Immunology

Accurate, sensitive quantification of B-lineage cells is critical for pharmacodynamic evaluation of B cell–targeted therapies in lupus nephritis (LN) clinical trials. While high-dimensional discovery platforms offer broad profiling, they often lack the sensitivity, quantitative rigor, and throughput needed for precise cell enumeration in renal trial needle biopsies. Traditional immunostaining is hampered by CD20-directed therapeutic masking or downregulation, inadequate sensitivity of CD19 in FFPE tissue, and confounding renal tubular CD138 expression. This protocol details an automated, fit-for-purpose, 5-plex sequential tyramide signal amplification (TSA)-based immunofluorescence assay (CD38, CD79a, CD19, Ki-67, CD138) developed on the Ventana Discovery Ultra platform for deployment on single tissue sections. The workflow anchors B-cell detection on CD79a to ensure sensitivity and utilizes CD38 as an obligate co-marker for CD138+ antibody-secreting cells (ASCs) to definitively exclude the CD138+ epithelial background. Following acquisition via fluorescence whole-slide imaging, a digital analysis pipeline utilizing InstanSeg-based automated segmentation rigorously classifies cell phenotypes to generate precise spatial densities (cells/mm²). This validated protocol maximizes data yield from scarce clinical biopsies while providing high-precision quantitative monitoring of longitudinal therapeutic depletion in the renal microenvironment.

The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus that predominantly spreads through cell-to-cell contact due to the limited infectivity of cell-free viruses. Among various modes of intercellular transmission, HTLV-1 biofilms emerge as adhesive structures, polarized at the cell surface, which encapsulate virions within a protective matrix. This biofilm is supposed to facilitate simultaneous virion delivery during infection. Yet, the molecular and functional intricacies of viral biofilms remain largely unexplored, despite their pivotal role in understanding retroviral pathogenesis. In this study, we optimized a protocol to isolate HTLV-1 biofilms from chronically infected T cells, facilitating their structural and molecular characterization using proteomic and super-resolution microscopy analyses. This protocol involves cultivating HTLV-1 chronically infected T cells at high density to facilitate the natural detachment of viral biofilms into the supernatant. Then, employing successive centrifugations, the cells are separated from the detached biofilms, and these structures are pelleted at medium speed (10,000× g). This method circumvents the need for mechanical, chemical, or enzymatic biofilm detachment, bypasses the use of ultracentrifugation, and enables us to resuspend the biofilms in the appropriate buffer for subsequent analyses such as western blotting or super-resolution microscopy imaging as presented.

Neutrophils, constituting 50%–70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies.

Innate lymphoid cells (ILCs) are a rare cell population subdivided into ILC1s, ILC2s, and ILC3s, based on transcription factor expression and cytokine production. In models of lung inflammation, the release of alarmins from the epithelium activates ILC2s and promotes the production of Th2-cytokines and the proliferation and migration of ILC2s within the lung. ILC2s are the innate counterpart to CD4⁺ Th2s and, as such, express Gata-3 and produce IL-4, IL-5, and IL-13. Due to the low number of ILCs and the lack of specific surface markers, flow cytometry is the most reliable technique for the identification and characterization of ILCs. In this protocol, multicolor flow cytometry is utilized to identify Lineage⁻Thy1.2⁺ ILCs. Intracellular cytokine staining further identifies ILC2s within the lung. This protocol presents a reliable method for promoting ILC2-mediated lung inflammation and for monitoring ILC2 biology.

Key features

• In this protocol, ILC2s are expanded via intranasal challenges with Alternaria alternata, a fungal allergen, or recombinant IL-33.

• Bronchoalveolar lavage (BAL) and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines.

• During lung inflammation, the percentage of ILC2s and eosinophils increases. ILC2s express greater levels of Gata-3 and Ki-67 and produce greater amounts of IL-5 and IL-13.

Graphical overview

Employing a novel mouse model of immune related adverse events (irAEs) induced by combination of anti-PD1 and anti-CTLA-4 antibodies, we visualized immune infiltration into the liver, lung, pancreas, and colon. Here, we describe the avidin-biotin conjugate (ABC) method used to stain T cells (CD4 and CD8), B cells (CD19), macrophages (F4/80), and cells bound by the in vivo administered rat anti-mouse antibodies for chromogenic immunohistochemistry (IHC). Using a biotinylated goat anti-rat antibody, we detected the localization of cells bound to the in vivo antibodies for PD-1 and CTLA-4. IHC has advantages over other techniques, namely antibody availability, resistance to photobleaching, and greater sensitivity. Additionally, detection and localization of in vivo antibodies can be used in mice models to infer their therapeutic efficacy, stability, and function.

Graphical abstract:

During adaptive immune responses, germinal centers (GC) appear as transient microstructures, in which antigen-specific B and T cells interact with each other. Because only the antigen-activated B and T cells, such as Plasmablasts or follicular T helper (Tfh) cells, are present in GC, the in depth-analysis of GC is of great interest. To identify the cells that reside within GC, the majority of studies use the expression of specific surface molecules for analysis by flow cytometry. To do so, the tissue has to be disrupted for the preparation of single-cell suspensions. Thereby, the local information regarding neighborhoods of B cells and T cells and their potential interaction is lost. To study GC in vivo within their original microenvironment, we established a protocol for the isolation of GC by laser microdissection. To enable the identification of GC for subsequent transcriptomic analysis, the degradation of mRNA was diminished by using frozen tissues and by establishing a rapid staining protocol. This procedure enables histological and transcriptomic analysis of individual GC even within one lymphoid organ.

Autoreactive T cells in autoantibody-mediated autoimmune diseases can be divided into two major subsets: (i) follicular T helper cells (Tfh) that provide T cell help in germinal centers (GC) and (ii) effector T (Teff) cells that immigrate into peripheral tissue sites such as the skin and mediate local inflammation. To study the sequence of events leading to the loss of tolerance in autoantibody-mediated autoimmune diseases it is required to investigate both T cell subsets simultaneously. This approach is hampered mainly because the appearance of skin inflammation in mouse models is a random process, which makes it difficult to define the location of inflammation at the right time point. To overcome this problem, we developed a scratching technique for ear skins that leads to the establishment of chronic autoimmune wounds in the mouse model for the pemphigoid-like disease epidermolysis bullosa acquisita. By defining the exact place where the skin wounds should form, this protocol enables a detailed analysis of skin-immigrating Teff cells. Of note, this protocol induces GC in draining lymph nodes in parallel so that Tfh cells in GC can be investigated concurrently. This protocol is not restricted to T cells and can be used for any other skin-immigrating inflammatory cells.

Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological fluids and tissues; however, this can be achieved by quantitating NET components (e.g., DNA or granule/histone proteins) or by directly visualizing them by microscopy, respectively. Direct visualization by confocal microscopy is preferably performed on formalin-fixed paraffin-embedded (FFPE) tissue sections stained with a fluorescent DNA dye and antibodies directed against myeloperoxidase (MPO) and citrullinated histone 3 (Cit-H3), two components of NETs, following paraffin removal, antigen retrieval, and permeabilization. NETs are defined as extracellular structures that stain double-positive for MPO and Cit-H3. Here, we propose a novel software-based objective method for NET volume quantitation in tissue sections based on the measurement of the volume of structures exhibiting co-localization of Cit-H3 and MPO outside the cell. Such a technique not only allows the unambiguous identification of NETs in tissue sections but also their quantitation and relationship with surrounding tissues.

Graphic abstract:

Graphical representation of the methodology used to stain and quantitate NETs in human lung tissue.