Healthy Donor Monocytes Research Articles

MiR-155-overexpressing monocytes resemble HLAhighISG15+ synovial tissue macrophages from patients with rheumatoid arthritis and induce polyfunctional CD4+ T-cell activation.

MicroRNAs (miRs) are known to regulate pro-inflammatory effector functions of myeloid cells, and miR dysregulation is implicated in rheumatoid arthritis (RA), a condition characterized by inflammation and destruction of the joints. We showed previously that miR-155 is increased in myeloid cells in RA and induces pro-inflammatory activation of monocytes and macrophages; however, its role at the interface between innate and adaptive immunity was not defined. Here, RNA-sequencing revealed that overexpression of miR-155 in healthy donor monocytes conferred a specific gene profile which bears similarities to that of RA synovial fluid-derived CD14+ cells and HLAhighISG15+ synovial tissue macrophages, both of which are characterized by antigen-presenting pathways. In line with this, monocytes in which miR-155 was overexpressed, displayed increased expression of HLA-DR and both co-stimulatory and co-inhibitory molecules, and induced activation of polyfunctional T cells. Together, these data underpin the notion that miR-155-driven myeloid cell activation in the synovium contributes not only to inflammation but may also influence the adaptive immune response.

Optimized Inhibition of GM-CSF in Preclinical Models of Anti-CD19 Chimeric Antigen Receptor T Cell Therapy

Anti-CD19 chimeric antigen receptor T (CART19) cell therapy has resulted in unprecedented outcomes in patients with relapsed/refractory B-cell malignancies, which led to the FDA approval for several indications. However, CART19 cell therapy is limited by the development of severe life-threatening toxicities, as well as by the limited rates of durable response. It has become apparent that myeloid cells contribute to the development of both CART cell toxicity and also to the inhibitory tumor microenvironment. We and others have identified that granulocyte-monocyte-colony-stimulating factor (GM-CSF) depletion results in decreased myeloid activation, reduced toxicities, and enhancement of CART19 cell therapy efficacy in pre-clinical models. Furthermore, we observed that GM-CSF knockout (GM-CSF k/o) in CART19 cells resulted in the improvement of their functions (in vitro and in vivo). These findings suggest that there is also a direct effect of GM-CSF on CART19 cells, which is independent of the GM-CSF impact on myeloid cell activation.To further evaluate this, we first examined GM-CSF receptor alpha (GM-CSFRα) expression by flow cytometry on resting and activated CART19 cells (using FMC63-41BBζ). When CART19 cells were stimulated with either anti-CD3/CD28 beads or lethally irradiated (120 Gy) CD19 + Nalm6 cells (B cell acute lymphoblastic leukemia cancer cell line), GM-CSFRα expression was upregulated upon both T cell receptor (TCR) (data not shown) and CAR stimulation (Figure 1A).Having demonstrated that GM-CSFRα is significantly upregulated on stimulated CART19 cells, we aimed to determine the impact of GM-CSF neutralization (clinical-grade anti-GM-CSF antibody, lenzilumab, 10 µg/mL) versus GM-CSFRα blockade (research-grade antibody, 10 µg/mL) on CART19 cell function and CART cell-monocyte interactions. An IgG isotype antibody was used as a control antibody. Neither the GM-CSF neutralizing antibody, nor GM-CSFRα blocking antibody, had any impact on CART19 cell antigen-specific killing against the CD19 + JeKo-1 cells (mantle cell lymphoma cancer cell line), in the presence or absence of CD14 + monocytes (ratio 1:1:1) isolated by magnetic beads from healthy donors (Figures 1B-C).Next, we compared the effects of GM-CSF neutralization versus GM-CSFRα blockade on CART19 cell antigen-specific proliferation. Here, CART19 cells were co-cultured with lethally irradiated CD19 + cell line JeKo-1 at 1:1 ratio in the presence of 10 µg/mL of the GM-CSF neutralizing antibody, increasing doses of the GM-CSFRα blocking antibody (10-100 µg/mL), or an IgG control. The absolute number of CART cells was measured by flow cytometry on day 5. GM-CSF neutralization did not affect CART19 cell proliferation, but GM-CSFRα blocking antibody significantly inhibited CART19 cell proliferation in a dose-dependent manner. Then, we assessed the effects of GM-CSF neutralizing antibody (20 µg/mL) versus GM-CSFRα blocking antibody (20 µg/mL) on CART19 cell antigen-specific proliferation in the presence of healthy donor monocytes (ratio 1:1:0.5) on day 3. Flow cytometric analysis revealed that GM-CSF neutralization, but not GM-CSFRα blockade, mitigated monocyte-suppression of CART19 antigen-specific proliferation (Figure 1E).In summary, our findings indicate significant differences on CART cell functions and CART cell-monocyte interactions when a specific cytokine, GM-CSF, is neutralized compared to blocking its receptor. Further mechanistic studies are ongoing to assess the functions of GM-CSFRα k/o and GM-CSF k/o CART cells. [Display omitted] DisclosuresSakemura: Humanigen: Patents & Royalties. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Behring: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees. Durrant: Humanigen Inc.: Current Employment. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Pyrin Inflammasome Activation Abrogates Interleukin-1 Receptor Antagonist, Suggesting a New Mechanism Underlying Familial Mediterranean Fever Pathogenesis.

