Dominant Clonotypes Research Articles

Single-cell T-cell receptor repertoire profiling in dogs

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

Chicken γδ T cells proliferate upon IL-2 and IL-12 treatment and show a restricted receptor repertoire in cell culture.

In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8-, and TCR1lowCD8- subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8- subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function.

Reverse Engineering of Antigenic Peptides in LGL to Decipher Disease Triggers

One of the hypotheses as to the pathogenesis of T-cell large granular lymphocytic leukemia (LGL) posits its origin from the initially polyclonal stimulation by unknown antigens, e.g., viral, autoimmune or tumor antigens. LGL evolves then via oligoclonal and ultimately monoclonal cytotoxic T-cell expansion. This process may be accompanied by acquisition of STAT3 (or rarely STAT5) mutations in 40% of cases. Identification of driver antigens is a key research question in LGL as it could both clarify its triggers as well as the mechanisms of cytopenias. We investigated a cohort of patients with this disease (n=195) by combining HLA allotypes and NGS-based TCR analysis to identify marker TCR Vβ clonotypes with the goal to reverse engineer or deduce peptides and therefore antigenic targets. For this purpose we used in silico and machine-learning modelling to capture structural features predictive of CDR3 Vβ epitope interactions in specific HLA allotype context. To focus on specific HLA/TCR interactions we have analyzed HLA for the presence of common haplotypes. First, we generated HLA haplotype calls using whole-exome sequencing data. We have found that HLA-A*02 was the most common class I allele, but not overrepresented in LGL vs controls. We have also identified HLA-B*44 as a risk allele present in 36% LGL with STAT3 mutation which was further enriched when considering HLA-A*02/HLA-B*44 together (Fig.1A). After TCR NGS we selected a threshold of 5.0% (based on study of 900 controls) to define an expanded (co)dominant clonotypes: 145 expanded clones were identified, of which 143 were unique/private. To identify possible consensus sequence across the CDR3 profile, we clustered the CDR3 AA sequences based on global alignment scores (Needleman-Wunsch) using BLOSUM62 substitution matrix and identified 9 clusters. However, multiple-sequence alignment within each CDR3 cluster did not show any distinct consensus sequence patterns overall and within cases sharing HLA-A*02. Cross-referencing with CDR3 databases of known conditions yielded 87 CDR3β (co)dominant LGL clonotypes. About 50% of those LGL clonotypes were shared in 2 common disease: type I diabetes, Celiac disease We also mined the VDJdb[2] for known CDR3-pHLA associations. Filtering to include high quality annotations, matches and HLA-A*02 restriction; LGL-derived TCR were found to recognize multiple epitopes. Prominent examples include CMV and EBV derived peptidic antigens from pp65, BRLF1, and BMLF1 (NLVPMVATV, RPPIFIRRL) present in 44% LGL expanded clonotypes (Fig.1B). In order to reverse engineer the identity of antigenic peptides based on the CDR3β sequence and HLA type (high affinity peptides restricted to HLA A2, a machine-learning method was developed as predictive modelling tool that quantifies CDR3β/peptide binding. The previously developed LLM trained on AA sequences was used for that purpose[3] whereby LLM embeddings serve as input to a deep CNN-based model, which is trained on IEDB pos/neg datasets[4]. This method was augmented with control CDR3 sequences as negative data points. As a result, our model showed an AUC of 0.94 in cross-validation. As a proof of concept of the proposed model in epitope discovery, CMV pp65-derived peptides were fitted into specific LGL CDR3 and CMV specific CDR3 clonotypes were found to match e.g., pp65 epitope NLVPMVATV among several others overlapping with the above-described findings. In conclusion, our analysis shows that a significant number of expanded clonotypes may be derived from known epitopes e.g., in autoimmune disease and viral infection pointing towards molecular mimicry. However, the specificity of a large fraction of the LGL spectrum remains unknown. For the discovery of corresponding peptides we generated ML-based antigen simulation method allowing to identify the best fitting peptides into the corresponding HLA allele and CDRVβ clonotype. Fig. Global map of co-occurrence of HLA allotypes showing the delta frequency compared to healthy controls and Sankey plot of identified CDR3β - Epitope associations (a) Using the whole-exome sequencing based HLA genotype calls, we calculated the frequency of co-occurrence of different HLA loci and investigated the differences in regards to controls (b) Using the VDJdb, we conducted sequence similarity based search and extracted the CDR3-pHLA annotations.

