Co-culture Of Peripheral Blood Mononuclear Cells Research Articles

Transcriptional profiling of circulating mononuclear cells from patients with chronic obstructive pulmonary disease receiving mesenchymal stromal cell infusions.

Chronic obstructive pulmonary disease (COPD) is an inflammatory airways disease with limited therapeutic options. We have previously shown that mesenchymal stromal cell (MSC) infusions are well tolerated in patients with COPD and reduce circulatory biomarkers associated with systemic inflammation and oxidative stress. This study aimed to delineate the underlying mechanisms further by characterizing the transcriptional networks in these patients and to explore the role of MSC‐derived paracrine factors in regulating these pathways. Allogeneic, bone marrow‐derived MSCs were systemically administered into patients with stable COPD (n = 9). Gene expression profiles from peripheral blood mononuclear cells (PBMCs) were analyzed across the first week after infusion. Paracrine mechanisms associated with these transcriptional changes were explored further by culturing patient PBMCs with MSC‐conditioned medium (MSC‐CM) or post‐MSC infusion (PI) plasma to measure the regulatory effects of soluble factors that may be derived from MSCs. MSC‐CM and PI‐plasma were characterized further to identify potential immunoregulatory candidates. MSC infusion elicited a strong but transient transcriptional response in patient PBMCs that was sustained up to 7 days. MSC infusion strongly downregulated transcriptional pathways related to interleukin (IL)‐8 and IL‐1β, which were also significantly inhibited in vitro following co‐culture of PBMCs with MSC‐CM and PI‐plasma. MSC‐derived soluble tumor necrosis factor receptor‐1, transforming growth factor‐β1, and extracellular vesicle‐associated microRNAs were identified as potential mechanisms promoting these changes, but depletion of these individual candidates revealed inconsistent results. MSC‐derived paracrine factors modulate important inflammatory pathways that are relevant to COPD pathogenesis. These data strengthen the hypothesis that therapies using MSCs and their secreted products may be beneficial to patients with COPD.

RANKL expression in chondrocytes and its promotion by lymphotoxin-α in the course of cartilage destruction during rheumatoid arthritis.

We investigated the expression and localization of the receptor activator nuclear factor κB ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-α on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-α concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-α, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-α, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-α concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-α increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-α, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-α stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-α could promote the expression of RANKL in human chondrocytes.

Mechanisms of Regulatory T Cell Recruitment By Mesenchymal Stem Cells in Vitro

Regulatory T cells (Treg) play a critical role in the maintenance of immune tolerance. The scarcity of clinical trials using adoptive Treg therapy in humans has been largely due to the difficulty in obtaining sufficient Treg numbers under GMP conditions. Due to the paucity of Treg in the peripheral blood, several groups are investigating the possibility of expanding these cells in vitro .We sought to investigate the potential of mesenchymal stem cells (MSC) to recruit Treg in vitro . Previous studies suggest that MSC-mediated immunomodulation may be partly driven by Treg induction and/or expansion. However, the mechanisms of Treg recruitment by MSC are so far poorly understood.We performed co-cultures of human peripheral blood mononuclear cells (PBMC) from healthy donors with allogeneic bone marrow (BM)-derived MSC expanded in xeno-free conditions. Our data show an increase in the absolute counts and frequency within the T cell pool of CD4+ CD25high Foxp3+ CD127low Treg cells (4 and 6fold, respectively) after a 14day coculture, both compared to PBMC cultured alone and to Day 0. This increase appears to be specific for Treg, as the levels of conventional CD4 T cells (Tcon) and CD8 T cells are not increased in the presence of MSC. Co-culture with MSC does not induce Treg proliferation, as assessed by Ki67 expression. In terms of susceptibility to apoptosis, MSC increase Bcl-2 levels within Treg but not within Tcon, further suggesting a preferential impact on Treg.The observation of increased Treg numbers after MSC co-culture in the absence of Treg proliferation suggests that MSC recruit Treg by inducing Tcon cells to acquire a Treg-like phenotype. In order to determine the mechanism of MSC-mediated Treg recruitment, we co-cultured either purified CFSE-labelled Treg or Tcon with MSC. In Treg:MSC co-cultures, cell numbers are maintained but Treg do not proliferate. In Tcon:MSC co-cultures, only the cells that acquire a Treg-like phenotype proliferate, whereas the cells that maintain a Tcon phenotype do not. These results indicate that the increase in Treg numbers after co-culture of PBMC with MSC is mainly due to the induction of Tcon-derived Treg-like cells, rather than to Treg expansion.In order to determine if these Treg-like cells induced by MSC also resemble Treg in terms of functional ability, we co-cultured PBMC or purified Tcon with MSC and FACSorted the resulting Treg-like populations after 10 days. The suppressive ability of these purified cells, as well as of unsorted cells from the bulk co-cultures, was assessed in suppression assays using autologous fresh Tcon labelled with CFSE as responder cells. We further compared their suppressive ability to that of Treg co-cultured with MSC, as well as of fresh Treg. Our data suggest that bulk co-cultures of PBMC:MSC, Tcon:MSC and Treg:MSC significantly suppress Tcon proliferation (% divided cells at 1:1 Tcon:Treg ratio compared to Tcon alone, Mann Whitney test: P=0.0022, P=0.0043 and P=0.0303, respectively). As for the functional ability of Treg purified after MSC co-culture, both induced Treg-like cells isolated from Tcon:MSC co-cultures and natural Treg isolated from Treg:MSC co-cultures significantly suppress Tcon proliferation (Mann Whitney test: P=0.0095 for both).We also assessed the expression of functional markers that are associated with Treg induction and/or expansion. After Tcon:MSC co-culture, Treg-like cells express higher levels of PD-1 and CD15s than the cells that maintain a Tcon phenotype, whereas the expression of GITR was similar within Treg-like and Tcon cells. Interestingly, the frequency of MSC expressing PD-1 ligand (PDL-1) increases after co-culture, both compared to Day 0 and to MSC cultured alone.In order to investigate which cytokines may potentially be driving the induction of Treg-like cells by MSC, we measured cytokine levels in co-culture supernatants. The anti-inflammatory cytokines IL-10 and TGF-β are increased, whilst the pro-inflammatory cytokines IFN-γ and TNF-α are reduced in PBMS:MSC co-cultures when compared to PBMC cultured alone.Overall, our data suggest that Treg recruitment by MSC is due to Tcon conversion into Treg-like cells, rather than Treg expansion. Our results demonstrate that MSC induce the generation of a Treg-like population, both in terms of the phenotypic hallmarks of Treg cells and of the functional ability to suppress autologous Tcon proliferation. DisclosuresNo relevant conflicts of interest to declare.

