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Administration of cannabinoid receptor 2 (CB2R) agonists in inflammatory and autoimmune disease and CNS injury models results in significant attenuation of clinical disease, and reduction of inflammatory mediators. Previous studies reported that CB2R signaling also reduces leukocyte migration. Migration of dendritic cells (DCs) to various sites is required for their activation and for the initiation of adaptive immune responses. Here, we report for the first time that CB2R signaling affects DC migration in vitro and in vivo, primarily through the inhibition of matrix metalloproteinase 9 (MMP-9) expression. Reduced MMP-9 production by DCs results in decreased migration to draining lymph nodes in vivo and in vitro in the matrigel migration assay. The effect on Mmp-9 expression is mediated through CB2R, resulting in reduction in cAMP levels, subsequent decrease in ERK activation, and reduced binding of c-Fos and c-Jun to Mmp-9 promoter activator protein 1 sites. We postulate that, by dampening production of MMP-9 and subsequent MMP-9–dependent DC migration, cannabinoids contribute to resolve acute inflammation and to reestablish homeostasis. Selective CB2R agonists might be valuable future therapeutic agents for the treatment of chronic inflammatory conditions by targeting activated immune cells, including DCs.

We reported previously that prostaglandin E2 (PGE2) up-regulates IL-23 in vitro in bone marrow-derived dendritic cells and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced up-regulation of Il23a gene expression. In this study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular Toll-like receptor ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC). Using the EP4 agonist PGE(1)OH in conjunction with TNFα, we found that PKA-induced phosphorylation of cAMP-response element-binding protein ((P)CREB) and EPAC-induced phosphorylation of C/AATT enhancer-binding protein β ((P)C/EBPβ) mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPβ involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPβ-induced IL-23p19 expression.

Background: Adoptive T cell therapy with gene-engineered T cells is an emerging cancer treatment strategy that has been applied successfully to the treatment of hematological cancers. We conducted a clinical trial to test proof of principle for this type of treatment in an epithelial cancer. Patients with human papillomavirus (HPV) 16-associated cancers were treated with gene-engineered T cells targeting HPV16 E7. Methods: The clinical trial was a phase I study with a 3 + 3 design and three dose levels (DL) of gene-engineered T cells (DL1: 1 x 109, DL2: 1 x 1010, DL3: 1 x 1011). Patients had metastatic HPV-16+ cancers from any primary tumor site. Treatment consisted of a one-time infusion of autologous T cells that were gene-engineered to express an HLA-A*02:01-restricted T-cell receptor (TCR) that targets HPV-16 E7 (E7 T cells). A lymphocyte-depleting conditioning regimen was administered before treatment. E7 T cell infusion was followed by high-dose systemic aldesleukin. Results: Twelve patients were treated (DL1, n=3; DL2, n=3; DL3, n=6). The age range was 31 to 59 years. The site of the primary cancer was vulva (n=1), head and neck (n=4), uterine cervix (n=5), and anus (n=2). Each patient had multiple metastases and had previously received 3 to 7 anti-cancer agents. The conditioning regimen consisted of cyclophosphamide 30 mg/Kg (n=6) or 60 mg/Kg (n=6) iv daily for 2 days overlapping with fludarabine 25 mg/m2 iv daily for 5 days. The E7 TCR was expressed by 90-99% of the infused T cells for each patient. E7 T cell cross-reactivity against healthy tissues was not identified. Cytokine-release syndrome was not observed. A single patient, at DL3, experience dose-limiting toxicity. Four patients experienced confirmed responses, and two patients experienced unconfirmed responses (i.e. met criteria for response at only one assessment) (Figure 1). Confirmed responses occurred in patients with cervical cancer, oropharyngeal cancer, vulvar cancer, and anal cancer. The duration of responses was 3 months (ongoing), 4 months, 8 months, and 9 months, respectively. These patients had previously received 7, 4, 7 and 3 prior anti-cancer agents, respectively. Three patients with confirmed responses had previously received PD-1 or PD-L1 checkpoint blockade. Four patients whose cancer progressed after E7 T cells received PD-1 or PD-L1 checkpoint blockade; none responded. Conclusions: Tumor regression can occur following treatment of an epithelial cancer with gene-engineered T cells. These findings support continued study of E7 T cells and possibly other types of gene-engineered T cells in epithelial cancers. Disclosures Adhikary: Kite Pharma: Employment. Schweitzer:Kite Pharma: Employment. Astrow:Kite Pharma: Employment. Hinrichs:Kite Pharma: Research Funding; NIH: Patents & Royalties: NIH patents related to cell therapy.

