scholarly journals Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αβ-engineered human CD4+ T cells

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz7809 ◽  
Author(s):  
Jan A. Rath ◽  
Gagan Bajwa ◽  
Benoit Carreres ◽  
Elisabeth Hoyer ◽  
Isabelle Gruber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cell Function ◽  
Xenograft Model ◽  
Cell Receptor ◽  
Target Cells ◽  
Antigen Specificity ◽  
Molecular Features ◽  
Mouse Xenograft Model

Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4+ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4+ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4+ and CD8+ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8+ T cell function and preserved less differentiated CD4+ and CD8+ T cells after tumor challenge. TCR8+CD4+ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.

scholarly journals O7 A bispecific VHH approach to leverage the potent and widely applicable tumor cytolytic capacity of Vγ9Vδ2 T cells

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A6.2-A7
Author(s):  
LA King ◽  
R Lameris ◽  
RC Roovers ◽  
P Parren ◽  
TD de Gruijl ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Xenograft Model ◽  
Cell Receptor ◽  
Target Cells ◽  
Mouse Xenograft Model ◽  
Vγ9vδ2 T Cells

Vγ9Vδ2-T cells include a unique and potent subset of T cells which play an important role in tumor defense. Vγ9Vδ2-T cells recognize and can lyse butyrophilin 3A1-expressing target cells with elevated levels of non-peptide phosphoantigens (pAg), induced by cell stress or malignancy. To date, Vγ9Vδ2-T cell based cancer immunotherapeutic approaches were well tolerated and in some cases capable of inducing relevant clinical responses. In an effort to improve the efficacy and consistency of Vγ9Vδ2-T cell based cancer immunotherapy, we designed a bispecific VHH that binds to both Vγ9Vδ2-T cells and EGFR expressed by tumor cells and results in the target-specific activation of Vγ9Vδ2-T cells and subsequent lysis of colorectal cancer cell lines and primary colorectal cancer samples both in vitro and in an in vivo mouse xenograft model. Of note, tumor cell lysis was independent of mutations in KRAS and BRAF that are known to impair the efficacy of clinically registered anti-EGFR monoclonal antibodies as well as common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific VHH, this immunotherapeutic approach can in principle be applied to a large group of cancer types.Disclosure InformationL.A. King: None. R. Lameris: None. R.C. Roovers: None. P. Parren: None. T.D. de Gruijl: None. H.J. van der Vliet: None.

scholarly journals Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioana Sandu ◽  
Dario Cerletti ◽  
Manfred Claassen ◽  
Annette Oxenius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Viral Infections ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

scholarly journals Dual TCR-alpha expression on MAIT cells as a potential confounder of TCR interpretation

2021 ◽  
Author(s):  
Sara Suliman ◽  
Lars Kjer-Nielsen ◽  
Sarah K. Iwany ◽  
Kattya Lopez Tamara ◽  
Liyen Loh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cell Receptor ◽  
Antigen Specificity ◽  
Mait Cells ◽  
Human T Cells ◽  
V Genes ◽  
Antigen Presenting ◽  
Β Chain

AbstractMucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant T cell receptor (TCR) α chain that uses TRAV1-2 joined to TRAJ33/20/12 and recognize metabolites from bacterial riboflavin synthesis bound to the antigen-presenting molecule, MR1. Recently, our attempts to identify alternative MR1-presented antigens led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered antigen specificity is likely to lead to altered affinity for the most potent known antigen, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), we performed bulk TCRα and β chain sequencing, and single cell-based paired TCR sequencing, on T cells that bound the MR1-5-OP-RU tetramer, but with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Whereas we initially interpreted these as diverse MR1-restricted TCRs, single cell TCR sequencing revealed that cells expressing atypical TCRα chains also co-expressed an invariant MAIT TCRα chain. Transfection of each non-TRAV1-2 TCRα chain with the TCRβ chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα chain expression in human T cells and competition for the endogenous β chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and non-canonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of antigen specificity.

