Journal Articles

Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor

Chimeric antigen receptor-T cell (CAR-T) therapy has been effective in the treatment of hematologic malignancies, but it has shown limited efficacy against solid tumors. Here we demonstrate an approach to enhancing CAR-T function in solid tumors by directly vaccine-boosting donor cells through their chimeric receptor in vivo. We designed amphiphile CAR-T ligands (amph-ligands) that, upon injection, trafficked to lymph nodes and decorated the surfaces of antigen-presenting cells, thereby priming CAR-Ts in the native lymph node microenvironment. Amph-ligand boosting triggered massive CAR-T expansion, increased donor cell polyfunctionality, and enhanced antitumor efficacy in multiple immunocompetent mouse tumor models. We demonstrate two approaches to generalizing this strategy to any chimeric antigen receptor, enabling this simple non-human leukocyte antigen-restricted approach to enhanced CAR-T functionality to be applied to existing CAR-T designs.

Author Info: (1) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815

Author Info: (1) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. (2) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (3) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (4) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (5) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (6) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (7) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (8) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (9) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (10) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (11) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (12) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (13) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (14) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (15) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (16) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. (17) David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. djirvine@mit.edu. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Engineering nanoparticles to locally activate T cells in the tumor microenvironment

Immunological tolerance of tumors is characterized by insufficient infiltration of cytotoxic T lymphocytes (CTLs) and immunosuppressive microenvironment of tumor. Tumor resistance to immune checkpoint inhibitors due to immunological tolerance is an ongoing challenge for current immune checkpoint blockade (ICB) therapy. Here, we report the development of tumor microenvironment-activatable anti-PDL1 antibody (alphaPDL1) nanoparticles for combination immunotherapy designed to overcome immunological tolerance of tumors. Combination of alphaPDL1 nanoparticle treatment with near-infrared (NIR) laser irradiation-triggered activation of photosensitizer indocyanine green induces the generation of reactive oxygen species, which promotes the intratumoral infiltration of CTLs and sensitizes the tumors to PDL1 blockade therapy. We showed that the combination of antibody nanoparticles and NIR laser irradiation effectively suppressed tumor growth and metastasis to the lung and lymph nodes in mouse models. The nanoplatform that uses the antibody nanoparticle alone both for immune stimulation and PDL1 inhibition could be readily adapted to other immune checkpoint inhibitors for improved ICB therapy.

Author Info: (1) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. Yantai Key Laborator

Author Info: (1) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia Medica, Shandong 264000, China. University of Chinese Academy of Sciences, Beijing 100049, China. (2) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. University of Chinese Academy of Sciences, Beijing 100049, China. (3) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. hjyu@simm.ac.cn ypli@simm.ac.cn. Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia Medica, Shandong 264000, China. (4) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. University of Chinese Academy of Sciences, Beijing 100049, China. (5) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (6) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (7) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (8) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (9) Institute of Biomedical Science and Children's Hospital, Fudan University, Shanghai 200032, China. (10) State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. hjyu@simm.ac.cn ypli@simm.ac.cn. School of Pharmacy, Yantai University, Shandong 264005, China.

Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells

Spotlight 

Bachem et al. showed that microbial production of short-chain fatty acids (SCFAs), specifically butyrate, rewired metabolism in antigen-activated CD8+ T cells by uncoupling oxidative phosphorylation from glycolysis, allowing for enhanced glycolysis and preferential fueling of oxidative phosphorylation through increased glutamine utilization and fatty acid catabolism. These metabolic adaptations promoted CD8+ T cells to transition to a memory phenotype, supporting long-term persistence and enhanced recall responses upon antigen re-encounter. This effect was partially dependent on T cell expression of SCFA receptors GPR41 and GPR43.