Distinct T Cell Receptor Gene Repertoires and T Cell Subset Distribution in Peripheral Blood and Bone Marrow of Patients with Multiple Myeloma

Mounting evidence suggests that the T cell repertoire in multiple myeloma (MM) is actively shaped by antigen selection. However, the molecular underpinnings of this observation are not fully understood, while relevant studies have been mostly based on immunophenotypic characterization of T cell subsets, with considerably less information regarding T cell receptor (TR) gene repertoire profiles. Moreover, most available information derives from the analysis of a single tissue compartment [i.e. peripheral blood (PB) or bone marrow (BM)], thus hindering addressing the potential impact of local T cell responses. Here we sought to overcome these limitations through paired analysis of PB and BM aspirates of 20 newly diagnosed patients with MM before the administration of any treatment, from whom we isolated the PB and BM mononuclear cell (PBMC/BMMC) fraction, as well as CD4 + and CD8 + T cells. TR beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4 + and CD8 + T cells and then subjected to paired-end next generation sequencing (NGS). Raw reads (n=14,369,410 | median 179,618/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ gene rearrangements were taken into consideration (n=9,212,507 | median 115,156/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 1,005,150 TRΒ distinct clonotypes were assessed (median=12,565/sample). Both CD4 + and CD8 + T cell populations displayed skewed TRBV repertoires (7 TRBV genes and 3 TRBJ genes accounting for about ~40% and ~60% of the repertoire) in both BM and PB. All patients displayed oligoclonal T cell expansions in both CD4 + and CD8 + T cells, albeit these were significantly (p<0.001) more pronounced in CD8 + T cells for both BM and PB samples. That said, oligoclonality was more pronounced in BM vs. PB for both CD4 and CD8 T cells, reaching statistical significance in the former: in particular, the median cumulative frequency of the most expanded clonotypes/sample was ~40% higher in CD4 + BM T cells vs. CD4 + PB T cells (p<0.001). Additionally, a significant shift was noticed in the major clonotype repertoire of CD4 + and CD8 + T cells in both PB and BM. In more detail, the 10 most expanded clonotypes/sample in PB had significantly lower frequency in BM, and, vice versa, the 10 most expanded clonotypes in BM were represented at diminished frequency or were absent in the PB in 12/20 patients. Flow cytometry analysis revealed distinct T cell subset composition in CD4 + or CD8 + T cells, also in different tissue compartments (BM vs. PB). Significant (p<0.001) differences concerned the over-representation of: (i) effector T cells (CD45RA +/CCR7 -, Temra) in CD8 + vs. CD4 + T cells in both BM and PB (median frequencies in BM and PB: 22.5% and 7% for CD8 + T cells; 6% and 1.6% for CD4 + T cells); (ii) CD8 + Temra cells in BM vs. PB (median frequencies of 22.5% vs. 7% in BM and PB, respectively; p<0.001); (iii) CD4 + vs. CD8 + central memory T cells (CD45RO +/CCR7 +, Tcm) in both PB and BM (median frequencies in PB and BM: 42.7% and 49.1% for CD4 + Tcm and 14% and 18.8% for CD8 + Tcm); (iv) CD8 + effector memory T cells (CD45RO +/CCR7 -, Tem) in BM vs. PB (median frequencies of 21.8% vs. 2.7% in BM and PB, respectively); (v) CD8 + naïve T cells (CD45RA +/CCR7 +, Tn) in PB vs. BM (median frequencies of 67.7% vs. 24.2% in PB and BM, respectively). In conclusion, oligoclonal expansions of CD4 + and, particularly, CD8 + T cells in MM that are more pronounced in the BM (at least for CD4 + cells) argue for selection by BM-biased antigens, a claim also supported by the significant differences in the relative frequency of dominant clonotypes in BM vs. PB. The distinctive T cell subset distribution in the BM, characterized by a significant increase of effector at the expense of memory T cells, likely reflects an antigen-driven albeit ineffective immune response.

