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1.
Cell ; 184(25): 6081-6100.e26, 2021 12 09.
Article in English | MEDLINE | ID: mdl-34861191

ABSTRACT

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunotherapy, Adoptive/methods , Pancreatic Neoplasms/therapy , Receptors, Chimeric Antigen/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Line, Tumor , HEK293 Cells , Humans , Inhibitor of Differentiation Proteins/immunology , Male , Mice , Mice, Knockout , Mice, Nude , Mice, SCID , Neoplasm Proteins/immunology , SOXC Transcription Factors/immunology
2.
Cell ; 162(4): 727-37, 2015 Aug 13.
Article in English | MEDLINE | ID: mdl-26276629

ABSTRACT

Chronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt's lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells. PAPERCLIP.


Subject(s)
Genomic Instability , Lymphoma, B-Cell/genetics , Malaria/complications , Malaria/genetics , Plasmodium chabaudi/physiology , Animals , B-Lymphocytes/pathology , Chronic Disease , Cytidine Deaminase/metabolism , DNA Replication , Genes, p53 , Germinal Center/parasitology , Malaria/parasitology , Malaria/pathology , Mice , Translocation, Genetic
3.
Nature ; 607(7918): 360-365, 2022 07.
Article in English | MEDLINE | ID: mdl-35676488

ABSTRACT

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.


Subject(s)
Cell- and Tissue-Based Therapy , Immunotherapy, Adoptive , Interleukin Receptor Common gamma Subunit , Neoplasms , Receptors, Interleukin-9 , Recombinant Fusion Proteins , T-Lymphocytes , Animals , Cell- and Tissue-Based Therapy/methods , Immunotherapy, Adoptive/methods , Interleukin Receptor Common gamma Subunit/genetics , Interleukin Receptor Common gamma Subunit/immunology , Interleukins/genetics , Interleukins/immunology , Melanoma/immunology , Mice , Neoplasms/genetics , Neoplasms/immunology , Pancreatic Neoplasms/immunology , Receptors, Interleukin-9/genetics , Receptors, Interleukin-9/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , STAT Transcription Factors/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
4.
Proc Natl Acad Sci U S A ; 121(10): e2317735121, 2024 Mar 05.
Article in English | MEDLINE | ID: mdl-38408246

ABSTRACT

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.


Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Receptors, Chimeric Antigen , Humans , Immunotherapy, Adoptive/methods , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Antigens, CD19 , Cell- and Tissue-Based Therapy , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism
6.
J Biol Chem ; 286(50): 42830-9, 2011 Dec 16.
Article in English | MEDLINE | ID: mdl-22025621

ABSTRACT

For optimal proteolytic function, the central core of the proteasome (core particle (CP) or 20S) has to associate with activators. We investigated the impact of the yeast activator Blm10 on proteasomal peptide and protein degradation. We found enhanced degradation of peptide substrates in the presence of Blm10 and demonstrated that Blm10 has the capacity to accelerate proteasomal turnover of the unstructured protein tau-441 in vitro. Mechanistically, proteasome activation requires the opening of a closed gate, which allows passage of unfolded proteins into the catalytic chamber. Our data indicate that gate opening by Blm10 is achieved via engagement of its C-terminal segment with the CP. Crucial for this activity is a conserved C-terminal YYX motif, with the penultimate tyrosine playing a preeminent role. Thus, Blm10 utilizes a gate opening strategy analogous to the proteasomal ATPases HbYX-dependent mechanism. Because gating incompetent Blm10 C-terminal point mutants confers a loss of function phenotype, we propose that the cellular function of Blm10 is based on CP association and activation to promote the degradation of proteasome substrates.


Subject(s)
Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Phenotype , Proteasome Endopeptidase Complex/genetics , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
7.
Curr Opin Immunol ; 74: 76-84, 2022 02.
Article in English | MEDLINE | ID: mdl-34798542

ABSTRACT

T cells engineered to express transgenes such as chimeric antigen receptors (CAR) or modified T cell receptors (TCR) represent a new pillar of cancer therapy. Use of CRISPR/Cas gene-editing tools now allows even stronger and more precise control over the fate and function of engineered T cell therapies, including multiplex genome editing to facilitate use of off-the-shelf allogeneic T cells and novel approaches which have the potential to overcome some of the limitations of canonical Cas9-mediated DNA cleavage. This review summarizes the CRISPR/Cas techniques that have been used in preclinical research and outlines those that currently being tested in clinical trials.


