-
Nature Mar 2023T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells. Here we developed a clinical-grade approach based on CRISPR-Cas9...
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Topics: Humans; Antigens, Neoplasm; Biopsy; Cell- and Tissue-Based Therapy; Cytokine Release Syndrome; Disease Progression; Encephalitis; Gene Editing; Gene Knock-In Techniques; Gene Knockout Techniques; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Mutation; Neoplasms; Patient Safety; Precision Medicine; Receptors, Antigen, T-Cell; T-Lymphocytes; Transgenes; HLA Antigens; CRISPR-Cas Systems
PubMed: 36356599
DOI: 10.1038/s41586-022-05531-1 -
Nature Jul 2018Decades of work have aimed to genetically reprogram T cells for therapeutic purposes using recombinant viral vectors, which do not target transgenes to specific genomic...
Decades of work have aimed to genetically reprogram T cells for therapeutic purposes using recombinant viral vectors, which do not target transgenes to specific genomic sites. 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 using homology-directed repair. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
Topics: Animals; Autoimmunity; CRISPR-Cas Systems; Cells, Cultured; Cellular Reprogramming; Gene Editing; Genome, Human; Humans; Interleukin-2 Receptor alpha Subunit; Male; Mice; Neoplasm Transplantation; Protein Engineering; Receptors, Antigen, T-Cell; T-Lymphocytes
PubMed: 29995861
DOI: 10.1038/s41586-018-0326-5 -
Blood May 2022Large granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired...
Large granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 T-cell receptor αβ [TCRαβ] T-LGL and 12 TCRγδ T-LGL). Seventy-six mutations were observed in 3 or more patients in the cohort, and out of those, STAT3, KMT2D, PIK3R1, TTN, EYS, and SULF1 mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole-exome cohort. Hotspot mutations in STAT3, PIK3R1, and FAS were detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2D and TET2 were observed. Moreover, STAT3 mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2D and SETD1B (P < .01 and P < .05, respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower absolute neutrophil count values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3 coiled-coil domain were characterized. STAT3-mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferon-γ signaling and decreased phosphatidylinositol 3-kinase-Akt signaling for STAT3-mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.
Topics: Amino Acid Transport Systems, Neutral; Exome; Eye Proteins; Genomics; Humans; Leukemia, Large Granular Lymphocytic; Mutation; Nerve Tissue Proteins; RNA Helicases; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; STAT3 Transcription Factor
PubMed: 35015834
DOI: 10.1182/blood.2021013164 -
Cell Research Jun 2020Naturally arising regulatory CD4 T (Treg) cells, which specifically express the transcription factor FoxP3 in the nucleus and CD25 and CTLA-4 on the cell surface, are a... (Review)
Review
Naturally arising regulatory CD4 T (Treg) cells, which specifically express the transcription factor FoxP3 in the nucleus and CD25 and CTLA-4 on the cell surface, are a T-cell subpopulation specialized for immune suppression, playing a key role in maintaining immunological self-tolerance and homeostasis. FoxP3 is required for Treg function, especially for its suppressive activity. However, FoxP3 expression per se is not necessary for Treg cell lineage commitment in the thymus and insufficient for full Treg-type gene expression in mature Treg cells. It is Treg-specific epigenetic changes such as CpG demethylation and histone modification that can confer a stable and heritable pattern of Treg type gene expression on developing Treg cells in a FoxP3-independent manner. Anomalies in the formation of Treg-specific epigenome, in particular, Treg-specific super-enhancers, which largely include Treg-specific DNA demethylated regions, are indeed able to cause autoimmune diseases in rodents. Furthermore, in humans, single nucleotide polymorphisms in Treg-specific DNA demethylated regions associated with Treg signature genes, such as IL2RA (CD25) and CTLA4, can affect the development and function of naïve Treg cells rather than effector T cells. Such genetic variations are therefore causative of polygenic common autoimmune diseases including type 1 diabetes and rheumatoid arthritis via affecting endogenous natural Treg cells. These findings on the transcription factor network with FoxP3 at a key position as well as Treg-specific epigenetic landscape facilitate our understanding of Treg cell development and function, and can be exploited to prepare functionally stable FoxP3-expressing Treg cells from antigen-specific conventional T cells to treat autoimmune diseases.