Dominant CD4+ T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

In people with HIV (PWH), the post-antiretroviral therapy (ART) window is critical for immune restoration and HIV reservoir stabilization. We employ deep immune profiling and Tcell receptor (TCR) sequencing and examine proliferation to assess how ART impacts Tcell homeostasis. In PWH on long-term ART, lymphocyte frequencies and phenotypes are mostly stable. By contrast, broad phenotypic changes in natural killer (NK) cells, γδ Tcells, B cells, and CD4+ and CD8+ Tcells are observed in the post-ART window. Whereas CD8+ Tcells mostly restore, memory CD4+ T subsets and cytolytic NK cells show incomplete restoration 1.4 years post ART. Surprisingly, the hierarchies and frequencies of dominant CD4 TCR clonotypes (0.1%-11% of all CD4+ Tcells) remain stable post ART, suggesting that clonal homeostasis can be independent of homeostatic processes regulating CD4+ Tcell absolute number, phenotypes, and function. The slow restoration of host immunity post ART also has implications for the design of ART interruption studies.

EuroClonality-NGS Recommendations for Evaluation of B-Cell Clonality Analysis by Next-Generation Sequencing: A Structured Approach with the DEPART Algorithm

Next-generation sequencing (NGS)-based clonality analysis allows in-depth assessment of the clonal composition of a sample with high sensitivity for detecting small clones. Within the EuroClonality-NGS Working Group, a protocol for NGS Ig clonality analysis was developed and validated previously. This NGS-based approach was designed to generate small amplicons, making it suitable for samples with suboptimal DNA quality, especially material derived from formalin-fixed, paraffin-embedded tissue. Using expert assessment of NGS Ig clonality results as a reference, a structured algorithmic approach to the assessment of NGS-amplicon-based B-cell clonality analysis was developed. A structured approach with the Detection of clonality through Evaluation of sample quality and assessment of Pattern, Abundance and RaTio (DEPART) algorithm was proposed, which consecutively evaluates sample quality, the pattern of the clonotypes present, the abundance of the most dominant clonotypes, and the ratio between the dominant clonotypes and the background to evaluate the different Ig gene targets. Specific issues with respect to evaluation of the various Ig targets as well as the integration of results of individual targets into a molecular clonality conclusion are discussed and illustrated with case examples. Finally, the importance of interpretation of NGS-based clonality results in clinical and histopathologic contexts is discussed. It is expected that these recommendations will have clinical utility to facilitate proper evaluation of clonality assessment.

Clonal expansion of intra-epithelial T cells in breast cancer revealed by spatial transcriptomics.

The spatial distribution of tumor-infiltrating lymphocytes (TIL) predicts breast cancer outcome and response to systemic therapy, highlighting the importance of an intact tissue structure for characterizing tumors. Here, we present ST-FFPE, a spatial transcriptomics method for the analysis of formalin-fixed paraffin-embedded samples, which opens the possibility of interrogating archival tissue. The method involves extraction, exome capture and sequencing of RNA from different tumor compartments microdissected by laser-capture, and can be used to study the cellular composition of tumor microenvironment. Focusing on triple-negative breast cancer (TNBC), we characterized T cells, B cells, dendritic cells, fibroblasts and endothelial cells in both stromal and intra-epithelial compartments. We found a highly variable spatial distribution of immune cell subsets among tumors. This analysis revealed that the immune repertoires of intra-epithelial T and B cells were consistently less diverse and more clonal than those of stromal T and B cells. T-cell receptor (TCR) sequencing confirmed a reduced diversity and higher clonality of intra-epithelial T cells relative to the corresponding stromal T cells. Analysis of the top 10 dominant clonotypes in the two compartments showed a majority of shared but also some unique clonotypes both in stromal and intra-epithelial T cells. Hyperexpanded clonotypes were more abundant among intra-epithelial than stromal T cells. These findings validate the ST-FFPE method and suggest an accumulation of antigen-specific T cells within tumor core. Because ST-FFPE is applicable for analysis of previously collected tissue samples, it could be useful for rapid assessment of intratumoral cellular heterogeneity in multiple disease and treatment settings.

Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing–based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein–Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing–based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

Distinct clonal identities of B-ALLs arising after lenolidomide therapy for multiple myeloma

AbstractPatients with multiple myeloma (MM) who are treated with lenalidomide rarely develop a secondary B-cell acute lymphoblastic leukemia (B-ALL). The clonal and biological relationship between these sequential malignancies is not yet clear. We identified 17 patients with MM treated with lenalidomide, who subsequently developed B-ALL. Patient samples were evaluated through sequencing, cytogenetics/fluorescence in situ hybridization (FISH), immunohistochemical (IHC) staining, and immunoglobulin heavy chain (IgH) clonality assessment. Samples were assessed for shared mutations and recurrently mutated genes. Through whole exome sequencing and cytogenetics/FISH analysis of 7 paired samples (MM vs matched B-ALL), no mutational overlap between samples was observed. Unique dominant IgH clonotypes between the tumors were observed in 5 paired MM/B-ALL samples. Across all 17 B-ALL samples, 14 (83%) had a TP53 variant detected. Three MM samples with sufficient sequencing depth (>500×) revealed rare cells (average of 0.6% variant allele frequency, or 1.2% of cells) with the same TP53 variant identified in the subsequent B-ALL sample. A lack of mutational overlap between MM and B-ALL samples shows that B-ALL developed as a second malignancy arising from a founding population of cells that likely represented unrelated clonal hematopoiesis caused by a TP53 mutation. The recurrent variants in TP53 in the B-ALL samples suggest a common path for malignant transformation that may be similar to that of TP53-mutant, treatment-related acute myeloid leukemia. The presence of rare cells containing TP53 variants in bone marrow at the initiation of lenalidomide treatment suggests that cellular populations containing TP53 variants expand in the presence of lenalidomide to increase the likelihood of B-ALL development.

Combined chemoradiotherapy and programmed cell death-ligand 1 blockade leads to changes in the circulating T-cell receptor repertoire of patients with non-small-cell lung cancer.

Combined chemoradiotherapy (CRT) and programmed cell death-ligand 1 (PD-L1) blockade is a new care standard for unresectable stage III non-small-cell lung cancer (NSCLC). Although this consolidation therapy has improved the overall survival of patients with NSCLC, the synergistic action mechanisms of CRT and immunotherapy on T cells remain unclear. In addition, there is a paucity of reliable biomarkers to predict clinical responses to therapy. In this study, we analyzed T-cell receptor (TCR) sequences in the peripheral blood of five patients with NSCLC. T-cell receptor analysis was undertaken before treatment, after CRT, and after PD-L1 blockade. Notably, we observed the expansion and alteration of the dominant T-cell clonotypes in all cases with a complete response. In contrast, neither expansion nor alteration of the TCR repertoire was observed in cases with progressive disease. T cell expansion was initiated after CRT and was further enhanced after PD-L1 blockade. Our findings suggest the systemic effect of CRT on circulating T cells in addition to the curative effect on limited tumor sites. Dynamic changes in circulating T-cell clonotypes could have a prognostic significance for combined CRT and PD-L1 blockade.

T-cell receptor diversity in minimal change disease in the NEPTUNE study.

Minimal change disease (MCD) is the major cause of childhood idiopathic nephrotic syndrome, which is characterized by massive proteinuria and debilitating edema. Proteinuria in MCD is typically rapidly reversible with corticosteroid therapy, but relapses are common, and children often have many adverse events from the repeated courses of immunosuppressive therapy. The pathobiology of MCD remains poorly understood. Prior clinical observations suggest that abnormal T-cell function may play a central role in MCD pathogenesis. Based on these observations, we hypothesized that T-cell responses to specific exposures or antigens lead to a clonal expansion of T-cell subsets, a restriction in the T-cell repertoire,and anelaboration of specific circulating factors that trigger disease onset and relapses. To test these hypotheses, we sequenced T-cell receptors in fourteen MCD, four focal segmental glomerulosclerosis (FSGS), and four membranous nephropathy (MN) patients with clinical data and blood samples drawn during active disease and during remission collected by the Nephrotic Syndrome Study Network(NEPTUNE). We calculated several T-cell receptor diversity metrics to assess possible differences between active disease and remission states in paired samples. Median productive clonality did not differ between MCD active disease (0.0083; range: 0.0042, 0.0397) and remission (0.0088; range: 0.0038, 0.0369). We did not identify dominant clonotypes in MCD active disease, and few clonotypes were shared with FSGS and MN patients. While these data do not support an obvious role of the adaptive immune system T-cells in MCD pathogenesis, further study is warranted given the limited sample size. A higher resolution version of the Graphical abstract is available as Supplementary information.

