Co-culture Of Peripheral Blood Mononuclear Cells Research Articles

PBMCs as Tool for Identification of Novel Immunotherapy Biomarkers in Lung Cancer.

Lung cancer (LC), including both non-small (NSCLC) and small (SCLC) subtypes, is currently treated with a combination of chemo- and immunotherapy. However, predictive biomarkers to identify high-risk patients are needed. Here, we explore the role of peripheral blood mononuclear cells (PBMCs) as a tool for novel biomarkers searching. We analyzed the expression of the cGAS-STING pathway, a key DNA sensor that activates during chemotherapy, in PBMCs from LC patients divided into best responders (BR), responders (R) and non-responders (NR). The PBMCs were whole exome sequenced (WES). PBMCs from BR and R patients of LC cohorts showed the highest levels of STING (p < 0.0001) and CXCL10 (p < 0.0001). From WES, each subject had at least 1 germline/somatic alteration in a DDR gene and the presence of more DDR gene mutations correlated with clinical responses, suggesting novel biomarker implications. Thus, we tested the effect of the pharmacological DDR inhibitor (DDRi) in PBMCs and in three-dimensional spheroid co-culture of PBMCs and LC cell lines; we found that DDRi strongly increased cGAS-STING expression and tumor infiltration ability of immune cells in NR and R patients. Furthermore, we performed FACS analysis of PBMCs derived from LC patients from the BR, R and NR cohorts and we found that cytotoxic T cell subpopulations displayed the highest STING expression. cGAS-STING signaling activation in PBMCs may be a novel potential predictive biomarker for the response to immunotherapy and high levels are correlated with a better response to treatment along with an overall increased antitumor immune injury.

Abstract 100: Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer

Abstract Background: Cancer immune evasion remains a major barrier in developing enduring therapies for epithelial ovarian cancer (EOC). Expression of programmed death-ligand 1 (PD-L1) is known for its immunosuppressive role in cancer, thus interferences to PD-L1 have shown marked efficacy against many advanced cancers. However, the mechanisms by which PD-L1-negative cancers evade immune response remain unelucidated. We have found chemokine (C-C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) to up-regulate in PD-L1-negative EOC. Therefore, the present study aims to investigate blockade of CCL5/CXCL10 signaling to overcome immune evasion of EOC. Methods: Isogenic BRCA2-mutated PEO1 and BRCA2-wild type PEO4 EOC cells were treated with IFNγ, olaparib, or both in combination for western blot and flow cytometric analyses of PD-L1 expression. The conditioned medium of PEO1 and PEO4 cells was analyzed for immune array analysis to determine and compare the levels of 36 cytokines. PEO1 and PEO4 cells were co-cultured with CD3/CD28 antibodies-activated human peripheral blood mononuclear cells (PBMCs). Then, PBMCs were stained with anti-CD8 or CD4 and CD25 antibodies for flow cytometric analyses of cytotoxic or helper T cells, respectively. PEO1 and PEO4 cells were co-cultured with activated PBMCs in the presence and absence of TAK-779 or AMG-487. Thereafter, PBMCs were stained with anti-CD4, CD25, and FOXP3 antibodies for flow cytometric analyses of regulatory T cells (Tregs). PEO1 and PEO4 cells were transfected with CCL5- or CXCL10-siRNA and subsequently co-cultured with activated SUPT1 cells or PBMCs. The conditioned medium was collected and analyzed for the levels of IFNγ by ELISA. Results: PEO1 cells exhibited an up-regulation of basal and IFNγ-induced PD-L1 compared with PEO4 cells. PEO4 cells displayed a pronounced increase in the CCL5 and IFNγ-induced CXCL10 compared with PEO1 cells. Co-culture of PBMCs with PEO1 and PEO4 cells led to a significant decrease in activated CD8+ effector T and CD4+ helper T cells. However, co-culture of PBMCs with PEO4 cells caused a significant increase in activated CD4+ helper T cells. Co-culture of PBMCs with PEO4 cells caused a significant increase in FOXP3+CD4+CD25+ Tregs. This increase in Tregs was significantly reduced by the treatment with the CCL5/CCR5 antagonist TAK-779 or the CXCL10/CXCR3 antagonist AMG-487. Silencing of CCL5 or CXCL10 in PEO4 cells by siRNA significantly increased IFNγ production by SUPT1 cells and PBMCs. Conclusions: PEO4 cells secrete CCL5 and IFNγ-induced CXCL10 to activate Tregs, enabling tumor cells to evade host immune response by dysregulating their microenvironment. PEO4 cells-induced Tregs are reduced by CCL5/CXCL10 signaling blockade with TAG-779 or AMG-487. Furthermore, silencing of CCL5 or CXCL10 in PEO4 cells with siRNA abolishes Treg-mediated immunosuppression and restores the antitumor activity of effector T cells. Citation Format: Annabelle C. Lin, Jake Moscarelli, Z. Ping Lin, Elena S. Ratner. Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 100.

