Immune repertoire analysis using RNA for tumor milieu analysis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE160302 | GEO | 2020/12/31
REPOSITORIES: GEO
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Project description:Immune repertoire analysis using RNA for tumor milieu analysis
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Project description:This study provides an in-depth analysis of the diversity of the CD4 TCR CDR3β repertoire specific to Influenza A (HA) and Epstein-Barr virus (EBNA) epitopes. Epitope-specific CD4 T cells from 13 healthy donors were enriched using a short-term culture step, isolated based on activation markers, and sequenced for their TCR CDR3β region using high-throughput sequencing. The frequency of each clonotype were then identified within the complete circulating CD4 T-cell CDR3β repertoire. For both epitopes, the clonotype distribution was markedly skewed, with a small number of highly expanded clones comprising approximately 60% of the repertoire, alongside numerous low-frequency clonotypes. VJ gene usage and motif preferences differed between the two peptides, highlighting epitope-specific TCR selectivity. The response was predominantly composed of private T-cell clonotypes. The proportion of public clonotypes can increase among donors sharing HLA class II molecules and reveals in HLA-unrelated donors the level of TCR promiscuity. Overall, our data demonstrate that CD4 T-cell responses to these viral epitopes are polyclonal and highly personalized. The modest overlap of clonotypes between donors, coupled with a long tail of low-frequency clones, suggests that the full diversity of the epitope-specific T-cell repertoire is likely broader than previously estimated.
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Project description:Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative source of potent immunogenic neoepitopes that might shape the T cell receptor (TR) gene repertoire in CLL. On these grounds, here we interrogated the TR gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures.The study group included 44 untreated CLL patients with who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=17; del(11q), n=10; del(13q), n=7; TP53mut, n=5; NOTCH1mut, n=5. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Bioinformatics analysis with a purpose-build pipeline and in silico prediction models were used for the profiling of the TR gene repertoire and the identification of neoepitope-specific TR clonotypes, respectively.
2023-01-02 | E-MTAB-12216 | biostudies-arrayexpress
Project description:Anti-leukemic treatment in chronic lymphocytic leukemia (CLL) impacts autologous T cells through various direct and indirect mechanisms. However, how first-line treatment with venetoclax and obinutuzumab impacts the TRB repertoire has not been studied. Here, we have used next-generation sequencing to longitudinally characterize the TRB repertoire of previously untreated CLL patients who received treatment with obinutuzumab and venetoclax in the HOVON-139/ GIVE trial. Following treatment, due to the emergence of a polyclonal population of small-sized, diverse clonotypes, TRB repertoire diversity increased (Shannon’s H; trial entry 5.5 [95%CI 5.2-6.2] vs end of induction treatment [EOiT] 6.2 [95%CI 5.7- 6.7], P=0.001). In contrast, treatment with obinutuzumab and venetoclax did not ameliorate TRB oligoclonality (EOiT, cumulative frequency of top 10 clonotypes 32.5% [95%CI 25.3-39.9], P=0.10; R+12 CF10 31.2% [95%CI 24.3-38.1], P=0.17), due to the persistence of large pre-treatment clonotypes. Patients with IGLV3-21R110 CLL had weaker recovery of TRB repertoire diversity (mean Shannon’s H 5.2 [95%CI 4.0-6.3] vs 6.8 [95%CI 6.3-7.3], P=0.01). TRB clonotypes that were shared among patients could be identified, a minority of which had known pathogenic or auto-antigenic affinity. The concurrent presence or future resurgence of MRD did not impact the TRB repertoire, but low TRB diversity (Poisson regression coefficient -0.32±0.15, P=0.046) and high oligoclonality (Poisson regression coefficient 0.02±0.009, P=0.02) were associated with an increased risk of infection. These data provide the groundwork for the future investigation of the fitness of autologous T cells for T cell-mediated immunotherapy, following treatment with obinutuzumab and venetoclax.