Abstract 5217: Defining the balance of anti-viral and anti-tumor T cell responses to oncolytic virus therapy using single cell approaches

Abstract Preclinical and clinical studies have shown that intratumoral oncolytic viruses (OVs) can potentiate host anti-tumor immunity and overcome resistance to immune checkpoint blockade, although clinical responses to OVs have been modest to date. While T cell infiltration of tumors is frequently cited as a measure of OV immunogenicity, this measure is non-specific as OVs elicit a strong virus-directed T cell response, and it remains unknown what proportion of T cells expanded by OV therapy are virus-specific vs. tumor-specific or how these T cells distribute across virus-treated and distant tumors. We employed oncolytic Newcastle disease virus (NDV) in a bilateral flank melanoma mouse model to identify and phenotypically characterize anti-viral and anti-tumor T cells using single cell (sc) RNA and T cell receptor (TCR) sequencing. Intratumoral NDV therapy to a single flank tumor resulted in increased infiltration of CD4+ and CD8+ T cells in the injected (enestic) and distant (non-enestic) tumors and increased the breadth of the TCR repertoire at both sites with preferential expansion of several dominant clonotypes. There was substantial expansion of the proportion of T cell clonotypes shared between enestic and nonenestic tumors as well as the spleen, indicative of inter-tumor TCR repertoire normalization. In both treated and distant tumors, we observed a significant increase in the frequency of convergent TCR clonotypes, i.e. TCRs encoded by different nucleotide sequences that converge on the same amino acid sequence, implying that the presence of these TCRs in tumors is non-random. Using scRNA and paired TCR sequencing, we demonstrate that NDV therapy leads to expansion of unique clusters of terminally differentiated and activated CD4+ and CD8+ T cells associated with distinct TCR-based clonotypes. Notably, the predominant phenotypic clusters were distinct between the enestic and non-enestic tumors. Enestic tumors were dominated by CD8+ T cells exhibiting a signature associated with terminal dysfunction (PDCD1, LAG3, TOX), while the predominant expanded CD8+ T cells in non-enestic tumors exhibited an activation signature associated with high expression of cytolytic markers. While phenotypic states were conserved for the dominant TCR clones shared across the enestic and non-enestic tumors, TCRs unique to the non-enestic tumor were predominantly associated with an activated T cell state. Taken together, these studies highlight that T cells expanded by OV therapy exhibit unique functional states and TCRs in treated and distant tumors and imply that virus- and tumor-specific T cells may be identified by distinct TCR repertoires and phenotypes. Understanding the balance between virus- and tumor-directed T cells elicited by OV therapy will be key to engineering OVs and designing combination strategies that drive stronger tumor-directed T cell response. Citation Format: Bharat Burman, Nicholas Ceglia, Daniel Hirschhorn, Sadna Budhu, Levi Mangarin, Anton Oseledchyk, Yonina Bykov, Andrew McPherson, Sohrab Shah, Jedd Wolchok, Taha Merghoub, Dmitriy Zamarin. Defining the balance of anti-viral and anti-tumor T cell responses to oncolytic virus therapy using single cell approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5217.

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation.

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition.

Histopathological and immunological spectrum in response evaluation of talimogene laherparepvec treatment and correlation with durable response in patients with cutaneous melanoma.

Talimogene laherparepvec (T-VEC) is an intralesional oncolytic virotherapy for patients with irresectable stage III–IVM1a cutaneous melanoma. Although this treatment is considered to mainly act through T cell-mediated mechanisms, prominent numbers of plasma cells after T-VEC treatment have been described. The aim was to investigate how often these plasma cells were present, whether they were relevant in the response to treatment, and if these or other histopathological features were associated with durable response to treatment. Histopathological (granulomas, perineural inflammation, etc.) and immunological features [e.g. B cells/plasma cells (CD20/CD138) and T cells (CD3,CD4,CD8)] were scored and correlated with durable tumor response [i.e. complete response (CR) persisting beyond 6 months after treatment]. Plasmacellular infiltrate was examined with next-generation sequencing and immunohistochemistry (IgG, IgM, IgA, and IgD). Plasma cells were present in all T-VEC injected biopsies from 25 patients with melanoma taken at 3–5 months after starting treatment. In patients with a durable response (n = 12), angiocentric features and granulomas were more frequently identified compared with patients without a (durable) response (n = 13); 75% versus 29% for angiocentric features (P = 0.015) and 58% versus 15% for granulomas (P = 0.041). There was a class switch of IgM to IgG with skewing to certain dominant Ig heavy chain clonotypes. An angiocentric granulomatous pattern in T-VEC injected melanoma lesions was associated with a durable CR (>6 months). Plasma cells are probably a relevant feature in the mechanism of response but were not associated with durable response.