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Adjuvant AS01 activates human monocytes for costimulation and systemic inflammation

BackgroundThe adjuvanted recombinant zoster vaccine (RZV) is highly effective even in adults over 80 years old. The high efficacy of RZV is attributed to its highly reactogenic adjuvant, AS01, but limited studies have been done on AS01’s activation of human immune cells. MethodsWe stimulated peripheral blood mononuclear cells (PBMC) with AS01 and used flow cytometry and RNA Sequencing (RNAseq) to analyze the impacts on human primary cells. ResultsWe found that incubation of PBMC with AS01 activated monocytes to a greater extent than any other cell population, including dendritic cells. Both classical and non-classical monocytes demonstrated this activation. RNASeq showed that TNF-ɑ and IL1R pathways were highly upregulated in response to AS01 exposure, even in older adults. ConclusionsIn a PBMC co-culture, AS01 strongly activates human monocytes to upregulate costimulation markers and induce cytokines that mediate systemic inflammation. Understanding AS01’s impacts on human cells opens possibilities to further address the reduced vaccine response associated with aging.

Butyrate Protects Barrier Integrity and Suppresses Immune Activation in a Caco-2/PBMC Co-Culture Model While HDAC Inhibition Mimics Butyrate in Restoring Cytokine-Induced Barrier Disruption.

Low-grade inflammation and barrier disruption are increasingly acknowledged for their association with non-communicable diseases (NCDs). Short chain fatty acids (SCFAs), especially butyrate, could be a potential treatment because of their combined anti-inflammatory and barrier- protective capacities, but more insight into their mechanism of action is needed. In the present study, non-activated, lipopolysaccharide-activated and αCD3/CD28-activated peripheral blood mononuclear cells (PBMCs) with and without intestinal epithelial cells (IEC) Caco-2 were used to study the effect of butyrate on barrier function, cytokine release and immune cell phenotype. A Caco-2 model was used to compare the capacities of butyrate, propionate and acetate and study their mechanism of action, while investigating the contribution of lipoxygenase (LOX), cyclooxygenase (COX) and histone deacetylase (HDAC) inhibition. Butyrate protected against inflammatory-induced barrier disruption while modulating inflammatory cytokine release by activated PBMCs (interleukin-1 beta↑, tumor necrosis factor alpha↓, interleukin-17a↓, interferon gamma↓, interleukin-10↓) and immune cell phenotype (regulatory T-cells↓, T helper 17 cells↓, T helper 1 cells↓) in the PBMC/Caco-2 co-culture model. Similar suppression of immune activation was shown in absence of IEC. Butyrate, propionate and acetate reduced inflammatory cytokine-induced IEC activation and, in particular, butyrate was capable of fully protecting against cytokine-induced epithelial permeability for a prolonged period. Different HDAC inhibitors could mimic this barrier-protective effect, showing HDAC might be involved in the mechanism of action of butyrate, whereas LOX and COX did not show involvement. These results show the importance of sufficient butyrate levels to maintain intestinal homeostasis.

Bi-directional epithelial-immune communication amplifies inflammasome activation and IL-6 release during SARS-CoV-2 infection

Abstract Increased levels of the pro-inflammatory cytokines IL-1β and IL-6 are linked to severe COVID-19. Here, we investigate the contributions of viral host cells, primary human airway epithelia (HAE), and immune bystanders, peripheral blood mononuclear cells (PBMCs), in the production of these cytokines. Despite having functional inflammasomes and supporting viral replication, HAE do not secrete IL-1β upon SARS-CoV-2 infection. PBMCs do not support productive viral replication, but the SARS-CoV-2 envelope (E) protein primes inflammasomes through stimulation of TLR2. To activate inflammasomes in PBMCs, SARS-CoV-2-infected HAE provide a second signal in the form of damage-associated molecular patterns (DAMPs), which includes mitochondrial and genomic DNA. Co-culture of PBMCs with infected epithelial cells stimulates IL-1β release, while addition of benzonase, STING, or caspase-1 inhibitors, but not NLRP3 inhibitors, abrogates this response. In turn, IL-1β stimulates IL-6 secretion from both populations with a robust response from HAE. Thus, while infection in either cell type in isolation does not yield IL-1β or IL-6, immune bystander detection of SARS-CoV-2 infection in the epithelium stimulates both IL-1β and IL-6 production, amplifying pro-inflammatory cytokine production through cell-cell communication. This circuit is reflected in patient data, as COVID-19 autopsy lungs show hallmarks of inflammasome priming and activation in myeloid populations lacking SARS-CoV-2 antigens. Meta-analysis of single cell RNA sequencing (scRNAseq) data reveals discordant, exacerbated priming of inflammasomes in myeloid cells from severe but not mild COVID-19 patients, indicating this circuit is intensified in severe disease. Supported by grants from NIH R56 AI158314, R01 AI158314, AI029564, AI141333 and CA232109 to J.P.-Y.T; NCI T32CA071341 to K.C.B.; UH3 HL123645, R01 HL136961, P30 DK 065988, P01 HL108808, and from the Cystic Fibrosis Foundation (CFF) BOUCHE19R0 to T.A. and R.C.B; CFF grant BOUCHE19XX0 to R.C.B. and K.O.; CFF grant OKUDA20G0 KO; NC Policy Collaboratory, NIH R01AI157253, and U19 AI100625 to M. T. H.; NSF GRFP 2019255197 to J.F.L. T.A. is a Japan Society for the Promotion of Science Overseas Research Fellow. Y.X. is supported by NSF DMS-191290 and NSF IOS 2107215. Y.X and Y.L.L. are supported by NIH grants R01DE026728 and R01DE031951. Some BSL3 work was performed in the Duke Regional Biocontainment Laboratory (Duke RBL) which received partial support for construction from NIH/NIAID (UC6AI058607).

