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1.
Nat Immunol ; 24(5): 869-883, 2023 05.
Article in English | MEDLINE | ID: mdl-37081150

ABSTRACT

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.


Subject(s)
CD8-Positive T-Lymphocytes , Immunotherapy, Adoptive , Interleukin-2 , Neoplasms, Experimental , CD8-Positive T-Lymphocytes/immunology , T-Cell Exhaustion , Lymphocytes, Tumor-Infiltrating/immunology , Interleukin-2/pharmacology , Interleukin-33 , Protein Engineering , Female , Animals , Mice , Mice, Inbred C57BL , Cell Line, Tumor , Neoplasms, Experimental/therapy , Programmed Cell Death 1 Receptor/metabolism
2.
Mol Ther ; 24(11): 1987-1999, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27439899

ABSTRACT

B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo, B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6-8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Ovarian Neoplasms/therapy , Receptors, Antigen/metabolism , T-Lymphocytes/immunology , V-Set Domain-Containing T-Cell Activation Inhibitor 1/immunology , Animals , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Ovarian Neoplasms/immunology , T-Lymphocytes/transplantation , Treatment Outcome , Xenograft Model Antitumor Assays
3.
Mol Ther ; 20(3): 633-43, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22127019

ABSTRACT

Cancer regression by gene-modified T cells bearing a chimeric antigen receptor (CAR) exodomain of mouse origin can be limited by the induction of transgene immunogenicity resulting in poor persistence and function in vivo. The development of functionally-active CAR of human origin can address this issue. Here, we constructed and evaluated fully human anti-mesothelin CARs comprised of a human mesothelin-specific single-chain antibody variable fragment (P4 scFv) coupled to T cell signaling domains. Primary human T cells expressing P4 CAR specifically produced proinflammatory cytokines, degranulated and exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells also mediated bystander killing of mesothelin-negative cancer cells during coculture. CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in the presence of soluble mesothelin in a xenogenic model of human ovarian cancer. Thus, primary human T cells expressing fully human anti-mesothelin CAR efficiently kill mesothelin-expressing tumors in vitro and in vivo and have the potential to overcome the issue of transgene immunogenicity that may limit CAR T cell trials that utilize scFvs of mouse origin.


Subject(s)
GPI-Linked Proteins/immunology , Ovarian Neoplasms/immunology , Receptors, Antigen, T-Cell/genetics , Single-Chain Antibodies/genetics , T-Lymphocytes/immunology , Animals , Bystander Effect/immunology , Cell Line , Cytotoxicity, Immunologic , Epitopes/immunology , Female , GPI-Linked Proteins/metabolism , Gene Order , Genetic Vectors/genetics , Humans , Lentivirus/genetics , Mesothelin , Mice , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/therapy , Receptors, Antigen, T-Cell/immunology , Single-Chain Antibodies/immunology , T-Lymphocytes/metabolism , Transduction, Genetic , Xenograft Model Antitumor Assays
4.
J Transl Med ; 9: 198, 2011 Nov 14.
Article in English | MEDLINE | ID: mdl-22082029

ABSTRACT

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines. METHODS: Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation. RESULTS: DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning(®) tissue-culture treated surface and Corning(®) ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media. CONCLUSIONS: This study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).


Subject(s)
Cell Culture Techniques/methods , Cell Extracts/pharmacology , Dendritic Cells/cytology , Dendritic Cells/metabolism , Interleukin-12/metabolism , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Cryopreservation , Culture Media/pharmacology , Dendritic Cells/drug effects , Humans , Hypochlorous Acid/pharmacology , Interleukin-12/biosynthesis , Lymphocyte Culture Test, Mixed , Oxidation-Reduction/drug effects , Phenotype , Time Factors , Trypsin/metabolism
5.
Hum Gene Ther ; 32(19-20): 1044-1058, 2021 10.
Article in English | MEDLINE | ID: mdl-34662233