Adaptability of Antibody-Coupled T Cell Receptor (ACTR) Engineered Autologous T Cells in Combination with Daratumumab over CAR-Based Approaches

Chimeric antigen receptor (CAR)-T cells have demonstrated profound clinical responses against CD19+ B cell malignancies, with ongoing trials in multiple myeloma (MM). Although BCMA CAR-T cells show promise in MM, development of CAR-T against other MM targets has been problematic due to target expression on T cells themselves. Specifically, efforts to develop CAR-T cell therapies targeting CD38 have been hampered by the upregulation of CD38 on activated T cells and consequent induction of CAR-T mediated autolysis that occurs during manufacturing. To overcome this challenge during CAR-T production, investigators have taken several approaches including multiple rounds of T cell expansion, addition of CD38 blocking antibody, and CD38 gene knockout in T cells prior to CAR transduction (1,2,3).The Antibody-Coupled T Cell Receptor (ACTR) technology is a universal, engineered T cell therapy that combines the Fc receptor, CD16, with endogenous T cell signaling and costimulatory domains (4). ACTR T cells function by engaging the Fc domain of therapeutic antibodies opsonized on target cells, resulting in T cell activation and tumor cell killing. In contrast to CAR-T cell constructs that express the antigen targeting single chain variable fragment (scFv), ACTR T cell activity is regulated through antibody administration and is therefore not susceptible to target-mediated autolysis during T cell production.The recent approval of the CD38-targeting antibody, daratumumab, has improved the treatment of multiple myeloma through its tumor targeting and immunomodulating capabilities. Here, we leveraged the ability of the ACTR T cell platform to decouple the targeting antibody from the engineered T cell signaling moieties, and evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. We demonstrated that in contrast to CD38-targeting CAR-T cells, ACTR T cells exhibited enhanced viability and expansion of the chimeric construct throughout the manufacturing process. We then evaluated the anti-myeloma activity of ACTR T cells in combination with daratumumab. ACTR T cells mediated robust cytotoxicity, cytokine production, and proliferation in response to a range of daratumumab-opsonized target cell lines. Notably, the target specific killing and cytokine production mediated by ACTR T cells in combination with daratumumab was greater than the activity mediated by daratumumab alone. Recognizing the augmented activity of ACTR T cells in combination with daratumumab, we systematically investigated the potential for on-target off-tumor toxicity. In a peripheral blood mononuclear cell coculture assay, ACTR T cells in combination with daratumumab selectively targeted CD38-expressing multiple myeloma cells over CD38-expressing normal B cells, T cells, and natural killer cells. Furthermore, despite low levels of CD38 expression reported on erythrocytes, ACTR T cells in combination with daratumumab did not mediate hemolysis. Taken together, our findings demonstrate the adaptability of the ACTR platform to function in combination with daratumumab, and bypass the challenges of scFv-based CAR-T cell production to safely and effectively target CD38-positive multiple myeloma.