Topics: Animals; Autoimmune Diseases; CTLA-4 Antigen; DNA Methylation; Epigenesis, Genetic; Forkhead Transcription Factors; Gene Expression Regulation; Humans; Interleukin-2 Receptor alpha Subunit; Polymorphism, Single Nucleotide; T-Lymphocytes, Regulatory
PubMed: 32367041
DOI: 10.1038/s41422-020-0324-7 -
Frontiers in Immunology 2021Hyperforin is a major active constituent of L. extract, which is widely used for the treatment of depressive disorders. Recent studies have reported that hyperforin...
Hyperforin is a major active constituent of L. extract, which is widely used for the treatment of depressive disorders. Recent studies have reported that hyperforin reduced inflammation in stroke and suppressed proliferation and differentiation in keratinocytes. Psoriasis is a chronic immune-mediated inflammatory skin disease in which the IL-23/IL-17 axis plays an important role. To investigate the underlying inflammatory mechanisms and response of hyperforin in psoriasis, we use imiquimod (IMQ)-induced mice model, cultured murine splenic γδ T cells, and HaCaT cells in this study. Data showed that hyperforin reduced epidermal thickness and decreased IMQ-induced pathological scores of cutaneous skin lesions in mice. Meanwhile we proved that hyperforin suppressed infiltration of CD3 T cells and downregulated expression of , , , , , antimicrobial peptides (AMPs) in the skin lesion. Hyperforin significantly inhibited imiquimod-induced splenomegaly, reduced serum levels of TNF-α and IL-6, and IL-17A in splenocytes and draining lymph nodes. Our study also suggested that hyperforin lessened the infiltration of γδ T cell and CCR6 γδ T cells in spleen and lymph nodes. Hyperforin also suppressed the typical psoriasis-like inflammatory responses and the infiltration of IL-17A cells in dermal γδ T cells of IMQ treated mice transferred with γδ T cells. studies, hyperforin reduced the expression and secretion of IL-17A in γδ T cells, and suppressed the activation of MAPK/STAT3 pathways in human keratinocyte HaCaT cells and γδ T cells. In conclusion, hyperforin alleviates IMQ-induced inflammation in psoriasis through suppressing the immune responses exerted by IL-17 A-producing γδ T cells and related cytokines by modulating MAPK/STAT3 pathways. Our study provided a novel therapeutic tragedy for psoriasis by which hyperforin attenuates psoriasis-related inflammatory responses.
Topics: Adoptive Transfer; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Genes, T-Cell Receptor; HaCaT Cells; Humans; Imiquimod; Interleukin-17; Intraepithelial Lymphocytes; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Phloroglucinol; Phosphorylation; Psoriasis; STAT3 Transcription Factor; Signal Transduction; Skin; Terpenes; Mice
PubMed: 34025642
DOI: 10.3389/fimmu.2021.635076 -
Cytometry. Part B, Clinical Cytometry May 2021Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay...
Flow cytometric evaluation of TRBC1 expression in tissue specimens and body fluids is a novel and specific method for assessment of T-cell clonality and diagnosis of T-cell neoplasms.
BACKGROUND
Flow cytometric detection of T-cell clonality is challenging. The current available methodology for T-cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T-cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells.
METHODS
Here, we used a single TRBC1 antibody, in conjunction with other T-cell associated markers, to detect T-cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T-cell neoplasm and 97 patients with no T-cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1.
RESULTS
We examined TRBC1 expression on neoplastic T-cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T-cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T-cell neoplasms, and arbitrarily dissected T-cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets.
CONCLUSIONS
Single TRBC1 antibody detection of T-cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Body Fluids; Female; Flow Cytometry; Humans; Immunophenotyping; Lymphoma, T-Cell; Male; Middle Aged; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; Young Adult
PubMed: 32333725
DOI: 10.1002/cyto.b.21881 -
Journal of Molecular Cell Biology Nov 2023Chemoresistance is a primary cause of treatment failure in pancreatic cancer. Identifying cell surface markers specifically expressed in chemoresistant cancer cells...
Chemoresistance is a primary cause of treatment failure in pancreatic cancer. Identifying cell surface markers specifically expressed in chemoresistant cancer cells (CCCs) could facilitate targeted therapies to overcome chemoresistance. We performed an antibody-based screen and found that TRA-1-60 and TRA-1-81, two 'stemness' cell surface markers, are highly enriched in CCCs. Furthermore, TRA-1-60+/TRA-1-81+ cells are chemoresistant compared to TRA-1-60-/TRA-1-81- cells. Transcriptome profiling identified UGT1A10, shown to be both necessary and sufficient to maintain TRA-1-60/TRA-1-81 expression and chemoresistance. From a high-content chemical screen, we identified Cymarin, which downregulates UGT1A10, eliminates TRA-1-60/TRA-1-81 expression, and increases chemosensitivity both in vitro and in vivo. Finally, TRA-1-60/TRA-1-81 expression is highly specific in primary cancer tissue and positively correlated with chemoresistance and short survival, which highlights their potentiality for targeted therapy. Therefore, we discovered a novel CCC surface marker regulated by a pathway that promotes chemoresistance, as well as a leading drug candidate to target this pathway.
