Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

Deoxycytidine kinase augments ATM-Mediated DNA repair and contributes to radiation resistance.

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [(18)F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.

Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene.

Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.

Role of autonomous androgen receptor signaling in prostate cancer initiation is dichotomous and depends on the oncogenic signal.

The steroid hormone signaling axis is thought to play a central role in initiation and progression of many hormonally regulated epithelial tumors. It is unclear whether all cancer-initiating signals depend on an intact hormone receptor signaling machinery. To ascertain whether cell autonomous androgen receptor (AR) is essential for initiation of prostate intraepithelial neoplasia (PIN), the response of AR-null prostate epithelia to paracrine and cell autonomous oncogenic signals was assessed in vivo by using the prostate regeneration model system. Epithelial-specific loss of AR blocked paracrine FGF10-induced PIN, whereas the add back of exogenous AR restored this response. In contrast, PIN initiated by cell-autonomous, chronic-activated AKT developed independent of epithelial AR signaling. Our findings demonstrate a selective role for AR in the initiation of PIN, dependent on the signaling pathways driving tumor formation. Insights into the role of hormone receptor signaling in the initiation of epithelial tumors may help define this axis as a target for chemoprevention of carcinomas.

PET probes for distinct metabolic pathways have different cell specificities during immune responses in mice.

Clinical tools that measure changes in immune cell metabolism would improve the diagnosis and treatment of immune dysfunction. PET, utilizing probes for specific metabolic processes, detects regions of immune activation in vivo. In this study we investigated the immune cell specificity of PET probes for two different metabolic pathways: [18F]-2-fluorodeoxyglucose ([18F]-FDG) for glycolysis and [18F]-2-fluoro-D-(arabinofuranosyl)cytosine ([18F]-FAC) for deoxycytidine salvage. We isolated innate and adaptive immune cells from tissues of mice challenged with a retrovirus-induced sarcoma and measured their ability to accumulate FDG and FAC. We determined that the two probes had distinct patterns of accumulation: FDG accumulated to the highest levels in innate immune cells, while FAC accumulated predominantly in CD8+ T cells in a manner that correlated with cellular proliferation. This study demonstrates that innate and adaptive cell types differ in glycolytic and deoxycytidine salvage demands during an immune response and that these differential metabolic requirements can be detected with specific PET probes. Our findings have implications for the interpretation of clinical PET scans that use [18F]-FDG or [18F]-FAC to assess immune function in vivo and suggest potential applications of metabolic PET to monitor the effects of targeted immune modulation.

Epithelial uptake of [18F]1-(2'-deoxy-2'-arabinofuranosyl) cytosine indicates intestinal inflammation in mice.

BACKGROUND & AIMS: Uptake of [18F]1-(2'-deoxy-2'-arabinofuranosyl)cytosine (D-FAC) is a trait of activated lymphocytes; its biodistribution predominates in the spleen, thymus, and bone marrow. In addition, D-FAC is taken up at high levels by the intestine. We analyzed the regional specificity of uptake and cell types that mediate it. METHODS: In mice, 3-dimensional isocontour regions of interest were drawn based on computed tomographic images to quantify intestinal signals from micro-positron emission tomography scans. To ascertain the cell type responsible, intestinal epithelium and immune cells were isolated and D-FAC uptake was analyzed in vitro. Mice deficient in mucosal homing (beta7 integrin-/-), enteric microbiota (germ-free), or active for immune colitis (G alpha i2-/- CD3+ transferred into Rag-/- recipients) were studied. RESULTS: Strong uptake of D-FAC was detected throughout the intestine, with greatest signal per region of interest in the duodenum. Fractionation of intestinal cell types after in vivo uptake revealed that the signal was almost entirely from epithelial cells. Among resident immune cell types, CD4+ T cells showed the greatest per-cell and total uptake. D-FAC uptake increased in both intestinal and systemic lymphoid sites during colitis. Compared with fluorodeoxyglucose, increased uptake of D-FAC in the small and large intestine occurred at an earlier stage of disease development. CONCLUSIONS: Uptake of D-FAC is a prominent trait of normal mouse intestinal epithelial cells, which is useful for their noninvasive visualization by positron emission tomography. Increased uptake of D-FAC reflects the activity of the epithelium and lymphocytes, providing a unique early marker of intestinal inflammation.

Normal immune development and glucocorticoid-induced thymocyte apoptosis in mice deficient for the T-cell death-associated gene 8 receptor.

