Antitumor Effects of Tumor-Derived Exosomes in Murine Hepatocellular Carcinoma Models.

Impact of the hypomethylating agent 5-azacytidine on dendritic cells function

Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide.1 It has been so for decades, notwithstanding a wide array of – mostly preventive – treatment modalities targeting known risk factors, such as hyperlipidemia, type 2 diabetes mellitus, hypertension, or obesity. Recent technical and conceptual advances have unveiled important contributions of the immune system in the pathophysiology of a variety of CVDs such as atherosclerosis, ischemic stroke, chronic heart failure, and other myocardial conditions like myocardial ischemia and reperfusion, viral myocarditis, and cardiac transplantation.2–4 In many of these disorders, so-called danger-associated molecular patterns (DAMPs), released from necrotic tissue and dying cells, can lead to the activation of certain immune cell populations such as monocytes/ macrophages, granulocytes, and T cells, thus aggravating ongoing inflammatory processes at the lesion site. Dendritic cells (DCs) are key modulators of immunity, pivotal in directing innate and adaptive immune responses against microbial, viral, but also modified self-antigens present at the sites of injury. Given the tissue trauma underlying various CVDs, it is not surprising that recent observations have allocated a regulatory role for DCs in CVD-associated immune responses. Interestingly, nondiseased arteries of young individuals were seen to host a network of resident vascular DCs (CD1a+ S100+ lag+ CD31− CD83− CD86−),5 representing a phenotype related to Langerhans cells in the skin. In agreement, monocyte-derived CD11c+ CD68+ dendritic cells could be detected in the atherosclerosis-prone lesser curvature and aortic sinus in inbred atherosclerosis-susceptible (C57Bl/6), but not resistant mouse strains (balb/c).6 Murine vascular resident DCs express an immature phenotype with low expression of costimulatory molecules, and are present in the subendothelial space with occasional probing into the vascular lumen. DCs have …

Although chemoimmunotherapy has led to higher response rates in chronic lymphocytic leukemia (CLL), most patients relapse due to emergence of resistant subclones. Here, we analyzed changes in immune cell (IC) populations of 29 subjects with relapsed/refractory CLL enrolled in PCYC-1108, a Phase 1b, open-label, parallel-group, nonrandomized, multicenter study of ibrutinib (PCI-32765) in combination with BR (O'Brien et al; ASCO 2012). Mononuclear cells were collected and cryopreserved at 4 time points: 1: just prior to therapy, 2: cycle 1 day 15 (d15), 3: at start of cycle 3 (d 60) and; 4: at the end of study (d 180). Based on a decrease in absolute lymphocyte count (ALC), 27/29 patients responded to this combination therapy. We analyzed residual B, T and monocyte populations and prognostic markers, CD38 and CD49d in each patient and grouped the cases based on percent reduction of CD19+CD5+ (“CLL B cells”) at all time points while on therapy. 10/29 cases (∼34%) showed > 50% reduction (Grp1) whereas 41% (12/29) showed greater retention of CLL cells (Grp2), and 7/29 cases (∼24%) showed variable changes (Grp 3). Median reduction in ALC in Grp1 was 97.8% (range: 68.3-99.75) vs 87.7% (range: 77.3- 99.6) for Grp2. Grp 1 cases showed a decrease in CD23+ and CD38+ CLL B cells at d15; although CD23+ cells decreased further CD38+ cells increased in 8/10 Grp1 cases. CD49d+ CLL cells remained stable in 6/10 Grp 1 and 11/12 Grp 2 cases but increased in 4/10 Grp1 cases. A marked increase in CD3+ and CD14+ cells accompanied the decrease in CLL B cells in cases in Grp1. Grp2 followed a different trend: CD3+ cells decreased until d60 and eventually increased, albeit mildly whereas their percentages of CD14+ cells did not change throughout therapy. Such dynamic changes in IC populations prompted us to assess the presence of dividing CLL cells, in the face of reduced ALC. Surprisingly, although Grp1 showed decreased percentages of CLL cells, 3/10 cases developed increased numbers of Ki-67+ CLL cells, achieving 19-43% at d180. In Grp2, only 0.1-4.0% Ki-67+ CLL B cells were found. BCR-induced phosphorylation of BTK was analyzed by phosphoflow. Both Grp1 and Grp2 exhibited increased BTK phosphorylation at d15 which decreased precipitously at subsequent time points. Together, these studies indicate that combining ibrutinib with BR effectively reduced numbers of circulating CLL cells in >93% of cases, but relative proportions of CLL cells only in ∼1/3 relapse/refractory CLL cases. Of note, the proportion of circulating CD3+, CD14+, and Ki-67+ CLL B cells, increased considerably in Grp 1 cases, possibly representing compensatory proliferative changes due to increased availability of immune niches and/or release of cells from niches. A post-protocol correlation of clinical parameters with changes in circulating immune cell populations is pending. Citation Format: Rajendra N. Damle, Sonal Temburni, Prachi Aggarwal, Susan O'Brien, Jacqueline Barrientos, Jennifer R. Brown, Ian W. Flinn, Paul Barr, Jan Burger, Jonathan W. Friedberg, Kanti R. Rai, Betty Chang, Danelle James, Joseph Buggy, Nicholas Chiorazzi. Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2013-3531

Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2

Dendritic Cells, Tolerance Induction and Transplant Outcome

Matched Skin and Sentinel Lymph Node Samples of Melanoma Patients Reveal Exclusive Migration of Mature Dendritic Cells

e14573 Background: With unique feature of self-immune tolerance and high radio-sensitivity, hepatocellular carcinoma (HCC) can gradually develop undesired effects. Iodine-125 (125I) seed, as a source of internal radiation therapy, may have a favorable adaptability and immunostimulation. In this study, we investigated whether 125I seeds can stimulate local tumor infiltrating lymphocytes (TILs) and whether a combination of 125I seeds and dendritic cells (DCs) loaded with tumor antigen (Ag+DCs) can enhance both local tumor control and abscopal effect in murine subcutaneous tumor models. The related mechanism of anti-tumor immune response was also explored. Methods: H22/Hepa1-6 HCC cells were examined for radiosensitivity and immunosensitivity. Cell apoptosis and expression of MHC-I (major histocompatibility complex class I) and PD-L1 (programmed death-ligand 1) were detected before and after irradiation. The tumor cells were injected subcutaneously as primary and abscopal tumor. Bone marrow-derived dendritic cells (BM-DCs) were induced from mouse bone marrow cells and incubated with tumor cell lysate. 125I seeds were implanted into the primary tumors, followed by intratumoral injection of Ag+DCs and intraperitoneal injection of αPD1 antibody (αPD1-ab). DC migration was detected by transwell chambers in vitro and CFSE (carboxyfluorescein succinimidyl ester)-labeled DCs in vivo. DC maturation was tested by flow cytometry. Analyses of tumor growth and survival rates were performed in vitro, and that of, TILs, T-cell proliferation, cytotoxicity test, immunostimulatory cytokines release, and immunological memory were performed in vivo. In situ vaccine and abscopal effect were verified in orthotopic mouse models of liver cancer and lung metastatic tumor. Results: 125I seeds alone could induce TILs and activate cytotoxic T cells (CTLs), but these effects were limited. The combination treatment of 125I seeds with Ag+DC administration inhibited primary tumor growth and significantly prolonged survival time in association with a significant increase in T-cell proliferation and interferon-γ release. In addition, triple-combination treatment with αPD1-ab amplified these responses, leading to significant regression of primary and metastatic tumors and successful stimulation of immunological memory. Conclusions: 125I seeds can safely activate anti-tumor immune response.125I seeds combined with Ag+DC administration is able to convert irradiated liver cancer into effective in situ vaccine. Furthermore, triple-combination therapy of 125I seeds, Ag+DC, and αPD1-ab can be a promising approach to activate systemic anti-tumor immunity, which is a potential novel individualized therapy for patients with solid cancers.

