Stromal fibroblasts shape the myeloid phenotype in normal colon and colorectal cancer and induce CD163 and CCL2 expression in macrophages

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production

Analysis of the Chloroplast Protein Kinase Stt7 during State Transitions

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b6f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b6f assembly and heme biogenesis

The F4/AS01B HIV-1 Vaccine Candidate Is Safe and Immunogenic, But Does Not Show Viral Efficacy in Antiretroviral Therapy-Naive, HIV-1-Infected Adults: A Randomized Controlled Trial

The impact of the investigational human immunodeficiency virus type 1 (HIV-1) F4/AS01(B) vaccine on HIV-1 viral load (VL) was evaluated in antiretroviral therapy (ART)-naive HIV-1 infected adults.This phase IIb, observer-blind study (NCT01218113), included ART-naive HIV-1 infected adults aged 18 to 55 years. Participants were randomized to receive 2 (F4/AS01(B)_2 group, N=64) or 3 (F4/AS01(B)_3 group, N=62) doses of F4/AS01(B) or placebo (control group, N=64) at weeks 0, 4, and 28. Efficacy (HIV-1 VL, CD4(+) T-cell count, ART initiation, and HIV-related clinical events), safety, and immunogenicity (antibody and T-cell responses) were evaluated during 48 weeks.At week 48, based on a mixed model, no statistically significant difference in HIV-1 VL change from baseline was demonstrated between F4/AS01(B)_2 and control group (0.073 log(10)copies/mL [97.5% confidence interval (CI): -0.088; 0.235]), or F4/AS01(B)_3 and control group (-0.096 log(10)copies/mL [97.5% CI: -0.257; 0.065]). No differences between groups were observed in HIV-1 VL change, CD4(+) T-cell count, ART initiation, or HIV-related clinical events at intermediate timepoints. Among F4/AS01(B) recipients, the most frequent solicited symptoms were pain at injection site (252/300 doses), fatigue (137/300 doses), myalgia (105/300 doses), and headache (90/300 doses). Twelve serious adverse events were reported in 6 participants; 1 was considered vaccine-related (F4/AS01(B)_2 group: angioedema). F4/AS01(B) induced polyfunctional F4-specific CD4(+) T-cells, but had no significant impact on F4-specific CD8(+) T-cell and anti-F4 antibody levels.F4/AS01(B) had a clinically acceptable safety profile, induced F4-specific CD4(+) T-cell responses, but did not reduce HIV-1 VL, impact CD4(+) T-cells count, delay ART initiation, or prevent HIV-1 related clinical events

CCL23 expression is induced by IL-4 in a STAT6-dependent fashion.

The chemokine CCL23 is primarily expressed in cells of the myeloid lineage but little information about its regulation is available. In this study, it is demonstrated that IL-4 and IL-13 induced CCL23 expression in human peripheral blood monocytes. GM-CSF had no effect on its own but synergized with IL-4, but not IL-13. CCL23 promoter reporter gene constructs were sensitive to IL-4 stimulation in the presence of the transcription factor STAT6. A canonical STAT6 binding site in the promoter region of the CCL23 gene was critical for the IL-4-inducible phenotype because reporter plasmids with a defective STAT6 binding site were unable to respond to IL-4 stimulation. In addition, two tandem copies of the STAT6 site conferred cytokine responsiveness to a heterologous minimal promoter. Furthermore, IL-4 inducibility of the CCL23 promoter was dependent on the absence of a negatively acting cis-element downstream of the STAT6 binding site. The negative function of this element was operative also on heterologous IL-4-inducible promoters. CCL23 was also expressed in skin from patients suffering from atopic dermatitis at higher levels than in normal individuals. However, no correlation between CCL23 expression in the serum and IgE levels as a diagnostic marker for atopy was found. Collectively, these data suggest a link between the inducible phenotype of CCL23 expression in monocytes by the prototype Th2 molecule pair IL-4/STAT6 and the increased number of CCL23-expressing cells in skin of atopic dermatitis patients

A novel low molecular weight inhibitor of dendritic cells and B cells blocks allergic inflammation.