The immunomodulatory effects of low molecular weight garlic protein in crosstalk between peripheral blood mononuclear cells and colon cancer cells

Garlic is an ancient plant whose medicinal usage has a long traditional history. It has been always advocated as a suitable candidate for immunomodulation. The purpose of the present study was to assess the immunomodulatory effects of Low Molecular Weight Garlic Proteins (LMWGP) on the proliferation of peripheral blood mononuclear cells (PBMCs), induction of regulatory T cells (Tregs), as well as cytokine secretion in the co-culture of PBMCs and colon cancer cell lines, SW48.Garlic cloves were initially extracted and then protein fractions were separated using gel filtration chromatography and analyzed with mass spectrometry (MS). The effects of LMWGP on the proliferation of PBMCs and induction of Tregs were later analyzed by flow cytometry. Cytokine secretion was also evaluated using the ELISA test.The findings revealed that the proliferation of PBMCs had significantly augmented in the presence of LMWGP (p ≤ 0.05). Moreover, this fraction had reduced induction of Tregs in the co-culture medium (p ≤ 0.05). ELISA results had further demonstrated a decline in cytokines, IL-10, and galectin-3, but an increment in IL-6 in the presence of LMWGP (p ≤ 0.05).According to co-culture study results, it was concluded that LMWGP could be assumed as a beneficial immunomodulatory supplement in herbal medicine for cancer.

Vibrio cholerae toxin coregulated pilus provokes inflammatory responses in Coculture model of Caco-2 and peripheral blood mononuclear cells (PBMC) leading to increased colonization.

The aim of this study was to assess the modulatory effect of TcpA in the expression of CEACAM1 adhesin molecule and IL-1, IL-8, and TNF-α pro-inflammatory cytokines in the Coculture model of Caco-2/PBMC (peripheral blood mononuclear cell) that can mimic the intestinal milieu. The TcpA gene from Vibrio cholerae ATCC14035 was cloned in pET-28a and transformed into Escherichia coli Bl-21. The recombinant TcpA-His6 protein was expressed and purified using Ni-column chromatography. The sequencing of transformed plasmid and Western blot analysis of purified protein confirmed the identity of rTcp. The cytotoxicity of different concentrations of recombinant protein for human colon carcinoma cell line (human colorectal adenocarcinoma cell [Caco-2 cell]) was assessed by MTT assay and showed viability of 92%, 82%, and 70%, for 10 µg/mL of TcpA after 24, 48, and 72 h, respectively. Co-cultures of Caco-2 and PBMCs were used to mimic the intestinal milieu and treated with different concentrations of rTcpA (1, 5, 10, and 50 µg/mL). Our data showed about 2.04-, 3.37-, 3.68-, and 42.7-fold increase in CEACAM1 gene expression, respectively, compared with the nontreated Caco-2/PBMC Coculture. Moreover, the expression of IL-1, IL-8, and TNF-α genes was significantly increased up to 15.75-, 7.04-, and 80.95-folds, respectively. In conclusion, V. cholerae TcpA induces statistically significant dose-dependent stimulatory effect on TNF-α, IL-,1, and IL-8 pro-inflammatory cytokines expression. Of these, TNF-α was much more affected which, consequently, elevated the CEACAM1 expression level in IECs. This suggests that TcpA protein is a critical effector as an inducer of increased adhesion potential of V. cholera as well as inflammatory responses of host intestinal tissue.

Suppression of T-cell Immune responses by Neutrophils in a direct contact manner

Abstract Neutrophils are protective against early Mycobacterium tuberculosis (Mtb) infection, but overactive neutrophil response is often associated with chronic lung inflammation, tissue damage and compromised T-cell immunity, implying potential immunosuppression by neutrophils in Mtb infection. To test this, we examined the effect of peripheral blood neutrophils on the immune responses of T-cells from the peripheral blood mononuclear cells (PBMC) of persons with latent tuberculosis infection. Co-cultures of PBMC with autologous neutrophils at different ratios inhibited Mtb-stimulated T-cell proliferation and cytokines IFN-γ, IL-17 and IL-10 production dose dependently without affecting cell viability as determined by resazurin assay for cell metabolism and annexin V/PI staining for cell death. Neutralization of IL-10 and TGF-β failed to rescue neutrophil suppression of T-cell IFN-γ production. Neutrophils suppressed IFN-γ mRNA expression as determined by qPCR. Neutrophils inhibited purified T-cells stimulated with immobilized anti-CD3 and anti-CD28 mAbs, suggesting direct effect on T-cells without the involvement of antigen presenting cells. Trans-well study showed that cell-cell contact is required for this suppression. Further assessment of granulocyte derived suppressor cell phenotype and blocking the interaction between immune checkpoint molecules, PD-1 and PD-L1, refuted their roles in suppression of T-cells responses in Mtb infection. Together, these data suggest that neutrophils suppress T-cell immune responses by direct cell-cell contact mechanism and identification of molecular details of such suppression will shed new insights into the pathogenesis of tuberculosis and other infectious diseases.

