scholarly journals 192 Pooled T cell receptor screening (POTS) provides unbiased, high-throughput method for TCR discovery

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A204-A204
Author(s):  
Jack Reid ◽  
Shihong Zhang ◽  
Ariunaa Munkhbat ◽  
Matyas Ecsedi ◽  
Megan McAfee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Ex Vivo ◽  
Cell Receptor ◽  
High Avidity ◽  
Reporter System ◽  
High Throughput Method

BackgroundT Cell Receptor (TCR)-T cell therapies have shown some promising results in cancer clinical trials, however the efficacy of treatment remains suboptimal. Outcomes could potentially be improved by utilizing highly functional TCRs for future trials. Current TCR discovery methods are relatively low throughput and rely on synthesis and screening of individual TCRs based on tetramer binding and peptide specificity, which is costly and labor intensive. We have developed and validated a pooled approach relying on directly cloned TCRs transduced into a fluorescent Jurkat reporter system (figure 1). This approach provides an unbiased, high-throughput method for TCR discovery.MethodsAs a model for POTS, T cells specific for a peptide derived adenovirus structural protein were sorted on tetramer and subjected to 10x single cell VDJ analysis. Pools of randomly paired TCR alpha and beta chains were cloned from the 10x cDNA into a lentiviral vector and transduced into a Jurkat reporter cells. Consecutive stimulations with cognate antigen followed by cell sorts were performed to enrich for functional TCRs. Full length TCRab pools were sequenced by Oxford Nanopore Technologies (ONT) and compared to a 10x dataset to find naturally paired TCRs.ResultsComparison between the ex vivo single cell VDJ sequencing and ONT sequencing of the transduced antigen specific TCRs showed more than 99% of the TCR pairs found in reporter positive Jurkat cells were naturally paired TCRs. The functionality of 8 TCR clonotypes discovered using POTS were compared and clone #2 showed the strongest response. Of the selected clonotypes, clone #2 showed a low frequency of 0.9% in the ex vivo single cell VDJ sequencing. After the first round of stimulation and sequencing, clone #2 takes up of 5% of all reporter-positive clones. The abundance of clone #2 further increased to 17% after another round of stimulation, sorting and sequencing, suggesting this method can retrieve and enrich for highly functional antigen specific TCRs.Abstract 192 Figure 1Outline of the POTS workflow.ConclusionsPOTS provides a high-throughput method for discovery of naturally paired, high-avidity T cell receptors. This method mitigates bias introduced by T cell differentiation state by screening TCRs in a clonal reporter system. Additionally, POTS allows for screening of low abundance clones when compared with traditional TCR discovery techniques. Pooled TCRs could also be screened in vivo with primary T cells in a mouse model to screen for the most functional and physiologically fit TCR for cancer treatment.

scholarly journals Increased Activation and Oligoclonality of Peripheral CD8+ T Cells in the Chronic Human Helminth Infection Alveolar Echinococcosis

2002 ◽  
Vol 70 (3) ◽  
pp. 1168-1174 ◽  
Author(s):  
Burkhard J. Manfras ◽  
Stefan Reuter ◽  
Thomas Wendland ◽  
Peter Kern
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Alveolar Echinococcosis ◽  
Ex Vivo ◽  
Active Role ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
And Function

ABSTRACT Alveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4+ and CD8+ T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8+ T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens and Echinococcus multilocularis vesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8+ but not CD4+ T cells, suggesting a persistent antigenic drive for CD8+ T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8+ T cells in AE.

scholarly journals Isolation and enrichment of mouse splenic T cells for ex vivo and in vivo T cell receptor stimulation assays

2021 ◽  
Vol 2 (4) ◽  
pp. 100961
Author(s):  
Clémence Grosjean ◽  
Julie Quessada ◽  
Mathis Nozais ◽  
Marie Loosveld ◽  
Dominique Payet-Bornet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Receptor Stimulation

Alloreactivity as a Source of High Avidity T Cell Clones Specific for Cancer Testis-Antigens Associated with Multiple Myeloma: Prospects for Adoptive Immunotherapy with Primary T Cells Redirected toward Multiple Myeloma by T Cell Receptor Gene Transfer.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3712-3712
Author(s):  
Holger Kronig ◽  
Kathrin Hofer ◽  
Julia Neudorfer ◽  
Christian Peschel ◽  
Helga Bernhard
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
High Avidity ◽  
Peptide Epitopes ◽  
Ct Antigen ◽  
Ct Antigens ◽  
Cancer Testis