ABSTRACT

T cell modification with genes that encode chimeric antigen receptors (CAR-T cells) has shown tremendous promise for the treatment of B cell malignancies. The successful translation of CAR-T cell therapy to other tumor types, including solid tumors, is the next big challenge. As the field advances from second- to next-generation CAR-T cells comprising multiple genetic modifications, more sophisticated methods and tools to engineer T cells are being developed. Viral vectors, especially γ-retroviruses and lentiviruses, are traditionally used for CAR-T cell engineering due to their high transduction efficiency. However, limited genetic cargo, high costs of production under good manufacturing practice (GMP) conditions, and the high regulatory demands are obstacles for widespread clinical translation. To overcome these limitations, different nonviral approaches are being explored at a preclinical or clinical level, including transposon/transposase systems and mRNA electroporation and nonintegrating DNA nanovectors. Genome editing tools that allow efficient knockout of particular genes and/or site-directed integration of the CAR and/or other transgenes into the genome are also being evaluated for CAR-T cell engineering. In this review, we discuss the development of viral and nonviral vectors used to generate CAR-T cells, focusing on their advantages and limitations. We also discuss the lessons learned from clinical trials using the different genetic engineering tools, with special focus on safety and efficacy.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Genetic Engineering , Genetic Vectors/genetics , Humans , Immunotherapy, Adoptive , Neoplasms/genetics , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Receptors, Chimeric Antigen/genetics , T-Lymphocytes
6.
J Exp Med ; 218(2)2021 02 01.
Article in English | MEDLINE | ID: mdl-33156338

ABSTRACT

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.


Subject(s)
Interleukin-15/genetics , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , Animals , Cell Line , Cell Line, Tumor , Female , Genetic Engineering/methods , Genetic Vectors/genetics , Genetic Vectors/immunology , Humans , Immunologic Memory/genetics , Immunologic Memory/immunology , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
7.
J Immunother Cancer ; 9(8)2021 08.
Article in English | MEDLINE | ID: mdl-34389616

ABSTRACT

BACKGROUND: The adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101. METHODS: Primary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups. RESULTS: In line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1+ Ki67- and LAG-3+ Ki67- CAR-T cells in the TME. CONCLUSIONS: This study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function.


Subject(s)
Neoplasms/immunology , Receptors, Chimeric Antigen/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Humans , Mice
8.
Cell Rep ; 36(3): 109412, 2021 07 20.
Article in English | MEDLINE | ID: mdl-34289354

ABSTRACT

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.


Subject(s)
BRCA1 Protein/deficiency , Inflammation/pathology , Membrane Proteins/metabolism , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Animals , BRCA1 Protein/metabolism , Cell Line, Tumor , Chemokine CCL5/metabolism , Chromatin/metabolism , DNA/metabolism , DNA Damage , Epigenesis, Genetic , Female , Gene Silencing , Humans , Immune Checkpoint Inhibitors/pharmacology , Inflammation/complications , Inflammation/immunology , Interferons/metabolism , Mice, Inbred C57BL , Neoplasm Grading , Neovascularization, Pathologic/pathology , Ovarian Neoplasms/complications , Ovarian Neoplasms/genetics , Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes/immunology , Transcription, Genetic , Vascular Endothelial Growth Factor A/metabolism
9.
Curr Opin Biotechnol ; 65: 75-87, 2020 10.
Article in English | MEDLINE | ID: mdl-32109718

ABSTRACT

Synthetic biology has been transformative to the treatment of advanced hematological malignancies by chimeric antigen receptor (CAR)-engineered T cells. A range of obstacles are now understood to limit the responses of solid epithelial-derived tumors to CAR therapy. For example, inefficient tumor homing and a fortified stroma can restrain the number of CAR-T cells reaching the tumor bed. Upon transendothelial migration across the tumor vasculature, CAR-T cells face a highly suppressive microenvironment that can quickly render them hypofunctional. Safety also remains a critical issue for advancing CAR therapy of solid tumors. Innovative CAR design as well as coengineering and combinatorial treatment strategies with oncolytic adenovirus, radiotherapy, vaccines, chemotherapy, small molecules and monoclonal antibodies hold tremendous potential to support CAR-T cell control of solid tumors, either by directly promoting CAR-T cell function, or/and by re-programming the TME and harnessing the endogenous immune system against the tumor.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Cell- and Tissue-Based Therapy , Humans , Immunotherapy, Adoptive , Neoplasms/therapy , Synthetic Biology , Tumor Microenvironment
10.
Mol Ther Oncolytics ; 16: 111-123, 2020 Mar 27.
Article in English | MEDLINE | ID: mdl-32021906