3009 Background: Engineered T-cell therapy has shown promise in B-cell malignancies and melanoma, but clinical investigation in epithelial cancers has been limited. Methods: We conducted a phase I/II clinical trial of T cells genetically engineered to express a T-cell receptor that targets an HLA-A*02:01-restricted epitope of E6 (E6 TCR T Cells) for patients with metastatic HPV-16+ carcinoma. The cell dose was escalated in cohorts of single patients (1 x 109, 1 x 1010, and 1-2 x 1011cells). Patients received a nonmyeloablative conditioning regimen of cyclophosphamide and fludarabine, a single infusion of E6 TCR T Cells, and systemic high-dose aldesleukin. Results: Twelve patients were treated, 9 at the highest cell dose, plus one retreatment. The cancer types were 6 cervical, 4 anal, 1 oropharyngeal, and 1 vaginal. No dose-limiting toxicity, autoimmune adverse events, or cytokine storm were observed. Two patients with anal cancer treated at the highest cell dose experienced partial tumor responses lasting 6 and 3 months after treatment. The patient with a 6-month response had complete regression of one tumor and partial regression of two tumors that were resected upon progression; she has no evidence of disease 22 months after treatment. T-cell receptor gene transfer efficiency was 45 and 51% in the responding patients, and 47-76% (median 61%) in the non-responding patients. Responding patients showed robust levels of E6 TCR T cell memory (30 and 46% of circulating T cells 1-month after treatment). Non-responding patients showed wide-ranging levels of E6 TCR T cell memory (range 4-53%, median 29%). Expression of programmed cell death protein 1 (PD-1) by circulating E6 TCR T Cells 1-month after treatment was low in all patients ( < 5%). The patient with a 6-month response had 7% E6 TCR T Cells in a resected tumor 10 months after treatment, 25% of which expressed PD-1. A patient with no response had no detectable E6 TCR T Cells in a resected tumor 3 months after treatment. Conclusions: E6 TCR T-cell therapy was safe at doses up to 2 x 1011 cells. Regression of metastatic HPV+ carcinoma occurred in two patients following treatment, suggesting that TCR T-cell therapy can mediate epithelial cancer regression. Clinical trial information: NCT02280811.

The proinflammatory cytokine IL-23 emerged recently as a key player in autoimmune diseases. Production of IL-23 by pathogen-activated dendritic cells (DC) is regulated by endogenous mediators such as prostaglandin E2 (PGE2) and interferon-γ (IFN-γ). PGE2 stimulates IL-23 and inhibits IL-12 production. In contrast, IFN-γ inhibits IL-23 and stimulates IL-12. In the presence of both PGE2 and IFN-γ, TLR-stimulated DC produce higher levels of both cytokines. Here we analyze the signaling pathways involved in this complex regulation. IRF1, induced by LPS and at high levels by IFN-γ, acts as a positive transcription factor for IL-12p40, and as a negative regulator for IL-23p19. The potent effect of IFN-γ on IRF1 correlates with the inhibition of IL-23 and stimulation of IL-12 in LPS-activated DC. In contrast, PGE2 abolishes IRF1 expression in LPS-stimulated DC, resulting in the inhibition of IL-12 and stimulation of IL-23 production. When both PGE2 and IFN-γ are present, the IRF1 levels are similar to LPS controls, allowing for both IL-23p19 and IL-12p40 expression. In addition, as previously reported by us, the stimulatory effect of PGE2 on p19 transcription is also mediated through the activation of the positive transcription factors CREB and C/EBP. Our findings suggest that the combined presence of PGE2 and IFN-γ in autoimmune conditions results in sustained production of both IL-12 and IL-23, which contribute to the maintenance of a proinflammatory environment.

