DataSheet_1_Microenvironmental and cell intrinsic factors governing human cDC2 differentiation and monocyte reprogramming.pdf

cDC2s occur abundantly in peripheral tissues and arise from circulating blood cDC2s. However, the factors governing cDC2 differentiation in tissues, especially under inflammatory conditions, remained poorly defined. We here found that psoriatic cDC2s express the efferocytosis receptor Axl and exhibit a bone morphogenetic protein (BMP) and p38MAPK signaling signature. BMP7, strongly expressed within the lesional psoriatic epidermis, cooperates with canonical TGF-β1 signaling for inducing Axl+cDC2s from blood cDC2s in vitro. Moreover, downstream induced p38MAPK promotes Axl+cDC2s at the expense of Axl+CD207+ Langerhans cell differentiation from blood cDC2s. BMP7 supplementation allowed to model cDC2 generation and their further differentiation into LCs from CD34+ hematopoietic progenitor cells in defined serum-free medium. Additionally, p38MAPK promoted the generation of another cDC2 subset lacking Axl but expressing the non-classical NFkB transcription factor RelB in vitro. Such RelB+cDC2s occurred predominantly at dermal sites in the inflamed skin. Finally, we found that cDC2s can be induced to acquire high levels of the monocyte lineage identity factor kruppel-like-factor-4 (KLF4) along with monocyte-derived DC and macrophage phenotypic characteristics in vitro. In conclusion, inflammatory and psoriatic epidermal signals instruct blood cDC2s to acquire phenotypic characteristics of several tissue-resident cell subsets.</p

The Major Birch Pollen Allergen Bet v 1 Induces Different Responses in Dendritic Cells of Birch Pollen Allergic and Healthy Individuals

<div><p>Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.</p></div

Kinetics of allergen uptake into iMoDCs of BP allergic and normal donors.

<p>Internalization of labeled Bet v 1 (Bet v 1–488) and labeled Api g 1 (Api g 1–610) by iMoDCs of allergic (A) and normal donors (B) was followed by live-cell fluorescence imaging. Results are representative of independent experiments of three different donors for each group (shown for donors AD1 and ND3). Exocytosis of fluorescent markers (panel A) and the absence of spillover of fluorescence (panel B) are depicted by framed display details.</p

Gene expression of iMoDCs after Bet v 1 stimulation or cross-linking of cell-bound IgE.

<p>iMoDCs of BP allergic (A) and normal donors (B) were stimulated with Bet v 1 (squares), anti-IgE (diamonds), or loaded with IgE before anti-IgE treatment (IgE+anti-IgE, circles). mRNA levels of IL-4, IL-13 and the NFκB related genes IL-1β and IL-6 were analyzed by real-time PCR. Error bars indicate SD. Results are representative of two independent experiments for each donor group (shown for donors AD1 and ND1). (C) Activation of NFκB was evaluated by detection of its inhibitory protein IκBα. The Western blots shown are representative of independent experiments for three donors of each group yielding similar results (shown for donors AD2 and ND2).</p

Gene expression induced by Bet v 1 and MFs in iMoDCs of BP allergic donors.

<p>Cells were sham-treated or treated with Bet v 1 (squares), control stimulus (MF, circles), or a combination of Bet v 1 and control stimulus (diamonds) for indicated time periods. mRNA levels of EGR-1 and-3, Th2 cytokines IL-4 and IL-13, Th1 chemokines CXCL10 and 11, and pro-inflammatory cytokines IL-1β and IL-6 were analyzed by real-time PCR. Expression levels, normalized to the average of housekeeping genes, are shown relative to unstimulated cells. Data for donor AD2 are shown, which are representative of independent experiments with mRNA from cells of three donors each performed in duplicates.</p

Phosphorylation of MAP kinases after activation by allergens.

<p>Cells of BP allergic (A) and normal donors (B) were stimulated with the allergens (each 20 μg/ml) or control stimulus (MFs) for the indicated time points and then lysed. Cell extracts were analyzed by Western blot using antibodies specific for the phosphorylated forms of PKCα, Erk1/2, and p38 MAPKs. Sample amounts were determined with antibodies to Erk1/2 and PKCα. The blots shown are representative of independent experiments with cells of three donors for each group yielding similar results (shown for donors AD2 and ND1).</p

Gene expression of MoDCs induced by Bet v 1.

<p>Cells of BP allergic (squares) and normal donors (triangles) were either sham-treated or treated with Bet v 1 for the indicated times. mRNA levels of EGR-1 and-3 and the Th2 cytokines IL-4 and IL-13 were analyzed by real-time PCR. Expression levels, normalized to the average of housekeeping genes, are shown relative to non-stimulated cells. Data are presented as mean values ± SEM (n = 7 in both groups) performed in duplicates. Significance is indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001).</p

B Cells and Ectopic Follicular Structures: Novel Players in Anti-Tumor Programming with Prognostic Power for Patients with Metastatic Colorectal Cancer

<div><p>Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor – liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.</p></div

Competitive binding of allergens to iMoDCs of BP allergic (AD) and normal donors (ND).

<p>(A) Uptake of labeled Api g 1 (20 μg/ml) in the presence of unlabeled Bet v 1 (shown for donors AD3 and ND3). (B) Cross-competition of labeled Bet v 1 with Api g 1 (Bet v 1*/Api g 1) and self-competition of Bet v 1 (Bet v 1*/Bet v 1) using 20μg/ml of labeled allergen and 200 μg/ml of competitor (donor AD3). Results in (A) and (B) show uptakes after 45 minutes of chase and are representative of four independent experiments with similar outcomes for both donor groups. (C) Densitometric quantification of the competition assays measuring a 1 mm² area.</p

Patient-specific imprint of CD45-positive cells at the site of CRCLM.

<p>(A) Massive infiltration of CD45-positive cells confined to the tumor – liver border of the metastases. Representative parts of the metastatic areas for two CRCLM patients are shown (red channel for CD45, blue channel for nuclei/DAPI). T: tumor; L: liver. Scale bar: 200 µm. (B) Different patterns of CD45-positive cell accumulations at the metastatic border. Representative images of various types of immune cell infiltrates are shown: (<i>a</i>) single cells; (<i>b</i>) large immune cell aggregates; (<i>c</i>) prominent ectopic follicle. In addition to the merged images (<i>a–c</i>, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (<i>d–f</i> for DAPI; <i>g–i</i> for CD45); the individual channels are shown in black/white, whereas merged images are shown in color. T: tumor; L: liver. Scale bar: 50 µm. (C) (<i>a</i>–<i>c</i>) Kaplan-Meier estimates for patients stratification based on the CD45-derived values at the border. Kaplan-Meier curves for RFS based on CD45 values for panel I, panel II, and their combination are shown giving patients' stratification into low and high risk groups (higher than median indicates low risk); p value of the log-rank test is indicated. Panel I: median is equal to 20.95, below median n = 6, above median n = 7; panel II: median is equal to 17.66, below median n = 10, above median n = 9; panel I+II: median is equal to 19.13, below median n = 16, above median n = 16. (<i>d</i>) Boxplots of CD45 data sets for patients without recurrence (No) versus patients with recurrence (Yes) at the time point of 17 months. The cut off was set according to the latest event occurrence (16.3 months) where no censoring has occurred, thereby, providing clear separation from the censored subjects (≥32.2 months) as visualized on (<i>b</i>). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (<i>b</i>) is indicated by dashed line; p value is shown (t test).</p