Aberrant pyrin inflammasome activity triggers familial Mediterranean fever (FMF) pathogenesis, but the exact mechanism remains elusive and an obstacle to efficient treatment. We undertook this study to identify pyrin inflammasome-specific mechanisms to improve FMF treatment and diagnostics in the future. Pyrin-specific protein secretion was assessed by proteome analysis in U937-derived macrophages, and specific findings were confirmed in pyrin inflammasome-activated monocytes from healthy blood donors and patients with FMF, stratified according to MEFV genotype categories corresponding to a suspected increase in FMF disease severity. Proteome data revealed a differential secretion pattern of interleukin-1 receptor antagonist (IL-1Ra) from pyrin- and NLRP3-activated U937-derived macrophages, which was verified by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Moreover, pyrin activation significantly reduced IL1RN messenger RNA expression (P < 0.001) and IL-1Ra secretion (P < 0.01) in healthy donor and FMF monocytes, respectively. Independent of MEFV genotype, unstimulated FMF monocytes from colchicine-treated patients secreted lower amounts of IL-1Ra compared to healthy donors (P < 0.05) and displayed decreased ratios of IL-1Ra:IL-1β (P < 0.05), suggesting a reduced antiinflammatory capacity. Our data show an inherent lack of IL-1Ra expression specific to pyrin inflammasome activation, suggesting a new mechanism underlying FMF pathogenesis. The reduced IL-1Ra levels in FMF monocytes suggest a diminished antiinflammatory capacity that potentially leaves FMF patients sensitive to proinflammatory stimuli, regardless of receiving colchicine therapy. Thus, considering the potential clinical consequence of reduced monocyte IL-1Ra secretion in FMF patients, we suggest further investigation into IL-1Ra dynamics and its potential implications for FMF treatment in the future.

38P The comparative transcriptomic analysis of monocytes of colorectal cancer patients and healthy donors

Studies in animal models and in patients with colorectal cancer (CRC) recently demonstrated that tumor-associated macrophages (TAM) suppress growth of primary tumor and metastasis. Circulating monocytes are recruited to the growing tumor and constitute the main plastic resource of TAM. However, the role of programming of circulating monocytes in the functional polarization and activation of pro- and anti-tumor functions of TAM is poorly understood.

The immunoregulatory function of peripheral blood CD71+ erythroid cells in systemic-onset juvenile idiopathic arthritis