In murine models of multiple sclerosis (experimental autoimmune encephalomyelitis or EAE), cannabinoid receptor 2 (CB2) agonists have been shown to be beneficial, reducing the number of CNS infiltrating leukocytes. Adhesion molecules, chemokines, and matrix metalloproteinases (MMPs), particularly MMP-9, play important roles in migration to the CNS. Antigen presenting cells such as dendritic cells (DC) and macrophages, as well as activated microglia are major producers of MMP-9. In this study, we investigated the effects of CB2 receptor agonists on DC migration and MMP-9 production utilizing both in vitro and in vivo migration assays. DC activated with cytokine cocktail (TNFα+IL-1β+IL-6+PGE2) in the presence or absence of CB2R agonist exhibited similar migratory capacity in transwell migration assays. However, the CB2R agonist treated cells migrated less in matrigel migration assays as compared to untreated cells. We found that this effect was due to reduced MMP-9 production in CB2R agonist treated cells. Also, in the in vivo migration assay using cytokine cocktail-activated DCs injected into mice footpads, we found significantly fewer CB2R agonist-treated DCs in the draining lymph nodes. Our data indicate that one of the mechanisms by which CB2R agonists attenuate inflammation is the inhibition of MMP-9 release which results in decreased migration of activated dendritic cells.

Previous studies showed that cannabinoids have anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), but the mechanism is elusive. In the present study, we investigated the effects of Gp1a (a new CB2 selective agonist) in EAE. EAE developed in a similar way in both vehicle and Gp1a treatment group, but mice treated with Gp1a recovered faster than the vehicle group. Cellular analysis was performed in spleen, brain and spinal cord. In spleen, there was no difference in numbers of CD3+ T cells, Th1 and Th17 cells, or macrophages. In contrast, CNS mononuclear cell analysis showed fewer CD3+ T cells, Th1 and Th17 cells, macrophages and activated microglia in spinal cord but not in brainin the Gp1a treatment group. Accordingly, for the Gp1a-treated group, histology studies also showed fewer CD3+ T cells, CD11b+ macrophages/microglia and less Iba-1 expression in spinal cord but not in brain. In vivo cell proliferation as measured through BrdU incorporation in CD3+CD45hi, CD11b+CD45hi and CD11b+CD45int mononuclear cells was not affected by Gp1a treatment. We hypothesize that Gp1a inhibits cell migration to the CNS. We also analyzed gene expression in brain and spinal cord following EAE development and found lower levels of VCAM-1, CXCL-10 and MMP9 in spinal cord. We also found that Gp1a inhibits MMP9 and TNFα production in BV.2 cells (a microglia cell line). We conclude that Gp1a improves EAE recovery through inhibiting immune cell migration into CNS.

Protective anti-inflammatory effects of DHA were reported in clinical studies and animal models of colitis, sepsis, and stroke. In the present study, we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) in CD4+ T cell stimulation and differentiation. Pretreatment of DC with DHA prevented LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of costimulatory molecules and lack of proinflammatory cytokine production (IL-12p70, IL-6 and IL-23). DHA-treated DC were poor stimulators of antigen-specific T cells in terms of proliferation and Th1/Th17 differentiation. This was associated with an increase in p27(kip1), a cell cycle arresting agent. In contrast, T cells co-cultured with DC-DHA express higher levels of TGFβ and Foxp3, without exhibiting a functional Treg phenotype. In addition, DHA prevents up-regulation of VCAM-1 and ICAM-1 in bEND.3 cells (mouse brain microvascular endothelial cell line). Here we report for the first time a beneficial effect of dietary n-3 fatty acids in experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. The beneficial effect of DHA in EAE was associated with reduced numbers of IFNγ- and IL-17-producing CD4+ T cells in both spleen and CNS.