Distinct Modes of Ongoing Antigen Interactions Shape Intraclonal Dynamics in Splenic Marginal Zone Lymphoma

Almost one-third of all splenic marginal zone lymphoma (SMZL) cases express B cell receptor immunoglobulin (BcR IG) encoded by the IGHV1-2*04 gene. Such cases display a distinctive profile of genomic aberrations (e.g. higher incidence of NOTCH2 and KLF2 mutations) and a more aggressive clinical course compared to SMZL cases utilizing other IGHV genes. Such skewing of the BcR IG gene repertoire implicates antigen selection in SMZL ontogeny. Although the supportive evidence is compelling, it mostly derives from low-throughput approaches, which are inherently limited in their capacity to capture the complexity of the BcR IG gene repertoire. This hinders the comprehensive assessment of the subclonal architecture of SMZL that could offer insight into the dynamics of antigen-IG interactions. Here, we sought to overcome this limitation through a high-throughput immunogenetic investigation of SMZL, focusing on the detailed characterization of somatic hypermutation (SHM) and intraclonal diversification (ID) profiles. Our study included 22 cases utilizing the IGHV1-2*04 gene and 36 cases utilizing other IGHV genes. IGHV-IGHD-IGHJ (IGH) gene rearrangements were PCR-amplified and libraries were sequenced on the Illumina MiSeq platform. Data was analyzed with the IMGT/HighV-QUEST and TRIP software as well as a novel bioinformatics/biostatistics pipeline. Clonotypes were defined as unique combinations of a given IGHV gene+VH CDR3 amino acid (aa) sequence. Only IGH gene rearrangement sequences assigned to the dominant clonotypes of each case were assessed. In detail, all nucleotide variants (nt vars, i.e. all sequences clustered in the same dominant clonotype yet displaying distinct SHM profiles) were identified and further analyzed. Starting from the most abundant nt var, a network was built representing its connections with all other nt vars. For this analysis, we introduce the terms ‘most relevant pathway’ (MRP) corresponding to the pathway including connected nt vars with the highest total number of IGH sequences; and ‘longest mutational pathways’ (LMP) corresponding to the pathways with the highest number of nt vars (Fig. 1). Different graph metrics assessed the impact of ID in different SMZL subgroups: the first one focuses on the ‘most relevant pathway’ and quantifies SHM convergence [ratio of the total number of IGH sequences corresponding to the nt vars of this pathway to the number of IGH sequences in the most abundant nt var]; while the second refers to the length of the ‘longest mutational pathways’.Cases lacking additional connected nt vars [length of the LMP=1; 3 IGHV1-2*04 cases and 4 non-IGHV1-2*04 cases] were excluded. Consequently, the analysis included 19 IGHV1-2*04 cases and 32 non-IGHV1-2*04 cases. Significant differences were noted in the SHM and ID profiles between groups; the IGHV1-2*04 group had significantly (p<0.01) higher convergence values ranging from 0.009 to 1.243 (median: 0.102), as opposed to the non-IGHV1-2*04 group (range: 0.002-1.13, median: 0.014), overall suggesting that stronger selective pressures act in SMZL cases expressing the IGHV1-2*04 versus others. Moreover, IGHV1-2*04 cases displayed significantly (p<0.01) longer mutational pathways (length range: 2-6, median: 3) compared to the other group (range: 2-5, median: 2), alluding to more pronounced ID arising due to ongoing SHM.Finally, all mutations leading to aa changes were analyzed in the context of ID networks. More recurrent aa mutations were identified amongst cases with higher levels of convergence. For instance, the VH FR2 M39I change, one of the most prominent recurrent SHMs in the IGHV1-2*04 group, was found in the most abundant nt var in 13/19 IGHV1-2*04 cases, while it was identified in nt vars with extra mutations in another 3 cases. Of interest, it was present at the end of the mutational pathways in these 16 cases, whilst in the other group it was present only in one case using the IGHV1-2*02 gene, and absent in the rest (p<0.01).In conclusion, in the first large-scale high-throughput immunogenetic analysis of SMZL we provide strong evidence for more pronounced antigenic pressure in cases utilizing IGHV1-2*04 versus other IGHV genes. Our findings highlight a unique subclonal architecture for IGHV1-2*04 SMZL and corroborate the hypothesis that this group may represent a distinct molecular variant of SMZL. [Display omitted] DisclosuresRossi: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Chatzidimitriou: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Stamatopoulos: Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