ABSTRACT

Therapy by adoptive transfer of ex vivo-expanded tumor-infiltrating or genetically modified T cells may lead to impressive clinical responses. However, there is a need to improve in vivo persistence and functionality of the transferred T cells, in particular, to face the highly immunosuppressive environment of solid tumors. Here, we investigate the potential of miR-155, a microRNA known to play an important role in CD8+ T cell fitness. We show that forced expression of miR-155 in tumor antigen-specific T cells improves the tumor control of B16 tumors expressing a low-affinity antigen ligand. Importantly, miR-155-transduced T cells exhibit increased proliferation and effector functions associated with a higher glycolytic activity independent of exogenous glucose. Altogether, these data suggest that miR-155 may optimize the antitumor activity of adoptively transferred low-affinity tumor-infiltrating lymphocytes (TILs), in particular, by rendering them more resistant to the glucose-deprived environment of solid tumors. Thus, transgenic expression of miR-155 may enable therapeutic targeting of self-antigen-specific T cells in addition to neoantigen-specific ones.

11.
Cancer Cell ; 35(6): 885-900.e10, 2019 06 10.
Article in English | MEDLINE | ID: mdl-31185212

ABSTRACT

We investigated the role of chemokines in regulating T cell accumulation in solid tumors. CCL5 and CXCL9 overexpression was associated with CD8+ T cell infiltration in solid tumors. T cell infiltration required tumor cell-derived CCL5 and was amplified by IFN-γ-inducible, myeloid cell-secreted CXCL9. CCL5 and CXCL9 coexpression revealed immunoreactive tumors with prolonged survival and response to checkpoint blockade. Loss of CCL5 expression in human tumors was associated with epigenetic silencing through DNA methylation. Reduction of CCL5 expression caused tumor-infiltrating lymphocyte (TIL) desertification, whereas forced CCL5 expression prevented Cxcl9 expression and TILs loss, and attenuated tumor growth in mice through IFN-γ. The cooperation between tumor-derived CCL5 and IFN-γ-inducible CXCR3 ligands secreted by myeloid cells is key for orchestrating T cell infiltration in immunoreactive and immunoresponsive tumors.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Chemotaxis, Leukocyte , Cytokines/metabolism , Dendritic Cells/metabolism , Lymphocyte Activation , Lymphocytes, Tumor-Infiltrating/metabolism , Macrophages/metabolism , Ovarian Neoplasms/metabolism , Animals , Antineoplastic Agents, Immunological/pharmacology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Chemokine CCL5/genetics , Chemokine CCL5/immunology , Chemokine CCL5/metabolism , Chemokine CXCL9/genetics , Chemokine CXCL9/immunology , Chemokine CXCL9/metabolism , Chemotaxis, Leukocyte/drug effects , Coculture Techniques , Cytokines/genetics , Cytokines/immunology , DNA Methylation , Dendritic Cells/drug effects , Dendritic Cells/immunology , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic , Humans , Immunotherapy/methods , Interferon-gamma/genetics , Interferon-gamma/immunology , Interferon-gamma/metabolism , Lymphocyte Activation/drug effects , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Macrophages/drug effects , Macrophages/immunology , Mice, Inbred C57BL , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Ovarian Neoplasms/therapy , Paracrine Communication , Receptors, CXCR3/genetics , Receptors, CXCR3/immunology , Receptors, CXCR3/metabolism , Signal Transduction
12.
Cancer Lett ; 266(1): 12-20, 2008 Jul 18.
Article in English | MEDLINE | ID: mdl-18378391

ABSTRACT

The heterodimeric transcription factor HIF-1 (hypoxia-inducible factor 1) represents the key mediator of hypoxia response. HIF-1 controls numerous genes of pivotal importance for cellular metabolism, angiogenesis, cell cycle regulation and inhibition of apoptosis. HIF-1 overexpression and enhanced transcriptional activity are linked to tumour initiation and progression. Malfunction of the HIF-1 signalling network has been associated with breast, ovarian and prostate cancers. Elevated reactive oxygen species (ROS), also observed in such tumours, have been implicated in HIF-1 signalling. Deciphering the role of ROS in cancer onset and their involvement in signalling networks should prove invaluable for the design of novel anticancer therapeutics.


