scholarly journals Reprogramming human T cell function and specificity with non-viral genome targeting

2017 ◽  
Author(s):  
Theodore L. Roth ◽  
Cristina Puig-Saus ◽  
Ruby Yu ◽  
Eric Shifrut ◽  
Julia Carnevale ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dna Sequences ◽  
Viral Genome ◽  
Viral Vectors ◽  
Cell Function ◽  
Experimental Manipulation ◽  
Target Cells ◽  
Human T Cell ◽  
Human T Cells

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2–5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

scholarly journals Zinc Aspartate Induces IL-16 Secretion and Apoptosis in Human T Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 246
Author(s):  
Dirk Reinhold ◽  
Karina Guttek ◽  
Annika Reddig ◽  
Linda Voss ◽  
Claudia Schubert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Diseases ◽  
T Cell Activation ◽  
Cell Activation ◽  
Caspase 3 ◽  
Cell Function ◽  
Human T Cell ◽  
Human T Cells ◽  
Zinc Aspartate

T cell activation mediates immunity to pathogens. On the flipside, T cells are also involved in pathological immune responses during chronic autoimmune diseases. We recently reported that zinc aspartate, a registered drug with high bioavailability, dose-dependently inhibits T cell activation and Th1/Th2/Th17 cytokine production of stimulated human and mouse T cells. To understand the suppressive effect of zinc on T cell function, we here investigated the influence of zinc aspartate on human T cells focusing on the secretion of immunosuppressive cytokines, induction of apoptosis, and caspase 3/7 activity. To this end, we monitored either freshly stimulated or pre-activated human T cells in the presence of zinc aspartate from 40–140 µM over a period of 72 h. Under both experimental conditions, we observed a dose-dependent suppression of human T cell proliferation. While IL-1ra, latent TGF-β1, and IL-10 were dose-dependently reduced, we, unexpectedly, detected elevated levels of IL-16 upon zinc supplementation. In addition, the number of cells with active caspase 3/7 and, consecutively, the amount of cells undergoing apoptosis, steadily increased at zinc aspartate concentrations exceeding 100 µM. Taken together, our findings suggest that zinc aspartate impairs T cell fitness and might be beneficial for the treatment of T cell-mediated autoimmune diseases.

scholarly journals RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration

2015 ◽  
Vol 20 (8) ◽  
pp. 943-956 ◽  
Author(s):  
Michael Freeley ◽  
Emily Derrick ◽  
Eugene Dempsey ◽  
Antje Hoff ◽  
Anthony Davies ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Lymphocyte Function ◽  
Rnai Screening ◽  
Chemically Modified ◽  
Human T Cell ◽  
Human T Cells

Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function.

Deltex1 suppresses T cell function and is a biomarker for diagnosis and disease activity of systemic lupus erythematosus

Rheumatology ◽  
2019 ◽  
Vol 58 (4) ◽  
pp. 719-728 ◽  
Author(s):  
Chuen-Miin Leu ◽  
Tzu-Sheng Hsu ◽  
Yu-Ping Kuo ◽  
Ming-Zong Lai ◽  
Po-Chun Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Disease Activity ◽  
Cell Function ◽  
Healthy Controls ◽  
Cell Functions ◽  
T Cell Function ◽  
Human T Cell ◽  
Human T Cells ◽  
Ifn Γ

Abstract Objective Deletion of Deltex1 (DTX1) in mice caused hyperactivation of T cells and lupus-like autoimmune syndromes, however, the association of DTX1 with human autoimmune diseases is totally unknown. This study investigated the role of DTX1 in human T cell functions and its correlation with disease activity in patients with SLE. Methods The influence of DTX1 on T cell function was evaluated using human primary cells. DTX1 expression in peripheral blood mononuclear cells (PBMCs) from healthy controls and SLE patients was measured by quantitative real-time PCR and the SLEDAI was used to assess disease activity. Results After stimulation with anti-CD3 and anti-CD28, silencing of DTX1 expression enhanced IFN-γ secretion by human T cells. The expression of DTX1 in PBMCs was significantly lower in 100 SLE patients than in 50 age- and sex-matched healthy controls (DTX1/glyceraldehyde 3-phosphate dehydrogenase, 0.452 vs 1.269, P < 0.001). The area under the receiver operator characteristics curve of the model was 0.737 (95% CI 0.658, 0.815). Intriguingly, a low DTX1 level in T cells led to high IFN-γ production in SLE patients and had a correlation with severe disease activity. In addition, low DTX1 expression in SLE patients was associated with active LN, lung involvement or hypocomplementaemia. Conclusion Knockdown DTX1 expression in human T cells reduced IFN-γ secretion. DTX1 expression in the PBMCs was significantly lower in SLE patients and had an inverse correlation with disease activity, indicating that the DTX1 level may be a good disease marker of SLE.

scholarly journals Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

2018 ◽  
Author(s):  
Beau R. Webber ◽  
Cara-lin Lonetree ◽  
Mitchell G. Kluesner ◽  
Matthew J. Johnson ◽  
Emily J. Pomeroy ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Checkpoint Inhibitors ◽  
Target Cells ◽  
Widespread Application ◽  
Base Editing ◽  
Highly Efficient ◽  
Human T Cell ◽  
Human T Cells ◽  
Car T