High-risk subtypes of chronic lymphocytic leukemia are detectable as early as 16 years prior to diagnosis

AbstractChronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 12% in aged individuals; however, the duration of MBL and the mechanisms of its evolution to CLL remain largely unknown. In this study, we sequenced the B-cell receptor (BcR) immunoglobulin heavy chain (IGH) gene repertoire of 124 patients with CLL and 118 matched controls in blood samples taken up to 22 years prior to diagnosis. Significant skewing in the BcR IGH gene repertoire was detected in the majority of patients, even before the occurrence of lymphocytosis and irrespective of the clonotypic IGH variable gene somatic hypermutation status. Furthermore, we identified dominant clonotypes belonging to major stereotyped subsets associated with poor prognosis up to 16 years before diagnosis in 14 patients with CLL. In 22 patients with longitudinal samples, the skewing of the BcR IGH gene repertoire increased significantly over time to diagnosis or remained stable at high levels. For 14 of 16 patients with available samples at diagnosis, the CLL clonotype was already present in the prediagnostic samples. Overall, our data indicate that the preclinical phase of CLL could be longer than previously thought, even in adverse-prognostic cases.

Cognate recognition of microbial antigens defines constricted CD4+ T cell receptor repertoires in the inflamed colon

Key aspects of intestinal Tcells, including their antigen specificity and their selection by the microbiota and other intestinal antigens, as well as the contribution of individual Tcell clones to regulatory and effector functions, remain unresolved. Here we tracked adoptively transferred Tcell populations to specify the interrelation of Tcell receptor repertoire and the gut antigenic environment. We show that dominant TCRα clonotypes were shared between interferon-γ- and interleukin-17-producing but not regulatory Foxp3+ Tcells. Identical TCRα clonotypes accumulated in the colon of different individuals, whereas antibiotics or defined colonization correlated with the expansion of distinct expanded Tcell clonotypes. Our results demonstrate key aspects of intestinal CD4+ Tcell activation and suggest that few microbial species exert a dominant effect on the intestinal Tcell repertoire during colitis. We speculate that dominant proinflammatory Tcell clones might provide a therapeutic target in human inflammatory bowel disease.

Receptor repertoires of murine follicular T helper cells reveal a high clonal overlap in separate lymph nodes in autoimmunity.

Follicular T helper cells (Tfh) are a specialized subset of CD4 effector T cells that are crucial for germinal center (GC) reactions and for selecting B cells to undergo affinity maturation. Despite this central role for humoral immunity, only few data exist about their clonal distribution when multiple lymphoid organs are exposed to the same antigen (Ag) as it is the case in autoimmunity. Here, we used an autoantibody-mediated disease model of the skin and injected one auto-Ag into the two footpads of the same mouse and analyzed the T cell receptor (TCR)β sequences of Tfh located in GCs of both contralateral draining lymph nodes. We found that over 90% of the dominant GC-Tfh clonotypes were shared in both lymph nodes but only transiently. The initially dominant Tfh clonotypes especially declined after establishment of chronic disease while GC reaction and autoimmune disease continued. Our data demonstrates a dynamic behavior of Tfh clonotypes under autoimmune conditions and emphasizes the importance of the time point for distinguishing auto-Ag-specific Tfh clonotypes from potential bystander activated ones.

