, we found persisting long-lived interaction occurring between subset #4 BcR IGs, thus recalling high affinity receptor-cognate antigen interactions associated with the induction of anergy. This scenario well fits with the anergic phenotype of the subset #4 leukemic cells, and thus provides a biochemical explanation for the indolent clinical course of this subset. In conclusion, though focusing on a particular CLL subset, the structural and biochemical analysis here presented describes a general model for autologous recognition that may epitomize the molecular events leading to the expansion of CLL B lymphocytes at large. It is conceivable that CLL-associated BcR IGs can each bind to a distinct internal epitope with the specific nature of the interaction dictated by diverse factors e.g. VDJ recombination, heavy and light chain pairing, SHM, and isotype switch. The strength and persistence of the autologous recognition can then lead to a specific outcome in the intracellular signaling process, ranging from proliferation to anergy. The structural diversity thus produced in the BcR IG development may be linked to and underlie the heterogeneity characterizing CLL at the biological and clinical level. Disclosures Stamatopoulos: Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Ghia:Pharmacyclics: Honoraria; Gilead: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria; Roche: Research Funding; GSK: Research Funding; AbbVie: Honoraria; Celgene: Honoraria; Adaptive Biotechnologies: Consultancy.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.4129.4129
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 6
    Online Resource
    Online Resource
    Automated Clustering Analysis of Immunoglobulin Sequences in Chronic Lymphocytic Leukemia Based on 3D Structural Descriptors
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 4365-4365
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 4365-4365
    Abstract: Immunoglobulins (Igs) are crucial for the defense against pathogens, but they are also important in many clinical and biotechnological applications. Their characteristics, and ultimately their function, depend on their three-dimensional (3D) structure; however, the procedures to experimentally determine it are extremely laborious and demanding. Hence, the ability to gain insight into the structure of Igs at large relies on the availability of tools and algorithms for producing accurate Ig structural models based on their primary sequence alone. These models can then be used to determine structural and eventually functional similarities between different Igs. An example of such a task is the clustering of Igs based on their structure to determine meaningful common features such as the possible existence of common molecular targets (antigens). Several approaches have been proposed in order to achieve an optimal solution to this task yet their results were hindered mainly due to the lack of efficient clustering methods based on the similarity of 3D structure descriptors. Here, we present a novel workflow for robust Ig 3D modeling and automated clustering. We validated our protocol in chronic lymphocytic leukemia (CLL), where the clonotypic Igs are critically implicated in the disease ontogeny and evolution. Indeed, immunogenetic studies on the clonotypic Igs have strongly implicated antigen selection in the pathogenesis of CLL, while also providing robust prognostic information. In the present study, we used the structure prediction tools PIGS and I-TASSER for creating the 3D models and the TM-align algorithm to superpose them. The innovation of the current methodology resides in the usage of methods adapted from 3D content-based search methodologies to determine the local structural similarity between the 3D models. The Fast Point Feature Histograms descriptors derived from the structurally aligned parts are used to compute a distance matrix, which is then used as input for the clustering procedure. Clustering analysis on the data is performed through the application of the agglomerative and density-based clustering approaches. The first method is unsupervised whereas the second belongs to the semi-supervised type, i.e. requires a predefined number of clusters. To evaluate the quality of the herein described workflow, we performed a supervised analysis of 125 Ig 3D models originating from 5 CLL stereotyped subsets i.e. subgroups sharing (quasi) identical IGs, namely subsets #1, #2, #4, #6, #8. The reasoning behind this choice was that (i) homologous Ig primary sequences can be reasonably anticipated to be reflected in overall similar 3D structures, hence providing a reference for evaluating the developed workflow; and, (ii) these subsets are well characterized at both the clinical and biological levels. Subset size distribution was as follows: subset #1 (IGHV clan I/IGKV1(D)-39), n=37; subset #2 (IGHV3-21/IGLV3-21), n=43; subset #4 (IGHV4-34/IGKV2-30), n=22; subset #6 (IGHV1-69/IGKV3-20), n=12; and, subset #8 (IGHV4-39/IGKV1(D)-39), n=11. Overall, we obtained a high level of clustering accuracy i.e. Ig 3D model clusters matched to a very high degree the subsets defined by Ig primary sequence similarity. In detail, 5 Ig 3D model clusters were produced by: (i) cluster 1 containing 37/37 (100%) subset #1 models and one (8.3%) subset #6 model, (ii) cluster 2 containing 43/43 (100%) subset #2 models, (iii) cluster 3 containing 21/22 (95.5%) subset #4 models, (iv) cluster 4 containing 11/12 (91.7%) #6 models, and, (v) cluster 5 containing 11/11 (100%) subset #8 models along with a single (4.5%) subset #4 model (subsets #4 and #8 concern IgG CLL, in itself a rarity for CLL). These findings support that the innovative workflow described here enables robust clustering of 3D models produced from Ig sequences from patients with CLL. Furthermore, they indicate that CLL classification based on stereotypy of Ig primary sequences is likely also verified at the Ig 3D structural level. Studies are ongoing for both addressing the minor discrepancies observed here and producing the unsupervised 3D clustering of the IGs from a large series of both stereotyped and non-stereotyped CLL cases. Disclosures Rosenquist: Gilead Sciences: Speakers Bureau. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.4365.4365
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 7
    Online Resource
    Online Resource
    Integrating multiple immunogenetic data sources for feature extraction and mining somatic hypermutation patterns: the case of “towards analysis” in chronic lymphocytic leukaemia
    Springer Science and Business Media LLC ; 2016
    In:  BMC Bioinformatics Vol. 17, No. S5 ( 2016-6)
    Details
    In: BMC Bioinformatics, Springer Science and Business Media LLC, Vol. 17, No. S5 ( 2016-6)
    Type of Medium: Online Resource
    ISSN: 1471-2105
    URL: Article
    DOI: 10.1186/s12859-016-1044-3
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
    detail.hit.zdb_id: 2041484-5
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 8
    Online Resource
    Online Resource
    CLL with Mutated IGHV4-34 Antigen Receptors Is Clinically Heterogeneous: Antigen Receptor Stereotypy Makes the Difference
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 5263-5263
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 5263-5263
    Abstract: The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with 〉 90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p 〈 0.0001). Regarding other genomic aberrations, they were either absent (NOTCH1 mutations) or rare (SF3B1 mutations, trisomy 12, del(11q), TP53 aberrations due to either del(17p) and/or TP53 mutations). The sole exception concerned a high frequency (14%) of TP53 aberrations in subset #29 (p 〈 0.05 compared with the other subsets), which is notable for M-CLL cases in general. Time to first treatment (TTFT) could be analyzed in 228 cases. IgG subsets #4 and #16 had significantly (p=0.036) longer TTFT (median TTFT: not yet reached) compared to the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). In conclusion, we have identified distinct clinicobiological profiles for different stereotyped IGHV4-34 M-CLL subsets, highlighting subsets #4 and #16 as particularly indolent, which is important for both medical and social reasons, especially considering that a significant proportion of patients in these subsets are diagnosed at younger ages. Our findings support the notion that BcR IG stereotypy refines prognostication in CLL, superseding the crude immunogenetic distinction based on SHM load only. Additionally, the observed heterogeneity suggests that not all M-CLL are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Disclosures Tausch: Gilead: Other: Travel support. Shanafelt:Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding; Celgene: Research Funding; Polyphenon E Int'l: Research Funding; Hospira: Research Funding; Janssen: Research Funding; Pharmactckucs: Research Funding; Cephalon: Research Funding. Niemann:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy; Novartis: Other: Travel grant. Langerak:InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). Hallek:Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding. Ghia:Janssen Pharmaceuticals: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.5263.5263
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 9
    Online Resource
    Online Resource
    Evidence for Epitope-Specific T Cell Responses in HIV-Associated Non Neoplastic Lymphadenopathy: High-Throughput Immunogenetic Evidence
    American Society of Hematology ; 2018
    In:  Blood Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1117-1117
    Details
    In: Blood, American Society of Hematology, Vol. 132, No. Supplement 1 ( 2018-11-29), p. 1117-1117
    Abstract: Non-neoplastic lymphadenopathy (NNL) associated with the human immunodeficiency virus (HIV) infection may develop concurrently with the onset of HIV viremia (acute retroviral syndrome) that can persist beyond the acute phase. Histopathological findings at this early phase mainly pertain to hyperplastic changes with large lymphoid follicles; with time, the number of lymphoid follicles diminishes, while plasma cells increase; at the extreme is a pattern characterized by sclerosis of the germinal centers in the residual follicles. HIV-specific CD8+ T cell responses have been reported and certain viral protein epitopes have been identified e.g. the p24 protein, a component of the HIV particle capsid. Overall, these findings reflect an ongoing immune response that is still incompletely characterized at the molecular level, particularly as it concerns the composition of the T cell receptor (TR) gene repertoire. In order to obtain a comprehensive view into the role of antigen selection in shaping T cell responses in HIV-associated NNL [HIV(+) NNL], we studied in-depth the TR repertoire in: (i) lymph node biopsy samples from 12 patients with HIV(+) NNL, (ii) lymph node samples from 5 non-HIV patients with reactive lymphadenopathy [HIV(-) RL] ; and, (iii) peripheral blood samples from 4 healthy, HIV-seronegative individuals without lymphadenopathy [healthy controls, HIV(-) HC]. Genomic DNA was isolated from either paraffin-embedded lymph nodes (for patients with lymphadenopathy) or blood mononuclear cells (for healthy individuals). TRBV-TRBD-TRBJ gene rearrangements were amplified according to the BIOMED2 protocol. PCR products were subjected to next generation sequencing (NGS) on the MiSeq Illumina Platform. NGS data analysis, interpretation and visualization was performed by a validated, in-house bioinformatics pipeline. Overall, we obtained: (i) 1,440,305 (mean: 120,025) productive rearrangement sequences in the HIV(+) NNL group; (ii) 702,533 (mean: 140,506) productive sequences in the HIV(-) RL group; and, (iii) 539,981 (mean: 134,995) productive sequences in HIV(-) HC cases. Rearrangements with identical TRBV gene usage and CDR3 sequence were defined as clonotypes. In total, we identified 15,553 unique clonotypes in patients with HIV(+) NNL (mean: 1,296, range: 337-6,212), 53,874 in HIV(-) RL (mean: 10,774, range: 3,336-16,304) and 220,069 clonotypes in HIV(-) HC cases (mean; 55,017, range: 35,430-68,916), indicating significant repertoire restriction in the former group. Indeed, this group was characterized by an increased level of oligoclonality compared to the other two groups: the mean values of the sum of relative frequencies for the 10 most frequent clonotypes were 80%, 19.6% and 16.5%, respectively. Seven of 12 HIV(+) NNL cases carried the same dominant clonotype (TRBV29-1, SVDPSGTGGEGYT) that was also found in the remaining 5 patients of this group, albeit at lower frequencies; in contrast, it was completely absent in the HIV(-) RL and HIV(-) HC groups. Regarding the TRBV gene repertoire, the TRBV29-1 gene was overrepresented (p 〈 0.005) in the HIV(+) NNL group, whereas the TRBV6-5 and TRBV19 genes were frequent in both groups of patients with lymphadenopathy (HIV-associated or not); finally, the TRBV5-1 was underrepresented (p 〈 0.005) in patients with lymphadenopathy (HIV-associated or not) compared to HIV(-) HC cases. Comparison of the present TR gene sequence dataset against public databases identified 2 clonotypes with an established reactivity against the p24 protein that were present in 2 different patients with HIV(+) NNL of the present cohort. In conclusion, the TR gene repertoire of patients with HIV(+) NNL displays increased level of clonality, distinct TRBV gene repertoire as well as a widely shared, specific dominant clonotype compared to HIV(-) RL cases or HIV(-) healthy controls. These findings allude to an antigen-driven, HIV-specific immune process, a claim also supported by the detection of clonotypes with established anti-HIVp24 reactivity in at least a fraction of the analyzed patients. Disclosures Gemenetzi: Gilead: Research Funding. Agathangelidis:Gilead: Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou:Gilead: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2018-99-118975
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2018
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 10
    Online Resource
    Online Resource
    Overexpression of the Histone Methyltransferase ΕΖΗ2 in Chronic Lymphocytic Leukemia Confers Protection from Apoptosis and Is Linked to Clinical Aggressiveness
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 1956-1956
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1956-1956
    Abstract: EZH2 is the enzymatic subunit of the polycomb repressive complex 2 (PRC2), which induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 over-expression has been reported in a broad range of hematopoietic and solid malignancies and associated with poor prognosis. Recently, we reported for the first time that EZH2 is expressed in CLL and, notably, that EZH2 mRNA and protein levels were up-regulated in clinically aggressive, IGHV-unmutated (U-CLL) stereotyped subset #1 versus indolent, IGHV-mutated (M-CLL) subset #4 and, moreover, that EZH2 expression in subset #1 is regulated by miR-101. Prompted by these preliminary observations, here we sought to investigate in more detail EZH2 expression patterns and functionality in CLL. First, using RQ-PCR, we extended our gene expression analysis to 141 CLL cases, including stereotyped subset #2, the largest stereotyped subset overall (~3% of all CLL) with noted clinical aggressiveness, though mostly concerning M-CLL. We found that bad-prognosis, U-CLL cases express higher EZH2 mRNA levels compared to more indolent, M-CLL cases (fold difference, FD 〉 2, p 〈 0.00001), albeit with variability in EZH2 mRNA levels within each mutational subgroup. Similar results were obtained by Western analysis for EZH2 protein expression, being significantly (p 〈 0.01) higher in U-CLL (n=28) vs M-CLL (n=28), again with intra-subgroup variability. Of note, EZH2 levels where low in the aggressive subset #2 cases, similar to M-CLL, but in sharp contrast to other aggressive, U-CLL cases, including stereotyped subsets #1, #6 and #8. We also extended miR-101 expression analysis to 20 U-CLL and 20 M-CLL cases, in addition to 8 subset #1 and subset #4 cases reported previously, and found that EZH2 mRNA levels were significantly anti-correlated (r=-0.6, p 〈 0.005) with miR-101 levels only in U-CLL, reinforcing the possibility of a regulation of EZH2 expression by miR-101. In order to explore if other polycomb and trithorax complex components, including chromatin modification enzymes and remodeling factors, are dysregulated in CLL, using PCR arrays we analysed the expression of 84 relevant genes in 10 U-CLL and 10 M-CLL cases. Focusing on the main PRC2 components (namely, SUZ12, EED, EZH1, RBBP4/7), we found that their levels were significantly correlated (r=0.49-0.78; p 〈 0.05) with EZH2 levels. Given the variability in EZH2 expression within both M-CLL and U-CLL, for further investigations into the functional impact of EZH2 in CLL, the studied cases were classified into ‘’EZH2 high’’ and ‘’EZH2 low’’ subgroups based on EZH2 mRNA levels (cut-off value determined by ROC curve analysis and Youden Index). In order to explore the effect of EZH2 expression on cell survival, we analyzed the viability of CD19+ CLL cells after 9 days in culture and found that CLL clones from 7 ‘’EZH2 high’’ cases displayed significantly (p 〈 0.0001) higher viability compared to 8 ‘’EZH2 low’’ cases. Moreover, ‘’EZH2 high’’ cases (n=19) displayed higher H3K27me3 levels compared to ‘’EZH2 low’’ cases (n=34) (p 〈 0.05). Next, we blocked EZH2 expression using siRNA in CLL cells from 3 ‘’EZH2 high’’ cases and observed downregulation of H3K27me3 levels along with time-dependent increase of cell apoptosis, indicating that EZH2 associates with a survival advantage to CLL cells. In line with this, when we treated CD19+ CLL cells from 6 ‘’EZH2 high’’ cases with EZH2 pharmacological inhibitors (GSK-343, EPZ-6438), we found that H3K27me3 levels were decreased in a time- and dose-dependent manner. Moreover, both inhibitors decreased CLL cell viability overtime, suggesting that the histone trimethylation catalytic activity of EZH2 is vital for CLL cell survival. Finally, we searched for potential clinical implications and found that ‘’EZH2 high’’ cases (n=45) showed significantly shorter time-to-first-treatment (p 〈 0.0001) in comparison to ‘’EZH2 low’’ cases (n=62). In conclusion, we demonstrate that EZH2 overexpression is associated with prolonged CLL cell survival and that pharmacological inhibition of EZH2 catalytic activity leads to apoptosis, highlighting a crucial role for EZH2 in CLL cell homeostasis. On these grounds, EZH2 emerges as a novel potential therapeutic target for specific subgroups of CLL. Disclosures Stamatopoulos: GlaxoSmithKline Pharmaceuticals Ltd: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.1956.1956
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...