Activation-Induced Cytidine Deaminase (AID)-Associated Multigene Signature to Assess Impact of AID in Etiology of Diseases with Inflammatory Component

Activation-induced cytidine deaminase (AID) is expressed in B cells within germinal centers and is critically involved in class switch recombination and somatic hypermutation of immunoglobulin loci. Functionally active AID can additionally be detected within ectopic follicular structures developed at sites of chronic inflammation. Furthermore, AID may target non-Ig genes in B- and non-B-cell background. Therefore, AID-associated effects are of increasing interest in disease areas such as allergy, inflammation, autoimmunity, and cancer

Atopic donor status does not influence the uptake of the major grass pollen allergen, Phl p 5, by dendritic cells

AbstractDendritic cells (DCs) are sentinels of the immune system for antigen recognition and uptake, as well as presentation to naïve T cells for stimulation or priming. Internalization and endocytic degradation of allergens by DCs are important steps required for T cell priming.In the current study we investigated binding and internalization of purified recombinant non-glycosylated grass pollen allergen, Phl p 5, and natural non-specific lipid transfer protein from sunflower, SF-nsLTP to human monocyte derived dendritic cells (MoDCs). Colocalization of Phl p 5 with low affinity (CD23) or high affinity receptor (FcεRI) was investigated by immunofluorescence staining. Likewise, localization of the allergens in early (EE) and late endosomes (LE) was detected by co-staining for early endosome antigen (EEA1) and lysosomal-associated membrane protein 1 (LAMP1).In our experimental setting we could demonstrate that Phl p 5 as well as SF-nsLTP bound to MoDCs from both, grass pollen allergic and non-allergic individuals. Competitive allergen uptake experiments demonstrated non-preferential and simultaneous uptake of Phl p 5 and SF-nsLTP by MoDCs. No overlap of signals from Phl p 5 and CD23 or FcεRI was detectable, excluding IgE-mediated uptake for this allergen. Both allergens, Phl p 5 and SF-nsLTP, were localized in early and late endosomes.The present study applied a set of methods to assess the allergen uptake by MoDCs in an in vitro model. No qualitative and quantitative differences in the allergen uptake of both, Phl p 5 and SF-nsLTP were detected in single and competitive assays

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

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

Ectopic follicular structures at the site of CRCLM.

<p>(A) When formed, ectopic follicles are attracted to the tumor – liver interface. Representative image of the large-scale metastatic area surrounded by ectopic follicles is shown; brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 2 mm. The higher-power views of follicles at border (<i>a</i>), proximal (<i>b</i>) and distant (<i>c</i>) to tumor portal veins are given. Scale bar: 50 µm. (B) Representative images of AID-positive ectopic follicular structures located at the tumor – liver border are shown (<i>a</i>, insert: the higher-power view); brown color, AID staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 50 µm. (C) The characteristic structure of a mature, AID-positive lymphoid follicle (<i>a-e</i>) is shown. Consequent slides were stained for CD20 using immunofluorescent (<i>a</i>, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (<i>b</i>, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (<i>c</i>), IgM (<i>d</i>), CD138 (<i>e</i>). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (<i>e</i>) was reduced by 60% in comparison to those shown on <i>a-d</i>. The corresponding AID-positive core of the follicle is indicated by asterisk (<i>c</i>, <i>e</i>). Example of follicle which is predominantly composed of IgM-positive B-cell subset (<i>f</i>, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (<i>a-d, f</i>) 20 µm, (<i>e</i>) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (<i>a</i>) follicular structure at border surrounded by CD138-positive plasma cells, (<i>b</i>) aggregate of infiltrating cells or (<i>c</i>) diffused cells around the portal veins distant to border which do not contain CD138-positive cells; brown color, CD138 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 50 µm.</p

CD20-positive B lymphocytes at the site of CRCLM.

<p>(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (<i>a</i>) ectopic follicle, (<i>c</i>) large cell aggregate and (<i>e</i>) non-organized cell population and the corresponding areas stained for CD20 are shown (<i>b</i>, <i>d</i>, <i>f</i>, respectively). Quantitative analysis was done using TissueQuest and HistoQuest software; percentage of positive cells is indicated. Scale bar: 100 µm. (B) Different organisation patterns of CD20-positive cells within the three regions of interest. Representative images (i) at the border: (<i>a</i>) single cells, (<i>b</i>) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (<i>c</i>) ectopic follicular structures; (ii) around the portal veins (<i>d</i>) proximate to the border and (<i>e</i>) distant to the border as well as (iii) within liver tissue (<i>f</i>) are shown. T: tumor; L: liver. Scale bar: 50 µm. (C) Kaplan-Meier estimates for patients stratification based on the CD20-derived values at the border. Kaplan-Meier curves for RFS based on CD20 values for panel I, panel II and panel I+II are shown giving patient 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 2.70, below median n = 5, above median n = 6; panel II: median is equal to 2.11, below median n = 25, above median n = 26; panel I+II: median is equal to 2.22, below median n = 31, above median n = 31. (<i>d</i>) Boxplots of CD20 data sets for patients without recurrence (No) versus patients with recurrence (Yes) at the time point of 24 months. The cut off was set according to the latest event occurrence (23.3 month) where no censoring has occurred, giving separation from the censored subjects (≥29.4 months) as visualized on (<i>b</i>). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (<i>b</i>); p value is shown (t test).</p

Prognostic effect of ectopic follicular structures allocated at the tumor – liver border.

<p>Kaplan-Meier estimates for patients stratification based on the ectopic follicle score at the border (<i>a</i>–<i>c</i>). Kaplan-Meier curves for RFS for panel I, panel II, and panel I+II show patient stratification into low (high number of follicular structures), intermediate (low number of follicular structures) and high risk (no follicular structures) groups; p value of the log-rank trend test is indicated. Panel I: low risk n = 5, intermediate risk n = 7, high risk n = 2; panel II: low risk n = 16, intermediate risk n = 17, high risk n = 18; panel I+II: low risk n = 21, intermediate risk n = 24, high risk n = 20. (<i>d</i>) Comparative assessment of contribution of <i>no</i>, <i>low</i>, and <i>high</i> ectopic follicle score to the total quantity for patient sub-groups without (No) and with (Yes) disease recurrence as estimated at the time point of 24 months where no censoring has occurred; p value of chi-square trend test is shown.</p