PM10 promotes an inflammatory cytokine response that may impact SARS-CoV-2 replication in vitro.

In the last decades, a decrease in air quality has been observed, mainly associated with anthropogenic activities. Air pollutants, including particulate matter (PM), have been associated with adverse effects on human health, such as exacerbation of respiratory diseases and infections. High levels of PM in the air have recently been associated with increased morbidity and mortality of COVID-19 in some regions of the world. To evaluate the effect of coarse particulate matter (PM10) on the inflammatory response and viral replication triggered by SARS-CoV-2 using in vitro models. Peripheral blood mononuclear cells (PBMC) from healthy donors were treated with PM10 and subsequently exposed to SARS-CoV-2 (D614G strain, MOI 0.1). The production of pro-inflammatory cytokines and antiviral factors was quantified by qPCR and ELISA. In addition, using the A549 cell line, previously exposed to PM, the viral replication was evaluated by qPCR and plaque assay. SARS-CoV-2 stimulation increased the production of pro-inflammatory cytokines in PBMC, such as IL-1β, IL-6 and IL-8, but not antiviral factors. Likewise, PM10 induced significant production of IL-6 in PBMCs stimulated with SARS-CoV-2 and decreased the expression of OAS and PKR. Additionally, PM10 induces the release of IL-1β in PBMC exposed to SARS-CoV-2 as well as in a co-culture of epithelial cells and PBMCs. Finally, increased viral replication of SARS-CoV-2 was shown in response to PM10. Exposure to coarse particulate matter increases the production of pro-inflammatory cytokines, such as IL-1β and IL-6, and may alter the expression of antiviral factors, which are relevant for the immune response to SARS-CoV-2. These results suggest that pre-exposure to air particulate matter could have a modest role in the higher production of cytokines and viral replication during COVID-19, which eventually could contribute to severe clinical outcomes.

Increased Galectin-9 Levels Correlate with Disease Activity in Patients with DMARD-Naïve Rheumatoid Arthritis and Modulate the Secretion of MCP-1 and IL-6 from Synovial Fibroblasts.

Background: Fibroblast-like synoviocytes (FLSs) are essential mediators in the expansive growth and invasiveness of rheumatoid synovitis, and patients with a fibroblastic-rich pauci-immune pathotype respond poorly to currently approved antirheumatic drugs. Galectin-9 (Gal-9) has been reported to directly modulate rheumatoid arthritis (RA) FLSs and to hold both pro- and anti-inflammatory properties. The objective of this study was to evaluate clinical and pathogenic aspects of Gal-9 in RA, combining national patient cohorts and cellular models. Methods: Soluble Gal-9 was measured in plasma from patients with newly diagnosed, treatment-naïve RA (n = 98). The disease activity score 28-joint count C-reactive protein (DAS28CRP) and total Sharp score were used to evaluate the disease course serially over a two-year period. Plasma and synovial fluid samples were examined for soluble Gal-9 in patients with established RA (n = 18). A protein array was established to identify Gal-9 binding partners in the extracellular matrix (ECM). Synovial fluid mononuclear cells (SFMCs), harvested from RA patients, were used to obtain synovial-fluid derived FLSs (SF-FLSs) (n = 7). FLSs from patients suffering from knee Osteoarthritis (OA) were collected from patients when undergoing joint replacement surgery (n = 5). Monocultures of SF-FLSs (n = 6) and autologous co-cultures of SF-FLSs and peripheral blood mononuclear cells (PBMCs) were cultured with and without a neutralizing anti-Gal-9 antibody (n = 7). The mono- and co-cultures were subsequently analyzed by flow cytometry, MTT assay, and ELISA. Results: Patients with early and established RA had persistently increased plasma levels of Gal-9 compared with healthy controls (HC). The plasma levels of Gal-9 were associated with disease activity and remained unaffected when adding a TNF-inhibitor to their standard treatment. Gal-9 levels were elevated in the synovial fluid of established RA patients with advanced disease, compared with corresponding plasma samples. Gal-9 adhered to fibronectin, laminin and thrombospondin, while not to interstitial collagens in the ECM protein array. In vitro, a neutralizing Gal-9 antibody decreased MCP-1 and IL-6 production from both RA FLSs and OA FLSs. In co-cultures of autologous RA FLSs and PBMCs, the neutralization of Gal-9 also decreased MCP-1 and IL-6 production, without affecting the proportion of inflammatory FLSs. Conclusions: In RA, pretreatment plasma Gal-9 levels in early RA were increased and correlated with clinical disease activity. Gal-9 levels remained increased despite a significant reduction in the disease activity score in patients with early RA. The in vitro neutralization of Gal-9 decreased both MCP-1 and IL-6 production in an inflammatory subset of RA FLSs. Collectively these findings indicate that the persistent overexpression of Gal-9 in RA may modulate synovial FLS activities and could be involved in the maintenance of subclinical disease activity in RA.