Bachem et al. showed that microbial production of short-chain fatty acids (SCFAs), specifically butyrate, rewired metabolism in antigen-activated CD8+ T cells by uncoupling oxidative phosphorylation from glycolysis, allowing for enhanced glycolysis and preferential fueling of oxidative phosphorylation through increased glutamine utilization and fatty acid catabolism. These metabolic adaptations promoted CD8+ T cells to transition to a memory phenotype, supporting long-term persistence and enhanced recall responses upon antigen re-encounter. This effect was partially dependent on T cell expression of SCFA receptors GPR41 and GPR43.

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation, and function. Here, we examined the impact of the microbiota on CD8(+) T cell memory. Antigen-activated CD8(+) T cells transferred into germ-free mice failed to transition into long-lived memory cells and had transcriptional impairments in core genes associated with oxidative metabolism. The microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8(+) T cells, and SCFAs were required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that butyrate uncoupled the tricarboxylic acid cycle from glycolytic input in CD8(+) T cells, which allowed preferential fueling of oxidative phosphorylation through sustained glutamine utilization and fatty acid catabolism. Our findings reveal a role for the microbiota in promoting CD8(+) T cell long-term survival as memory cells and suggest that microbial metabolites guide the metabolic rewiring of activated CD8(+) T cells to enable this transition.

Author Info: (1) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (2) Department of

Author Info: (1) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (2) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (3) Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, Australia. (4) Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, Australia. (5) Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, Australia. (6) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (7) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (8) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (9) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (10) Institute of Systems Immunology, University of Wurzburg, 97070 Wurzburg, Germany. (11) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (12) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (13) Immunology Division, Walter and Eliza Hall Institute for Medical Research, Parkville, VIC 3010, Australia. (14) Department of Immunology, Max-Planck Institute for Infection Biology, Berlin, Germany. (15) Department of Immunology, Max-Planck Institute for Infection Biology, Berlin, Germany; Centre for Biosecurity and Tropical Infectious Diseases, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia. (16) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (17) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (18) Department of Microbiology, Monash University, Clayton, VIC 3800, Australia. (19) Department of Microbiology, Monash University, Clayton, VIC 3800, Australia. (20) Cancer Immunology Program, Peter MacCallum Cancer Centre, Parkville, VIC 3010, Australia. (21) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (22) Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, Australia. (23) Department of Microbiology, Monash University, Clayton, VIC 3800, Australia. (24) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. (25) Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address: sbedoui@unimelb.edu.au.

A multipeptide vaccine plus toll-like receptor agonists LPS or polyICLC in combination with incomplete Freund's adjuvant in melanoma patients

BACKGROUND: Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8(+) T cell responses, whereas addition of an incomplete Freund's adjuvant (IFA) would reduce magnitude and persistence of immune responses. PATIENTS AND METHODS: Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNgamma ELIspot assay ex vivo or after one in vitro stimulation (IVS). RESULTS: Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. CONCLUSIONS: LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. TRIAL REGISTRATION: The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

Author Info: (1) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville

Author Info: (1) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville, VA, 22908, USA. Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA. (2) Department of Public Health Sciences/Division of Translational Research & Applied Statistics, University of Virginia, Charlottesville, VA, USA. (3) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville, VA, 22908, USA. (4) Department of Public Health Sciences/Division of Translational Research & Applied Statistics, University of Virginia, Charlottesville, VA, USA. (5) Department of Medicine/Division of Hematology/Oncology, University of Virginia, Charlottesville, VA, USA. (6) Department of Public Health Sciences/Division of Translational Research & Applied Statistics, University of Virginia, Charlottesville, VA, USA. (7) Department of Public Health Sciences/Division of Translational Research & Applied Statistics, University of Virginia, Charlottesville, VA, USA. (8) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville, VA, 22908, USA. (9) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville, VA, 22908, USA. (10) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville, VA, 22908, USA. (11) Department of Medicine/Division of Hematology/Oncology, University of Virginia, Charlottesville, VA, USA. (12) Department of Surgery/Division of Surgical Oncology and the Human Immune Therapy Center, Cancer Center, University of Virginia, 1352 Pinn Hall, P.O. Box 801457, Charlottesville, VA, 22908, USA. cls8h@virginia.edu.