We reported previously that PGE2 promotes IL-23p19 expression in dendritic cells (DC) treated with LPS. The effect is mediated through EP4 through the induction of cAMP. This study is focused on the signaling pathways involved in PGE1OH (an EP4 agonist)-induced p19 expression. EP4 activates adenylate cyclase and increases intracellular cAMP which leads to activation of PKA and EPAC. We found that adenylate cyclase, cAMP, and EPAC play a role in IL-23p19 induction. Using chemical inhibitors of PKA, PI3K, MAPK, and GSK3, we did not see a decrease in IL-23p19 expression. Previous studies reported the involvement of NFkB/c-Rel, ATF-2, SMAD-3 and AP-1 in p19 expression. Since two CRE-binding sites have been identified in the p19 promoter, we focused on CREB as a potential transcription factor activated by PGE1OH. Luciferase experiments including vectors containing mutated CRE sites and expressing dominant negative versions of CREB demonstrated the essential role CREB plays in IL-23p19 expression. In a transcription factor assay we found increased CREB binding activity in nuclear extracts from cells treated with PGE1OH. In addition, a ChIP assay confirmed CREB binding to the p19-promoter in the presence of PGE1OH. Overall, we identified CREB as a new transcription factor required for PGE1OH induction of IL-23p19 expression.

Recently cannabinoids are being studied in treatment of various animal models of CNS disorders such as MS, AD, PD and stroke. They are also being used in treating neuropathic pain in patients with spinal cord injury (SCI). Two cannabinoid receptors have been cloned; CB1, mainly expressed in the CNS and CB2, predominantly expressed in the periphery including immune cells. Previous studies from our laboratory showed that treatment with the CB2 specific agonist O-1966 improved recovery of motor and bladder function in mouse model of acute spinal cord injury. In SCI, increased infiltration and activation of inflammatory immune cells that follow the initial injury exacerbate neuronal damage. In this study we analyzed expression of inflammatory molecules that serve as markers for activation and infiltration of immune cells and their modulation by the CB2 agonist treatment. A murine thoracic spinal cord contusion model of SCI was used and spinal cords were analyzed for expression of various cytokines and chemokines. We found suppression of a number of molecules in spinal cords that were subjected to the CB2 agonist treatment. There was decreased expression of the CXCL family chemokine members CXCL1, 2, 3, 9 and 11, the CC family chemokine members CCL 8, 11, 20, T cell activation cytokines: IFN-γ, IL-23, IL-22 and some of the Toll-like receptors such as TLR1, 4, 6, and 7. Thus, it appears that CB2 agonist treatment may aid in recovery in acute SCI via its immunomodulating effects.

Previously, we have shown that PGE2 inhibits IL-12p35 expression & promotes IL-23p19 expression in dendritic cells (DC) treated with LPS or Poly I:C. The effect is mediated through EP4 and to a lesser degree EP2 receptors and induction of cAMP. This study is focused on essential transcriptional factors and signaling pathways involved in PGE1-OH (EP4 agonist) induced p19 expression. Previous studies reported involvement of NFkB (c-Rel), ATF-2, SMAD-3 & AP-1. CREB is a target of cAMP-activated PKA and two CREB binding sites have been identified in the p19 promoter. Therefore, CREB is a potential transcription factor activated by PGE. Experiments with p19-luciferase constructs containing wt & CREB-mutated sites indicate the importance of CREB sites in p19 transcriptional activity. PGE2/PGE1-OH also induced STAT-3 tyrosine phosphorylation in DC and in the DC cell line. Blocking STAT-3 phosphorylation with the chemical inhibitor S3I-201 and the specific peptide inhibitor PpYLKTK-mts reduced p19 expression in DC. Although no STAT-3 binding site was found in the p19 promoter, STAT-3 was proposed to act as a co-activator for c-Rel to induce p19 expression. These data show for the first time that CREB and STAT-3 are important regulators of PGE2-induced p19 expression in DC. These observations show the versatility that prostaglandins have to differentially regulate expression patterns of cytokines that orchestrate an immune response.

Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in clinical studies and animal models of colitis, sepsis, and stroke. In the present study we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) and CD4+ T cells. Our studies show that pretreatment with DHA prevents LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of MHCII and costimulatory molecules, and high levels of endocytosis. Exposure to DHA inhibits the production of proinflammatory cytokines and chemokines such as IL-6, TNFα, CCL-4, and IL-12p70. In addition, we report here for the first time that DHA has a similar inhibitory effect on IL-23 and IL-27, two other members of the IL-12 family. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet. We also investigated the effects of DHA on T cells and found that DC treated with DHA (DC-DHA) have poor stimulatory capacity for T cells. T cells activated by DC-DHA also showed low levels expression of IFNα and IL-17, and high levels of Foxp3. Based on the inhibition of proinflammatory cytokine release and reduction in T cell stimulatory capacity, we conclude that DC are important targets for the anti-inflammatory activity of n-3 fatty acids.

A few studies have been published showing effects of cannabinoids in modulating immune response from T helper cell type 1 (Th1) to T helper cell type 2 (Th2) in a model of Legionella pneumophila infection. Cannabinoids act through two cloned receptors, CB1 and CB2. The CB1 receptor is expressed primarily in the CNS, whereas the CB2 receptor is highly expressed in immune cells in the periphery. Previous results from our laboratories indicated that the CB2 agonist had an anti-inflammatory effect on dendritic cells (DCs) and T cells. DCs differentiated in the presence of CB2 agonist produced less IL-12 upon LPS stimulation and T cells produced less IFN-gamma when co-cultured with these DCs in syngeneic culture in the presence of specific antigen. Here, we report that CB2 agonist treatment of DCs resulted in reduced STAT-1 phosphorylation. Also, treatment of polyclonally activated CD4+ T cells resulted in reduced expression of IL-12 receptor beta 2 chain, which correlated with decreased IFN-gamma production. Our data lead us to believe that CB2 agonists can modulate immune responses by acting directly on CD4+ T cells or through their effects on DCs. This work is supported by NIH/NIAID R01AI05230, and training grant T32 DA07237 (SA)

Background Acute and chronic inflammation play essential roles in inflammatory/autoimmune conditions. Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in animal models of colitis, sepsis, and stroke. Since dendritic cells (DC) represent the essential cellular link between innate and adaptive immunity and have a prominent role in tolerance for self-antigens, we sought to investigate the impact of DHA on DC maturation and proinflammatory cytokine production. Methods Murine bone marrow-derived DC were treated with DHA and stimulated with various toll-like receptor (TLR) ligands. Flow cytometry was used to determine the levels of surface maturation markers and endocytic activity. Cytokine expression and secretion were measured by real-time RT-PCR and ELISA assays. PPARγ and NFκB activity in nuclear extracts were determined by binding to specific oligonucleotide sequences using ELISA-based assays. In vivo effects of DHA were assessed in splenic DC from LPS-inoculated mice maintained on a DHA-enriched diet. Results DHA maintained the immature phenotype in bone marrow-derived DC by preventing the upregulation of MHCII and costimulatory molecules (CD40, CD80 and CD86) and maintaining high levels of endocytic activity. DHA inhibited the production of pro-inflammatory cytokines, including the IL-12 cytokine family (IL-12p70, IL-23, and IL-27), from DC stimulated with TLR2, 3, 4, and 9 ligands. DHA inhibition of IL-12 expression was mediated through activation of PPARγ and inhibition of NFκBp65 nuclear translocation. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet. Conclusions Exposure of bone marrow-derived DC to DHA resulted in the maintenance of an immature phenotype and drastic reduction in proinflammatory cytokine release. DHA inhibited the expression and secretion of the IL-12 cytokine family members (IL-12p70, IL-23 and IL-27), which play essential roles in the differentiation of the proinflammatory Th1/Th17 effector cells. The effect of DHA on IL-12 expression was mediated through activation of PPARγ and inhibition of NFκB. Inhibition of IL-12 and IL-23 expression was also evident in splenic DC from mice fed a DHA-enriched diet, suggesting that dietary DHA acts as an anti-inflammatory agent in vivo.