免疫学

A rigorous determination of effector contributions of tumor-infiltrating immune cells is critical for identifying targetable molecular mechanisms for the development of novel cancer immunotherapies. A tumor/immune cell–admixture model is an advantageous strategy to study tumor immunology as the fundamental methodology is relatively straightforward, while also being adaptable to scale to address increasingly complex research queries. Ultimately, this method can provide robust experimental information to complement more traditional murine models of tumor immunology. Here, we describe a tumor/macrophage-admixture model using bone marrow–derived macrophages to investigate macrophage-dependent tumorigenesis. Additionally, we provide commentary on potential branch points for optimization with other immune cells, experimental techniques, and cancer types.

Bronchopulmonary dysplasia (BPD) and pulmonary hypertension associated with BPD (BPD-PH) are of multifactorial origin and share common risk factors. Most murine models of BPD expose newborn pups to only one of these risk factors—more commonly postnatal hyperoxia—thereby mimicking the vital increased fraction of inspired oxygen (FiO₂) that preterm infants in neonatal intensive care units often require. To improve representation of the multifactorial origins of BPD and BPD-PH, we established a double hit model, combining antenatal systemic inflammation followed by postnatal hyperoxia. On embryonic day 14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg of lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized and exposed to gas with either an FiO₂ of 0.21 (room air) or 0.65 (hyperoxia 65%). In our BPD and BPD-PH double hit model, we can obtain multiple readouts from individual pups that include echocardiography, lung histology and immunohistochemistry, ex vivo X-ray micro computed tomography, and pulmonary and plasmatic immunity by RNA, protein, or flow cytometry.

Graphical abstract:

Figure 1. Murine double hit model of cardiopulmonary disease. On embryonic day (E)14, pups are exposed to systemic maternal inflammation via a single intraperitoneal injection of 150 µg/kg lipopolysaccharide to the dam. Within 24 h after birth, pups and dams are randomized to be exposed to gas with either a fraction of inspired oxygen (FiO₂) of 0.21 (air; 21% O₂) or 0.65 (hyperoxia; 65% O₂) for a maximum of 28 days. According to the murine stage of lung development (Schittny, 2017), experimental endpoints include postnatal day (D)3, D5, D14, D28, and D60.

Abdominal surgeries are frequently associated with the development of post-surgical adhesions. These are irreversible fibrotic scar bands that appear between abdominal organs and the abdominal wall. Patients suffering from adhesions are at risk of severe complications, such as small bowel obstruction, chronic pelvic pain, or infertility. To date, no cure exists, and the understanding of underlying molecular mechanisms of adhesion formation is incomplete. The current paradigm largely relies on sterile injury mouse models. However, abdominal surgeries in human patients are rarely completely sterile procedures. Here, we describe a modular surgical procedure for simultaneous or separate induction of sterile injury and microbial contamination. Combined, these insults synergistically lead to adhesion formation in the mouse peritoneal cavity. Surgical trauma is confined to a localized sterile injury of the peritoneum. Microbial contamination of the peritoneal cavity is induced by a limited perforation of the microbe-rich large intestine or by injection of fecal content. The presented protocol extends previous injury-based adhesion models by an additional insult through microbial contamination, which may more adequately model the clinical context of abdominal surgery.

Graphical abstract:

Microbiome studies are quickly gaining momentum. Since most of the resident microbes (consisting of bacteria, fungi, and viruses) are difficult to culture, sequencing the microbial genome is the method of choice to characterize them. It is therefore important to have efficient methodology for gDNA isolation of gut microbes. Mouse models are widely used to understand human disease etiology while avoiding human ethics-related complications. However, the widely used kit-based methods are costly, and sometimes yields (in terms of quality and quantity) are sub-optimal. To overcome this problem, we developed a straightforward, standardized DNA isolation procedure from mouse cecal content for further microbiome-related studies. The reagents we used to standardize the procedure are readily available even in a not-so-well-equipped laboratory, and the reagents are not expensive. The yield and quality of the DNA are also better than those obtained by the readily available kit-based methods.

Additionally, we modified the kit-based method of RNA isolation from the colon tissue sample of the mouse for better yield. Churning the tissue with liquid nitrogen at the beginning of the procedure improves RNA quality and quantity.

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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.