Topics: Humans; Cell Line, Tumor; Drug Resistance, Neoplasm; Pancreatic Neoplasms; Gene Expression Profiling
PubMed: 37327088
DOI: 10.1093/jmcb/mjad039 -
Frontiers in Immunology 2022The aryl hydrocarbon receptor (AhR) is a widely studied ligand-activated cytosolic transcriptional factor that has been associated with the initiation and progression of... (Review)
Review
The aryl hydrocarbon receptor (AhR) is a widely studied ligand-activated cytosolic transcriptional factor that has been associated with the initiation and progression of various diseases, including autoimmune diseases, cancers, metabolic syndromes, and allergies. Generally, AhR responds and binds to environmental toxins/ligands, dietary ligands, and allergens to regulate toxicological, biological, cellular responses. In a canonical signaling manner, activation of AhR is responsible for the increase in cytochrome P450 enzymes which help individuals to degrade and metabolize these environmental toxins and ligands. However, canonical signaling cannot be applied to all the effects mediated by AhR. Recent findings indicate that activation of AhR signaling also interacts with some non-canonical factors like Kruppel-like-factor-6 (KLF6) or estrogen-receptor-alpha (Erα) to affect the expression of downstream genes. Meanwhile, enormous research has been conducted to evaluate the effect of AhR signaling on innate and adaptive immunity. It has been shown that AhR exerts numerous effects on mast cells, B cells, macrophages, antigen-presenting cells (APCs), Th1/Th2 cell balance, Th17, and regulatory T cells, thus, playing a significant role in allergens-induced diseases. This review discussed how AhR mediates immune responses in allergic diseases. Meanwhile, we believe that understanding the role of AhR in immune responses will enhance our knowledge of AhR-mediated immune regulation in allergic diseases. Also, it will help researchers to understand the role of AhR in regulating immune responses in autoimmune diseases, cancers, metabolic syndromes, and infectious diseases.
Topics: Humans; Receptors, Aryl Hydrocarbon; Metabolic Syndrome; Hypersensitivity; Autoimmune Diseases; Allergens; Immunity
PubMed: 36601108
DOI: 10.3389/fimmu.2022.1057555 -
Nature Immunology Apr 2021The diversity of regulatory T (T) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt T cells with...
The diversity of regulatory T (T) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt T cells with elevated expression of β-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt T cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-β-catenin signaling in human and murine T cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt T cells coexpressing multiple pro-inflammatory cytokines. Binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in T cells. Activation of β-catenin signaling interferes with this function and promotes the disease-associated RORγt T phenotype.
Topics: Animals; Case-Control Studies; Cell Proliferation; Cells, Cultured; Cellular Reprogramming; Colitis, Ulcerative; Colitis-Associated Neoplasms; Crohn Disease; Cytokines; Disease Models, Animal; Epigenesis, Genetic; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 1-alpha; Humans; Lymphocyte Activation; Mice, Inbred C57BL; Mice, Transgenic; Nuclear Receptor Subfamily 1, Group F, Member 3; Phenotype; T Cell Transcription Factor 1; T-Lymphocytes, Regulatory; Wnt Signaling Pathway; Mice
PubMed: 33664518
DOI: 10.1038/s41590-021-00889-2 -
Brain, Behavior, & Immunity - Health Feb 2022Inflammatory bowel diseases (IBD) are chronic intestinal disorders characterized by dysregulated immune responses to resident microbiota in genetically susceptible... (Review)
Review
Inflammatory bowel diseases (IBD) are chronic intestinal disorders characterized by dysregulated immune responses to resident microbiota in genetically susceptible hosts. The activation of the cholinergic system has been proposed for the treatment of IBD patients according to its potential anti-inflammatory effect in vivo. The α-7-nicotinic-acetylcholine receptor (α7nAChR) is involved in the inhibition of inflammatory processes, modulating the production of cytokines, suppressing dendritic cells and macrophage activity, leading to the suppression of T cells. In this review, we address the most recent studies and clinical trials concerning cholinergic signaling and its therapeutic potential for inflammatory bowel diseases.
PubMed: 34977822
DOI: 10.1016/j.bbih.2021.100401