High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Inhaled TLR9 Agonist Renders Lung Tumors Permissive to PD-1 Blockade by Promoting Optimal CD4+ and CD8+ T-cell Interplay.

Currently approved inhibitors of the PD-1/PD-L1 pathway represent a major advance for the treatment of lung cancers, yet they are ineffective in a majority of patients due to lack of preexisting T-cell reactivity. Here, we show that a TLR9 agonist delivered by inhalation is able to prime T-cell responses against poorly immunogenic lung tumors and to complement the effects of PD-1 blockade. Inhaled TLR9 agonist causes profound remodeling in tumor-bearing lungs, leading to the formation of tertiary lymphoid structures adjacent to the tumors, CD8+ T-cell infiltration into the tumors, dendritic cell expansion, and antibody production. Inhalation of TLR9 agonist also increased the pool of functional PD-1lowT-bethigh effector CD8+ T cells in tumor-bearing lungs. Effector CD8+ T cells generated by inhaled TLR9 agonist treatment were licensed by PD-1 blockade to become highly functional CTLs, leading to a durable rejection of both lung tumors and tumor lesions outside the lungs. CD4+ T cells activated in response to inhaled TLR9 play a critical role in this process by controlling the proliferation, preventing exhaustion, and guiding the differentiation of optimally functional CTLs. This study characterizes a strategy to apply localized TLR9 stimulation to a tumor type not accessible for direct injection, a strategy that may expand the therapeutic potential of PD-1 blockade in non-small cell lung cancer.Significance: These findings demonstrate that local delivery of a toll-like receptor 9 agonist can change the immune content of an entire organ and enhance the efficacy of immune checkpoint inhibition.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4943/F1.large.jpg Cancer Res; 78(17); 4943-56. ©2018 AACR.

Sphingosine-1-Phosphate Signaling and Metabolism Gene Signature in Pediatric Inflammatory Bowel Disease: A Matched-case Control Pilot Study.

Goal: The aim of this study was to investigate gene expression levels of proteins involved in sphingosine-1-phosphate (S1P) metabolism and signaling in a pediatric inflammatory bowel disease (IBD) patient population. Background: IBD is a debilitating disease affecting 0.4% of the US population. The incidence of IBD in childhood is rising. Identifying effective targeted therapies that can be used safely in young patients and developing tools for selecting specific candidates for targeted therapies are important goals. Clinical IBD trials now underway target S1PR1, a receptor for the pro-inflammatory sphingolipid S1P. However, circulating and tissue sphingolipid levels and S1P-related gene expression have not been characterized in pediatric IBD. Methods: Pediatric IBD patients and controls were recruited in a four-site study. Patients received a clinical score using PUCAI or PCDAI evaluation. Colon biopsies were collected during endoscopy. Gene expression was measured by qRT-PCR. Plasma and gut tissue sphingolipids were measured by LC-MS/MS. Results: Genes of S1P synthesis (SPHK1, SPHK2), degradation (SGPL1), and signaling (S1PR1, S1PR2, and S1PR4) were significantly upregulated in colon biopsies of IBD patients with moderate/severe symptoms compared with controls or patients in remission. Tissue ceramide, dihydroceramide, and ceramide-1-phosphate (C1P) levels were significantly elevated in IBD patients compared with controls. Conclusions: A signature of elevated S1P-related gene expression in colon tissues of pediatric IBD patients correlates with active disease and normalizes in remission. Biopsied gut tissue from symptomatic IBD patients contains high levels of pro-apoptotic and pro-inflammatory sphingolipids. A combined analysis of gut tissue sphingolipid profiles with this S1P-related gene signature may be useful for monitoring response to conventional therapy.

S1P Lyase Regulation of Thymic Egress and Oncogenic Inflammatory Signaling.