Subject(s)
Hypoxia-Inducible Factor 1/physiology , Neoplasms/etiology , Reactive Oxygen Species/metabolism , Animals , Cell Hypoxia , Gene Expression Regulation, Neoplastic , Humans , Mice , MicroRNAs/physiology , Models, Biological , Neoplasms/metabolism , Signal Transduction , Transcription, Genetic
13.
J Immunother Cancer ; 6(1): 71, 2018 07 13.
Article in English | MEDLINE | ID: mdl-30005714

ABSTRACT

Adoptive transfer of T cells transduced with Chimeric Antigen Receptors (CAR) are now FDA-approved for the treatment of B-cell malignancies. Yet, the functionality of the endogenous TCR in CART cells has not been fully assessed. Here, we demonstrate that CART cells progressively upregulate Fas, FasL, DR5 and TRAIL, which result in their programmed cell death, independently of antigen-mediated TCR or CAR activation. CART cell apoptosis occurs even when the CAR contains a single (co-)activatory domain such as CD3ζ, CD28 or 4-1BB. Importantly, the dominant role of the Fas and DR5 pathways in CART cell apoptosis is demonstrated by the significant rescue of CART cells upon in vivo blockade by combined Fas-Fc and DR5-Fc recombinant proteins. These observations are of crucial importance for the long-term persistence of CART cells and for the development of new applications including the combined TCR and CAR activation against solid tumors.


Subject(s)
Immunotherapy, Adoptive , Melanoma, Experimental/therapy , Receptors, TNF-Related Apoptosis-Inducing Ligand/immunology , Skin Neoplasms/therapy , fas Receptor/immunology , Animals , Cell Death , Fas Ligand Protein/immunology , Female , Melanoma, Experimental/pathology , Mice, Inbred C57BL , Receptors, Chimeric Antigen/immunology , Skin Neoplasms/pathology , TNF-Related Apoptosis-Inducing Ligand/immunology , Tumor Burden
14.
J Immunother Cancer ; 6(1): 92, 2018 09 25.
Article in English | MEDLINE | ID: mdl-30253808

ABSTRACT

After publication of this article [1], it was noticed that 3 authors were missed from the author list.

15.
Oncoimmunology ; 6(9): e1326442, 2017.
Article in English | MEDLINE | ID: mdl-28932632

ABSTRACT

Cancer immunotherapy relies upon the ability of T cells to infiltrate tumors. The endothelium constitutes a barrier between the tumor and effector T cells, and the ability to manipulate local vascular permeability could be translated into effective immunotherapy. Here, we show that in the context of adoptive T cell therapy, antitumor T cells, delivered at high enough doses, can overcome the endothelial barrier and infiltrate tumors, a process that requires local production of C3, complement activation on tumor endothelium and release of C5a. C5a, in turn, acts on endothelial cells promoting the upregulation of adhesion molecules and T-cell homing. Genetic deletion of C3 or the C5a receptor 1 (C5aR1), and pharmacological blockade of C5aR1, impaired the ability of T cells to overcome the endothelial barrier, infiltrate tumors, and control tumor progression in vivo, while genetic chimera mice demonstrated that C3 and C5aR1 expression by tumor stroma, and not leukocytes, governs T cell homing, acting on the local endothelium. In vitro, endothelial C3 and C5a expressions were required for endothelial activation by type 1 cytokines. Our data indicate that effective immunotherapy is a consequence of successful homing of T cells in response to local complement activation, which disrupts the tumor endothelial barrier.

16.
Nat Commun ; 7: 12602, 2016 09 07.
Article in English | MEDLINE | ID: mdl-27600764

ABSTRACT

Enteropathy-associated T-cell lymphoma (EATL), a rare and aggressive intestinal malignancy of intraepithelial T lymphocytes, comprises two disease variants (EATL-I and EATL-II) differing in clinical characteristics and pathological features. Here we report findings derived from whole-exome sequencing of 15 EATL-II tumour-normal tissue pairs. The tumour suppressor gene SETD2 encoding a non-redundant H3K36-specific trimethyltransferase is altered in 14/15 cases (93%), mainly by loss-of-function mutations and/or loss of the corresponding locus (3p21.31). These alterations consistently correlate with defective H3K36 trimethylation. The JAK/STAT pathway comprises recurrent STAT5B (60%), JAK3 (46%) and SH2B3 (20%) mutations, including a STAT5B V712E activating variant. In addition, frequent mutations in TP53, BRAF and KRAS are observed. Conversely, in EATL-I, no SETD2, STAT5B or JAK3 mutations are found, and H3K36 trimethylation is preserved. This study describes SETD2 inactivation as EATL-II molecular hallmark, supports EATL-I and -II being two distinct entities, and defines potential new targets for therapeutic intervention.