Abstract Cancer testis (CT)-antigens belong to a class of tumor antigens that are aberrantly expressed in a variety of hematological malignancies including multiple myeloma. Owing to their restricted gene expression, CT-antigens represent potential target antigens for immunotherapeutical approaches such as vaccination and adoptive T cell transfer. As the CT-antigens are self antigens, the majority of CT-antigen-specific autologous T cells display a low avidity T cell receptor (TCR), which often results in a weak tumor recognition efficiency. Our group has been focusing on the isolation of highly avid T cells against CT-antigens that are expressed in multiple myeloma, in particular MAGE-C1, MAGE-C2, and NY-ESO-1. The experimental approach was based on the stimulation of allo-restricted cytotoxic T cells, because highly avid T cells recognizing peptide epitopes in context with foreign HLA-alleles are not depleted in the thymus. HLA-A2-negative T cells were stimulated with HLA-A2-positive allogeneic dendritic cells that had been exogenously loaded with HLA-A2-binding peptides derived from NY-ESO-1, MAGE-C1 or MAGE-C2. Using this technique we were able to isolate allo-HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones with peptide-dominant binding against known and novel peptide epitopes derived from NY-ESO-1, MAGE-C1 and MAGE-C2. The expanded peptide-specific CTL clones lysed HLA-A2-positive myeloma cell lines expressing NY-ESO-1, MAGE-C1 and MAGE-C2, respectively. Of note, the MAGE-C1-specific T cells crossreacted with the corresponding MAGE-C2 peptide due to the existing sequence homology between MAGE-C1 and MAGE-C2. Current experiments focus on redirecting primary T cells toward myeloma cells by retroviral gene transfer of CT-antigen-specific TCRs. The establishment of a set of high avidity TCRs specific for CT-antigens facilitates the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of multiple myeloma.

scholarly journals Development of a CD8 co-receptor independent T-cell receptor specific for tumor-associated antigen MAGE-A4 for next generation T-cell-based immunotherapy

2021 ◽  
Vol 9 (3) ◽  
pp. e002035
Author(s):  
Kathrin Davari ◽  
Tristan Holland ◽  
Laura Prassmayer ◽  
Giulia Longinotti ◽  
Kenneth P Ganley ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
In Vivo Studies ◽  
High Avidity ◽  
Cell Repertoire

BackgroundThe cancer-testis antigen MAGE-A4 is an attractive target for T-cell-based immunotherapy, especially for indications with unmet clinical need like non-small cell lung or triple-negative breast cancer.MethodsAn unbiased CD137-based sorting approach was first used to identify an immunogenic MAGE-A4-derived epitope (GVYDGREHTV) that was properly processed and presented on human leukocyte antigen (HLA)-A2 molecules encoded by the HLA-A*02:01 allele. To isolate high-avidity T cells via subsequent multimer sorting, an in vitro priming approach using HLA-A2-negative donors was conducted to bypass central tolerance to this self-antigen. Pre-clinical parameters of safety and activity were assessed in a comprehensive set of in vitro and in vivo studies.ResultsA MAGE-A4-reactive, HLA-A2-restricted T-cell receptor (TCR) was isolated from primed T cells of an HLA-A2-negative donor. The respective TCR-T-cell (TCR-T) product bbT485 was demonstrated pre-clinically to have a favorable safety profile and superior in vivo potency compared with TCR-Ts expressing a TCR derived from a tolerized T-cell repertoire to self-antigens. This natural high-avidity TCR was found to be CD8 co-receptor independent, allowing effector functions to be elicited in transgenic CD4+ T helper cells. These CD4+ TCR-Ts supported an anti-tumor response by direct killing of MAGE-A4-positive tumor cells and upregulated hallmarks associated with helper function, such as CD154 expression and release of key cytokines on tumor-specific stimulation.ConclusionThe extensive pre-clinical assessment of safety and in vivo potency of bbT485 provide the basis for its use in TCR-T immunotherapy studies. The ability of this non-mutated high-avidity, co-receptor-independent TCR to activate CD8+ and CD4+ T cells could potentially provide enhanced cellular responses in the clinical setting through the induction of functionally diverse T-cell subsets that goes beyond what is currently tested in the clinic.

scholarly journals Antigen-specific T-cell receptor signatures of cytomegalovirus infection

2018 ◽  
Author(s):  
Alina Huth ◽  
Xiaoling Liang ◽  
Stefan Krebs ◽  
Helmut Blum ◽  
Andreas Moosmann
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Response ◽  
Ex Vivo ◽  
Cell Receptor ◽  
T Cell Response ◽  
The Body ◽  
T Cell Epitopes ◽  
Virus Reactivation