CD19 CAR T Cells Following Autologous Transplantation in Poor Risk Relapsed and Refractory B cell non-Hodgkin Lymphoma

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or chemorefractory diffuse large B-cell lymphoma (rel/ref DLBCL). Only 50% of patients are cured with this approach. We investigated whether CD19-specific chimeric antigen receptor (CAR) T cells administered following HDT-ASCT may enhance progression-free survival (PFS). METHODS: Eligibility for this study includes poor-risk rel/ref aggressive B-NHL chemosensitive to salvage therapy with: 1) FDG-PET (+) or 2) bone marrow involvement. Patients underwent BEAM conditioned HDT-ASCT and followed by 19-28z CAR-T cells on days +2 and +3. RESULTS: Of 15 subjects treated on study, dose-limiting toxicity was observed at both dose levels (5 x106 and 1 x107 19-28z CAR-T/kg). Ten of 15 subjects experienced CAR T cell-induced neurotoxicity and/or cytokine-release syndrome (CRS), which were associated with greater CAR T cell persistence (p=0.05) but not peak CAR T cell expansion. Serum IFN-g elevation (p<0.001) and possibly IL-10 (p=0.07) were associated with toxicity. The 2-year PFS is 30% (95% CI: 20-70%). Two subjects with progression of disease (POD) were CD19 (-) on re-biopsy. Subjects given decreased naive-like (CD45RA+CCR7+) CD4+ and CD8+ CAR T cells experienced superior PFS (p=0.02 and 0.04, respectively). There was no association between CAR T cell peak expansion, persistence or cytokine changes and PFS. CONCLUSIONS: 19-28z CAR T cells following HDT-ASCT was associated with a high-incidence of reversible neurotoxicity and CRS. Following HDT-ASCT, effector CD4+ and CD8+ immunophenotypes may improve disease control. Phenotype selection and/or multiple infusions may be the focus of the next clinical trial. This study is registered at www.clinicaltrials.gov as #NCT01840566.

Author Info: (1) Medicine, Memorial Sloan-Kettering Cancer Center, United States sauterc@mskcc.org. (2) medicine, mskcc, United States. (3) Memorial Sloan-Kettering Cancer Center, United States

Author Info: (1) Medicine, Memorial Sloan-Kettering Cancer Center, United States sauterc@mskcc.org. (2) medicine, mskcc, United States. (3) Memorial Sloan-Kettering Cancer Center, United States. (4) MSKCC, United States. (5) Memorial Sloan-Kettering Cancer Center, United States. (6) Memorial Sloan Kettering Cancer Center, United States. (7) Brentjens Research, MSKCC, United States. (8) Memorial Sloan Kettering Cancer Center, United States. (9) Cellular Therapeutics Center, Memorial Sloan Kettering Cancer Center, United States. (10) Memorial Sloan Kettering Cancer Center, United States. (11) Memorial Sloan Kettering Cancer Center, United States. (12) Department of Pathology, Memorial Sloan-Kettering Cancer Center, United States. (13) Memorial Sloan Kettering Cancer Center. (14) medicine, University of Miami Sylvester Comprehensive Cancer Center, United States. (15) Adult Bone Marrow Transplant Service, Memorial Sloan Kettering Cancer Center, United States. (16) Memorial Sloan-Kettering Cancer Center, United States. (17) Memorial Sloan Kettering Cancer Center, United States. (18) Pediatrics, Memorial Sloan-Kettering Cancer Center, United States. (19) Leukemia Service, Memorial Sloan-Kettering Cancer Center, United States. (20) Immunology, MSKCC, United States. (21) Medicine, Memorial Sloan Kettering Cancer Center, United States.

