Chimeric antigen receptor (CAR) T cells have demonstrated promising efficacy, particularly in hematologic malignancies. One challenge regarding CAR T cells in solid tumors is the immunosuppressive ...tumor microenvironment (TME), characterized by high levels of multiple inhibitory factors, including transforming growth factor (TGF)-β. We report results from an in-human phase 1 trial of castration-resistant, prostate cancer-directed CAR T cells armored with a dominant-negative TGF-β receptor (NCT03089203). Primary endpoints were safety and feasibility, while secondary objectives included assessment of CAR T cell distribution, bioactivity and disease response. All prespecified endpoints were met. Eighteen patients enrolled, and 13 subjects received therapy across four dose levels. Five of the 13 patients developed grade ≥2 cytokine release syndrome (CRS), including one patient who experienced a marked clonal CAR T cell expansion, >98% reduction in prostate-specific antigen (PSA) and death following grade 4 CRS with concurrent sepsis. Acute increases in inflammatory cytokines correlated with manageable high-grade CRS events. Three additional patients achieved a PSA reduction of ≥30%, with CAR T cell failure accompanied by upregulation of multiple TME-localized inhibitory molecules following adoptive cell transfer. CAR T cell kinetics revealed expansion in blood and tumor trafficking. Thus, clinical application of TGF-β-resistant CAR T cells is feasible and generally safe. Future studies should use superior multipronged approaches against the TME to improve outcomes.
T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8
T cell ...differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8
T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8
T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8
T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8
T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8
T cell fate decisions.
Genetic aberrations responsible for soft-tissue sarcoma formation in adults are largely unknown, with targeted therapies sorely needed for this complex and heterogeneous family of diseases. Here we ...report that that the Hippo pathway is deregulated in many soft-tissue sarcomas, resulting in elevated expression of the effector molecule Yes-Associated Protein (YAP). Based on data gathered from human sarcoma patients, a novel autochthonous mouse model, and mechanistic analyses, we determined that YAP-dependent expression of the transcription factor forkhead box M1 (FOXM1) is necessary for cell proliferation/tumorigenesis in a subset of soft-tissue sarcomas. Notably, FOXM1 directly interacts with the YAP transcriptional complex via TEAD1, resulting in coregulation of numerous critical pro-proliferation targets that enhance sarcoma progression. Finally, pharmacologic inhibition of FOXM1 decreases tumor size in vivo, making FOXM1 an attractive therapeutic target for the treatment of some sarcoma subtypes.
Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies ...have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors.
•ARNT promotes adult hematopoietic stem cell viability through regulation of BCL-2 and VEGF-A expression.•Fetal liver hematopoietic progenitors experience hypoxia and loss of hypoxia-induced transcription decreases their survival.
Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the ...serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.
Hypoxia inducible factors (HIFs) regulate adaptive responses to changes in oxygen (O
2) tension during embryogenesis, tissue ischemia, and tumorigenesis. Because HIF-deficient embryos exhibit a ...number of developmental defects, the precise role of HIF in early vascular morphogenesis has been uncertain. Using para-aortic splanchnopleural (P-Sp) explant cultures, we show that deletion of the HIF-β subunit (ARNT) results in defective hematopoiesis and the inhibition of both vasculogenesis and angiogenesis. These defects are rescued upon the addition of wild-type Sca-1
+ hematopoietic cells or recombinant VEGF.
Arnt
−/−
embryos exhibit reduced levels of VEGF protein and increased numbers of apoptotic hematopoietic cells. These results suggest that HIF coordinates early endothelial cell emergence and vessel development by promoting hematopoietic cell survival and paracrine growth factor production.
Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies
. In this strategy, the T cell genome is modified by integration of viral vectors ...or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells
. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.
DNA methylation is a key epigenetic mechanism that controls T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to ...5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines. TET2 mutations have been identified in angioimmunoblastic T cell lymphomas (AITL), which are derived from CD4+ T follicular helper (TFH) cells. CD4+ TFH cells are a T cell subset that provides essential help for the germinal center reaction and class switching. Here we demonstrate that TET2 regulates murine CD4+ TFH differentiation in vivo . At steady-state, mice with a T cell specific deletion of TET2 (TET2cKO) have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss promotes CD4+ TFH cell expansion. Examination of the Peyer's patches (a site of TFH differentiation in response to gut microbes) of wild-type and TET2cKO mice demonstrate an increase in the absolute number of the CD4+ TFH (CXCR5+PD1+) population in TET2cKO mice. Furthermore, there is an increase in the frequency and absolute number of germinal center B cells (characterized by PNA+GL7+ B cells) and an increase in IgA+ B cells in the Peyer's patches of TET2cKO mice compared to wild-type mice. Ongoing studies are examining the mechanism by which TET2 controls CD4+ TFH expansion. Our data indicate that TET2 is an important regulator of CD4+ TFH cells. Understanding the role of TET2 in normal CD4+ TFH differentiation and its contribution to T cell lymphomagenesis will lead to important insights into the epigenetic and molecular mechanisms underlying AITL pathogenesis
No relevant conflicts of interest to declare.
Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has achieved tremendous success treating B cell malignancies; however, some patients fail to respond due to poor autologous T cell ...fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR T cell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR T cells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the T cell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of T cells from patients with leukemia that previously failed CAR T cell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient T cells, providing a strategy for increasing patient responses to CAR T cell therapy.
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•CTLA4 deletion enhances CART efficacy and surface CAR expression under stress•Deletion of CTLA4 permits unopposed CD28 signaling in CAR T cells•CTLA4 deletion rescues the function of nonresponding CLL patient CAR T cells•PD-1 deficiency nullifies CD28 signaling enabled by CTLA4 deletion in CAR T cells
CD19-directed CAR T cell therapy is an effective treatment for B cell malignancies, but some patients fail to respond. Agarwal et al. demonstrate that deletion of CTLA4 enhances anti-tumor efficacy and surface CAR expression in models of leukemia and lymphoma and in CLL patient CAR T cells. Interestingly, deletion of PDCD1 or of PDCD1 and CTLA4 did not promote anti-tumor efficacy of CART19 cells.
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