RATIONALE AND OBJECTIVE: During allergic lung inflammation dendritic cells (DCs) direct the generation and function of effector T-helper type 2 cells. T-helper type 2 cells not only orchestrate the inflammatory processes in the tissue by inducing the accumulation and activation of proinflammatory cells but also induce IgE production by B cells. Thus, inhibitors of DC function should have therapeutic benefits in patients with allergies. METHODS AND MEASUREMENTS: VAF347, a novel low molecular weight immunomodulator, is described and acts as an antiinflammatory compound by a dual mode of action. RESULTS: VAF347 inhibited the function of human monocyte-derived DCs to induce T-cell proliferation and cytokine production. Mechanistically, this effect may be due to reduced expression of CD86, HLA-DR, and interleukin 6 by DCs. In addition, the compound inhibited IgE synthesis in an isotype-specific fashion by human B lymphocytes. In a mouse model of antigen-induced eosinophilic inflammation, VAF347 blocked lung eosinophilia, mucus hyperplasia, and serum IgE levels, representing the hallmarks of allergic lung inflammation. The biological effects in vivo are most likely mediated by the immunoregulatory role of VAF347 on DCs because allergic lung inflammation was also inhibited in B-cell-deficient mice. CONCLUSION: VAF347 represents a novel type of immunomodulator by affecting two major pathways in allergic airway pathogenesis: dendritic cell-mediated T-helper-cell activation and induction of IgE production by human B lymphocytes

3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response.Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associatedmacrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME,along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlyingdisease progression. This particularly holds true for prediction of therapeutic response(s) to standardtherapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a cultureplatform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture,which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancerassociatedfibroblasts (CAF) and monocyteWe gratefully acknowledge Vítor E. Santo for the discussions in the initial phase of the work. We acknowledge support from the Innovative Medicines Initiative (IMI) Joint Undertaking (grant agreement no. 115188), MINECO/FEDER, Spain (Severo Ochoa SEV2015-0522, BIO2014-59614-JIN, RYC-2015-17935), Fundacio Pri- vada Cellex, Fundacion Mig-Puig and CERCA program, Spain; S.P. Rebelo et al. / Biomaterials 163 (2018) 185e197 195 iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundaçao para a Ci ~ encia e Tecnologia (FCT)/ Ministerio da Ciencia e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is also acknowledged. CP and MFE are recipients of PhDs fellowships SFRH/BD/52202/2013 and SFRH/BD/52208/2013, respectively, funded by FCT, PortugalPostprint (published version

Peritumoral CD90+CD73+ cells possess immunosuppressive features in human non-small cell lung cancer.

BACKGROUND Although T cell abundance in solid tumours is associated with better outcomes, it also correlates with a stroma-mediated source of immune suppression driven by TGFβ1 and poor overall survival. Whether this also is observed in non-small cell lung cancer (NSCLC) is unknown. METHODS We utilized molecular analysis of The Cancer Genome Atlas (TCGA) NSLCC cohort to correlate immune activation (IA) gene expression and extracellular matrix/stromal (ECM/stromal) gene expression with patient survival. In an independent cohort of NSCLC samples, we used flow cytometry to identify mesenchymal subsets and ex vivo functional studies to characterize their immune regulatory function. FINDINGS We observed a high enrichment in a core set of genes defining an IA gene expression signature in NSCLC across TCGA Pan-cancer cohort. High IA signature score correlates with enrichment of ECM/stromal gene signature across TCGA NSCLC datasets. Importantly, a higher ratio of ECM/stromal to IA gene signature score was associated with shorter overall survival. In tumours resected from a separate cohort of NSCLC patients, we identified CD90+CD73+ peritumoral cells that were enriched in the ECM/stromal gene signature, which was amplified by TGFβ1. IFNγ and TNFα-primed peritumoral CD90+CD73+ cells upregulate immune checkpoint molecules PD-L1 and IDO1 and secrete an array of cytokines/chemokines including TGFβ1. Finally, immune primed peritumoral CD90+CD73+ cells suppress T cell function, which was relieved following combined blockade of PD-L1 and TGFβ1 with IDO1 inhibition but not PD-L1 or anti-CD73 alone. INTERPRETATION Our findings suggest that targeting PD-L1 together with independent biological features of the stroma may enhance host antitumor immunity in NSCLC. FUNDING LW and HY are supported by a 4-year China Scholarship Council award. This work was funded, in part, by a grant from the Cancer League of Bern, Switzerland to SRRH. Laser scanning microscopy imaging was funded by the R'Equip grant from the Swiss National Science Foundation Nr. 316030_145003