Instructor, Stanford Institutes of Medicine
Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling.
Science (New York, N.Y.)
2021; 372 (6537)
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.
View details for DOI 10.1126/science.aba1786
View details for PubMedID 33795428
The Emerging Landscape of Immune Cell Therapies.
2020; 181 (1): 46–62
Cell therapies present an entirely new paradigm in drug development. Within this class, immune cell therapies are among the most advanced, having already demonstrated definitive evidence of clinical benefits in cancer and infectious disease. Numerous features distinguish these "living therapies" from traditional medicines, including their ability to expand and contract in proportion to need and to mediate therapeutic benefits for months or years following a single application. Continued advances in fundamental immunology, genetic engineering, gene editing, and synthetic biology exponentially expand opportunities to enhance the sophistication of immune cell therapies, increasing potency and safety and broadening their potential for treatment of disease. This perspective will summarize the current status of immune cell therapies for cancer, infectious disease, and autoimmunity, and discuss advances in cellular engineering to overcome barriers to progress.
View details for DOI 10.1016/j.cell.2020.03.001
View details for PubMedID 32243795
Pharmacologic control of CAR-T cell function using dasatinib.
2019; 3 (5): 711–17
View details for PubMedID 30814055
c-Jun overexpression in CAR T cells induces exhaustion resistance.
Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.
View details for DOI 10.1038/s41586-019-1805-z
View details for PubMedID 31802004
Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration
JOURNAL OF EXPERIMENTAL MEDICINE
2021; 218 (1)
Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.
View details for DOI 10.1084/jem.20192378
View details for Web of Science ID 000608148300004
View details for PubMedID 32970800
Tuning the Antigen Density Requirement for CAR T Cell Activity.
Insufficient reactivity against cells with low antigen density has emerged as an important cause of CAR resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and the CAR construct in axicabtagene-ciloleucel (CD19-CD28z) outperforms that in tisagenlecleucel (CD19-4-1BBz) against antigen low tumors. Enhancing signal strength by including additional ITAMs in the CAR enables recognition of low antigen density cells, while ITAM deletions blunt signal and increase the antigen density threshold. Further, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBz CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunological synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBz-CARs with enhanced capacity to recognize antigen low targets while retaining a superior capacity for persistence.
View details for DOI 10.1158/2159-8290.CD-19-0945
View details for PubMedID 32193224
Neurotoxicity Associated with a High-Affinity GD2 CAR-Letter.
Cancer immunology research
2018; 6 (4): 494–95
View details for PubMedID 29610423
Roles of transient receptor potential channels in regulation of vascular and epithelial barriers.
Transient receptor potential (TRP) channels are a ubiquitously expressed multi-family group of cation channels that are critical to signaling events in many tissues. Their roles have been documented in many physiologic and pathologic conditions. Nevertheless, direct studies of their roles in maintain barrier function in endothelial and epithelia are relatively infrequent. This seems somewhat surprising considering that calcium ion concentrations are known to regulate barrier function. This short review provides an introduction to TRP channels and reviews some of the work in which investigators directly studied the role of TRP channels in endothelial permeability to electric current, solute, or leukocytes during the inflammatory response.
View details for DOI 10.1080/21688370.2017.1331722
View details for PubMedID 28581893
Cutting Edge: CD99 Is a Novel Therapeutic Target for Control of T Cell-Mediated Central Nervous System Autoimmune Disease
JOURNAL OF IMMUNOLOGY
2016; 196 (4): 1443-1448
Leukocyte trafficking into the CNS is a prominent feature driving the immunopathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Blocking the recruitment of inflammatory leukocytes into the CNS represents an exploitable therapeutic target; however, the adhesion molecules that specifically regulate the step of leukocyte diapedesis into the CNS remain poorly understood. We report that CD99 is critical for lymphocyte transmigration without affecting adhesion in a human blood-brain barrier model. CD99 blockade in vivo ameliorated experimental autoimmune encephalomyelitis and decreased the accumulation of CNS inflammatory infiltrates, including dendritic cells, B cells, and CD4(+) and CD8(+) T cells. Anti-CD99 therapy was effective when administered after the onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest that it may serve as a novel therapeutic target for controlling neuroinflammation.