Subject(s)
CRISPR-Cas Systems , Neoplasms , CRISPR-Cas Systems/genetics , Gene Editing/methods , Humans , Immunotherapy , Immunotherapy, Adoptive/methods , Neoplasms/genetics , Neoplasms/therapy , T-Lymphocytes
8.
J Exp Med ; 214(3): 815-831, 2017 03 06.
Article in English | MEDLINE | ID: mdl-28179379

ABSTRACT

The RAG recombinase (RAG1/2) plays an essential role in adaptive immunity by mediating V(D)J recombination in developing lymphocytes. In contrast, aberrant RAG1/2 activity promotes lymphocyte malignancies by causing chromosomal translocations and DNA deletions at cancer genes. RAG1/2 can also induce genomic DNA insertions by transposition and trans-V(D)J recombination, but only few such putative events have been documented in vivo. We used next-generation sequencing techniques to examine chromosomal rearrangements in primary murine B cells and discovered that RAG1/2 causes aberrant insertions by releasing cleaved antibody gene fragments that subsequently reintegrate into DNA breaks induced on a heterologous chromosome. We confirmed that RAG1/2 also mobilizes genomic DNA into independent physiological breaks by identifying similar insertions in human lymphoma and leukemia. Our findings reveal a novel RAG1/2-mediated insertion pathway distinct from DNA transposition and trans-V(D)J recombination that destabilizes the genome and shares features with reported oncogenic DNA insertions.


Subject(s)
DNA Damage , DNA-Binding Proteins/physiology , Homeodomain Proteins/physiology , Mutagenesis, Insertional , Animals , B-Lymphocytes/metabolism , Genomic Instability , Immunoglobulins/genetics , Mice , Mice, Inbred C57BL , Receptors, Antigen, T-Cell/genetics , Translocation, Genetic , V(D)J Recombination
9.
PLoS One ; 8(7): e69208, 2013.
Article in English | MEDLINE | ID: mdl-23861962

ABSTRACT

The Aicda gene encodes Activation-Induced cytidine Deaminase (AID), an enzyme essential for remodeling antibody genes in mature B lymphocytes. AID is also responsible for DNA damage at oncogenes, leading to their mutation and cancer-associated chromosome translocation in lymphoma. We used fate mapping and AID(GFP) reporter mice to determine if AID expression in the mouse extends beyond lymphocytes. We discovered that AID(cre) tags a small fraction of non-lymphoid cells starting at 10.5 days post conception (dpc), and that AID(GFP+) cells are detectable at dpc 11.5 and 12.5. Embryonic cells are tagged by AID(cre) in the submandibular region, where conditional deletion of the tumor suppressor PTEN causes squamous papillomas. AID(cre) also tags non-lymphoid cells in the embryonic central nervous system. Finally, in the adult mouse brain, AID(cre) marks a small fraction of diverse neurons and distinct neuronal populations, including pyramidal cells in cortical layer IV.


Subject(s)
Cell Lineage , Cytidine Deaminase/metabolism , Lymphocytes/cytology , Lymphocytes/enzymology , Aging/metabolism , Animals , Brain/enzymology , Brain/pathology , Embryonic Development , Integrases/metabolism , Mice , PTEN Phosphohydrolase/metabolism , Papilloma/pathology , Skin/metabolism
10.
Cell Rep ; 3(1): 138-47, 2013 Jan 31.
Article in English | MEDLINE | ID: mdl-23291097

ABSTRACT

Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle.


Subject(s)
DNA Breaks , G2 Phase/genetics , Immunoglobulin Class Switching/genetics , Recombination, Genetic , Replication Protein A/metabolism , S Phase/genetics , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Chromatin Immunoprecipitation , Cytidine Deaminase/metabolism , DNA Breaks, Double-Stranded , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Genetic Loci , Histones/metabolism , Mice , Models, Biological , Protein Binding/genetics , Protein Serine-Threonine Kinases/metabolism , Rad51 Recombinase/metabolism , Tumor Suppressor Proteins/metabolism
11.
J Exp Med ; 210(1): 115-23, 2013 Jan 14.
Article in English | MEDLINE | ID: mdl-23254285

ABSTRACT

DNA double-strand breaks (DSBs) are byproducts of normal cellular metabolism and obligate intermediates in antigen receptor diversification reactions. These lesions are potentially dangerous because they can lead to deletion of genetic material or chromosome translocation. The chromatin-binding protein 53BP1 and the histone variant H2AX are required for efficient class switch (CSR) and V(D)J recombination in part because they protect DNA ends from resection and thereby favor nonhomologous end joining (NHEJ). Here, we examine the mechanism of DNA end resection in primary B cells. We find that resection depends on both CtBP-interacting protein (CtIP, Rbbp8) and exonuclease 1 (Exo1). Inhibition of CtIP partially rescues the CSR defect in 53BP1- and H2AX-deficient lymphocytes, as does interference with the RecQ helicases Bloom (Blm) and Werner (Wrn). We conclude that CtIP, Exo1, and RecQ helicases contribute to the metabolism of DNA ends during DSB repair in B lymphocytes and that minimizing resection favors efficient CSR.


Subject(s)
B-Lymphocytes/physiology , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Exodeoxyribonucleases/metabolism , Immunoglobulin Isotypes/genetics , Recombination, Genetic , Animals , Base Sequence , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases , Endonucleases , Exodeoxyribonucleases/genetics , Histones/genetics , Histones/metabolism , Immunoglobulin Isotypes/metabolism , MRE11 Homologue Protein , Mice , Mice, Mutant Strains , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RecQ Helicases/genetics , RecQ Helicases/metabolism , Tumor Suppressor p53-Binding Protein 1 , V(D)J Recombination , Werner Syndrome Helicase
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