AN ANTICANCER DRUG COCKTAIL OF Three Kinase Inhibitors Improved Response to a DENDRITIC CELL-BASED CANCER VACCINE

Monocyte-derived dendritic cell (moDC)-based cancer therapies intended to elicit antitumor T-cell responses have limited efficacy in most clinical trials. However, potent and sustained antitumor activity in a limited number of patients highlights the therapeutic potential of moDCs. In vitro culture conditions used to generate moDCs can be inconsistent, and moDCs generated in vitro are less effective than natural DCs. Based on our study highlighting the ability for certain kinase inhibitors to enhance tumor antigenicity, we therefore screened kinase inhibitors for their ability to improve DC immunogenicity. We identified AKT inhibitor MK2206, DNA-PK inhibitor NU7441, and MEK inhibitor trametinib as the compounds most effective at modulating moDC immunogenicity. The combination of these drugs, referred to as MKNUTRA, enhanced moDC activity over treatment with individual drugs while exhibiting minimal toxicity. An evaluation of 335 activation and T-cell suppressive surface proteins on moDCs revealed that MKNUTRA treatment more effectively matured cells and reduced the expression of tolerogenic proteins as compared with control moDCs. MKNUTRA treatment imparted to ICT107, a glioblastoma (GBM) DC-based vaccine that has completed Phase II trials, an increased ability to stimulate patient-derived autologous CD8+ T cells against the brain tumor antigens IL13Ralpha2(345-354) and TRP2(180-188). In vivo, treating ICT107 with MKNUTRA, prior to injection into mice with an established GBM tumor, reduced tumor growth kinetics. This response was associated with an increased frequency of tumor-reactive lymphocytes within tumors and in peripheral tissues. These studies broaden the application of targeted anticancer drugs and highlight their ability to increase moDC immunogenicity.

Author Info: (1) Medical Oncology Division, University of Colorado Anschutz Medical Campus Anschutz Medical Campus. (2) Medical Oncology Division, University of Colorado Anschutz Medical Campus

Author Info: (1) Medical Oncology Division, University of Colorado Anschutz Medical Campus Anschutz Medical Campus. (2) Medical Oncology Division, University of Colorado Anschutz Medical Campus Anschutz Medical Campus. (3) Medical Oncology Division, University of Colorado Anschutz Medical Campus. (4) Research, ImmunoCellular Therapeutics, Ltd. (5) Medical Oncology Division, University of Colorado Anschutz Medical Campus eduardo.davila@ucdenver.edu.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human beta2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Author Info: (1) Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. (2) Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA. (3)

Author Info: (1) Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. (2) Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA. (3) Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA. (4) Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA.

The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells

Immunotherapy with chimeric antigen receptor (CAR)-engineered T cells can be effective against advanced malignancies. CAR T cells are "living drugs" that require technologies to enable physicians (and patients) to maintain control over the infused cell product. Here, we demonstrate that the tyrosine kinase inhibitor dasatinib interferes with the lymphocyte-specific protein tyrosine kinase (LCK) and thereby inhibits phosphorylation of CD3zeta and zeta-chain of T cell receptor-associated protein kinase 70 kDa (ZAP70), ablating signaling in CAR constructs containing either CD28_CD3zeta or 4-1BB_CD3zeta activation modules. As a consequence, dasatinib induces a function-off state in CD8(+) and CD4(+) CAR T cells that is of immediate onset and can be sustained for several days without affecting T cell viability. We show that treatment with dasatinib halts cytolytic activity, cytokine production, and proliferation of CAR T cells in vitro and in vivo. The dose of dasatinib can be titrated to achieve partial or complete inhibition of CAR T cell function. Upon discontinuation of dasatinib, the inhibitory effect is rapidly and completely reversed, and CAR T cells resume their antitumor function. The favorable pharmacodynamic attributes of dasatinib can be exploited to steer the activity of CAR T cells in "function-on-off-on" sequences in real time. In a mouse model of cytokine release syndrome (CRS), we demonstrated that a short treatment course of dasatinib, administered early after CAR T cell infusion, protects a proportion of mice from otherwise fatal CRS. Our data introduce dasatinib as a broadly applicable pharmacologic on/off switch for CAR T cells.

Author Info: (1) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (2) Center for Cell Engineering and Immunology Program, Sloan Kettering Institute

Author Info: (1) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (2) Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. (3) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (4) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (5) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (6) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (7) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (8) Center for Cell Engineering and Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. (9) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. (10) Medizinische Klinik und Poliklinik II, Universitatsklinikum Wurzburg, 97080 Wurzburg, Germany. hudecek_m@ukw.de.

CTL-Derived Exosomes Enhance the Activation of CTLs Stimulated by Low-Affinity Peptides

Cytotoxic T cells (CTLs) bind to peptides presented by MHC I (pMHC) through T cell receptors of various affinities. Low-affinity CTLs are important for the control of intracellular pathogens and cancers; however, the mechanisms by which these lower affinity CTLs are activated and maintained are not well understood. We recently discovered that fully activated CTLs stimulated by strong-affinity peptides in the presence of IL-12 are able to secrete exosomes that, in turn, stimulate bystander CTLs without requiring the presence of antigen. We hypothesized that exosomes secreted by high-affinity CTLs could strengthen the activation of low-affinity CTLs. Naive OT-I CD8+ cells were stimulated with altered N4 peptides of different affinities in the presence or absence of Exo. The presence of Exo preferentially increased cell proliferation and enhanced the production of IFNgamma in CTLs stimulated by low-affinity peptides. The expression of granzyme B (GZB) was augmented in all affinities, with higher GZB production in low-affinity stimulated CTLs than in high-affinity stimulated ones. Exosomes promoted the rapid activation of low-affinity CTLs, which remained responsive to exosomes for a prolonged duration. Unexpectedly, exosomes could be induced quickly (24 h) following CTL activation and at a higher quantity per cell than later (72 h). While exosome protein profiles vary significantly between early exosomes and their later-derived counterparts, both appear to have similar downstream functions. These results reveal a potential mechanism for fully activated CTLs in activating lower-affinity CTLs that may have important implications in boosting the function of low-affinity CTLs in immunotherapy for cancers and chronic viral infections.

Author Info: (1) Department of Animal and Avian Sciences, University of Maryland, College Park, MD, United States. (2) Department of Animal and Avian Sciences, University of Maryland, College P

Author Info: (1) Department of Animal and Avian Sciences, University of Maryland, College Park, MD, United States. (2) Department of Animal and Avian Sciences, University of Maryland, College Park, MD, United States. (3) Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, United States. (4) Department of Animal and Avian Sciences, University of Maryland, College Park, MD, United States.

Induction of memory-like dendritic cell responses in vivo

Dendritic cells (DCs), a vital component of the innate immune system, are considered to lack antigen specificity and be devoid of immunological memory. Strategies that can induce memory-like responses from innate cells can be utilized to elicit protective immunity in immune deficient persons. Here we utilize an experimental immunization strategy to modulate DC inflammatory and memory-like responses against an opportunistic fungal pathogen that causes significant disease in immunocompromised individuals. Our results show that DCs isolated from protectively immunized mice exhibit enhanced transcriptional activation of interferon and immune signaling pathways. We also show long-term memory-like cytokine responses upon subsequent challenge with the fungal pathogen that are abrogated with inhibitors of specific histone modifications. Altogether, our study demonstrates that immunization strategies can be designed to elicit memory-like DC responses against infectious disease.

Author Info: (1) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at

Author Info: (1) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (2) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (3) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (4) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (5) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (6) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (7) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. (8) Department of Biology, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. floyd.wormley@utsa.edu. The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, 78249, USA. floyd.wormley@utsa.edu.

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