Angiogenic potential of human mesenchymal stromal cell and peripheral blood mononuclear cell co-cultures

Angiogenic potential of human mesenchymal stromal cell and peripheral blood mononuclear cell co-cultures

Synergistic efficacy of curcumin and anti-programmed cell death-1 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) ranks near the top in the global list of malignancies causing cancer-related death. Recently, combination therapy has gained popularity in treating this cancer. We tried to investigate the efficacy of combined treatment with curcumin and anti-programmed cell death-1 (anti-PD-1) in HCC. Hep3B cells were treated with different concentrations of curcumin, followed by determination of Hep3B cell proliferation and programmed cell death ligand-1 (PD-L1) expression. Then, Hep3B cells were co-cultured with peripheral blood mononuclear cells (PBMCs), after which the Hep3B cell growth and immune activity were detected following treatment with curcumin and/or anti-PD-1. Besides, we investigated the effect of transforming growth factor beta 1 (TGF-β1) on lymphocyte activation and the interaction between E1A binding protein P300 (P300), histone acetylation, TGF-β1, and thrombin. Additionally, the synergistic role of curcumin and anti-PD-1 in mouse models of HCC was studied. Curcumin retarded Hep3B cell growth and reduced surface PD-L1 expression in Hep3B cells. After co-culture of Hep3B cells and PBMCs, curcumin had a synergistic effect with anti-PD-1 to slow Hep3B cell proliferation, activate lymphocytes, inhibit immune evasion, and down-regulate TGF-β1 expression. Functionally, curcumin inhibited thrombin to reduce P300-induced histone acetylation in the TGF-β1 promoter region, and anti-PD-1 suppressed binding of PD-1 and PD-L1 to promote immune activity; the combination of the two showed better in vitro anti-cancer effects. In vivo, curcumin combined with anti-PD-1 also lowered HCC growth rate and improved the tumor microenvironment. In conclusion, the combination of curcumin and anti-PD-1 is synergistically effective in the treatment of HCC treatment.

Multi-Species Probiotic Modulates Cytokine Production and the Interplay between Immune and Colon Cancer Cells

The current study aimed to investigate the effect of a multi-species probiotic (MSP) on cytokine production by human peripheral blood mononuclear cells (PBMCs) and their immune dialogue with HT-29 colon cancer cells. PBMCs were incubated with MSP and their effect on cell proliferation and TNFα, IL-1β, IL-2, IL-6, IFNγ, IL-10, and IL-1ra production was evaluated. The impact of MSP on the cytokine production by PBMC stimulated by HT-29 cells was detected. Not-stimulated PBMC incubated with MSP showed increased production of TNFα, IL-1β, IL-6, and IL-10, but no change in IL-6, IFNγ, and IL-1ra. The stimulatory effect of MSP on lipopolysaccharide (LPS)-promoted PBMC was less pronounced for TNFα, IL-1β, and IFNγ, and the IL-6 production was decreased; phorbol 12-myristate 13- acetate (PMA)-induced IL-2 and IFNγ secretion was inhibited. The addition of MSP to co-cultures of PBMC and HT-29 cancer cells caused a remarkable increase in TNFα and IL-1β secretion, with no change in remaining cytokines. The multi-species probiotics modulated cytokine production by PBMC and affected the cross-talk between PBMC and HT-29 cancer cells. We conclude that probiotics may serve as supplements to the therapeutic strategies applied for the treatment of chronic inflammatory and malignant diseases, especially colorectal cancers.

The Role of Mesenchymal Stem Cells in Decreasing Interleukin-12 Human Systemic Lupus Erythematosus

BACKGROUND: Systemic lupus erythematosus (SLE) disease is characterized by a loss of self-tolerance leading to a local tissue inflammation up to a massive systemic organ-spesific inflammation. Mesenchymal stem cells (MSCs) present immunomodulatory properties to control the over-activating immune responses in SLE through several mechanisms. However, the capability of MSCs to decrease interleukin (IL)-12 production in in vitro remains unclear.
 AIM: The aim of this study was to investigate the role of MSCs in decreasing the level of IL-12 derived from peripheral blood mononuclear cells (PBMCs) of SLE patients.
 METHODS: This study used a post-test control group design using a coculture of PBMCs from SLE and healthy patients with MSCs as the subjects. This study included five groups: sham (Sh), control (C), and treatment groups (T) treated by a co-culture MSCs with PBMCs at ratio dose of 1:1 (T1), 1:25 (T2), and 1:50 (T3), respectively, for 72 hours of incubation. The IL-12 levels was analysed by cytometric bead array (CBA) of flow cytometry.
 RESULTS: This study showed a significant decrease of IL-12 levels (p < 0.05) in T1 and T2 after 72 hours incubation of co-culture MSCs with PBMCs from SLE patient.
 CONCLUSION: MSCs could decrease the level of IL-12 in PBMCs of human SLE to control the inflammation of SLE disease.