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Transplantation of hematopoietic material into recipient mice is an assay routinely used to determine the presence and function of hematopoietic stem and progenitor cells (HSPCs) in vivo. The principle of the method is to transplant donor cells being tested for HSPCs into a recipient mouse following bone marrow ablation and testing for reconstitution of hematopoiesis. Congenic mouse strains where donor and recipient differ by a distinct cell surface antigen (commonly CD45.1 versus CD45.2) are used to distinguish between cells derived from the donor and any residual recipient cells. Typically, the transplantation is performed using bone marrow cells, which are enriched for HSPCs. Here, we describe an analogous procedure using hematopoietic material from spleen, allowing detection of functional progenitors and/or stem cells in the spleen that can occur under certain pathologies. Key to the success of this procedure is the prior removal of mature T cells from the donor sample, to minimize graft versus host reactions. As such, this protocol is highly analogous to standard bone marrow transplant procedures, differing mainly only in the source of stem cells (spleen rather than bone marrow) and the recommendation for T cell depletion to avoid potential immune incompatibilities.

Graphical abstract:

Schematic overview for assessment of stem cells in spleen by transplantation. Single cell suspensions from spleens are depleted of potentially pathogenic mature T lymphocytes by magnetic bead immunoselection using biotinylated antibodies against CD4 and CD8, followed by streptavidin magnetic beads, which are subsequently removed by using a magnet (MojoSort, Biolegend). Successful T cell depletion is then evaluated by Fluorescence Activated Cell Sorting (FACS). T-cell depleted cell suspension is injected intravenously through the retro-orbital sinus into lethally irradiated recipients. Recipients are analyzed for successful engraftment by FACS analysis for the presence of donor-derived mature hematopoietic lineages in the peripheral blood. A second serial transplantation can be used to document the presence of long-term reconstituting stem cells in the periphery of the original donor mice.

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.

Here, we describe a combinatorial approach in reverse vaccinology to identify immunogenic class I major histocompatibility complex (MHC) displayed epitopes derived from a morbillivirus named pestes des petits ruminants (PPRV). The protocol describes an in silico prediction of immunogenic epitopes using an IEDB tool. The predicted peptides were further analysed by molecular docking with mouse class I MHC (H-2K^b), to assess their binding affinity, and their immunogenicity was validated, using acellular and cellular assays. Finally, an enumeration of the expanded PPRV-specific CD8⁺ T cells in infected or immunized mice against the immunogenic peptides was performed ex vivo. Synthetic peptide derivatives from different structural and non-structural proteins of PPRV were used to measure the extent of stabilized H2-K^b, using an ELISA based acellular assay and TAP deficient RMA/s cells. Fluorescently labelled H2-K^b-tetramers were generated by displacing a UV photocleavable conditional ligand with the PPRV-peptides. The resulting reagents were used to identify and enumerate virus-specific CD8⁺ T cells in immunized or PPRV-infected mice. The combinatorial approach described here could be used to identify immunogenic epitopes of any pathogen, autoantigens, as well as cancer antigens.

Graphic abstract:

Figure 1. General schematic to identify immunogenic peptides and their stabilization on MHC I molecule.

Cholangiocytes are epithelial cells lining the intrahepatic and extrahepatic bile ducts. Cholangiocytes perform key physiological functions in the liver. Bile synthesized by hepatocytes is secreted into bile canaliculi, further stored in the gallbladder, and finally discharged into the duodenum. Due to liver injury, biliary epithelial proliferate in response to endogenous or exogenous signals leading to cholangiopathies, inflammation, fibrosis, and cholangiocarcinoma. Cholangiocytes exhibit anatomical and functional heterogeneity, and understanding such diversified functions will potentially help in finding effective therapies for various cholestatic liver diseases. To perform such functional studies, effective cholangiocyte isolation and culture procedures are needed. This protocol will aid in easy isolation and expansion of cholangiocytes from the liver.

Solitary chemosensory epithelial cells are scattered in most mucosal surfaces. They are referred to as tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. They are the primary source of IL-25 in the epithelium and are also engaged in acetylcholine generation. We recently demonstrated that nasal solitary chemosensory (brush) cells can generate robust levels of cysteinyl leukotrienes in response to stimulation with calcium ionophore, aeroallergens, and danger-associated molecules, such as ATP and UTP, and this mechanism depends on brush cell expression of the purinergic receptor P2Y2. This protocol describes an effective method of nasal brush cell isolation in the mouse. The method is based on physical separation of the mucosal layer of the nasal cavity and pre-incubation with dispase, followed by digestion with papain solution. The single cell suspension obtained this way contains a high yield of brush cells for fluorescence-activated cell sorting (FACS), RNA-sequencing, and ex vivo assays.

Graphic abstract:

Workflow of nasal digestion for brush cell isolation.