T-cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor transcriptionally upregulated by glucocorticoids (GCs) and implicated by overexpression studies in psychosine-mediated inhibition of cytokinesis and in GC-induced apoptosis. To examine the physiological function of TDAG8, we generated knockout (KO) mice by homologous recombination. An enhanced green fluorescent protein reporter was knocked into the disrupted tdag8 locus to allow the analysis of TDAG8 expression in living cells. Interestingly, we found that during thymocyte development, TDAG8 expression resembled the dynamic regulation described for known modulators of GC-induced apoptosis, including Bcl-2, Notch1, and GC receptor. TDAG8 was expressed in double-negative cells, was downregulated at the double-positive transition, and was upregulated in single-positive thymocytes. However, despite this striking expression pattern, maturation and selection of thymocytes, as well as major immune functions, were not affected in TDAG8 KO mice. In contrast to previous overexpression results, TDAG8 was dispensable for psychosine-induced formation of multinucleated cells. Furthermore, TDAG8 KO thymocytes showed normal apoptosis following in vivo and in vitro GC treatment. These results, while establishing a useful reporter strain to study T-lymphocyte maturation, argue against a critical role for TDAG8 in immune development, psychosine-mediated inhibition of cytokinesis, and GC-induced cell death.

Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A.

Intracellular trafficking and spatial dynamics of membrane receptors critically regulate receptor function. Using microscopic and subcellular fractionation analysis, we studied the localization of the murine G protein-coupled receptor G2A (muG2A). Evaluating green fluorescent protein-tagged, exogenously expressed as well as the endogenous muG2A, we observed that this receptor was spontaneously internalized and accumulated in endosomal compartments, whereas its surface expression was enhanced and stabilized by lysophosphatidylcholine (LPC) treatment. Monensin, a general inhibitor of recycling pathways, blocked LPC-regulated surface localization of muG2A as well as muG2A-dependent extracellular signal-regulated kinase (ERK) activation and cell migration induced by LPC treatment. Mutation of the conserved DRY motif (R-->A) enhanced the surface expression of muG2A, resulting in its resistance to monensin inhibition of ERK activation. Our data suggest that intracellular sequestration and surface expression regulated by LPC, rather than direct agonistic activity control the signaling responses of murine G2A toward LPC.

Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.

G2A is a G-protein-coupled receptor (GPCR) involved in immune regulation. Previous studies have shown that lysophosphatidylcholine (LPC), a bioactive lipid associated with atherosclerosis and autoimmunity, acts through G2A to induce diverse biologic effects. Production of LPC during cell apoptosis serves as a chemotactic signal for macrophage recruitment. Here we demonstrate that macrophage chemotaxis to LPC is dependent on G2A function. Wild-type but not G2A-deficient mouse peritoneal macrophages migrated toward LPC. RNAi-mediated knockdown of G2A in J774A.1 macrophages abolished LPC-induced chemotaxis, whereas overexpression of G2A significantly enhanced this process. Mutation of the conserved DRY motif of G2A resulted in loss of chemotaxis to LPC, suggesting a requirement for G-protein signaling. Unlike most GPCRs, including the chemokine receptors, coupling to G(i) is not required for LPC/G2A-mediated chemotaxis, but coupling to G(q/11) and G(12/13) is necessary as judged by inhibition with dominant negative forms of these alpha subunits or with regulators of G-protein signaling (RGS) constructs. Collectively, these data establish that pertussis toxin-insensitive G2A signaling regulates macrophage chemotaxis to LPC. Defects in this signaling pathway may be related to the pathogenesis of systemic autoimmune disease.

A phosphorylation site in Bruton's tyrosine kinase selectively regulates B cell calcium signaling efficiency by altering phospholipase C-gamma activation.

Loss of function of Bruton's tyrosine kinase (Btk) causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptide-specific antibodies, we identified a tyrosine phosphorylation site (Y617) near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppressed Btk-mediated phospholipase Cgamma2 activation and calcium response in DT-40 cells, whereas Akt activation was not affected. The Btk Y617E mutant could partially restore conventional B cell development and proliferation in Btk(-)/Tec(-) mice but failed to rescue CD5(+) B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway.

T cell chemotaxis to lysophosphatidylcholine through the G2A receptor.

G2A is an immunoregulatory G protein-coupled receptor predominantly expressed in lymphocytes and macrophages. Ectopic overexpression studies have implicated G2A as a receptor for the bioactive lysophospholipid, lysophosphatidylcholine (LPC). However, the functional consequences of LPC-G2A interaction at physiological levels of receptor expression, and in a cellular context relevant to its immunological role, remain largely unknown. Here, we show impaired chemotaxis to LPC of a T lymphoid cell line in which G2A expression was chronically down-regulated by RNA interference technology. Rescuing this phenotype by reconstitution of the physiological level of receptor expression further supports a functional connection between LPC-G2A interaction and cellular motility. Overexpression of G2A in the T lymphoid cell line significantly enhanced chemotaxis to LPC. It also modified migration toward the LPC-related molecule, lysophosphatidic acid, indicating the possibility of crosstalk between G2A and endogenous lysophosphatidic acid receptors. The role of G2A in LPC-mediated cell migration may be relevant to the autoimmune syndrome associated with genetic inactivation of this G protein-coupled receptor in mice. The experimental system described here can be useful for understanding the structural requirements for LPC recognition by G2A and the signaling pathways regulated by this ligand-receptor pair.