Progress in treatments for hepatocellular carcinoma (HCC) has improved the prognosis of patients with HCC. However, HCC is usually associated with cirrhosis and often recurs even after complete treatment of the tumors in the remaining part of the cirrhotic liver. Thus, there is a strong need for the development of a new intervention therapy that suppresses the occurrence or recurrence effectively with fewer side effects. Immunotherapy may be such a treatment and several clinical trials have been performed for HCC treatment. Dendritic cells (DCs) are known as professional antigen-presenting cells characterized by their potent ability to elicit immune responses to tumor-specific T cells against tumor-associated antigens. Programmed cell death 1 (PD-1) has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1-PD-ligand interactions has been shown to partially restore T cell function. In this study, we evaluated the efficacy of the combination of DC-based vaccine and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. In protection model, mice were injected with DCs or/and PD-1-antibody before the murine HCC tumor cell (BNL cell) challenge. 40% of mice treated with both DCs and PD-1-antibody rejected tumor challenge, whereas other groups observed a palpable tumor in all mice tested. In therapeutic model, tumor-bearing mice were inoculated with DCs or/and PD-1-antibody. Significant suppression of outgrowth of the established tumors was observed in the DCs and anti-PD-1 combination treatment group (DCs + anti-PD-1, 138.00 ± 56.66 mm2 vs control, 402.33 ± 40.63 mm2 on day 42, P = 0.0073 vs controls). Immunohistochemical analyses of therapeutic model showed marked infiltration of CD4+ cells, CD8+ cells and CD11c+ cells in the established BNL tumors of mice treated with both DCs and PD-1-antibody. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of DCs or/and PD-1-antibody treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both DCs and anti-PD-1 were used as effector cells (30.0% ± 2.8% for BNL and 7.3% ± 0.1% for YAC-1, effector to target ratio; E/T=40). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by DC vaccine. The combination of DC vaccine and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. Citation Format: Eiichi Hayashi, Junichi Eguchi, Masashi Sakaki, Hiroyoshi Doi, Risa Oomori, Atsushi Kajiwara, Hitoshi Yoshida, Shigeaki Ishii, Kazumasa Hiroishi, Michio Imawari. Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2014-2810

Objective: To examine whether microglia differentiate to dendritic cells (DC) and those DCs can acquire the full characteristic phenotype of mature stimulatory DCs to participate in functional responses. Methods: Isolated microglia were stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and further with lipopolysaccharide (LPS) and/or tumor necrosis factor-α (TNF-α), then phenotypical changes were analysed by their surface antigen expression, cytokine profile and ability to present antigens. Results: Microglia acquire DC markers in the presence of GM-CSF. These GM-CSF-stimulated microglia still do not fully differentiate into mature DCs because of the low expression of mature DC marker CD83 and the high expression of immature DC marker CD209. However, TNF-α further induces their differentiation into DC-like cells by up-regulating the expression of DC-associated antigens. These cells differentiate into CD11c+, CD83high and CD209low mature DCs upon LPS stimulation, effectively present antigen to CD4+ T cells in an antigen-specific manner, and produce DC-like cytokines such as interleukin (IL)-23 and IL-12 as well as pro-inflammatory cytokines such as IL-6 and TNF-α. Conclusions: The resident microglia may become mature DCs and function as stimulatory DCs to induce inflammatory autoimmune responses in the CNS. (Clin. Exp. Neuroimmunol. doi: 10.1111/j.1759-1961.2010.00016.x, January 2011)

Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction

AB0018 ACCUMULATION OF FUNCTIONALLY MATURE CD1C+ DENDRITIC CELLS CONTRIBUTES TO SYNOVIAL INFLAMMATION IN INFLAMMATORY ARTHRITIS