Subject(s)
Enteropathy-Associated T-Cell Lymphoma/genetics , Histone-Lysine N-Methyltransferase/genetics , Intestinal Neoplasms/genetics , Enteropathy-Associated T-Cell Lymphoma/classification , Gene Expression Regulation, Neoplastic/physiology , Genetic Predisposition to Disease , Genomics , Humans , Intestinal Neoplasms/classification , Mutation
17.
Curr Opin Immunol ; 33: 55-63, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25665467

ABSTRACT

T cells play a critical role in tumor immune surveillance as evidenced by extensive mouse-tumor model studies as well as encouraging patient responses to adoptive T cell therapies and dendritic cell vaccines. It is well established that the interplay of tumor cells with their local cellular environment can trigger events that are immunoinhibitory to T cells. More recently it is emerging that the tumor vasculature itself constitutes an important barrier to T cells. Endothelial cells lining the vessels can suppress T cell activity, target them for destruction, and block them from gaining entry into the tumor in the first place through the deregulation of adhesion molecules. Here we review approaches to break this tumor endothelial barrier and enhance T cell activity.


Subject(s)
Lymphocyte Activation/immunology , Neoplasms/immunology , Neoplasms/pathology , Neovascularization, Pathologic/immunology , T-Lymphocytes/immunology , Animals , Humans , Immunotherapy, Adoptive/methods , Lymphocyte Activation/drug effects , Molecular Targeted Therapy , Neoplasms/metabolism , Neoplasms/therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/therapy , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism
18.
Biomed Res Int ; 2015: 712438, 2015.
Article in English | MEDLINE | ID: mdl-26448945

ABSTRACT

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.


Subject(s)
Biomarkers, Tumor/metabolism , CD8-Positive T-Lymphocytes/pathology , Glypicans/metabolism , Membrane Proteins/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/mortality , Adult , Aged , Aged, 80 and over , Female , Gene Expression Regulation, Neoplastic , Humans , Lymphocyte Activation , Middle Aged , Neoplasm Staging , Ovarian Neoplasms/pathology , Pennsylvania/epidemiology , Prevalence , Risk Factors , Survival Rate , Up-Regulation
19.
Int J Biochem Cell Biol ; 53: 389-98, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24880093

ABSTRACT

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.


Subject(s)
CD8-Positive T-Lymphocytes/pathology , Chromosomal Proteins, Non-Histone/biosynthesis , DNA-Binding Proteins/biosynthesis , Neoplasm Staging , Ovarian Neoplasms/genetics , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Chemokine CXCL9/biosynthesis , Female , Gene Expression Regulation, Neoplastic , Humans , Ovarian Neoplasms/pathology , Transcription Factors
20.
Hum Gene Ther ; 25(8): 730-9, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25003657

ABSTRACT

The ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon, and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T lymphocytes specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8(+) T cells with specificity for the ErbB2(369-377) peptide, we performed one round of in vitro peptide stimulation of CD8(+) T cells isolated from an HLA-A2(+) patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2-reactive T cells with strong ErbB2-specific, antitumor effector functions. We then stimulated these ErbB2-reactive T cells with ErbB2(+) HLA-A2(+) tumor cells in vitro and sorted tumor-activated ErbB2(369-377) peptide T cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2(369-377) peptide specificity. Primary human CD8(+) T cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2(369-377) peptide containing HLA-A2 tetramers, and efficiently recognized target cells pulsed with low nanomolar concentrations of ErbB2(369-377) peptide as well as nonpulsed ErbB2(+) HLA-A2(+) tumor cell lines in vitro. In a novel xenograft model, ErbB2-redirected T cells also significantly delayed progression of ErbB2(+) HLA-A2(+) human tumor in vivo. Together, these results support the notion that redirection of normal T-cell specificity by TCR gene transfer can have potential applications in the adoptive immunotherapy of ErbB2-expressing malignancies.


Subject(s)
Breast Neoplasms/therapy , Receptor, ErbB-2/immunology , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigen Presentation , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Line, Tumor , Dendritic Cells/immunology , Female , Genetic Engineering , Humans , Immunotherapy , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Receptor, ErbB-2/metabolism , Tumor Burden/immunology
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