Downregulation of HMGB1 in thymoma cells affects T cell differentiation.

Thymoma is the most common anterior mediastinal tumor and is closely associated with myasthenia gravis (MG). Our previous study showed that the expression of Th17 cells increased and the expression of Treg decreased in MG-associated thymoma tissues and peripheral blood. High mobility group box 1 (HMGB1) is an inflammatory mediator and participates in the pathogenesis of various autoimmune diseases. However, its function in thymoma is still unclear. Material and methods: We first analyzed immune indices in peripheral blood of patients with MG-associated thymoma and patients with thymoma alone. Next, we explored the expression of HMGB1 in MG-associated thymoma and thymoma alone tissues. Furthermore, we transfected si-HMGB1 in thymoma cell line Thy0517 and co-cultured Thy0517 with peripheral blood mononuclear cells (PBMC). In this study, the levels of IgG, C3, C4, CRP and globulins in peripheral blood of patients with MG-associated thymoma were different from those of patients with thymoma alone (p < 0.05). The expression of HMGB1 in MG-associated thymoma tissues was higher than thymoma alone. Co-culture of Thy0517 and PBMC showed that the percentage of Th17 cells in PBMC was lower than that in the control group, and the percentage of Treg cells was higher than that in the control group. These findings demonstrate that HMGB1 is involved in the mechanism of abnormal Th17/Treg cell differentiation in thymoma and affects the occurrence of immune abnormalities in MG-associated thymoma.

Potentiation of Antitumor Activity by Antibody Drugs and Mushroom-Derived β-Glucans in Natural Killer Cell-Mediated Tumoricidal Activities against Non-Hodgkin's B-Cell Lymphoma.

β-glucans are polysaccharides that activate innate immunity. We herein investigated whether P-glucans promote the immunological effects of antibody drugs against malignant tumor cells using human peripheral blood mononuclear cells (PBMCs). Rituximab bound to CD20-specific lymphoma and exhibited cytotoxic activity in the presence of human mononuclear cells, but not neutrophils. The addition of Sparassis crispa (cauliflower mushroom)-derived β-glucan (SCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) to co-cultures of PBMCs and Raji lymphoma cells further promoted antibody-dependent cell-mediated cytotoxicity (ADCC). The GM-CSF treatment increased β-glucan receptor expression on adherent cells in PBMCs. A co-stimulation with GM-CSF and SCG of PBMCs induced an increase in the number of spreading cells and the activation of natural killer (NK) cells. The enhancement in ADCC was abolished by the removal of NK cells, indicating that SCG and GM-CSF increased ADCC against lymphoma by activating β-glucan receptor-expressing cells in PBMCs and enhancing NK cell activity. The synergistic mechanisms of action of mushroom-derived β-glucans and biopharmaceuticals, including recombinant cytokines and antibodies, in the treatment of malignant tumor cells provide important insights into the clinical efficacy of β-glucans from mushrooms.

Epithelial-derived galectin-9 containing exosomes contribute to the immunomodulatory effects promoted by 2'-fucosyllactose and short-chain galacto- and long-chain fructo-oligosaccharides.

Early life exposure to non-digestible oligosaccharides (NDO) or microbial components is known to affect immune development. NDO in combination with a TLR9 agonist mimicking bacterial triggers (CpG) promoted the secretion of galectins through unknown pathways. We aimed to study the contribution of exosomes in epithelial galectin secretion and subsequent immunoregulation upon exposure to a mixture of NDO by inhibiting exosome biogenesis. Human intestinal epithelial cells (IEC) (FHs 74 Int or HT-29) were apically exposed to 2'-fucosyllactose (2'FL) and short-chain galacto- and long-chain fructo-oligosaccharides (GF), alone or with CpG. Basolaterally, non-activated or αCD3/CD28-activated peripheral blood mononuclear cells (PBMC) were added. After 24h incubation, IEC were washed and incubated in fresh medium to analyze epithelial-derived galectin secretion. Additionally, before exposure to NDO and CpG, IEC were exposed to GW4869 to inhibit exosome biogenesis. After 24h of incubation, IEC were washed and incubated for additional 24h in the presence of GW4869, after which epithelial-derived galectin secretion was studied. Also, epithelial-derived exosomes were isolated to study the presence of galectins within the exosomes. Compared to CpG alone, exposure to 2'FL/GF mixture and CpG, significantly enhanced Th1-type IFNγ, and regulatory IEC-derived galectin-9 secretion in the HT-29/PBMC model. Similarly, in the FHs 74 Int/PBMC co-culture, 2'FL/GF induced immunomodulatory effects in the absence of CpG. Interestingly, galectin-9 and -4 were present in CD63-expressing exosomes isolated from HT-29 supernatants after IEC/PBMC co-culture. Exposure to GW4869 suppressed 2'FL/GF and CpG induced epithelial-derived galectin-9 secretion, which subsequently prevented the rise in IL-10 and reduction in IL-13 secretion observed in the HT-29/PBMC co-culture model upon exposure to 2'FL/GF and CpG. Exposure to 2'FL/GF and CpG or 2'FL/GF promoted Th1-type regulatory effects in HT-29/PBMC or FHs 74 Int/PBMC co-culture respectively, while Th2-type IL-13 was reduced in association with increased galectin-9 release. Galectin-9 and -4 were present in exosomes from HT-29 and the inhibition of exosome biogenesis inhibited epithelial-derived galectin secretion. This, also affected immunomodulatory effects in IEC/PBMC co-culture suggesting a key role of galectin expressing IEC-derived exosomes in the mucosal immune regulation induced by NDO.