CD71+ erythroid cells (CECs) are recognized to have an immunoregulatory function via direct cell–cell interaction and soluble mediators. Circulating CECs appear in newborns or patients with hemolytic and cardiopulmonary disorders. To assess the biological role of CECs in systemic inflammation, we studied the gene expression and function in systemic-onset juvenile idiopathic arthritis (SoJIA). Peripheral blood mononuclear cells of SoJIA patients expressed upregulated erythropoiesis-related genes. It represented the largest expansion of CECs during active phase SoJIA among other inflammatory diseases. Despite the opposing roles of erythropoietin and hepcidin in erythropoiesis, both serum levels were in concert with the amounts of SoJIA-driven CECs. Circulating CECs counts in inflammatory diseases were positively correlated with the levels of C-reactive protein, IL-6, IL-18, or soluble TNF receptors. Co-culture with active SoJIA-driven CECs suppressed secretions of IL-1β, IL-6, and IL-8 from healthy donor monocytes. The top upregulated gene in SoJIA-driven CECs was ARG2 compared with CECs from cord blood controls, although cytokine production from monocytes was suppressed by co-culture, even with an arginase inhibitor. CECs are driven to the periphery during the acute phase of SoJIA at higher levels than other inflammatory diseases. Circulating CECs may control excessive inflammation via the immunoregulatory pathways, partly involving arginase-2.

The Oxidative Response of Human Monocytes to Surface Modified Commercially Pure Titanium.

Cellular responses to implanted biomaterials are key to understanding osseointegration. The aim of this investigation was to determine the in vitro priming and activation of the respiratory burst activity of monocytes in response to surface-modified titanium. Human peripheral blood monocytes of healthy blood donors were separated, then incubated with surface-modified grade 2 commercially pure titanium (CPT) disks with a range of known surface energies and surface roughness for 30- or 60-min. Secondary stimulation by phorbol 12-myrisate 13-acetate (PMA) following the priming phase, and luminol-enhanced-chemiluminescence (LCL) was used to monitor oxygen-dependent activity. Comparison among groups was made by incubation time using one-way ANOVA. One sample from each group for each phase of the experiment was viewed under scanning electron microscopy (SEM) and qualitative comparisons made. The results indicate that titanium is capable of priming peripheral blood monocytes following 60-min incubation. In contrast, 30 min incubation time lead to reduced LCL on secondary stimulation as compared to cells alone. At both time intervals, the disk with the lowest surface energy produced significantly less LCL compared to other samples. SEM examination revealed differences in surface morphology at different time points but not between differently surface-modified disks. These results are consistent with the hypothesis that the titanium surface characteristics influenced the monocyte activity, which may be important in regulating the healing response to these materials.

Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme a Reductase (Statins) Suppress Differentiation and Reduce LPS/IFNγ-Induced Cytokine Production in Human Monocyte/Macrophage Culture

We examined the effects of 72-h exposure to atorvastatin and rosuvastatin in concentrations of 2-10 nM on the cytokine expression in LPS/IFNγ-activated monocyte/macrophages derived from peripheral blood monocytes of healthy donors by culturing in the presence of GM-CSF. Pretreatment with statins was found to inhibit cytokine production in monocytes/macrophages after activation, while the level of cytokine mRNA in cells did not decrease. The number of cells containing active caspase-3 decreased in the culture. Culturing of monocytes/macrophages with statins was accompanied by changes in cell morphology and deceleration of cell growth. Cellular effects of "lipophilic" atorvastastin were observed at lower concentration compared to "hydrophilic" rosuvastatin.

Modulatory effect of rapamycin and tacrolimus on monocyte-derived dendritic cells phenotype and function

BackgroundImmunosuppressive-drugs are needed after solid organ transplantation to prevent allograft rejection but induce severe side effects. Understanding the alloimmune response is critical to modulate it and to achieve graft operational tolerance. The role of regulatory T cells and tolerogenic dendritic cells (Tol-DCs) is undoubtedly essential in tolerance induction. Tacrolimus is considered as the cornerstone of immunosuppression in solid organ transplantation. mTOR inhibitor such as rapamycin are thought to induce tolerance and are used as anticancer drugs in several cancers. The aim of this study was to better understand the effect of these immunosuppressive drugs on the differentiation, maturation and function of human monocyte derived dendritic cells (DCs). Material and methodsDCs were differentiated from monocytes of healthy donors with either rapamycin (Rapa-DCs) or tacrolimus (Tac-DCs). The phenotype was evaluated by flow cytometry analysis. The production of pro- and anti-inflammatory cytokines was assessed by ELISA. The mRNA expression level of IDO and PD-L1 was assessed by RTqPCR. Mixed leukocytes reactions were performed to analyse suppressive activity of DCs. ResultsRapa-DC were characterised by a lower expression of the co-stimulatory molecules and CD83 than control-DCs (CTR-DC) (p < 0.05). In contrast, tacrolimus had no effect on the expression of surface markers compared to CTR-DCs. Rapamycin reduced both IL-12 and IL-10 secretions (p < 0.05). Rapa-DCs had a suppressive effect on CD4+ allogenic T cells compared to CTR-DCs (p < 0.05). However, neither Rapa-DCs nor Tac-DCs favoured the emergence of a CD4+CD25highFoxp3+ population compared to CTR-DCs. Surprisingly, Rapa-DCs had a reduced expression of IDO and PD-L1 compared to Tac-DCs and CTR-DCs. ConclusionRapa-DCs exhibit an incomplete phenotypic tolerogenic profile. To our knowledge this is the first paper showing a reduction of expression of pro-tolerogenic enzyme IDO in DCs. Tacrolimus does not change the phenotypical or functional characteristics of moDCs.