AbstractCytomegalovirus (CMV) is a prevalent human pathogen. The virus cannot be eliminated from the body, but is kept in check by CMV-specific T cells. Patients with an insufficient T-cell response, such as transplant recipients, are at high risk of developing CMV disease. However, the CMV-specific T-cell repertoire is complex, and is not yet clear which T cells protect best against virus reactivation and disease. Here we present a highly resolved characterization of CMV-specific CD8+ T cells based on enrichment by specific peptide stimulation and mRNA sequencing of their T-cell receptor β chains (TCRβ). Our analysis included recently identified T-cell epitopes restricted through HLA-C, whose presentation is resistant to viral immunomodulation, and well-studied HLA-B-restricted epitopes. In 8 healthy virus carriers, we identified a total of 1052 CMV-specific TCRβ chains. HLA-C-restricted, CMV-specific TCRβ clonotypes theex vivoT-cell response, and contributed the highest-frequency clonotype of the entire repertoire in 2 of 8 donors. We analyzed sharing and similarity of CMV-specific TCRβ sequences and identified 63 public or related sequences belonging to 17 public TCRβ families. In our cohort and in an independent cohort of 352 donors, the cumulative frequency of these public TCRβ family members was a highly discriminatory indicator of carrying both CMV infection and the relevant HLA type. Based on these findings, we propose CMV-specific TCRβ signatures as a biomarker for an antiviral T-cell response to identify patients in need of treatment and to guide future development of immunotherapy.

scholarly journals Differential expression profile of gluten-specific T cells identified by single-cell RNA-seq

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258029
Author(s):  
Ying Yao ◽  
Łukasz Wyrozżemski ◽  
Knut E. A. Lundin ◽  
Geir Kjetil Sandve ◽  
Shuo-Wang Qiao
Keyword(s):  
T Cells ◽  
Celiac Disease ◽  
T Cell ◽  
Single Cell ◽  
Differential Expression ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Effector Memory ◽  
Fatty Acid Transport ◽  
Rna Seq

Gluten-specific CD4+ T cells drive the pathogenesis of celiac disease and circulating gluten-specific T cells can be identified by staining with HLA-DQ:gluten tetramers. In this first single-cell RNA-seq study of tetramer-sorted T cells from untreated celiac disease patients blood, we found that gluten-specific T cells showed distinct transcriptomic profiles consistent with activated effector memory T cells that shared features with Th1 and follicular helper T cells. Compared to non-specific cells, gluten-specific T cells showed differential expression of several genes involved in T-cell receptor signaling, translational processes, apoptosis, fatty acid transport, and redox potentials. Many of the gluten-specific T cells studied shared T-cell receptor with each other, indicating that circulating gluten-specific T cells belong to a limited number of clones. Moreover, the transcriptional profiles of cells that shared the same clonal origin were transcriptionally more similar compared with between clonally unrelated gluten-specific cells.

scholarly journals Linking T cell receptor sequence to transcriptional profiles with clonotype neighbor graph analysis (CoNGA)

2020 ◽  
Author(s):  
Stefan A. Schattgen ◽  
Kate Guion ◽  
Jeremy Chase Crawford ◽  
Aisha Souquette ◽  
Alvaro Martinez Barrio ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Graph Analysis ◽  
Theoretic Approach ◽  
Neighbor Graph

AbstractMulti-modal single-cell technologies capable of simultaneously assaying gene expression and surface phenotype across large numbers of immune cells have described extensive heterogeneity within these complex populations, in healthy and diseased states. In the case of T cells, these technologies have made it possible to profile clonotype, defined by T cell receptor (TCR) sequence, and phenotype, as reflected in gene expression (GEX) profile, surface protein expression, and peptide:MHC (pMHC) binding, across large and diverse cell populations. These rich, high-dimensional datasets have the potential to reveal new relationships between TCR sequence and T cell phenotype that go beyond identification of features shared by clonally related cells. In order to uncover these connections in an unbiased way, we developed a graph-theoretic approach---clonotype neighbor-graph analysis or “CoNGA”---that identifies correlations between GEX profile and TCR sequence through statistical analysis of a pair of T cell similarity graphs, one in which cells are linked based on gene expression similarity and another in which cells are linked by similarity of TCR sequence. Applying CoNGA across diverse human and mouse T cell datasets uncovered known and novel associations between TCR sequence features and cellular phenotype including the classical invariant T cell subsets; a novel defined population of human blood CD8+ T cells expressing the transcription factors HOBIT and HELIOS, NK-associated receptors, and a biased TCR repertoire, representing a potential previously undescribed lineage of “natural lymphocytes”; a striking association between usage of a specific V-beta gene segment and expression of the EPHB6 gene that is conserved between mouse and human; and TCR sequence determinants of differentiation in developing thymocytes. As the size and scale of single-cell datasets continue to grow, we expect that CoNGA will prove to be a useful tool for deconvolving complex relationships between TCR sequence and cellular state in single-cell applications.

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