Is There an Influence of Electrically Stimulated Osteoblasts on the Induction of Osteoclastogenesis?

Bone is a highly dynamic tissue characterized mainly by the interactions of osteoblasts and osteoclasts. When the healing ability of bone regeneration is disturbed, targeted biophysical stimulations such as electrical stimulation are applied. In this study the indirect effects of electrically stimulated human osteoblasts on osteoclastogenesis were investigated to better understand detailed cellular interactions. Therefore, two different cell developmental stages were examined: peripheral blood mononuclear cells (PBMCs) as precursors and pre-osteoclasts as differentiated cells. Previously, over a 21-day period, human osteoblasts were stimulated with a low-frequency alternating electric field. The supernatants were collected and used for an indirect co-culture of PBMCs and pre-osteoclasts. The cellular viability and the induction of differentiation and activity were analyzed. Further, the secretion of relevant osteoclastic markers was examined. Supernatants of 7 d and 14 d stimulated osteoblasts led to a decrease in the viability of PBMCs and an increased number of cells containing actin ring structures. Supernatants from osteoblasts stimulated over 7 d induced PBMC differentiation and pre-osteoclastic activation. Furthermore, pre-osteoclasts showed varying mRNA transcripts of MCP-1, ACP5, CA2, and CASP8 when cultivated with media from osteoblasts. Supernatants from day 21 did not influence PBMCs at all but increased the viability of pre-osteoclasts. We could show that different time points of stimulated osteoblasts have varying effects on the cells and that changes can be observed due to the differentiation stages of the cells. Through the effects of the indirect stimulation, it was possible to underline the importance of studying not only osteoblastic differentiation and mineralization behavior under electric stimulation but also analyzing changes in osteoclastogenesis and the activity of osteoclasts.

414.6: Porcine ULBP1 Disruption Does Not Significantly Reduce Human NK Cell Activation and Cytotoxicity in an in Vitro Model

Background: Pig-to-human xenotransplantation has the potential to address the critical organ shortage. Genetically-engineered pig organs have significantly prolonged survival in human and nonhuman primates by overcoming hyperacute rejection. Human natural killer (NK) cell-mediated acute xenograft rejection of porcine endothelial cells has been established. Porcine UL-Binding Protein (ULBP1) has been identified as a porcine ligand of human NK cell activating receptor NKG2D; interactions presumably lead to NK cell activation and ultimately porcine cell cytotoxicity. We sought to improve pig-to-human compatibility by eliminating ULBP1 ligand in an immortalized porcine liver-derived endothelial cell line (ipLDEC) with five-gene knockout (5GKO). The 5GKO cell line has mutations in GGTA1/CMAH/β4galNT2/SLA-I α-chain/β2M: three genes encoding enzymes responsible for xenoantigen production and two genes encoding swine leukocyte antigen class I. Methods: CRISPR/Cas9 technology was used to knockout the porcine ULBP1 gene in the 5GKO cell line to create a ULBP1-KO/5GKO cell line. Human peripheral blood mononuclear cells (PBMCs) from four donors were activated with human IL-2 for five days. Human PBMCs were co-cultured with 5GKO, ULBP1-KO/5GKO, and Wild-Type (WT) ipLDECs. Cells were stained with antibodies to CD45, CD3, CD56, and CD107a. Flow cytometry analysis was gated on the CD3-CD56+ NK cell population and monitored for expression of CD107a, a marker of NK cell degranulation. Calcein AM cytotoxicity assay was performed to determine human NK cell-mediated ipLDECs cell death. WT, 5GKO, ULBP1-KO/5GKO, HLA-G+/5GKO (positive control) were incubated in a calcein solution for 30 minutes at a concentration of 106 cells/mL. Stained porcine cells were co-cultured with activated human PBMCs at a 10:1 E:T ratio for 4 hours. Co-culture supernatant was harvested and calcein release was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm to calculate cytotoxicity percentages. Results: Porcine ULBP1 gene mutation was confirmed by Sanger sequencing (Figure 1a), establishing a porcine ULBP1-KO/5GKO cell line. There is no statistically significant difference in NK cell activation between co-cultures of human PBMCs (n=4) with pULBP1-KO/5GKO and 5GKO ipLDEC using CD107a. (Figure 1b). There is no statistically significant difference in NK-induced cytotoxicity between co-cultures of human PBMCs (n=6) with porcine ULBP1-KO/5GKO and 5GKO cell lines using Calcein AM assays (Figure 1c). As expected, HLA-G+/5GKO cytotoxicity was decreased (p = 0.0168) compared to WT (Figure 1c). Conclusion: We established a porcine ULBP1-KO/5GKO cell line. Our studies demonstrate no statistically significant difference in NK cell activation and NK cell-mediated cytotoxicity between 5GKO and ULBP1-KO/5GKO cell lines. Our studies show that ULBP1 is not a crucial ligand for human NKG2D. Future studies will target different human NK cell activating porcine ligands.