Novel adoptive T-cell immunotherapy using a WT1-specific TCR vector encoding silencers for endogenous TCRs shows marked antileukemia reactivity and safety

Blood ◽  
2011 ◽  
Vol 118 (6) ◽  
pp. 1495-1503 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Sachiko Okamoto ◽  
Jun An ◽  
Kozo Nagai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Xenograft Model ◽  
Genetically Engineered ◽  
Adoptive T Cell Therapy ◽  
Vector System ◽  
Human Leukemia ◽  
Antigen Specificity ◽  
T Cell Therapy ◽  
Mouse Xenograft Model

Abstract Adoptive T-cell therapy for malignancies using redirected T cells genetically engineered by tumor antigen-specific T-cell receptor (TCR) gene transfer is associated with mispairing between introduced and endogenous TCR chains with unknown specificity. Therefore, deterioration of antitumor reactivity and serious autoimmune reactivity are major concerns. To address this problem, we have recently established a novel retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). In this study, to test the clinical application of siTCR gene therapy for human leukemia, we examined in detail the efficacy and safety of WT1-siTCR–transduced T cells. Compared with conventional WT1-TCR (WT1-coTCR) gene-transduced T cells, these cells showed significant enhancement of antileukemia reactivity resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Notably, WT1-siTCR gene-transduced T cells were remarkably expandable after repetitive stimulation with WT1 peptide in vitro, without any deterioration of antigen specificity. WT1-siTCR gene–transduced T cells from leukemia patients successfully lysed autologous leukemia cells, but not normal hematopoietic progenitor cells. In a mouse xenograft model, adoptively transferred WT1-siTCR gene-transduced T cells exerted distinct antileukemia efficacy but did not inhibit human hematopoiesis. Our results suggest that gene-immunotherapy for leukemia using this WT1-siTCR system holds considerable promise.

scholarly journals Induction of T Cell Senescence by Cytokine Induced Bystander Activation

2021 ◽  
Vol 2 ◽  
Author(s):  
Attiya A. Abbas ◽  
Arne N. Akbar
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Immune Cell ◽  
Critical Role ◽  
The Elderly ◽  
Cell Receptor ◽  
Antigen Specificity ◽  
Tcr Signaling ◽  
Bystander Activation

As people around the world continue to live longer, maintaining a good quality of life is of increasing importance. The COVID-19 pandemic revealed that the elderly are disproportionally vulnerable to infectious diseases and Immunosenescence plays a critical role in that. An ageing immune system influences the conventional activity of T cells which are at the forefront of eliminating harmful foreign antigens. With ageing, unconventional end-stage T cells, that exhibit a senescent phenotype, amass. These senescent T cells deviate from T cell receptor (TCR) signaling toward natural killer (NK) activity. The transition toward innate immune cell function from these adaptor T cells impacts antigen specificity, contributing to increased susceptibility of infection in the elderly. The mechanism by which senescent T cells arise remains largely unclear however in this review we investigate the part that bystander activation plays in driving the change in function of T cells with age. Cytokine-induced bystander activation may offer a plausible explanation for the induction of NK-like activity and senescence in T cells. Further understanding of these specific NK-like senescent T cells allows us to identify the benefits and detriments of these cells in health and disease which can be utilized or regulated, respectively. This review discusses the dynamic of senescent T cells in adopting NK-like T cells and the implications that has in an infectious disease context, predominately in the elderly.

scholarly journals Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 867
Author(s):  
Ling Wu ◽  
Joanna Brzostek ◽  
Shvetha Sankaran ◽  
Qianru Wei ◽  
Jiawei Yap ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Model ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
Barr Virus ◽  
Car T ◽  
Epstein Barr ◽  
Lower Magnitude

Chimeric antigen receptor T cells (CAR-T) utilize T cell receptor (TCR) signaling cascades and the recognition functions of antibodies. This allows T cells, normally restricted by the major histocompatibility complex (MHC), to be redirected to target cells by their surface antigens, such as tumor associated antigens (TAAs). CAR-T technology has achieved significant successes in treatment of certain cancers, primarily liquid cancers. Nonetheless, many challenges hinder development of this therapy, such as cytokine release syndrome (CRS) and the efficacy of CAR-T treatments for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, which has been less studied. To dissect CAR signaling, we designed a CAR that targets an epitope from latent membrane protein 2 A (LMP2 A) of the Epstein–Barr virus (EBV) presented on HLA*A02:01. Because of this, CAR and TCR signaling can be compared directly, allowing us to study the involvement of other signaling molecules, such as coreceptors. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer, but at a lower magnitude, than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. The distinct CAR signaling processes could provide explanations for clinical behavior of CAR-T therapy and suggest ways to improve the technology.