Profibrotic Activation of Human Macrophages in Systemic Sclerosis

Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts. Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates. The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor β under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05). These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

Pro-fibrotic Activation of Human Macrophages in Systemic Sclerosis

Abstract Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in Systemic sclerosis (SSc), and we have shown that macrophages constitute the dominant inflammatory signature in SSc tissue. However, little is known about how these cells contribute to fibrotic activation in SSc. Using a bioinformatics approach, we show that the gene expression profile of blood-derived human SSc macrophages is significantly enriched in SSc patient skin. We characterize the immunophenotype of human SSc macrophages as pro-fibrotic, demonstrating that these cells are activated under basal conditions, expressing elevated levels of surface markers associated with activation and releasing CCL2, IL-6, and TGF-beta. We also show that STAT3 is phosphorylated in macrophage from SSc patient compared to the healthy donor, and the phosphorylation is attenuated after IL-6 blockade, suggesting that increased expression of IL-6 by SSc macrophages accounts, at least in part, for increased signaling. Moreover, our results suggest that activation of SSc macrophages arises from soluble factors, as differentiation of healthy donor monocytes in SSc patient-derived plasma confers the immunophenotype of SSc patient macrophages. For the first time, we show that co-culture of human SSc macrophages with SSc fibroblasts induces fibroblast activation. Collectively, these studies implicate macrophages as likely drivers of fibrosis in SSc and suggest therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

IFN-α Suppresses Myeloid Cytokine Production, Impairing IL-12 Production and the Ability to Support T-Cell Proliferation.

Interferon-α (IFN-α) can suppress production of T-cell polarizing cytokines or induce inhibitory antigen-presenting cells that suppress T-cell activation. Previous studies showed that IFN-α therapy fails to boost virus-specific T-cell immunity in patients with chronic hepatitis B virus infection. Our aim was to determine whether IFN-α exposure alters human antigen-presenting cell function in vivo. We investigated the immunomodulatory effects using peripheral blood mononuclear cells from healthy donors exposed to IFN-α and chronic hepatitis B (CHB) patients starting IFN-α therapy. IFN-α increased HLA-DR, CD80, CD86, and PD-L1 expression on healthy donor monocytes. In contrast to the activated phenotype, IFN-α inhibited Toll-like receptor-induced cytokine production and monocyte-induced T-cell proliferation. In CHB patients, peg-IFN treatment induced an interferon-stimulated gene signature in monocytes and increased HLA-DR, CD80, CD86, and PD-L1 expression. As early as 3 days after CHB patients started treatment, IFN-α inhibited monocyte cytokine production and T-cell stimulation ex vivo. IFN-α-mediated inhibition of IL-12 production, rather than inhibitory receptor expression, was responsible for inhibition of T-cell proliferation. Addition of IL-12 restored T-cell proliferation to baseline levels. Understanding how professional antigen-presenting cells respond to immunomodulation is important for both new innate and adaptive-targeted immunotherapies. NCT00962871.