Chemokine Assay Matrix Defines the Potency of Human Bone Marrow Mesenchymal Stromal Cells

Potency analysis of mesenchymal stromal cells (MSCs) is required for their use in advanced clinical trials. Assay matrix strategy evaluating more than a single property of MSCs is an emerging strategy in potency analysis. Here we developed an assay matrix approach focusing on the secretory chemokine responses of MSCs using multiplex analytical method. MSCs’ innate fitness in secreting matrix of chemokines is correlated with their metabolic fitness in differential degrees. In addition, innately secreting chemokines are correlated among themselves in a unique pattern. MSC’s matrix chemokine responses to exogenous stimulation of IFNγ and/or TNFα are distinct. However, the combination of IFNγ and TNFα is superior than individual stimulations in eliciting robust and broad matrix chemokine responses of MSCs. Correlation matrix analysis has identified that chemokine responses to IFNγ and/or TNFα display unique correlative secretion patterns. MSC and peripheral blood mononuclear cells coculture analysis has identified the correlation matrix responses of chemokines that predicted immune suppression. In addition, MSC-mediated blocking of T-cell proliferation predominantly correlates with chemokines in an inverse manner. Knockdown of chemokines has demonstrated that MSC-sourced inherent chemokines do not actively play a role in T-cell suppression and thus are the bystander predictors of T-cell suppression. The present analysis of MSC’s matrix chemokine responses can be deployed in the advanced potency analysis of MSCs.

Abstract 1113: NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells

Abstract Background: NUC-3373 is a phosphoramidate modification of fluorodeoxyuridine-monophosphate (FUDR-MP), the active anti-cancer metabolite of fluorouracil (5-FU), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373 is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, reduce generation of toxic metabolites, and deliver higher levels of active metabolite FUDR-MP to tumors. Our previous work demonstrated that NUC-3373 causes colorectal cancer (CRC) cell lines to release damage associated molecular patterns (DAMPs)- molecular signals that activate circulating immune cells leading to immunogenic cell death (ICD). NUC-3373 promotes activation of co-cultured NK-92 MI cells, a natural killer cell line, by upregulating degranulation and IFN-γ production and downregulating the immune checkpoint molecule TIGIT. This study aims to test the hypothesis that NUC-3373 induced DAMPs promote ICD in co-cultures of CRC cells with peripheral blood mononuclear cells (PBMCs). We also explore the dynamics of PD-L1 and cytokine expression and combination with a PD-1 checkpoint inhibitor. Methods: HCT116 cells were exposed to increasing concentrations of NUC-3373 or vehicle control for 24 hours prior to co-culture with healthy patient-derived PBMCs. At the time of co-culture, cells were treated with 10 υg/ml nivolumab to assess the effect of an anti-PD-1 antibody being combined with NUC-3373. CRC and PBMC co-cultures were continued for up to 72 hours. Gene expression of PD-L1 and cytokines (TNF-α, IFN-γ, and IL-2) was assessed by RT-PCR. PD-L1 surface expression was determined by flow cytometry. Confluence was determined by automated cytometry analysis (Celigo) and cell viability of CRC cells was determined by sulforhodamine-B assay (SRB). Results: NUC-3373 increased the gene expression of cytokines and PD-L1 over 48 hours of co-culture. Surface expression of PD-L1 was increased on both PBMCs and CRC cells in co-culture, as well as on CRC cells cultured without PBMCs, indicating that global increase in PD-L1 is NUC-3373 mediated. CRC cells pre-treated with NUC-3373 and co-cultured with PBMCs displayed a reduction in cell viability compared to monocultured CRC cells. Furthermore, CRC and PBMC co-cultures treated with anti-PD-1 antibody showed increased cytotoxicity when CRC cells were pre-treated with NUC-3373. Conclusions: NUC-3373 induces DAMPs and PD-L1 expression in CRC cells and promotes pro-immune cytokine production from PBMCs resulting in ICD in vitro. Furthermore, the addition of anti-PD-1 antibody to co-cultures of NUC-3373-treated CRC cells and PMBCs enhanced this ICD. With an improved clinical safety profile (NCT03428958), including less haematologic toxicity, and a more convenient dosing regimen than 5-FU, NUC-3373 is an attractive combination partner for immune checkpoint inhibitors. Citation Format: Oliver James Read, Jennifer Bré, David J. Harrison. NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1113.

Mesoporous silica nano-adjuvant triggers pro-inflammatory responses in Caco-2/peripheral blood mononuclear cell (PBMC) co-cultures.

The aim of this study was to evaluate the cytotoxicity and immune-stimulatory effect of Mesoporous silica nanoparticle (MSN) Nano-adjuvant on pro-inflammatory cytokines and pattern recognition receptors (PRR) genes expression in Caco-2/PBMC co-culture model. MSNs were synthesized and characterized by scanning electron microscope (SEM), Brunauer Emmett Teller (BET) and Barrett Joyner Halenda (BJH) techniques. The BET specific surface area of MSNs was around 947 m2/g and the total pore volume and average pore diameter were 1.5 cm3/g and 8.01 nm, respectively. At the concentration of 10 µg/mL, MSN showed a low and time-dependent cytotoxicity on Caco-2 cells, while no cytotoxic effect was observed for 0.1 and 1 µg/mL concentrations after 24, 48 and 72 h. The expression of pro-inflammatory cytokines genes (IL-1, IL-8 and TNF-α) in co-cultures treated with different concentrations of MSN showed a dose-dependent significant increase up to 17.44, 2.722 and 4.34 folds, respectively, while the expression augmentation of IL-1 gene was significantly higher than the others. This indicates slight stimulation of intestinal inflammation. Different concentrations of MSN significantly increased TLR4 and NOD2 expression to 4.14 and 2.14 folds, respectively. NOD1 was not affected significantly. It can be concluded that MSN might increase protective immune responses against antigens as a vaccine adjuvant candidate. It seems that stimulation of TNF-α, IL-1, and IL-8 expression in enterocytes probably transpires through the agonistic activity of MSN for TLRs including TLR4, while NOD2-associated signaling pathways are also involved. This study provides an overall picture of MSN as a novel and potent oral adjuvant for mucosal immunity.

Nitro-fatty acids decrease type I interferons and monocyte chemoattractant protein 1 in ex vivo models of inflammatory arthritis

BackgroundInflammatory arthritis including rheumatoid arthritis (RA) and spondyloarthritis (SpA) is characterized by inflammation and destruction of the joints. Approximately one third of patients do not respond to first-line treatments. Nitro-fatty acids are bioactive lipids with anti-inflammatory properties and tissue-protective functions. The nitro-fatty acid 10-NO2-oleic acid (10-NO2-OA) is being tested in clinical trials for patients with fibrotic and inflammatory conditions. Here, we tested whether 10-NO2-OA could inhibit immune reactions involved in the inflammatory and joint destructive processes in inflammatory arthritis.MethodsSynovial fluid and blood samples were obtained from 14 patients with active RA or SpA. The in vitro models consisted of synovial fluid mononuclear cells (SFMCs) cultured for 48 h, SFMCs cultured for 21 days, and fibroblast-like synovial cells (FLSs) co-cultured with peripheral blood mononuclear cells (PBMCs) for 48 h. Cells were treated with or without 10-NO2-OA or the tumor necrosis factor alpha (TNFα) inhibitor etanercept. Supernatants were analyzed for type I interferon, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase 3 (MMP3) and tartrate resistant acid phosphatase (TRAP).ResultsIn SFMCs cultured for 48 h, 10-NO2-OA dose-dependently decreased the secretion of bioactive type I interferons and MCP-1 but not MMP3 (P = 0.032, P = 0.0001, and P = 0.58, respectively). Both MCP-1 and MMP3 were decreased by etanercept (P = 0.0031 and P = 0.026, respectively). In SFMCs cultured for 21 days, 10-NO2-OA significantly decreased the production of MCP-1 but not TRAP (P = 0.027 and P = 0.1523, respectively). Etanercept decreased the production of TRAP but not MCP-1 (P < 0.001 and P = 0.84, respectively). In co-cultures of FLSs and PBMCs, 10-NO2-OA decreased the production of MCP-1 (P < 0.0001). This decrease in MCP-1 production was not seen with etanercept treatment (P = 0.47).Conclusion10-NO2-OA decreased the release of MCP-1 in three models of inflammatory arthritis. Of particular interest, 10-NO2-OA inhibited type I interferon, and 10-NO2-OA was more effective in reducing MCP-1 production in cultures dominated by FLSs compared with etanercept. Our results encourage clinical investigations of 10-NO2-OA in patients with inflammatory arthritis.

Human umbilical cord mesenchymal stem cells regulate CD54 and CD105 in vascular endothelial cells and suppress inflammation in Kawasaki disease

ObjectiveThe objective was to evaluate the expression levels of CD31+CD54+ and CD31+CD105+ endothelial microparticles (EMPs) before and after intravenous immunoglobulin (IVIG) treatment of Kawasaki disease (KD). To explore the role of human umbilical cord mesenchymal stem cells (hucMSCs) in inhibiting endothelial inflammation in KD, the effects of hucMSCs on the expression of CD54 and CD105 in endothelial cells in KD were analyzed in vivo and in vitro. MethodsThe concentrations of IL-1β and VEGF in the peripheral blood of KD or healthy children were detected, and the distributions of CD31+CD54+ and CD31+CD105+ EMPs in platelet-poor plasma (PPP) were analyzed by flow cytometry. Human umbilical vein endothelial cells (HUVECs) were first cocultured with the patients’ peripheral blood mononuclear cells (PBMCs). Next, HUVECs were cocultured with hucMSCs after stimulation with inactivated serum from patients. Cell proliferation and migration activities were assessed, and the expression of CD54, CD105 and IL-1β was analyzed. In an in vivo study, hucMSCs were transplanted into KD mice. The locations and expression levels of CD54, CD105 and IL-1β in the heart tissues of mice were analyzed. ResultsThe levels of IL-1β and CD31+CD54+ EMPs were significantly higher before IVIG treatment and 2 weeks after treatment in KD patients (P < 0.01). However, the levels of VEGF and CD31+CD105+ EMPs increased significantly in KD only after IVIG treatment (P < 0.01). KD-inactivated serum stimulation combined with cocultivation of PBMCs can activate inflammation in HUVECs, leading to reduced cell proliferation and migration activities. Cocultivation also increased the expression of CD54 and decreased the expression of CD105 (P < 0.001). Cocultivation with hucMSCs can reverse these changes. Additionally, hucMSC transplantation downregulated the expression of IL-1β and CD54 and significantly upregulated the expression of CD105 in KD mice. ConclusionThe expression levels of CD31+CD54+ and CD31+CD105+ EMPs showed inconsistent changes at different KD statuses, providing potential markers for clinical application. HucMSCs suppress inflammation and regulate the expression levels of CD54 and CD105 in vascular endothelial cells in KD, possibly providing a new basis for stem cell therapy for KD.