Massive surge of mRNA expression of clonal B-cell receptor in patients with COVID-19

BackgroundAntibody production is one of the primary mechanisms for recovery from coronavirus disease 2019 (COVID-19). It is speculated that massive clonal expansion of B cells, which can produce clinically meaningful neutralizing antibodies, occurs in patients who recover on the timing of acquiring adaptive immunity. MethodsTo evaluate fluctuations in clonal B cells and the size of the clones, we chronologically assessed the B-cell receptor (BCR) repertoire in three patients with COVID-19 who recovered around 10 days after symptom onset. ResultsWe focused on the three dominant clonotypes (top 3) in each individual. The percentage frequencies of the top 3 clonotypes increased rapidly and accounted for 27.8 % on day 9 in patient 1, 10.4 % on day 12 in patient 2, and 10.8 % on day 11 in patient 3, respectively. The frequencies of these top 3 clonotypes rapidly decreased as the patients’ clinical symptoms improved. Furthermore, BCR network analysis revealed that accumulation of clusters composed of similar complementarity-determining region 3 (CDR3) sequences were rapidly formed, grew, and reached their maximum size around 10 days after symptom onset. ConclusionsBCR repertoire analysis revealed that a massive surge of some unique BCRs occurs during the acquisition of adaptive immunity and recovery. The peaks were more prominent than expected. These results provide insight into the important role of BCRs in the recovery from COVID-19 and raise the possibility of developing neutralizing antibodies as COVID-19 immunotherapy.

Quantitative Assessment of TCR Repertoire and Allo-Reactivity of Human Acute Gvhd Tissue-Infiltrating T Cells

Abstract Introduction: Due to difficulty in isolating T cells from human tissues, the diversity of allo-reactive T cells that are infiltrating human acute GVHD (aGVHD) tissues remains largely uninvestigated. Here, we report the analysis of comprehensive T cell repertoire and allo-reactivity of human aGVHD tissue-infiltrating T cells. Patients and Methods: Skin (n = 7), stomach (n = 3) and colon (n = 2) biopsy samples and blood samples (n = 8) were obtained from eight post-transplant patients when the patients were supposed to develop aGVHD and prior to the fist-line treatment of aGVHD. Total RNA was extracted from tissue and blood samples, and T cell receptor (TCR)-β gene deep sequencing was performed (~100000 reads for each sample). To estimate the TCR-β repertoire diversity, inverse Simpson's diversity index (1/Ds) were calculated. T cells were isolated from tissues and cloned by limiting dilution. This study was approved by the ethics committees of Nagoya University. Results: In the skin of patient #1, the relative abundance of the most and second most frequent clonotypes were 74.9% and 22.8%, respectively, suggesting that only two T cell clones accounted for approximately 98% of GVHD skin-infiltrating T cells. Two T cell clones, termed clones A and B, were isolated from the skin of patient #1. Both clones lysed patient peripheral blood mononuclear cells, but failed to lyse donor cells. Thus, clones A and B were allo-reactive cytotoxic T lymphocytes (CTLs). The IFN-γ production of clone B was significantly increased when stimulated by COS cells transfected with HLA-DRB1*14:54 cDNA, suggesting that the clone B recognized the mismatched patient's HLA-DRB1*14:54 molecule. To confirm the relative abundance of clones A and B in his skin, the TCR-β usage and CDR3 were determined. Surprisingly, clones A and B were the most and second most frequent skin-infiltrating T cell clones. Taken together, these data suggested that a limited allo-reactive CTL clones were involved in GVHD development of patient #1. In all samples, the mean 1/Ds of skin, stomach, colon and all tissues were 9.4 (range, 1.2-21.3), 7.5 (1.9-14.9), 3.3 (1.0-5.5) and 7.9 (1.0-21.3), respectively, while that of all blood samples was 247 (18.8-888.7) (P 10% frequency) clonotypes in a certain tissue were detected in other tissue(s) of the same patient at an extremely low frequency ( 10% frequency) clonotypes in the skin of a certain patient were detected in those of other patients with an extremely low frequency ( Conclusion: In humans, a limited allo-reactive CTL clones, which differ from tissue to tissue and from patient to patient, may induce aGVHD. The degree of skewing of tissue-infiltrating T cell repertoire may be predictive of GVHD outcome. Download : Download high-res image (62KB) Download : Download full-size image Disclosures Kiyoi: Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; MSD K.K.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding.