Three-dimensional human germinal centers of different sizes in patients diagnosed with lymphadenitis show comparative constant relative volumes of B cells, T cells, follicular dendritic cells, and macrophages.

Germinal centers (GCs) are some of the most important structures in the human immune system. As such, their cell types and functions have been thoroughly investigated. B cells, T cells, follicular dendritic cells (FDCs), and macrophages have widely been found to typically be aggregated in GCs. However, the amount of space occupied by each of these cell types has yet to be investigated. In this study, we conducted confocal laser-based 3D cell-volume quantification of typical GC cells under reactive conditions in lymphadenitis and investigated how volume proportions change during GC development. For this investigation, we used anti-CD3 (T cells), anti-CD20 and anti-Pax5 (B cells), anti-CD23 (FDCs), anti-CD68 (macrophages), and DAPI (nuclear staining). We detected average proportions of about 11% CD3, 9% CD20, 6% CD23, and 2% CD68 in the largest possible regions of interest within GCs. Interestingly, these values remained steady relatively independent of GC size. The remarkably low B cell proportion can be attributed to technical constraints given the use of the CD20 antibody in 3D. Applying the B cell marker Pax5, we found that about 44% of the volume was occupied by B cells after extrapolating the volume of B cell nuclei to that of whole B cells. We concluded that Pax5 is more suitable than anti-CD20 for 3D B cell quantification in GCs. The substantial unstained volume in GCs raises the question of whether other cell types fill these open spaces. Our 3D investigation enabled a unique morphological and volumetric evaluation of GC cells that balance their overall volumes in GCs.

Holistic View on the Structure of Immune Response: Petri Net Model.

The simulation of immune response is a challenging task because quantitative data are scarce. Quantitative theoretical models either focus on specific cell-cell interactions or have to make assumptions about parameters. The broad variation of, e.g., the dimensions and abundance between lymph nodes as well as between individual patients hampers conclusive quantitative modeling. No theoretical model has been established representing a consensus on the set of major cellular processes involved in the immune response. In this paper, we apply the Petri net formalism to construct a semi-quantitative mathematical model of the lymph nodes. The model covers the major cellular processes of immune response and fulfills the formal requirements of Petri net models. The intention is to develop a model taking into account the viewpoints of experienced pathologists and computer scientists in the field of systems biology. In order to verify formal requirements, we discuss invariant properties and apply the asynchronous firing rule of a place/transition net. Twenty-five transition invariants cover the model, and each is assigned to a functional mode of the immune response. In simulations, the Petri net model describes the dynamic modes of the immune response, its adaption to antigens, and its loss of memory.

The E3 Ligase TRIM25 Impairs Apoptotic Cell Death in Colon Carcinoma Cells via Destabilization of Caspase-7 mRNA: A Possible Role of hnRNPH1.

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.

New definitions of human lymphoid and follicular cell entities in lymphatic tissue by machine learning.

Histological sections of the lymphatic system are usually the basis of static (2D) morphological investigations. Here, we performed a dynamic (4D) analysis of human reactive lymphoid tissue using confocal fluorescent laser microscopy in combination with machine learning. Based on tracks for T-cells (CD3), B-cells (CD20), follicular T-helper cells (PD1) and optical flow of follicular dendritic cells (CD35), we put forward the first quantitative analysis of movement-related and morphological parameters within human lymphoid tissue. We identified correlations of follicular dendritic cell movement and the behavior of lymphocytes in the microenvironment. In addition, we investigated the value of movement and/or morphological parameters for a precise definition of cell types (CD clusters). CD-clusters could be determined based on movement and/or morphology. Differentiating between CD3- and CD20 positive cells is most challenging and long term-movement characteristics are indispensable. We propose morphological and movement-related prototypes of cell entities applying machine learning models. Finally, we define beyond CD clusters new subgroups within lymphocyte entities based on long term movement characteristics. In conclusion, we showed that the combination of 4D imaging and machine learning is able to define characteristics of lymphocytes not visible in 2D histology.

3D connectomes of reactive and neoplastic CD30 positive lymphoid cells and surrounding cell types.

Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas in Western Europe. It is diagnosed on the basis of histological sections by pathologists using a light microscope. The tumor cells, the Hodgkin- and Reed Sternberg cells (HRS), are visualized by morphology and positive response for the CD30-antigen. The same antigen can also be detected by immunohistochemistry on a reactive counterpart, showing CD30+ cells in special immunoreactions, such as inflammations of lymph nodes (lymphadenitis). CD30+ cells in reactive and neoplastic conditions are surrounded by lymphocytes and histiocytes, forming a micromilieu that enables the survival of the tumor cells, as well as their reactive counterparts. This study deals with an investigation of CD30+-surrounding cells using a confocal laser technology, visualizing the contacts of reactive and neoplastic CD30+ cells with CD68+ macrophages and CD163+ macrophages as well as to PD1+ lymphocytes and B cells (CD20+). CD4 immunostains were not included, because CD4+ cells were too numerous for clear dissection of single cells. 3D images visualized the, so-called, connectomes. Clear differences in the number of contacts between CD30-reactive and neoplastic cells (HRS) with macrophages and B lymphocytes were visible. Lymphadenitis and Mixed Cellularity type of classical Hodgkin Lymphoma (cHL) differed in that Mixed Cellularity (MC) cHL had more connections to macrophages (CD163+) and lower number of connections to B cells (CD20+). The connectomes of both Hodgkin variants MCcHL and Nodular Sclerosis cHL (NScHL) mainly differed in the number of contacts to CD163+ macrophages, which was higher in MCcHL. Investigating the volumes of CD30+ -reactive and neoplastic cells, we found out that reactive cells showed lesser volumes, which correlated with the number of contacts. The comparison between 2D and 3D images, including 3D prints, demonstrated clear advantages of the 3D method. 3D images visualized significantly more and clearly defined intercellular contacts. Complicated cellular networks and their contacts became especially evident in volume and surface evaluations, as well as in 3D prints.

Detection of follicular regions in actin-stained whole slide images of the human lymph node by shock filter.

Human lymph nodes play a central part of immune defense against infection agents and tumor cells. Lymphoid follicles are compartments of the lymph node which are spherical, mainly filled with B cells. B cells are cellular components of the adaptive immune systems. In the course of a specific immune response, lymphoid follicles pass different morphological differentiation stages. The morphology and the spatial distribution of lymphoid follicles can be sometimes associated to a particular causative agent and development stage of a disease. We report our new approach for the automatic detection of follicular regions in histological whole slide images of tissue sections immuno-stained with actin. The method is divided in two phases: (1) shock filter-based detection of transition points and (2) segmentation of follicular regions. Follicular regions in 10 whole slide images were manually annotated by visual inspection, and sample surveys were conducted by an expert pathologist. The results of our method were validated by comparing with the manual annotation. On average, we could achieve a Zijbendos similarity index of 0.71, with a standard deviation of 0.07.

3D investigation shows walls and wall-like structures around human germinal centres, probably regulating T- and B-cell entry and exit.

This study deals with 3D laser investigation on the border between the human lymph node T-zone and germinal centre. Only a few T-cells specific for antigen selected B-cells are allowed to enter germinal centres. This selection process is guided by sinus structures, chemokine gradients and inherent motility of the lymphoid cells. We measured gaps and wall-like structures manually, using IMARIS, a 3D image software for analysis and interpretation of microscopy datasets. In this paper, we describe alpha-actin positive and semipermeable walls and wall-like structures that may hinder T-cells and other cell types from entering germinal centres. Some clearly defined holes or gaps probably regulate lymphoid traffic between T- and B-cell areas. In lymphadenitis, the morphology of this border structure is clearly defined. However, in case of malignant lymphoma, the wall-like structure is disrupted. This has been demonstrated exemplarily in case of angioimmunoblastic T-cell lymphoma. We revealed significant differences of lengths of the wall-like structures in angioimmunoblastic T-cell lymphoma in comparison with wall-like structures in reactive tissue slices. The alterations of morphological structures lead to abnormal and less controlled T- and B-cell distributions probably preventing the immune defence against tumour cells and infectious agents by dysregulating immune homeostasis.

3D image analysis reveals differences of CD30 positive cells and network formation in reactive and malignant human lymphoid tissue (classical Hodgkin Lymphoma).

AIMS: The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues. MATERIALS AND RESULTS: Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL. CONCLUSION: In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.

3D analysis of morphological alterations of the fibroblastic reticular cells in reactive and neoplastic human lymph nodes.

Histopathological methods based on 2 µm thin sections are routinely used in pathological anatomical diagnosis. Many medical disciplines already rely on a 3D representation, regarding visualization and imaging techniques. Pathology in particular uses different tissue visualizations to make the final diagnosis. Thereby, a standard 2D histological section only represents a flat snapshot of a three-dimensional complex cell system. Despite that, 3D cell analysis is not yet standardly used in clinical routine. This work used 3D analysis systems to investigate the morphological alterations of the fibroblastic reticular cell network inside human lymph nodes during neoplastic transformation and evaluates the added value of 3D visualizations in tissue interpretation. We investigated the surface and volume quotient, cell cross-linking and percentage cell volume of the fibroblastic reticular cell (FRC) network inside Lymphadenopathy (follicular hyperplasia) (LAD), Follicular Lymphoma Grade 1 (FL1), Nodular Sclerosis classical Hodgkin Lymphoma (NScHL) and Angioimmunoblastic T-Cell Lymphoma (AITL). We found that the average quotient of LAD and FL1 differed from those of NScHL and AITL, indicating that the surface and volume quotient changes in the course of neoplastic transformation. This is probably due to an increased network convolution, while the total cell volume remains the same at about 2%. In conclusion, this paper describes the tumor-related morphological changes of the FRC network, which would have been difficult to achieve without the use of 3D analysis systems.