View details for DOI 10.4049/jimmunol.1501634
View details for Web of Science ID 000369632300002
View details for PubMedID 26773145
View details for PubMedCentralID PMC4744533
CD99-like 2 (CD99L2)-deficient mice are defective in the acute inflammatory response
EXPERIMENTAL AND MOLECULAR PATHOLOGY
2015; 99 (3): 455-459
CD99-Like 2 (CD99L2) is a Type I glycoprotein expressed on leukocytes and endothelial cells as well as other cell types. It is related to CD99, although it shows only 38% sequence identity. CD99L2 has been shown to play a role in leukocyte extravasation in mice under various inflammatory conditions using anti-CD99L2 antibodies and, in one case by targeted deletion of CD99L2. We report here studies on an independently made CD99L2 "knockout mouse" that extend our knowledge of the role of CD99L2 in inflammation. CD99L2 deficiency did not affect the total or relative numbers of circulating leukocyte subsets, red blood cells, or platelets. Neither did CD99L2 deficiency affect the expression of ICAM-1, PECAM, or CD99 on endothelial cells. Mice lacking CD99L2 had a defective inflammatory response in the thioglycollate peritonitis model with a greater than 80% block in neutrophil infiltration and a nearly complete block in monocyte emigration into the peritoneal cavity measured 16h after the inflammatory challenge. The mice will be a useful resource to study the role of CD99L2 in various acute and chronic inflammatory diseases.
View details for DOI 10.1016/j.yexmp.2015.08.011
View details for Web of Science ID 000366954500010
View details for PubMedID 26321243
View details for PubMedCentralID PMC4679605
TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response
JOURNAL OF EXPERIMENTAL MEDICINE
2015; 212 (11): 1883-1899
Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca(2+)]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca(2+)]i regulates TEM and the channels mediating this ↑[Ca(2+)]i are unknown. Buffering ↑[Ca(2+)]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca(2+)]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca(2+) channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca(2+)]i required for TEM at a step downstream of PECAM homophilic interactions.
View details for DOI 10.1084/jem.20150353
View details for Web of Science ID 000366926800016
View details for PubMedID 26392222
View details for PubMedCentralID PMC4612081
Requirement of myeloid cell-specific Fas expression for prevention of systemic autoimmunity in mice
ARTHRITIS AND RHEUMATISM
2012; 64 (3): 808-820
The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway, and its role in mediating lymphoproliferation has been extensively examined. The present study was undertaken to investigate the impact of myeloid cell-specific loss of Fas.Mice with Fas flanked by loxP sites (Fas(flox/flox) ) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (Cre(LysM) ), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for Cre(LysM) Fas(flox/flox) mice was verified by polymerase chain reaction and flow cytometric analysis. Flow cytometric analysis was also used to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood, and spleen. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure serum cytokine/chemokine and immunoglobulin levels. Renal damage or dysfunction was examined by immunohistochemical and immunofluorescence analysis.Cre(LysM) Fas(flox/flox) mice exhibited a systemic lupus erythematosus (SLE)-like disease that included leukocytosis, splenomegaly, hypergammaglobulinemia, antinuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1(low) and Gr-1(intermediate) blood monocytes and splenic macrophages and, in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in Cre(LysM) Fas(flox/flox) mice.Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice, reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor of systemic autoimmunity.
View details for DOI 10.1002/art.34317
View details for Web of Science ID 000300835900025
View details for PubMedID 22143975
View details for PubMedCentralID PMC3290732
Cyclin-dependent kinase inhibitor p21, via its C-terminal domain, is essential for resolution of murine inflammatory arthritis
ARTHRITIS AND RHEUMATISM
2012; 64 (1): 141-152
The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis.Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay.Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages.These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.
View details for DOI 10.1002/art.33311
View details for Web of Science ID 000298598100018
View details for PubMedID 21898359
View details for PubMedCentralID PMC3253189