Modulatory effect of Vibrio cholerae toxin co-regulated pilus on mucins, toll-like receptors and NOD genes expression in co-culture model of Caco-2 and peripheral blood mononuclear cells (PBMC)

Vibrio cholerae, the causative agent of cholera, tend to colonize the small intestine as a Gram-negative pathogen. The intestinal mucus layer forms mucin physical barrier, consisted of high molecular weight proteins. Regarding the role of toxin–coregulated pilus (TCP) as one of the most important colonization factors of V. cholerae, this experimental study was designed to determine the role of TcpA in induction of mucin production and its regulatory effect on innate immunity molecules including toll like receptors (TLRs) and Nucleotide-binding oligomerization domain-containing proteins (NODs) using Caco2- PBMC co-cultures as an interactive model. The rTcpA protein of V. cholerae was expressed in BL21 Escherichia coli, purified using Ni-column chromatography and confirmed by western blotting. Nontoxic doses of rTcpA was determined on Caco-2 cell lines and different concentrations of rTcpA (1, 5, 10 and 50 μg/mL) showed a statistically significant effect on the expression of muc genes (MUC3 and MUC4) in a dose-dependent manner. This finding is supposed to facilitate physical adhesion and colonization of V. cholerae in intestinal lumen. The rTcpA moderately stimulated the expression of tlr4 and overexpressed tlr1, both of which are supposed to induce a mucosal protective response against bacterial infection. NOD2 was significantly increased which suggests that it may contribute in pro-inflammatory responses observed in cholera disease. No change in NOD1 expression was seen which might be attributed to the non-invasive nature of V. cholerae as an intestinal pathogen. In conclusion, the rTcpA protein of V. cholerae showed a statistically significant modulatory effect on the human gut epithelium gene expression which would help promising protection in prophylaxis applications.

Abstract 4519: Photochemically-primed immune cell enrichment and anti-tumor responses to pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is among the deadliest forms of cancer. Currently, it is the third leading cause of cancer deaths in the United States. At diagnosis, the majority of patients present with advanced primary or metastatic disease that is unresectable. PDAC pathophysiology is challenging for current therapies as immunosuppressive desmoplastic stroma limits responsiveness to treatments including immunotherapy. The latter condition is making a vast proportion of PDAC tumors being immunologically cold as defined by the lack of effective immune infiltration to the tumor. Thus, there is an unmet need for improved treatment strategies. Photodynamic therapy (PDT) is an approved modality that utilizes light, a photo-responsive photosensitizer (PS), and oxygen to generate reactive oxygen species that will kill nearby cells or prime neighboring cells to be more responsive to traditional therapies, and thereby produce a synergistic outcome. We aimed to investigate PDT-induced immunogenicity in PDAC tumors. In this study, we applied PDT in an immunocompetent mouse (C57BL/6 mice), PDAC tumor model and assessed tumour-infiltrating lymphocyte (TIL) infiltration at different time points. Visudyne® used as the PS. Multicolor flow cytometric analysis showed gradual increase of T and B cell infiltration at 1h time point to 5 days after PDT. T cell subset analysis showed a higher infiltration of CD8+ T cells with time. Localization of immune cells using immunofluorescence assays further confirmed latter findings. We observed a presence of proliferating T cells in the T cell zone of Spleen B cell follicles 1h after treating with PDT suggesting an early T cell activation. Activated dendritic cells were localized in the spleens treated with PDT compared to the untreated control. We further evaluated the anti-tumour immune reactivity of CD8+ T cells treated with PDT in an in vitro 3D system consisting of human PDAC cell line, MiaPaCa2, pancreatic fibroblast and human peripheral blood mononuclear cell co-culture. Tumor reactivity by CD8+ T cells was evaluated at baseline, 3 days, 7 days and 2 weeks in co-culture after PDT by staining for the surface expression of degranulation marker CD107a. There was a continuous CD107a upregulation in CD8+ T cells from day 3 to 2 week in co-culture treated with PDT compared to untreated controls demonstrating enhanced PDT priming of T cells, in vitro. In addition, there was an effective tumor cell killing in PDT treated immune-tumour co-cultures. Our data shows a possibility of triggering an immediate and effective immune-response to PDT in PDAC tumors enhancing the immunogenicity of this tumor type. Citation Format: Pushpamali De Silva, Mohammad Ahsan Saad, Assiris P. Camargo, Joseph Swain, Akilan Palanasami, Tayyaba Hasan. Photochemically-primed immune cell enrichment and anti-tumor responses to pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4519.

Abstract B49: Effects of exercise on ovarian cancer initiation and progression

Abstract Epidemiology studies support a strong association between physical activity and lower risk of cancer. Exercise is a promising nonpharmacologic strategy in preventing cancer and mitigating adverse effects of cancer and anticancer therapies. However, we have not been able to optimize the efficacy of exercise training as an anticancer intervention due to the lack of knowledge regarding the biologic mechanisms of action, the effective doses, and the biomarkers for predicting and monitoring responses of exercise-based intervention. Our objective is to utilize mouse models to determine the effects and underlying mechanisms of exercise on ovarian cancer initiation and progression. C57BL/6 mice ran on a treadmill for 45min daily to mimic human exercise activity. The control groups were placed on treadmill for 45min daily. After a month of this exercise regiment, both groups were injected with cancer cells derived from ovarian tumors of Dicer-/-; Pten-/-; Tp53R172H mice. Tumor progression was monitored through IVIS imaging system. Blood and peritoneal fluid samples were tested for cytokine levels. Tissue samples were collected and gene expression levels were quantified via qPCR. Peritoneal macrophages were isolated after acute exercise and were used in flow cytometry analysis or cocultured with cancer cells. Our model resembled the biologic changes of exercise in human. Eight-week treadmill-running exercise suppressed early tumor formation and tumor spreading. Exercise altered visceral fat tissue gene expression profile, which may inhibit cancer-induced cachexia and metabolic imbalance. In addition, one-hour exercise induced the acute proinflammatory activation of peritoneal macrophages. Coculture of peritoneal macrophages and PBMC (peripheral blood mononuclear cells) or NK (natural killer) cells from the exercise group and cancer cells reduced survival of cancer cells. Chronic and acute exercise can both suppress ovarian cancer tumorigenesis in mouse models. The underlying mechanisms involve enhancing anticancer immune system responses and adjusting metabolic balance. More research is needed to determine the specific mechanisms by which chronic and acute exercise affects ovarian cancer. Citation Format: Fangfang Bi, Madeline Morrisson, Sarah Cady, Alexandra Cerchia, Kevin Yang, Ivy Ling, Yang Yang-Hartwich. Effects of exercise on ovarian cancer initiation and progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B49.

Abstract A09: Activation of the CD45 receptor on leukocytes by the C24D peptide results in immune-mediated head and neck squamous cell carcinoma apoptosis

Abstract Objectives: CD45 is a highly conserved receptor-linked protein tyrosine phosphatase that is expressed on all leucocytes. C24D, a novel 24-amino acid dimer peptide, previously shown to activate immune-modulated breast cancer cell killing, binds to CD45, triggering leukocyte activation against cancer cells. The aim of this study was to evaluate the efficacy of C24D in activating leukocytes against head and neck squamous cell carcinoma (HNSCC). Methods: We performed a series of in vitro studies using 3 HNSCC cell lines (Cal33, FaDu, and A431). HNSCC cells were cocultured with peripheral blood mononuclear cells (PBMC) from healthy donors and subsequently treated with either C24D peptide or a scrambled control version. C24D activity was evaluated by tumor cell density and apoptosis analysis. Leukocyte activation by the peptide was evaluated by interferon gamma (IFN-γ) secretion. Results: Cocultures of HNSCC cells and PBMCs treated with C24D resulted in a substantial decrease in tumor cell density and increased apoptosis. IFN-γ levels were significantly elevated in the C24D treated PBMCs cocultured with HNSCC, suggesting leukocyte activation. Conclusions: C24D peptide activates leukocytes via CD45, causing immune cell-mediated killing of HNSCC cell lines. Citation Format: Aviram Mizrachi, Elad Hikri, Amit Ritter, Julia Dick-Lipovetsky, Sivan Simchi, Aron Popovtzer, Rinat Yerushalmi, Gideon Bachar, Anat Raiter. Activation of the CD45 receptor on leukocytes by the C24D peptide results in immune-mediated head and neck squamous cell carcinoma apoptosis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A09.

AB0039 ROLE OF MESENCHYMAL STEM CELLS ISOLATED FROM DENTAL PULP (DPSCS) IN IMMUNOREGULATION PROCESSES MEDIATED BY PROGRAMMED DEATH-LIGAND 1 (PD-L1)

Background:Stem cells isolated from dental pulp (DPSCs) are characterized by a high rate of proliferation, low immunogenicity and a high ability to differentiate in different lineages (i.e. osteogenic, chondrogenic, adipogenic, myogenic and neural commitment). Their multipotency can be attributed to the peculiar embryological origin from the neural crest. DPSCs represent a promising stem cell resource since they hold a low ethical impact and can be easily isolated through routine dental procedures. These cells own immuno-modulatory properties, exerted through the activation of different mechanisms, including the Fas / FasL pathway, as well as through the release of soluble factors. Currently, other molecular mechanisms are under consideration such as PD-1 / PD-L1 (Programmed Death 1 and its Ligand) which are supposed to be involved in the induction and / or maintenance of immune tolerance.Objectives:The aim of this research was to investigate whether the stimulation of PD-L1 in DPSCs can affect the immunomodulatory effects of these stem cells on peripheral blood mononuclear cells (PBMCs). Furthermore, the expression of PD-L1 was also assayed after the induction of osteogenic differentiation of DPSCs in order to evaluate a possible application of DPSCs in autoimmune inflammatory osteo-erosive diseases.Methods:Immuno-selection was performed on DPSCs, isolated from waste material, against the stemness markers c-Kit and STRO-1, to obtain a pure stem cell population. Then, STRO-1+/c-Kit+ DPSCs, were co-cultured either directly and indirectly with peripheral blood mononuclear cells (PBMCs) from healthy adult donors, previously activated by anti-CD3 and anti-CD28 antibodies. Co-cultures of PBMCs with amniotic fluid stem cells (AFSCs) and bone marrow mesenchymal stem cells (BM-MSCs) were also set up. The expression of PD-1 in PBMCs as well as of PD-L1 in DPSCs, AFSCs, BM-MSCs and PBMCs, was evaluated by Western Blot (WB) and immunofluorescence (IF) analyses, before and after osteogenic differentiation. Osteogenic differentiation of DPSCs, after 30 days of induction, was verified by IF and WB, of osteopontin, osteocalcin and RUNX2 markers. Interleukin-2 (IL-2) expression levels in PBMCs were analyzed by Real-Time PCR analysis.Results:Our data highlight that, after direct and indirect co-culture with activated PBMCs, PD-L1 expression was up-regulated not only in DPSCs, but also in BM-MSCs and AFSCs (Figure 1), thus suggesting that 1) this is a common ability of mesenchymal stem cells and 2) this event can be also mediated by soluble factors release. Moreover, when evaluating the effects of DPSCs co-culture on PBMCs an increased expression of cleaved caspase 3 was observed, together with a decreased expression of IL-2 - a growth factor essential for the proliferation and survival of T cells (Figure 2). These findings showed how DPSCs can modulate the immune system by PD-L1 up-regulation. On the other hand, it is noteworthy that, after reaching osteogenic commitment, DPSCs down-regulated the expression of PD-L1, allowing to hypothesize that PD-L1 expression is strictly related to the maintenance of stemness.Figure 1.Figure 2.Conclusion:Taken together, our findings suggest that the expression of PD-L1 in DPSCs is involved in the modulation of immune response and pave the way for further investigations on the role of PD-1/PD-L1 pathway in controlling inflammation and immune response when applied to the treatment of autoimmune inflammatory diseases.

The Inhibitory Mechanisms of Tumor PD-L1 Expression by Natural Bioactive Gallic Acid in Non-Small-Cell Lung Cancer (NSCLC) Cells.

Non-small-cell lung cancer (NSCLC) is the most common lung cancer subtype and accounts for more than 80% of all lung cancer cases. Epidermal growth factor receptor (EGFR) phosphorylation by binding growth factors such as EGF activates downstream prooncogenic signaling pathways including KRAS-ERK, JAK-STAT, and PI3K-AKT. These pathways promote the tumor progression of NSCLC by inducing uncontrolled cell cycle, proliferation, migration, and programmed death-ligand 1 (PD-L1) expression. New cytotoxic drugs have facilitated considerable progress in NSCLC treatment, but side effects are still a significant cause of mortality. Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a phenolic natural compound, isolated from plant derivatives, that has been reported to show anticancer effects. We demonstrated the tumor-suppressive effect of GA, which induced the decrease of PD-L1 expression through binding to EGFR in NSCLC. This binding inhibited the phosphorylation of EGFR, subsequently inducing the inhibition of PI3K and AKT phosphorylation, which triggered the activation of p53. The p53-dependent upregulation of miR-34a induced PD-L1 downregulation. Further, we revealed the combination effect of GA and anti-PD-1 monoclonal antibody in an NSCLC-cell and peripheral blood mononuclear–cell coculture system. We propose a novel therapeutic application of GA for immunotherapy and chemotherapy in NSCLC.