3448 Macrophages, APOL1 Genotype, &amp; Immunometabolism in CVD (MAGIC

OBJECTIVES/SPECIFIC AIMS: This study aims to understand the potential immunomudualtory effect of APOL1 variants in auto-antigen activated myeloid cells by assessing lysosomal integrity in activated cells expressing APOL1. The primary stimuli were: 1. ssRNA hY3 as a proxy for the Ro immune complex; 2. in an bulk RNA seq model, interferon-response gene, Siglec 1, as a read out of interferon activity. The primary outcomes were: 1. Myeloid cell APOL1 expression both in primary macrophage cultures and ex-vivo patient derived macrophages; 2. Lysosome integrity as measured by fluorescence intensity of lysotracker dye on light microscopy. METHODS/STUDY POPULATION: All recruited subjects provided written informed consent as per the NEW YORK UNIVERSITY Division of Rheumatology-wide Specimen and Matched Phenotype Linked Examination (SAMPLE) protocol. Subjects were African American; SLE subjects met 4 American College of Rheumatology criteria for SLE. Healthy donor monocytes representing each genotype in duplicate (reference allele: G0/G0; heterozygote variant: RV/G0; and homozygote variant RV/RV) were cultured with GM-CSF to yield macrophages which were incubated in serum free media or with hY3 ssRNA (TLR 7/8 agonist) to yield inflammatory M1 macrophages. Fold increase of APOL1 in untreated vs hY3 treated macrophages was measured using qPCR. Live cells were then cultured on glass chamber slides with DNA dye, DAPI, and Lysotracker red, a fluorescent dye that stains acidic lysosomes. As a proof of concept, interferon response gene, Signlec1, and APOL1 transcriptional activity in peripheral blood monocytes (PBMCs) were measured and correlated in 17 SLE patients by RNA seq. RESULTS/ANTICIPATED RESULTS: Regardless of genotype, hY3 increased APOL1 expression by 29 (+/−18.4) fold (P = 0.007 vs no treatment). Genotyping of the qPCR product showed concordance with the chromosomal DNA with the RV heterozygotes expressing both alleles. To examine lysosomal membrane integrity, live hY3-treated macrophages were stained with lysosotracker dye and fluorescence intensity was measured. Compared to reference allele carrying macrophages, each additional variant allele corresponded with a lesser degree of lysosome compartment staining. In SLE PBMCs, we found that APOL1 was highly expressed, and significantly correlated with Siglec1 (F=10.5; P = 0.005) supporting an association between circulating interferons and APOL1 accumulation in monocytes. DISCUSSION/SIGNIFICANCE OF IMPACT: Given that the “cytokine milieu” in SLE elicits APOL1 expression, induces inflammatory cell metabolic rewiring, and stimulates autophagy thereby exposing defects in autophagic flux, this gene-environment interaction may underpin the relationship between chronic inflammation and heightened APOL1 polymorphism-attributed cardiovascular risk. These data support further inquiry into the intersection between chronic autoimmunity and APOL1’s functional role in the vascular microenvironment. The in vitro studies herein extend our prior work by demonstrating a mechanistic link between SLE-associated inflammation, APOL1 risk variant status and CVD via a lysosomal defect which converges on common autophagic and metabolic pathways in mononuclear cells.

Toll-like receptor 2 is overexpressed in Familial Mediterranean fever patients and is inhibited by colchicine treatment.

To study the role of Toll-like receptor (TLR) 2 in Familial Mediterranean fever (FMF) inflammatory process. TLR2 expression on monocytes of FMF attack-free patients (n=20) and the effect of sera of FMF patients with an acute attack (n=9) on TLR2 expression on monocytes of healthy donors were studied by flow cytometry (FACS). TLR2 expression was also studied in THP-1cells, and TLR2 downstream signaling was studied by ELISA for the secretion of IL-1β and pro-inflammatory cytokines or by western blotting to measure nuclear factor (NF)-κB. FMF attack-free patients had increased CD14+TLR2+ cell count as compared to healthy donors. High-dose colchicine treatment (≥2mg/d) inhibited this increased expression in FMF patients. Colchicine invitro also inhibited TLR2 expression on THP-1cells. Sera from FMF patients with an acute attack induced TLR2 expression by both monocytes of healthy donors and THP-1cells as well as pro-inflammatory cytokine secretion by healthy monocytes, while colchicine inhibited this induction. Pam2CSK4 increased interleukin-1β (IL-1β) secretion by peripheral blood mononuclear cells (PBMCs) of healthy donors, and this activation was inhibited by colchicine. THP-1cells presented elevated NF-κB expression when cultured with Pam2CSK4, whereas colchicine inhibited this elevation. TLR2 activation was upregulated in monocytes of FMF patients, and colchicine inhibited this upregulation both in-vitro and in-vivo. This indicates that elevated expression of TLR2 promotes the production of pro-inflammatory cytokines, which may contribute to uncontrolled inflammation in FMF.

Abstract 4623: Transcriptional analysis of the functional differences between M1 and M2 macrophages to identify new targets for myeloid cell modulation in pancreatic tumors

Abstract Macrophages exhibit dynamic plasticity in their function in response to the accumulating genetic alterations that occur during tumorigenesis, resulting in the establishment of an immune tolerized tumor microenvironment. However, depending on their environmental cues, they can switch between two functional phenotypes: pro-inflammatory/anti-tumorigenic (M1) or anti-inflammatory/pro-tumorigenic (M2). The importance of these two macrophage subsets is well-recognized; however, identifying and distinguishing M1 from M2 macrophages in patient tumors still remains a challenge. Prior studies have utilized transcriptome profiling of artificially skewed M1 and M2 macrophages from healthy donors to identify individual genes or gene clusters unique to each macrophage subset. However, these studies are limited to surface marker expression and gene expression profiles alone to distinguish subsets and lack functional validation of M1 and M2 macrophages. Surface marker and gene expression profiling of human M1 and M2 macrophages provide an incomplete characterization, as functionality is an important feature distinguishing these macrophage subsets. To improve the characterization of M1 and M2 macrophages, we performed a thorough three-step validation of artificially skewed M1 and M2 macrophages from healthy donor monocytes using surface marker expression, gene expression profiling, and multi-analyte ELISA analyses. Using our validation assays, we tested previously published protocols for their efficacy in generating M1 and M2 macrophages. While M1 macrophages using these protocols generated as expected M1 characteristics, M2 macrophages lacked hallmark functional features, particularly IL-10 secretion. This necessitated the generation of an optimized protocol to skew M1 and M2 macrophages that exhibited distinctive surface marker expression, gene expression profiles, and cytokine and chemokine production for their respective subset. We then performed RNA-sequencing to interrogate the transcriptional landscape of functional M1 and M2 macrophages and have found 1,472 transcripts that have at least a two-fold change in differential expression between M1 and M2 macrophages across 4 different donors. These results represent the most in-depth characterization of human M1 and M2 macrophages and provide insights into better biomarkers and targets for novel immunotherapy approaches. Citation Format: Kimberline R. Yang, Todd D. Armstrong, Elizabeth M. Jaffee. Transcriptional analysis of the functional differences between M1 and M2 macrophages to identify new targets for myeloid cell modulation in pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4623. doi:10.1158/1538-7445.AM2017-4623

Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-\u03b3 and multiple toll-like receptor agonists

Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.

Immature myeloid cells (IMCs) increased in the peripheral blood of patients with alcohol use disorder (AUD) suppress the bactericidal activity of monocytes

Abstract AUD patients have an increased risk of serious opportunistic infections. IMCs have been described as suppressor cells for host antibacterial defenses. In this study, IMCs isolated from AUD patients were examined for their inhibitory functions on the bactericidal activity of healthy donor (HD) monocytes. IMCs and monocytes were isolated from the peripheral blood of 10 AUD patients admitted to Osaka Medical Collage and 5 HD. HD monocytes (1 × 105 cells/ml) were stimulated with heat-killed Enterococcus faecalis, then cultured in media supplemented with the conditioned media of patient IMCs (24 hr-culture fluids, 10% v/v, 1 × 106 cells/ml). Twenty-four hrs after the cultivation, monocytes were harvested and tested for their bactericidal activities against E. faecalis. In some cases, patient IMCs, cultured with 5 μg/ml of retinoic acid (RA) for 24 hrs, were utilized for the experiments. Also, both groups of IMCs were analyzed for RA receptor (RAR) expression by flow cytometry. In the results, as compared to HD peripheral blood IMCs, the numbers of IMCs increased in AUD patients. HD monocytes were shown to be bactericidal against E. faecalis. However, the bactericidal activity of HD monocytes was not demonstrated when they were cultured with media supplemented with the conditioned media of patient IMCs. HD IMCs differentiated to monocytes or dendritic cells after cultivation with RA. However, AUD patient IMCs were shown to be not susceptible to RA treatment (patient IMCs, RAR−; HD IMCs, RAR+). These results suggest that, through the production of some soluble factors active against killing functions of monocytes, patient IMCs increase the susceptibility of AUD patients to opportunistic infections.

The intracellular C5 system is critical to DAMP sensing and cellular responses in human monocytes

Abstract Cholesterol crystal (CC)-induced inflammasome activation is a key contributor to atherosclerosis. Phagocytosis of CC causes lysosomal damage, assembly of the NLRP3 inflammasome, activation of caspase-1 and release of mature IL-1β. We have demonstrated that complement activation by CC in serum leads to C5a-C5aR1 driven CR3 up-regulation, increased uptake and IL1B gene expression in monocytes. Intracellular complement activation now emerges as critical regulator of cellular responses for induction of Th1 cells. In this study we investigated if CC-induced NLRP3 activation and IL-1β release in monocytes involves intracellular C5 activation. We observed that resting monocytes contained stores of C5 and C5a that increased upon CC uptake. Reduction of intracellular C5 by siRNA treatment abrogated IL-1β secretion to the same extent as NLRP3 inhibition. We confirmed the importance of the intracellular C5a-C5aR1 axis using monocytes from a serum C5-deficient patient, which produced intracellular C5 and detectable C5a and mounted equally strong IL-1β response when compared to healthy donor monocytes. Monocytes expressed also active Factors B and D and contained C3 and C3 activation fragments suggesting that an intracellular C5 convertase could mediate intracellular C5 activation. This notion is supported by the finding that knockdown of Factor B and usage of a cell-permeable convertase inhibitor both reduced CC-induced IL-1β. These results indicate that intracellular C3/C5 convertase-driven C5 activation is required for DAMP sensing, such as for CC, and suggest a critical role of intracellular complement in sterile inflammation and related disease states – and, importantly, that this novel axis can be pharmacologically targeted.

Downregulation of Blood Monocyte HLA-DR in ICU Patients Is Also Present in Bone Marrow Cells.

BackgroundThe downregulation of blood monocyte HLA-DR expression also occurs in tissue infiltrative cells in a context of acute clinical inflammation, especially sepsis. This context favors the development of secondary infections and results from various mechanisms. Little is known about HLA-DR expression on bone marrow (BM) cells of the monocyte lineage, the source of circulating monocytes. This study analyzed the BM HLA-DR expression in ICU patients compared to BM monocytes from non-ICU patients and to blood monocytes of control healthy donors. A potential dysfunction of myeloid differentiation was investigated in a sub-population of these ICU patients to characterize the phenotype of the immature forms of monocytes and granulocytes in BM.Methods and FindingsBM and blood were drawn from 33 ICU and 9 non-ICU patients having a BM analysis to precise the etiology of abnormal low count in blood cells. The data were compared with blood cells of 28 control donors. Flow cytometry was used for both HLA-DR expression and phenotyping of immature forms of monocytes and granulocytes. HLA-DR expression was downregulated in both blood and BM monocyte in ICU patients compared to BM of non-ICU patients and blood of control donors. Amplitude of HLA-DR downregulation was comparable in septic and non-septic ICU patients. The phenotype of immature forms of monocytes and granulocytes in BM (n = 11) did not show abnormal myeloid (monocyte + granulocyte) differentiation.ConclusionThe downregulation of HLA-DR in BM monocyte lineage is present in ICU patients without major changes in myeloid cells. It may result from a regulation mediated by soluble and/or neuro-endocrine factors present in BM cell microenvironment.