scholarly journals Herpes Simplex Virus Remodels T-Cell Receptor Signaling, Resulting in p38-Dependent Selective Synthesis of Interleukin-10

2007 ◽  
Vol 81 (22) ◽  
pp. 12504-12514 ◽  
Author(s):  
Derek D. Sloan ◽  
Keith R. Jerome
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
T Cell ◽  
T Cell Receptor ◽  
Intracellular Signaling ◽  
Cell Function ◽  
Cell Receptor ◽  
Interleukin 10 ◽  
Simplex Virus

ABSTRACT Herpes simplex virus (HSV)-specific T cells are essential for viral clearance. However, T cells do not prevent HSV latent infection or reactivation, suggesting that HSV has the potential to modulate T-cell function. T-cell receptor (TCR) stimulation is a potent and specific means of activating T cells. To investigate how HSV affects T-cell function, we have analyzed how HSV affects TCR-stimulated intracellular signaling and cytokine synthesis in mock-infected and HSV-infected T cells. Mock-infected T cells stimulated through the TCR synthesized a broad range of cytokines that included the proinflammatory cytokines tumor necrosis factor alpha, gamma interferon, and interleukin-2. In contrast, HSV-infected T cells stimulated through the TCR selectively synthesized interleukin-10, a cytokine that suppresses cellular immunity and favors viral replication. To achieve selective interleukin-10 synthesis, HSV differentially affected TCR signaling pathways. HSV inhibited TCR-stimulated formation of the linker for activation of the T-cell signaling complex, and HSV inhibited TCR-stimulated NF-κB activation. At the same time, HSV activated the p38 and JNK mitogen-activated protein kinases as well as the downstream transcription factors ATF-2 and c-Jun. HSV did not inhibit TCR-stimulated activation of STAT3, a transcription factor involved in interleukin-10 synthesis. The activation of p38 was required for interleukin-10 synthesis in HSV-infected T cells. The ability of HSV to differentially target intracellular signaling pathways and transform an activating stimulus into an immunosuppressive response represents a novel strategy for pathogen-mediated immune modulation. Selective, TCR-stimulated interleukin-10 synthesis may play an important role in HSV pathogenesis.

scholarly journals Fas ligand mediates activation-induced cell death in human T lymphocytes.

1995 ◽  
Vol 181 (1) ◽  
pp. 71-77 ◽  
Author(s):  
M R Alderson ◽  
T W Tough ◽  
T Davis-Smith ◽  
S Braddy ◽  
B Falk ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Fas Ligand ◽  
Significant Proportion ◽  
Cell Receptor ◽  
Target Cells ◽  
Activated T Cells ◽  
Activation Induced Cell Death ◽  
Fas L

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

scholarly journals In Vivo Selection of T-Cell Receptor Junctional Region Sequences by HLA-A2 Human T-Cell Lymphotropic Virus Type 1 Tax11-19 Peptide Complexes

2001 ◽  
Vol 75 (2) ◽  
pp. 1065-1071 ◽  
Author(s):  
Mineki Saito ◽  
Graham P. Taylor ◽  
Akiko Saito ◽  
Yoshitaka Furukawa ◽  
Koichiro Usuku ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Virus Type ◽  
Cell Receptor ◽  
Antigen Specificity ◽  
Human T Cell ◽  
Cdr3 Region

ABSTRACT Using HLA-peptide tetrameric complexes, we isolated human T-cell lymphotrophic virus type 1 Tax peptide-specific CD8+ T cells ex vivo. Antigen-specific amino acid motifs were identified in the T-cell receptor Vβ CDR3 region of clonally expanded CD8+ T cells. This result directly confirms the importance of the CDR3 region in determining the antigen specificity in vivo.

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.

Sign in / Sign up

Export Citation Format

Share Document