Abstract A17: Enhanced immune infiltration and antitumor immune reactivity in response to optical priming in pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) is one of the most lethal malignancies with a survival rate of approximately 5% at five years. At diagnosis, the majority of patients present with advanced primary or metastatic disease that is unresectable. PDAC pathophysiology is challenging for current therapies as immunosuppressive desmoplastic stroma limits responsiveness to treatments including immunotherapy. Latter condition is making vast proportion of PDAC tumors immunologically cold as defined by the lack of effective immune infiltration to the tumor. Thus, there is an unmet need for improved treatment strategies. Photodynamic therapy (PDT) is an approved modality that utilizes light, a photoresponsive photosensitizer (PS), and oxygen to generate reactive oxygen species that will kill nearby cells or prime neighboring cells to be more responsive to traditional therapies, and thereby produce a synergistic outcome. We aimed to investigate PDT-induced immunogenicity in PDAC tumors. In this study, we applied PDT in an immunocompetent mouse (C57BL/6 mice), PDAC tumor model and assessed tumor-infiltrating lymphocyte (TIL) infiltration at different time points. Visudyne was used as the PS. Multicolor flow cytometric analysis showed gradual increase of T- and B-cell infiltration at 1h time point to 5 days after PDT. T-cell subset analysis showed a higher infiltration of CD8+ T cells with time. Localization of immune cells using immunofluorescence assays further confirmed latter findings. We observed a presence of proliferating T cells in the T cell zone of spleen B cell follicles 1h after treating with PDT, suggesting an early T-cell activation. Activated dendritic cells were localized in the spleens treated with PDT compared to the untreated control. We further evaluated antitumor immune reactivity of CD8+ T cells treated with PDT in an in vitro 3D system consisting of human PDAC cell line, MiaPaCa2, pancreatic fibroblast, and human peripheral blood mononuclear cell coculture. Tumor reactivity by CD8+ T cells was evaluated at baseline, 3 days, 7 days, and 2 weeks in coculture after PDT by staining for the surface expression of degranulation marker CD107a. There was a continuous CD107a upregulation in CD8+ T cells from day 3 to 2 week in coculture treated with PDT compared to untreated controls, demonstrating enhanced PDT priming of T cells, in vitro. In addition, there was effective tumor cell killing in PDT-treated immune-tumor cocultures. Our data show a possibility of triggering an immediate and effective immune response to PDT in PDAC tumors, enhancing the immunogenicity of this tumor type. Citation Format: Pushpamali De Silva, Mohammad Ahsan Saad, Assiris P. Camargo, Joseph Swain, Akilan Palanasami, Girgis Obaid, Shalin Shetty, Tayyaba Hasan. Enhanced immune infiltration and antitumor immune reactivity in response to optical priming in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A17.

The IL-1 Antagonist Anakinra Attenuates Glioblastoma Aggressiveness by Dampening Tumor-Associated Inflammation.

Background: The recombinant IL-1 receptor antagonist anakinra—currently approved for the treatment of autoinflammatory diseases—blocks IL-1β-mediated inflammatory signaling. As inflammation is a major driver of cancer, we hypothesized that anakinra might be able to mitigate glioblastoma (GBM) aggressiveness. Methods: Primary GBM or T98G cells were incubated alone or with peripheral blood mononuclear cells (PBMCs) and were subsequently treated with IL-1β and/or anakinra. T cells were obtained by magnetic bead isolation. Protein and mRNA expression were quantified by SDS-PAGE, qRT-PCR, and ELISA, respectively. Cell proliferation and apoptosis were analyzed via flow cytometry. Chemotaxis was studied via time-lapse microscopy. Results: Upon IL-1β stimulation, anakinra attenuated proinflammatory gene expression in both GBM cells and PBMCs, and mitigated tumor migration and proliferation. In a more lifelike model replacing IL-1β stimulation by GBM–PBMC co-culture, sole presence of PBMCs proved sufficient to induce a proinflammatory phenotype in GBM cells with enhanced proliferation and migration rates and attenuated apoptosis. Anakinra antagonized these pro-tumorigenic effects and, moreover, reduced inflammatory signaling in T cells without compromising anti-tumor effector molecules. Conclusion: By dampening the inflammatory crosstalk between GBM and immune cells, anakinra mitigated GBM aggressiveness. Hence, counteracting IL-1β-mediated inflammation might be a promising strategy to pursue.

Dental follicle mesenchymal stem cells regulate responses in sepsis

Objective: Sepsis-induced immune alterations are associated with secondary infections and increased risk of death. The use of mesenchymal stem cells (MSCs) has been described as a novel therapeutic strategy. We evaluated the immunomodulatory effects of human dental follicle (DF-MSCs) on lymphocytes of sepsis and septic shock patients. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples of sepsis, septic shock and healthy subjects. PBMCs were co-cultured in the presence and absence of DF-MSCs with or without interferon-gamma (IFN-γ) for 72 hours. CD4+CD25+FoxP3+regulatory T (Treg) cell frequency, lymphocyte proliferation, cytokine levels and apoptosis were evaluated via flow cytometry. Results: DF-MSCs significantly suppressed proliferation of lymphocytes in sepsis group compared to septic shock group (p<0.005). DF-MSCs remarkably increased Treg ratio in sepsis compared to control group (p<0.05). Reduction of lymphocyte apoptosis in cocultures of DF-MSCs and PBMC was significant in both sepsis and septic shock groups. IFN-γ stimulation of DF-MSCs ameliorated shift in the T-cell subsets from Th2 to Th1 phenotype in septic shock. Conclusion: Our findings revealed that DF-MSCs have immunoregulatory effects both in sepsis and septic shock, by reducing interleukin-4 (IL-4) and increasing IFN-γ levels. This immunoreactivity regulation may open new therapeutic approaches for septic shock patients.