Dendritic Cells in Fundamental and Clinical Immunology

BackgroundConflicting data exist on the role of pulmonary dendritic cells (DCs) and their maturation in patients with chronic obstructive pulmonary disease (COPD). Herein, we investigated whether disease severity and smoking status could affect the distribution and maturation of DCs in lung tissues of patients undergoing elective pneumectomy or lobectomy for suspected primary lung cancer.Materials and MethodsA total of 75 consecutive patients were included. Spirometry testing was used to identify COPD. Lung parenchyma sections anatomically distant from the primary lesion were examined. We used flow cytometry to identify different DCs subtypes—including BDCA1-positive myeloid DCs (mDCs), BDCA3-positive mDCs, and plasmacytoid DCs (pDCs)—and determine their maturation markers (CD40, CD80, CD83, and CD86) in all participants. We also identified follicular DCs (fDCs), Langerhans DCs (LDCs), and pDCs in 42 patients by immunohistochemistry.ResultsCOPD was diagnosed in 43 patients (16 current smokers and 27 former smokers), whereas the remaining 32 subjects were classified as non-COPD (11 current smokers, 13 former smokers, and 8 never smokers). The number and maturation of DCs did not differ significantly between COPD and non-COPD patients. However, the results of flow cytometry indicated that maturation markers CD40 and CD83 of BDCA1-positive mDCs were significantly decreased in smokers than in non-smokers (P = 0.023 and 0.013, respectively). Immunohistochemistry also revealed a lower number of LDCs in COPD patients than in non-COPD subjects.ConclusionsCigarette smoke, rather than airflow limitation, is the main determinant of impaired DCs maturation in the lung.

The cAMP-dependent signaling pathways that orchestrate dendritic cell (DC) maturation remain to be defined in detail. Although cAMP was previously thought to signal exclusively through protein kinase A (PKA), it is now clear that cAMP also activates exchange protein activated by cAMP (Epac), a second major cAMP effector. Whether cAMP signaling via PKA is sufficient to drive DC maturation or whether Epac plays a role has not been examined. In this study, we used cAMP analogs to selectively activate PKA or Epac in human monocyte-derived DCs and examined the effect of these signaling pathways on several hallmarks of DC maturation. We show that PKA activation induces DC maturation as evidenced by the increased cell-surface expression of MHC class II, costimulatory molecules, and the maturation marker CD83. PKA activation also reduces DC endocytosis and stimulates chemotaxis to the lymph node-associated chemokines CXCL12 and CCL21. Although PKA signaling largely suppresses cytokine production, the net effect of PKA activation translates to enhanced DC activation of allogeneic T cells. In contrast to the stimulatory effects of PKA, Epac signaling has no effect on DC maturation or function. Rather, Epac suppresses the effects of PKA when both pathways are activated simultaneously. These data reveal a previously unrecognized crosstalk between the PKA and Epac signaling pathways in DCs and raise the possibility that therapeutics targeting PKA may generate immunogenic DCs, whereas those that activate Epac may produce tolerogenic DCs capable of attenuating allergic or autoimmune disease.

Obesity is understood to be an inflammatory condition characterized in part by changes in resident immune cell populations in adipose tissue. However, much of this knowledge has been obtained through experimental animal models. Epigenetic mechanisms, such as DNA methylation may be useful tools for characterizing the changes in immune cell populations in human subjects. In this study, we introduce a simple and intuitive method for assessing cellular infiltration by blood into other heterogeneous, admixed tissues such as adipose tissue, and apply this approach in a large human cohort study. Associations between higher leukocyte infiltration, measured by evaluating a distance measure between the methylation signatures of leukocytes and adipose tissue, and increasing body mass index (BMI) or android fat mass (AFM) were identified and validated in independent replication samples for CD4 (pBMI = 0.009, pAFM = 0.020), monocytes (pBMI = 0.001, pAFM = 4.3 × 10-4 ), and dendritic cells (pBMI = 0.571, pAFM = 0.012). Patterns of depletion with increasing adiposity were observed for plasma B (pBMI = 0.430, pAFM = 0.004) and immature B (pBMI = 0.022, pAFM = 0.042) cells. CD4, dendritic, monocytes, immature B, and plasma B cells may be important agents in the inflammatory process. Finally, the method used to assess leukocyte infiltration in this study is straightforwardly extended to other cell types and tissues in which infiltration might be of interest.