159 METFORMIN DOWNREGULATES PD-L1 EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA BY INHIBITING IL-6 SIGNALING PATHWAY

Abstract ESCC is an aggressive cancer with poor prognosis in China. Metformin with low cost and toxicity have proved to have anti-cancer effects in many kinds of cancers, while its role in combination of immune checkpoint blockade and mechanism in ESCC has seldom been studied. This study was designed to evaluate the potential role of combinatorial anti-tumor effect with metformin and immunomodulatory monoclonal antibodies (mAbs) targeting programmed cell death protein-1 (PD-1) in vitro and in vivo. Methods Reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemistry (IHC) assays were used to study the effects of metformin on the expression levels of PD-L1. In addition, T cell activation and killing assay were performed in the co-culture system of ESCC cell and peripheral blood mononuclear cells (PBMCs) treated with metformin or IL-6 to evaluate the function of inhibiting PD-L1 expression. In vivo assay, we used NPIdKOTM mice model, which was re-constructed replaced immune system by transplanting PBMCs through intravenously injecting. Mice were treated with metformin alone as well as in combination with anti-PD-1 mAbs. Results PD-L1 expression were downregulated by metformin via JAK2 and STAT3 signaling pathway. The results showed that metformin promoted CD3/CD28 induced ERK phosphorylation at a dose dependent measure. And IL-2 was increased in the supernatant of KYSE-450/PBMC coculture system after metformin treatment. Both of which are indicators of T cell activation. T cell mediated tumor killing assay showed that metformin inducing PD-L1 downregulation could protect T cell function, and it could be reversed by IL-6. It showed that metformin enhanced its efficacy in TE-7 cells-harboring mice in the combination treatment group, reflected by tumor sizes assay, and in vivo imaging system. Conclusion Various approaches are under way to expand the benefits and improve the efficacy of these immune checkpoint inhibitors. Another approach is combining immunotherapy with existing anticancer therapies. In this regard, we have evaluated the mechanism of metformin inhibiting PD-L1 expression in ESCC. This finding indicated that metformin significantly improved the antitumor effects by anti-PD-1 blockade without detectable toxicity and suggested that metformin has strong potential to be used in combination with immunotherapy.

Small molecule tyrosine kinase inhibitors modulated blood immune cell counts in patients with oncogene-driven NSCLC

BackgroundLack of biomarkers and in vitro models has contributed to inadequate understanding of the mechanisms underlying the inferior clinical response to immune checkpoint inhibitors (ICIs) in patients with oncogene-driven non-small cell lung cancer (NSCLC).MethodsThe effect of small molecule tyrosine kinase inhibitors (TKIs) on peripheral blood mononuclear cells (PBMCs) in 34 patients with oncogene-driven NSCLC (cohort A) was compared with those from 35 NSCLC patients without oncogene-driven mutations received ICI (cohort B) or from 22 treatment-naïve NSCLC patients (cohort C). Data for each blood biomarker were summarized by mean and standard deviation and compared by Wilcoxon rank sum tests or Kruskal-Wallis tests with significance at 2-sided p value < 0.05. Co-culture of PBMCs and pleural effusion-derived tumor cells from individual patients with oncogene-driven NSCLC was used to determine the in vitro cytotoxicity of TKI and ICI.ResultsExcept for low CD3% in cohort A, there were no significant differences in other 12 blood biomarkers among the 3 cohorts at baseline. TKI treatment in cohort A was associated with significant increase in CD3% and decrease in total and absolute neutrophils (p < 0.05). In cohort B, patients with good clinical response to ICI treatment (N = 18) had significant increases in absolute lymphocyte counts (ALCs), CD4 and/or CD8 cell counts. Conversely, those patients with poor clinical response to ICI (N = 17) had significant decreases in these cell counts. Of the 27 patients with pre- and post-treatment blood samples in cohort A, 11 had poor clinical response to TKIs and decreased lymphocyte counts. Of the remaining 16 patients who had good clinical response to TKI therapy, 10 (62.5%) patients had decreased, and 6 (37.5%) patients had increased lymphocyte counts. Multicolor immunophenotyping of PBMCs revealed ICI treatment activated additional immune cell types that need further validation. We confirmed that TKI treatment could either antagonize or enhance the effect of ICIs in the co-culture assay using patient’s tumor cells and PBMCs.ConclusionsTo the best of our knowledge, this is the first study showing that TKIs can have various effects on blood immune cells, which may affect their response to ICIs. Further validation of the blood biomarker and in vitro assay is warranted.