2024-12-18 | GSE268787 | GEO
Project description:Fibrosis is the final path of nearly every form of chronic disease and accounts for up to 45% of all deaths in the developed world. However, antifibrotic therapies that target fibrogenic cells are lacking. We tested whether specific immunization against ADAM12 can elicit an antigen-specific cytotoxic T cell response to ameliorate liver fibrosis. In this study, we performed T cell receptor (TCR) alpha- and beta-chain repertoire sequencing on fibrotic livers to further characterize the T cell response and to detect potential TCR clonotypes. We observed TCR clonality of liver-infiltrating T cells from v-A12- and control-vaccinated mice with minimal overlap to v-CTRL mice in two α-chain sequences. However, the vast majority of expanded clones from v-A12-vaccinated animals showed a unique sequence pattern. Moreover, there was no overlap in the β-chain sequences between v-A12-vaccinated and control mice, suggesting a vaccination-induced expansion of antigen-specific TCR clonotypes.
2022-09-15 | GSE208635 | GEO
Project description:Generating balanced populations of CD8+ effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of how a diverse CD8+ T cell repertoire differentiates remains unclear. We identified several hundred TCR clonotypes that constitute the polyclonal response against a single antigen and found that a majority of TCR clonotypes were highly biased towards memory or effector fate. TCR-intrinsic biases were not stochastic and were dominant over environmental cues. Differential gene expression analysis of memory- or effector-biased TCR clonotypes showed bifurcation of differential fates at the early effector stage. Additionally, phylogenetic analysis revealed that memory-biased clonotypes retain their fate preferences in subclonal populations but effector-biased subclones can switch to memory fate. Our study highlights that the polyclonal CD8+ T cell response is a composite of unbiased and biased clonotypes with varying capacity to incorporate environmental cues in their cell fate decisions.
2025-02-27 | GSE241403 | GEO
Project description:Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein Ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in twenty-nine healthy donors. Sequencing of the CDR3β TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes, as well as a mostly hydrophobic composition of the CDR3β region according to their auto-reactivity. FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T-cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T-cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies
2024-01-22 | GSE253521 | GEO
Project description:During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire. SAP deficiency resulted in both a significant loss of an immatureGzma+Blk+Etv5+Tox2+γδT17 precursor population, and a significant increase inCd4+Cd8+Rorc+Ptcra+Rag1+thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αβ T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data suggest that SAP-dependent γδ/αβ T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire.
2024-07-08 | GSE262064 | GEO
Project description:TCR- and BCR-sequencing (TCR/BCR-seq) are two important technologies in studying the immune repertoire of samples such as PBMCs or tumors. In their most common form, these assays combine multiplex PCR of the repertoire using primers targeting regions of the V(D)J and the constant region with next-generation sequencing (NGS). The data produced by this assay provide a slew of information regarding immune repertoire(s) including the presence critical clonotypes, repertoire diversity, variable (V) gene usage, analysis of public clonotypes, etc. One issue that can arise during generation of the TCR/BCR-seq data is sequence bias during the PCR or NGS steps. To combat this, unique molecular identifiers (UMIs) have been used to identify and eliminate sequence bias. However, UMI fragments can be long and very diverse, resulting in the UMI sequences interfering with any of the multitude of primers during multiplex PCR. Here, we introduce Validator Barcodes (VBCs), a set of eight short barcodes (6-9 nucleotides in length). This compact set of barcodes improves PCR efficiency and facilitates PCR primer designs. Also, like UMIs, the VBCs may be used to estimate the number of template molecules (RNA or DNA). Using VBC-labeled primers for TCR and BCR repertoire profiling from PBMCs produces highly comparable results and similarly template values to those obtained through UMI-based assay counts. Overall, VBCs are a useful and simpler alternative to UMIs in assaying TCR and BCR repertoires.
2025-05-16 | GSE297422 | GEO
Project description:We developed an improved library prep protocol and standardized the data analysis pipeline for accurate repertoire profiling. In addition, two metrics were implemented to assess repertoire clone properties. We then studied systemically the effects of two adjuvants, CpG and Alum, on the Ig heavy chain repertoire using the ovalbumin (OVA) challenged mouse model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in terms of sequence mutation frequency, IGHV gene usage, CDR3 length, rescaled Hill numbers for clonal diversity, and clone selection strength. As a result, Ig repertoires of different tissues or isotypes exhibited distinguishable profiles at the non-immunized steady state. Adjuvanted immunizations further resulted in statistically significant alterations in Ig repertoire compared with PBS or OVA alone immunized groups. Lastly, we applied unsupervised machine learning techniques – multiple factor analysis and clustering – to identify Ig repertoire signatures in different compartments and under varying immunizations.
2023-04-07 | GSE228856 | GEO