Repertoire Analysis Research Articles

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5’ reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Abstract A008: Adaptive immune receptor repertoire characterization highlights the importance of interplay between B and T cells in disease progression

Abstract Understanding the adaptive immune system is integral to disease progression studies, development of diagnostic biomarkers, identification of therapeutic targets, and discerning varied treatment responses. Despite growing interest in profiling B and T cells in cancer research, the interaction between these cells and also the crucial roles of γδ T cells often go overlooked. In this research project, we explored the role of crucial yet frequently ignored γδ T cells in cancer research, using immune repertoire data from bladder cancer patients. We also studied the synergy between B and T cells in the immune repertoires of COVID-19 patients.Roche has developed immunoPETE (immune repertoire Primer Extension Target Enrichment), a DNA- based target enrichment assay to capture adaptive immune repertoire data*. immunoPETE efficiently identifies the heavy chains of B cells, alongside αβ and γδ T cells in one reaction, facilitating normalized clone counts across the adaptive immune system. To explore the significance of γδ T cells, we profiled the immune repertoires of 24 Non-Muscle Invasive Bladder Cancer patients treated with Bacillus Calmette-Guerin (BCG). Additionally, we evaluated the interplay between B and T cells in 117 peripheral blood immune repertoire from healthy and COVID-19 afflicted individuals, ranging from mild to severe cases.Immune repertoire data analysis indicates that underrepresentation of T cell receptor delta locus (TRD) is associated with higher risk of bladder cancer recurrence or progress. Urine pellet DNA was available for a subset of the patients in this study. Comparing immune repertoires from PBMC and urine revealed that urine repertoires represent the tumor repertoire more accurately than PBMC. Analysis of the immune repertoires from the COVID-19 patients linked disease severity with immune repertoire measures across all three cell types, highlighting the importance of multiple lymphocyte classes in disease progression. Using immunoPETE technology, which is capable of capturing the heavy chain of multiple immune cell types simultaneously in a single reaction, we have demonstrated that both B cell and T cell compartments provide distinct and valuable information in cancer and infectious diseases, each responding uniquely to the disease. Additionally, we emphasized the significance of γδ T cells in predicting cancer prognosis. Note: immunoPETE is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Hossein Asgharian, Hamid Mirebrahim, Dilduz Telman, Hahn Zhao, Ulrich Schlecht, Sylvie McNamara, Rajiv Dua, Jan Berka, Patrick Bogard, Dinesh Kumar. Adaptive immune receptor repertoire characterization highlights the importance of interplay between B and T cells in disease progression [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A008.

Abstract A075: Circulating T-cell receptor repertoire analysis improves cancer early detection

Abstract Introduction: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current ctDNA-based approaches detect only a minority of early-stage cancers. For liquid biopsy to realize its full potential for cancer early detection, sensitivity for earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy by exploiting tumor recognition by T cells through sequencing of the circulating T-cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 471 lung cancer patients (85% stage I) and 600 subjects without cancer enriched for older individuals with a history of smoking. We performed TCR beta chain sequencing to yield a median of 101,899 TCR clonotypes per sample (median 81,602 cancer / 112,966 controls) and built a TCR sequence similarity graph to cluster clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using an SVM model into a cancer score. Cancer RFU levels were also correlated to imputed subject HLA types for 32 HLA Class I and 38 Class II alleles. The model was evaluated by 10-fold cross-validation and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes and to 17 lung cancer related protein biomarkers in 86 subjects. Results: We identified 372 cancer-associated TCR RFUs at FDR≤0.1, including 198 enriched in cancer samples (fold-change range 1.03 to 3.13) and 174 enriched in controls (fold- change 0.96 to 0.30). Levels of 265/372 (71%) RFUs were correlated to presence of an HLA allele at FDR ≤ 0.1. Of the total 2,770 significant HLA-RFU associations, 479 (17%) involved an HLA Class I allele, while 2,291 (83%) involved an HLA Class II allele, highlighting a potential role for CD4 helper or regulatory T cell antigen recognition in the cancer RFUs found. The RFU cancer score achieved a cross-validated test ROC AUC of 0.71 for cancer status prediction (0.71 Stage I / 0.70 Stage II-IV) with 49% of Stage I cancers detected at a specificity of 80%. Importantly, the cancer score was not dependent on sample source site, technical or biological variation in TCR repertoire depth, or lung cancer histology subtype. The TCR RFU score also distinguished lung cancer patients from control subjects with other conditions such as benign lung nodules and COPD. We observed an up to ∼20%-point gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins in a potential multi-analyte cancer screening test. By contrast, TCR analysis did not provide additional sensitivity for stage II-IV disease. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. A similar study for the detection of early colorectal cancer and advanced adenomas is under way. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jasper Braun, María Antonia Fortuño, María-del-Mar Ocón, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M Montuenga, Roman Yelensky. Circulating T-cell receptor repertoire analysis improves cancer early detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A075.

Characterization of the T-cell receptor repertoire associated with lymph node metastasis in colorectal cancer

PurposeColorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with lymph node (LN) metastasis playing a pivotal role in disease progression. This study aimed to explore the T-cell receptor (TCR) repertoire among CRC patients, distinguishing those with LN metastasis from those without, in order to uncover potential biomarkers for predicting metastasis.MethodsWe analyzed the TCR repertoire in CRC patients with and without LN metastasis. A classification model utilizing random forest analysis was developed to assess the predictive potential of the TCR repertoire.ResultsThe findings demonstrated a significant increase in the number of V-J combinations and immune CDR3 sequences in patients with LN metastasis compared to the control group. The classification model achieved high accuracy in differentiating patients with LN metastasis, with AUC values ranging from 0.514 to 0.794. Specific V-J combinations and CDR3 sequences were identified as significant predictors of the model’s predictive accuracy.ConclusionThese results suggest that the TCR repertoire is altered in CRC patients exhibiting LN metastasis, potentially influencing disease progression. This study highlights the importance of TCR repertoire analysis as a non-invasive biomarker for predicting LN metastasis in CRC patients.

T-cell Receptor Repertoire Analysis in the Context of Transarterial Chemoembolization Synergy with Systemic Therapy for Hepatocellular Carcinoma

T-cell Receptor Repertoire Analysis in the Context of Transarterial Chemoembolization Synergy with Systemic Therapy for Hepatocellular Carcinoma

RBIO-06. STEREOTACTIC RADIOSURGERY ENHANCES T-CELL RECEPTOR REPERTOIRES AND INDUCES IMMUNOGENICITY IN BRAIN METASTASIS

Abstract BACKGROUND Brain metastasis is a frequent and deadly complication of systemic malignancy. The incidence rate is increasing and the prognosis remains poor, in contrast to recent advances in the management of systemic malignancy. Our previous work revealed an immunosuppressive microenvironment in brain metastasis and correlation between T-cell status and patient outcomes, shared across the primary tumor histologies. Therefore, we hypothesize that induction of immunogenicity has potential to improve brain metastasis patient outcome. METHODS Pre-operative stereotactic radiosurgery (SRS) (n = 44) and post-operative SRS (n = 47) brain metastasis specimens were collected from an ongoing clinical trial (MDACC protocol 2018-0552) designed to determine optimal timing of SRS for the treatment of brain metastasis. The patient materials were extracted DNA and RNA, and subjected to T-cell receptor (TCR) repertoire sequencing and mRNA-seq to interrogate radiation-induced immunogenicity. RESULTS In a preliminary analysis of the first 21 specimens, Gene Set Enrichment Analysis revealed that SRS significantly enhanced multiple immune-related signaling pathways including interferon response, along with apoptotic pathways, in brain metastasis. We also found that TCR signaling and PD-1 signaling was upregulated by radiotherapy. TCR repertoire analysis further identified increased density and diversity of TCRs induced by SRS treatment. Profiling of the remaining sample set is currently ongoing. CONCLUSIONS In light of our previous finding regarding the correlation between immune status and outcomes of patients with brain metastases, reversing immunosuppression in brain metastasis could be a key factor on patient prognosis and treatment. Our TCR repertoire and mRNA sequencing analyses with pre-operative and post-operative SRS brain metastasis cohorts support a role for radiation-induced immunogenicity, pointing to a prospective therapeutic approach for brain metastasis management.

The role of polyreactive memory B cells in systemic lupus erythematosus.

In systemic lupus erythematosus (SLE), the production of autoantibodies is a crucial characteristic, and B cells play a significant role in its pathogenesis. B cells are the immune cells most associated with the genetic predispositions of SLE, and recent clinical studies showing that anti-CD19 CAR-T cell therapy induces drug-free remission have underscored the importance of B cells in SLE. Meanwhile, various B cell subsets exist across different stages of differentiation, from naive B cells to plasma-cells, and identifying the important subpopulations within SLE remains a critical future challenge. Years of B cell repertoire analyses have revealed the importance of polyreactive B cell receptors (BCRs) and autoantibodies that react to a various self-antigens and microbial antigens. Particularly, memory B cells with polyreactive BCRs, which play a crucial role in biological defense during the fetal stage, are characteristically differentiated in SLE. Type I interferon-mediated expression of CXCL13 and IL21 in CD4+ T cells is associated with the development of polyreactive memory B cells. The expansion of the polyreactive B cell repertoire, vital for defending against infections such as viruses, may exert an intrinsic function in SLE.

Evolving antibody response to SARS-CoV-2 antigenic shift from XBB to JN.1.

The continuous evolution of SARS-CoV-2, particularly the emergence of BA.2.86/JN.1 lineage replacing XBB, necessitates re-evaluation of vaccine compositions 1-3. Here, we provide a comprehensive analysis of the humoral immune response to XBB and JN.1 human exposure. We demonstrate the antigenic distinctiveness of XBB and JN.1 lineages in SARS-CoV-2-naive individuals, and JN.1 infection elicits superior plasma neutralization against its subvariants. We highlight KP.3's strong immune evasion and receptor binding capability, supporting its foreseeable prevalence. Extensive analysis of the BCR repertoire, isolating ~2000 RBD-specific antibodies with their targeting epitopes characterized by deep mutational scanning (DMS), underscores the superiority of JN.1-elicited memory B cells 4,5. Class 1 IGHV3-53/3-66-derived neutralizing antibodies (NAbs) contribute majorly within wildtype-reactive NAbs against JN.1. However, KP.2 and KP.3 evade a substantial subset, even those induced by JN.1, advocating for booster updates to KP.2/KP.3. JN.1-induced Omicron-specific antibodies also demonstrate high potency across Omicron. Escape hotspots of these NAbs have already been mutated, resulting in higher immune barrier to escape, considering probable recovery of escaped NAbs. Additionally, the prevalence of IGHV3-53/3-66-derived antibodies, and their capability of competing with all Omicron-specific NAbs suggests their inhibitory role on the activation of Omicron-specific naive B cells, potentially explaining the heavy immune imprinting in mRNA-vaccinated individuals 6-8. These findings delineate the evolving antibody response to Omicron antigenic shift from XBB to JN.1, and highlight the importance of developing JN.1-lineage, especially KP.2/KP.3-based vaccine boosters.

Unravelling T-cell dynamics and immune responses in initial and recurrent uveitis.

This study aimed to identify novel serological targets and investigate immune responses in patients with non-infectious uveitis, focusing on differences between initial onset and recurrent episodes. Differential gene expression analysis, immunocyte typing and T-cell receptor (TCR) gene analysis were conducted on RNA-sequenced peripheral blood samples from healthy individuals (n = 6) and non-infectious uveitis patients (n = 12), divided into 6 patients each at initial onset and recurrent stages. Peripheral blood T-cell types were analysed using flow cytometry. Bioinformatics methods included tools for RNA sequencing data processing, CIBERSORT for immune cell type prediction and specialized software for TCR repertoire analysis. Findings indicated that individuals with recurrent uveitis demonstrated a stronger adaptive immune response and a more pronounced immune imbalance compared to those with initial onset. Memory T cells were predominant in recurrent episodes, suggesting their potential role as biomarkers for disease progression. Significant differences in TCR diversity and V(D)J gene usage were observed between the various uveitis groups and healthy controls. Importantly, 38 uveitis-specific TCR sequences showed substantial expansion in the uveitis patients compared to controls. An elevated expansion of these specific TCR sequences was associated with an increased risk of uveitis development. The study highlights the critical role of adaptive immune responses and specific immune cell types in the pathogenesis of recurrent uveitis. Identification of the uveitis-specific TCR repertoire set could provide deeper insights into the disease and facilitate the development of targeted therapies for uveitis patients.

Mycosis fungoides - differentiation from inflammation and detection of circulating tumor cells with the EuroClonality-NGS assay.

Mycosis fungoides (MF) is a rare malignancy characterized by the presence of circulating tumor cells (CTCs) in a subgroup of patients. Reliably distinguishing MF from inflammatory skin conditions (IF) is a challenging task. This study aimed to evaluate the potential benefits of next-generation sequencing (NGS)-based T-cell receptor (TR) rearrangement repertoire analysis for detecting clonal rearrangements in MF and IF. Skin biopsies and blood samples from 33 MF patients (diagnosed according to WHO-EORTC criteria) and 10 IF patients were analyzed using TRB- and TRG-NGS. 27/33 MF patients were early-stage IA (n=19), IB (n=8), and six had advanced stages (IIB, n=5; IIIA, n=1). Analysis applying the standard abundance thresholds identified at least one clonal rearrangement in the skin DNA of 32/33 MF (97%) and 9/10 IF cases (90%). To enhance specificity, an abundance- and distribution-based approach was applied, considering only rearrangements that significantly stood out from the physiological background as clonal (MF 29/33, IF 1/10), allowing for a highly sensitive (88%) and specific (90%) discrimination between MF and IF. CTCs were detected in 11/24 (46%) early-stage and 3/5 (60%) late-stage MF patients. NGS-based TR repertoire analysis is a highly sensitive and specific method for the differential diagnosis of early-stage MF compared to IF, and for the sensitive molecular detection of CTCs.

Characteristics of the IgM Repertoires in the Peripheral Blood of Early Rheumatoid Arthritis Patients.

Rheumatoid arthritis is a common autoimmune disease, but little is known about the characteristics of the B cell repertoires in the peripheral blood. In this study, the peripheral IgM repertoires of early rheumatoid arthritis (ERA) patients were analyzed by high-throughput sequencing and bioinformatics analyses. Clonal expansion was observed in IgM repertoires of ERA patients. Interestingly, a subset of the dominant clones in ERA repertoires showed self- and poly-reactivity to several autoantigens. The clones were also present in IgM repertoires of healthy adults but they were not expanded, suggesting that may stem from the natural auto-reactive B cell repertoire. Additionally, the ERA repertoires exhibited a greater extent of somatic hypermutations, particularly in the ERA dominant clones, resulting in an enrichment of amino acids important for antigen-antibody interaction. The in-depth analysis of B-cell repertoires improved our knowledge of the IgM repertoires in early rheumatoid arthritis, offering potential insights into the disease's pathogenesis.

Multiplexed Antibody Sequencing and Profiling of the Human Hemagglutinin-specific Memory B Cell Response following Influenza Vaccination.

Influenza virus is a highly contagious respiratory pathogen causing between 9.4 and 41 million infections per year in the United States in the last decade. Annual vaccination is recommended by the World Health Organization, with the goal to reduce influenza severity and transmission. Ag-specific single B cell sequencing methodologies have opened up new avenues into the dissection of the Ab response to influenza virus. The improvement of these methodologies is pivotal to reduce the associated costs and optimize the operational workflow and throughput, especially in the context of multiple samples. In this study, PBMCs and serum samples were collected longitudinally from eight influenza vaccinees either vaccinated yearly for four consecutive influenza seasons or once for one season. Following the serological and B cell profiling of their polyclonal Ab response to a panel of historical, recent, and next-generation influenza vaccine hemagglutinin (HA) and virus strains, a single multiplexed Ag-specific single B cell sequencing run allowed to capture HA-specific memory B cells that were analyzed for preferential Ig H chain/L chain pairing, isotype/subclass usage, and the presence of public BCR clonotypes across participants. Binding and functional profiles of representative private and public clonotypes confirmed their HA specificity, and their overall binding and functional activity were consistent with those observed at the polyclonal level. Collectively, this high-resolution and multiplexed Ab repertoire analysis demonstrated the validity of this optimized methodology in capturing Ag-specific BCR clonotypes, even in the context of a rare B cell population, such as in the case of the peripheral Ag-specific memory B cells.

Prevalent and persistent new-onset autoantibodies in mild to severe COVID-19

Autoantibodies have been shown to be implied in COVID-19 but the emerging autoantibody repertoire remains largely unexplored. We investigated the new-onset autoantibody repertoire in 525 healthcare workers and hospitalized COVID-19 patients at five time points over a 16-month period in 2020 and 2021 using proteome-wide and targeted protein and peptide arrays. Our results show that prevalent new-onset autoantibodies against a wide range of antigens emerged following SARS-CoV-2 infection in relation to pre-infectious baseline samples and remained elevated for at least 12 months. We found an increased prevalence of new-onset autoantibodies after severe COVID-19 and demonstrated associations between distinct new-onset autoantibodies and neuropsychiatric symptoms post-COVID-19. Using epitope mapping, we determined the main epitopes of selected new-onset autoantibodies, validated them in independent cohorts of neuro-COVID and pre-pandemic healthy controls, and identified sequence similarities suggestive of molecular mimicry between main epitopes and the conserved fusion peptide of the SARS-CoV-2 Spike glycoprotein. Our work describes the complexity and dynamics of the autoantibody repertoire emerging with COVID-19 and supports the need for continued analysis of the new-onset autoantibody repertoire to elucidate the mechanisms of the post-COVID-19 condition.

Single-cell transcriptomics reveal potent extrafollicular B cell response linked with granzyme K+ CD8 T cell activation in lupus kidney

ObjectivesB and T cells constitute the majority of infiltrating lymphocytes in the kidney and represent the local perpetrators in lupus nephritis (LN), but the underlying pathogenic mechanisms are not well...

Analysis of B-cell receptor repertoire to evaluate the immunogenicity of SARS-CoV-2 RBD mRNA vaccine: MAFB-7256a (DS-5670d).

A monovalent Omicron XBB.1.5 mRNA RBD analogue vaccine, MAFB-7256a (DS-5670d), was newly developed and approved in Japan in the Spring of 2024 for the prevention of COVID-19. However, clinical efficacy data for this vaccine are currently lacking. We previously established the Quantification of Antigen-specific Antibody Sequence (QASAS) method to assess the response to SARS-CoV-2 vaccination at the mRNA level using B-cell receptor (BCR) repertoire assays and the Coronavirus Antibody Database (CoV-AbDab). Here, we used this method to evaluate the immunogenicity of MAFB-7256a. We analyzed repeated blood samples using the QASAS method from three healthy volunteers before and after MAFB-7256a vaccination. BCR response increased rapidly one week post-vaccination and then decreased, as with conventional vaccine. Notably, the matched sequences after MAFB-7256a vaccination specifically bound to the receptor-binding domain (RBD), with no sequences binding to other epitopes. These results validate that MAFB-7256a is an effective vaccine that exclusively induces antibodies specific for the RBD, demonstrating its targeted immunogenic effect.

APRIL/BAFF upregulation is associated with clonal B-cell expansion in Hunner-type interstitial cystitis.

Hunner-type interstitial cystitis (HIC) is a chronic inflammatory disease of the urinary bladder with an unknown etiology. We conducted comprehensive immunogenomic profiling of bladder specimens obtained by biopsy and cystectomy from 37 patients with HIC. Next-generation RNA sequencing demonstrated abundant plasma cell infiltration with frequent light chain restriction in HIC-affected bladder tissue. Subsequent analysis of the B-cell receptor repertoire revealed spatial and temporal expansion of B-cell clones. The extent of B-cell clonal expansion was significantly correlated with the gene expression levels of TNFSF13 and TNFSF13B, which encode APRIL and BAFF, respectively. These findings indicate that APRIL and BAFF are the key regulators of clonal B-cell expansion in HIC and might serve as therapeutic targets in this debilitating disease. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Single-cell analysis of the T-cell receptor repertoire in untreated myeloma patients suggests potential myeloma-reactive CD8+ T-cells are shared between blood and marrow.

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Diversification of Antibodies: From V(D)J Recombination to Somatic Exon Shuffling.

Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.

A-259 TCR Vβ repertoire analysis and immunophenotyping to detect target molecules in patients with mycosis fungoides and Sézary syndrome

A-259 TCR Vβ repertoire analysis and immunophenotyping to detect target molecules in patients with mycosis fungoides and Sézary syndrome

A-229 Identification of clones by TCR Vβ repertoire analysis supports diagnosis of leukemic cutaneous T-cell lymphoma

A-229 Identification of clones by TCR Vβ repertoire analysis supports diagnosis of leukemic cutaneous T-cell lymphoma