Abstract 534: Trastuzumab increases uptake and cross-presentation of HER2-derived antigens by dendritic cells

Abstract Introduction: The purpose of this study was to identify the effect of trastuzumab on the uptake and presentation of HER2-derived peptides by dendritic cells (DCs). Clinical trials for HER2-derived peptide vaccines have shown improved outcomes for patients receiving a combination of vaccine and trastuzumab suggesting potential synergy between the two. We hypothesized that antigen processing and cross-presentation would be enhanced via trastuzumab facilitating antigen presenting cell (APC) uptake of HER2 shed from breast cancer cells. Methods: An ELISA assay was used to evaluate HER2 shedding in multiple cancer cell lines (SKBR3, SKOV3, BT474, MDA-MB-231). To look for uptake of HER2 shed from breast cancer cells into culture media by APCs, DCs were generated by incubating healthy donor monocytes in media supplemented with GM-CSF, IL-4, and TNF-α. These DCs were co-incubated with SKBR3, BT474, and SKOV3 (high HER2 expressers) as well as MDA-MB-231 (low HER2 expresser) in the presence of trastuzumab. Rituximab was used as an isotype control to determine trastuzumab specificity and an Fc blocker was used to evaluate the importance of the Fc receptor in the uptake mechanism. Cells were then stained for DC markers, including CD11c and HLA-DR, permeabilized and stained for HER2 to detect uptake, and then analyzed using flow cytometry. To evaluate antigen cross-presentation, antigen-specific T cells were expanded by co-culturing healthy donor PBMCs with DCs that have been pulsed with SKBR3 cells or trastuzumab-treated SKBR3 cells. DCs pulsed with E75 peptide (HER2, aa: 369-377) were used as a control. After T cell activation and expansion, the number of E75-specfic CD8+ T cells was enumerated using an E75-dextramer assay. Results: When compared to SKOV3 and MBA-MB-231 cells, SKBR3 and BT474 cells shed a significantly higher concentration of HER2 into the growth media. DCs took up HER2 when co-cultured with cell lines with higher levels of HER2 shedding (SKBR3, BT474). Trastuzumab treatment of SKBR3 resulted in a 50% increase in HER2 uptake by DCs at 24 hours (p = 0.0014). This finding persisted among all trastuzmab doses. Treatment with rituximab showed no significant increase in HER2 uptake when compared to untreated SKBR3. The effect of trastuzumab on uptake was abrogated by adding an Fc blocking agent. DCs treated with SKBR3 and trastuzumab also led to an increase in the generation of E75-CTLs as measured using a dextramer assay. Conclusions: Trastuzumab treatment leads to increased uptake of soluble HER2 by DCs in vitro. This effect is specific to trastuzumab and is Fc receptor mediated. Increased HER2 uptake led to increased E75-CTL generation secondary to increased cross-presentation of E75 by DCs. These data have global implications for HER2-targeting vaccine approaches. Specifically, by enhancing antigen presentation, trastuzumab can augment the immune response initiated by a peptide vaccine. Citation Format: Victor A. Gall, Anne V. Philips, Na Qiao, Karen C. Dwyer, Alexander A. Perakis, Mao Zhang, Pariya Sukhumalchandra, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. Trastuzumab increases uptake and cross-presentation of HER2-derived antigens by dendritic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 534.

Characteristics of morphological differences of detergent-resistant membrane domains isolated from different cells and investigated by atomic force microscopy

Planar rafts and caveolae are specific membrane clusters that contain high concentrations of cholesterol and lipids consisting of saturated fatty acids. These clusters are resistant to detergents and are known as “detergent-resistant membrane domains” (DRMs). Their morphology and size were studied by atomic force microscopy (AFM). Planar rafts extracted by Lubrol WX from monocytes of healthy donors are 150.6 ± 68.6 nm in diameter and 5.7 ± 2.9 nm in height, while caveolae are 87.3 ± 46.1 nm in diameter and 9.4 ± 5.4 nm in height. Significant differences in size and morphology were found between DRMs isolated from monocytes of healthy donors and patients with myocardial infarction, as well as between DRMs of monocytes and endothelial cells. The morphology dynamics of the isolated planar rafts and caveolae indicates that they quickly aggregate during storage; therefore, in order to assess the actual DRM size and morphology it is necessary to investigate them immediately after isolation.