Sphingosine-1-phosphate (S1P) is a potent lipid signaling molecule that regulates pleiotropic biological functions including cell migration, survival, angiogenesis, immune cell trafficking, inflammation, and carcinogenesis. It acts as a ligand for a family of cell surface receptors. S1P concentrations are high in blood and lymph but low in tissues, especially the thymus and lymphoid organs. S1P chemotactic gradients are essential for lymphocyte egress and other aspects of physiological cell trafficking. S1P is irreversibly degraded by S1P lyase (SPL). SPL regulates lymphocyte trafficking, inflammation and other physiological and pathological processes. For example, SPL located in thymic dendritic cells acts as a metabolic gatekeeper that controls the normal egress of mature T lymphocytes from the thymus into the circulation, whereas SPL deficiency in gut epithelial cells promotes colitis and colitis-associated carcinogenesis (CAC). Recently, we identified a complex syndrome comprised of nephrosis, adrenal insufficiency, and immunological defects caused by inherited mutations in human SGPL1, the gene encoding SPL. In the present article, we review current evidence supporting the role of SPL in thymic egress, inflammation, and cancer. Lastly, we summarize recent progress in understanding other SPL functions, its role in inherited disease, and SPL targeting for therapeutic purposes.

Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs.

Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL.

S1pping fire: Sphingosine-1-phosphate signaling as an emerging target in inflammatory bowel disease and colitis-associated cancer.

Inflammatory bowel disease (IBD) is a complex disease that involves unpredictable and destructive inflammation in the gastrointestinal tract resulting in gastrointestinal symptoms, infection, and tissue destruction, and which can be associated with an increased risk of colon cancer. The underlying cause of IBD involves disruption of the innate and adaptive immune mechanisms that maintain homeostasis between the gut mucosa and its environment. Elucidating how the homeostatic mechanisms controlling gut mucosal immunity and inflammation are disrupted in IBD represents the first steps to identifying novel therapeutic targets. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is enriched in the blood and lymph, and functions in innate and adaptive immunity. S1P signaling regulates inflammation via its impact on the trafficking, differentiation, and effector functions of bone marrow-derived immune cells. S1P also activates nuclear factor kappa B and signal transducer and activator of transcription 3 inflammatory pathways. S1P is generated by the ubiquitously expressed lipid kinase, sphingosine kinase (SphK)1 and its tissue-restricted homolog, SphK2. S1P is irreversibly degraded by S1P lyase, which is highly expressed in enterocytes. Recent studies targeting S1P metabolism and signaling have shown promise in preclinical models of IBD and have shed light on the mechanisms by which S1P signaling impacts IBD. The evidence suggests that targeting S1P signaling and metabolism may represent a novel strategy in treating IBD and it may reduce colon cancer risk by interrupting the progression from inflammation to carcinogenesis.

P2Y2 receptor promotes intestinal microtubule stabilization and mucosal re-epithelization in experimental colitis.

P2Y(2) receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y(2) stimulates PGE(2) release by IECs, suggesting a role in wound healing. For this study, we have used the non-cancerous IEC-6 cell line. IEC-6 cell migration was determined using Boyden chambers and the single-edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2-thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti-vinculin and anti-acetylated-α-tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y(2) agonist 2-thioUTP on remission. We showed that P2Y(2) increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin α(v) . Following P2Y(2) activation, we demonstrated that GSK3ß activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y(2) activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs.

P2Y(2) receptor expression is regulated by C/EBPß during inflammation in intestinal epithelial cells.

Inflammatory bowel diseases are characterized by relapses and remission periods during which numerous factors, including stress factors and nucleotides, are mobilized to re-establish intestinal mucosal homeostasis. We have previously found that expression of the P2Y(2) nucleotide receptor is increased in colonic tissue isolated from inflammatory bowel disease patients as well as in a mouse model of colitis, and that P2Y(2) transcription is regulated in part by nuclear factor κB (NF-κB) p65. Transcription factor DNA-binding site analysis identified three potential CCAAT/enhancer-binding protein ß (C/EBPß) binding sites in the P2Y(2) proximal promoter. We then assessed the role of C/EBP transcription factors in the regulation of P2Y(2) in intestinal epithelial cells (IECs). We identified a region between -229 and -220 bp upstream of the transcription initiation site as a DNA-binding site for C/EBPß, by electrophoretic mobility and supershift assays. Mutagenesis of this site decreased C/EBPß-dependent P2Y(2) expression, as assessed by luciferase assays. In vivo, C/EBPß as well as P2Y(2) expression was increased in colonic IECs isolated from mice with dextran sulfate sodium-induced acute colitis. In contrast, P2Y(2) expression was decreased in C/EBPß-deficient mice treated with dextran sulfate sodium. Although C/EBPß was sufficient to induce P2Y(2) transcription, the effect of C/EBPß and NF-κB p65 on receptor transcription was synergistic. Chromatin immunoprecipitation assays revealed that both proteins simultaneously bind to the P2Y(2) promoter. Thus, we have identified C/EBPß as a novel regulator of P2Y(2) expression.