Chimeric antigen receptor engineered T cell (CAR-T) immunotherapy has shown efficacy against a subset of hematological malignancies1,2, yet its autologous nature and ineffectiveness against epithelial and solid cancers limit widespread application. To overcome these limitations, targeted nucleases have been used to disrupt checkpoint inhibitors and genes involved in alloreactivity3–6. However, the production of allogeneic, “off-the-shelf” T cells with enhanced function requires multiplex genome editing strategies that risk off-target effects, chromosomal rearrangements, and genotoxicity due to simultaneous double-strand break (DSB) induction at multiple loci7–10. Moreover, it has been well documented that DSBs are toxic lesions that can drive genetic instability11,12. Alternatively, CRISPR/Cas9 base editors afford programmable enzymatic nucleotide conversion at targeted loci without induction of DSBs13,14. We reasoned this technology could be used to knockout gene function in human T cells while minimizing safety concerns associated with current nuclease platforms. Through systematic reagent and dose optimization, we demonstrate highly efficient multiplex base editing and consequent protein knockout in primary human T cells at loci relevant to the generation of allogeneic CAR-T cells including the T cell receptor α constant (TRAC) locus, β-2 microglobulin (B2M), and programmed cell death 1 (PDCD1). Multiplex base edited T cells equipped with a CD19 CAR killed target cells more efficiently; and importantly, both DSB induction and translocation frequency were greatly reduced compared to cells engineered with Cas9 nuclease. Collectively, our results establish a novel multiplex gene editing platform to enhance both the safety and efficacy of engineered T cell-based immunotherapies.

scholarly journals Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Josephine F. Reijneveld ◽  
Mira Holzheimer ◽  
David C. Young ◽  
Kattya Lopez ◽  
Sara Suliman ◽  
...  
Keyword(s):  
T Cells ◽  
Fatty Acid ◽  
Immune System ◽  
T Cell ◽  
Cross Reactivity ◽  
Lipid Binding ◽  
Human Immune System ◽  
Human T Cell ◽  
Human T Cells ◽  
Pure Compounds

AbstractThe cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 721-730 ◽  
Author(s):  
H Segall ◽  
I Lubin ◽  
H Marcus ◽  
A Canaan ◽  
Y Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Lymphocytes ◽  
Scid Mice ◽  
Human Antibody ◽  
Adoptive Therapy ◽  
Activation Markers ◽  
Human T Cell ◽  
Human T Cells

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.

scholarly journals Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Peter A. Szabo ◽  
Hanna Mendes Levitin ◽  
Michelle Miron ◽  
Mark E. Snyder ◽  
Takashi Senda ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interferon Response ◽  
Functional Responses ◽  
Multiple Tumor ◽  
Human T Cell ◽  
Human T Cells

Abstract Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8+ T cells and an interferon-response state for CD4+ T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8+ compared to CD4+ T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.

scholarly journals Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation

2020 ◽  
Vol 4 (21) ◽  
pp. 5343-5356
Author(s):  
Laura R. E. Becher ◽  
Wendy K. Nevala ◽  
Shari Lee Sutor ◽  
Megan Abergel ◽  
Michele M. Hoffmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clonal Expansion ◽  
Antigen Receptors ◽  
Fab Fragment ◽  
Public And Private ◽  
T Cell Clones ◽  
Human T Cell ◽  
Cell Clones ◽  
Human T Cells

Abstract Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

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.

Inhibitory Effects of Lactic Acid on Human Antigen-Specific CD8+ T-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3844-3844
Author(s):  
Marina Kreutz ◽  
Karin Fischer ◽  
Petra Hoffmann ◽  
Simon Volkl ◽  
Matthias Edinger ◽  
...  
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
T Cell ◽  
Cd8 T Cells ◽  
Acid Production ◽  
Cell Function ◽  
Immune Escape ◽  
Lactic Acid Production ◽  
Target Cells ◽  
Thymidine Uptake

Abstract A characteristic feature of inflammatory lesions or tumor sites is local acidosis, which is attributed to the local increase in lactic acid production. We studied the effect of such an acidic environment on the immune functions of antigen-specific CD8+ T-cells by incubating the cells in the presence of various concentrations of lactic acid for up to 48h. CD8+ T-cells were isolated from healthy donors and expanded by weekly stimulation with autologous dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. The obtained T cell population consisted of at least 90% CD8+ and about 60% Melan-A specific T cells, as determined by Melan-A multimer staining. Incubation of CD8+ T cells with up to 20mM lactic acid for 24h did not cause T-cell apoptosis or cell death as determined by combined annexin/propidium iodide staining. However, the proliferative capacity of CD8+ T cells, as determined by 3H-thymidine uptake, was strongly inhibited. Similar results were obtained when we determined cytokine production and cytotoxic activity of the cells after a 24h culture period in 5-20 mM lactic acid. Production of both, IL-2 and IFN-gamma was strongly diminished in comparison to untreated cells, as determined by intracellular staining after stimulation with PMA/ionomycin for 5h in the presence of monensin. Analysis of the antigen-specific cytolytic capacity revealed that CD8+ T cells pre-cultured with lactic acid were less effective in killing antigen-loaded T2 target cells as compared to untreated CD8+ T cells. In parallel, the intracellular contents of the cytotoxic effector molecules granzyme-B and perforin was diminished. Re-adjusting the pH of the medium to a physiological value of pH7.4 could partially revert the effect of lactic acid. Treatment of CD8+ T cells with sodium lactate instead of lactic acid had no inhibitory effect. We conclude, that lactic acid is an important modulator of CD8+ T-cell function and may contribute, together with other factors, to immune escape mechanisms in the tumor environment.

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