P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP-1-dependent manner in epithelial cells.

BACKGROUND: Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y(6) receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5'-diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis-like disease and we characterized the signaling events linking P2Y(6) to CXCL8 expression in IEC. METHODS: Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real-time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays. RESULTS: Mutation of the AP-1 site in the CXCL8 core promoter abolished the UDP-stimulating effect. The c-fos/c-jun dimer was identified as the AP-1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y(6) required PKCδ activation upstream of the signaling pathway composed of MEK1/2-ERK1/2 and c-fos. UDP administration to mice suffering from colitis-like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation. CONCLUSIONS: This study not only describes the P2Y(6) signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y(6) to reduce intestinal inflammation.

P2Y2 receptor transcription is increased by NF-kappa B and stimulates cyclooxygenase-2 expression and PGE2 released by intestinal epithelial cells.

Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y(2) mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn's disease and ulcerative colitis. However, the transcriptional events regulating P2Y(2) receptor (P2Y(2)R) expression are not known. We have identified a putative transcription start site in the P2Y(2)R gene and demonstrated acetylation of Lys(14) on histone H3 and Lys(8) on histone H4, thus suggesting that the chromatin associated with the P2Y(2) promoter is accessible to transcription factors. We also showed that the transcription factor NF-kappaB p65 regulates P2Y(2)R transcription under both proinflammatory and basal conditions. A NF-kappaB-responsive element was identified at -181 to -172 bp in the promoter region of P2Y(2). Hence, activation of P2Y(2)R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE(2) secretion by intestinal epithelial cells. These findings demonstrate that P2Y(2)R expression is regulated during intestinal inflammation through an NF-kappaB p65-dependent mechanism and could contribute not only to inflammatory bowel disease but also to other inflammatory diseases by regulating PG release.

Intestinal inflammation increases the expression of the P2Y6 receptor on epithelial cells and the release of CXC chemokine ligand 8 by UDP.

Epithelial cells participate in the immune response of the intestinal mucosa. Extracellular nucleotides have been recognized as inflammatory molecules. We investigated the role of extracellular nucleotides and their associated P2Y receptors in the secretion of cytokines by epithelial cells. The effect of intestinal inflammation on P2Y(6) receptor expression was determined by PCR in the mouse, rat, and human. Localization of the P2Y(6) receptor was determined by immunofluorescence microscopy in the colon of normal and dextran sulfate sodium-treated mice. The effect of P2Y(6) activation by UDP on cytokine expression and release by epithelial cells was determined using a combination of Western blots, luciferase assays, RT-PCR, cytokine Ab arrays, and ELISA. Inflammation up-regulates P2Y(2) as well as P2Y(6) receptor expression in the mucosa of the colon of colitic mice. In vitro, we demonstrated that UDP could be released by Caco-2/15 cells. We have confirmed the increased expression of P2Y(6) by challenging intestinal epithelial cell-6 and Caco-2/15 cells with TNF-alpha and IFN-gamma and showing that stimulation of epithelial cells by UDP results in an increased expression and release of CXCL8 by an ERK1/2-dependent mechanism. The increase in CXCL8 expression was associated with a transcriptional activation by the P2Y(6) receptor. This study is the first report demonstrating the implication of P2Y receptors in the inflammatory response of intestinal epithelial cells. We show for the first time that P2Y(6), as well as P2Y(2), expression is increased by the stress associated with intestinal inflammation. These results demonstrate the emergence of extracellular nucleotide signaling in the orchestration of intestinal inflammation.