Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

Abstract

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses [1][2][3] . The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system [4][5][6] . Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells 4,7,8 . These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages 9,10 . Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens 7 . While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life 6 . In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses 11 .Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-α knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice 12,13 . As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines 1,3 . Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination. Video LinkThe video component of this article can be found at https://www.jove.com/video/3960/ Protocol 1. NALT Collection and Culturing 1. Euthanize mice using approved IACUC guidance. Avoid use of inhalant anesthetics that may affect NALT. Transfer mice to an aseptic workspace or biosafety cabinet. Remove the lower jaw of the mouse and clean the upper palate area with alcohol and iodine wipes. 2. Use a No. 11 surgical blade in surgical knife handle to carefully cut and excise the upper palate by following the inside contour of the mouse incisors and molar teeth. 3. Gen...

Mucosal vaccination by the intranasal route. Nose-associated lymphoid tissue (NALT)—Structure, function and species differences

“…These two types of antigen-presenting cells formed a dense network. In conclusion, the high density of DC and macrophages in the mucosa of the nose forms the structural basis for an effective antigen uptake, a prerequisite for successful nasal vaccination protocols as documented by Cisney by combining surgical removal of NALT with vaccination protocols [54].…”

“…The efficacy of intranasal vaccination was dependent on NALT as after surgical removal the mice were not protected from toxic shock. In a more recent paper the same group studied NALT by ex vivo culturing NALT and as an alternative to gene knockout mice surgical removal of NALT was used, which is a difficult technique in the mouse [54].…”

Immunology‐Guided Biomaterial Design for Mucosal Cancer Vaccines

“…Expanded antigen‐specific T cells exit the LN via the draining lymphatics and ultimately enter the blood to circulate throughout the body and home to target tissues. Importantly, priming can occur in both, LNs and in MALT, although beyond the intestinal PPs, BALT, and the NALT, MALT has not been studied in detail or reported in every mucosal tissue. The presence of tissue‐specific cues during priming, for which peripheral tissue‐derived migratory DC are key, favors homing to specific tissues, in a process termed imprinting .…”

Collection and Processing of External Secretions and Tissues of Mouse Origin

“…A video illustration of these procedures (Cisney et al, 2012) can be accessed at the following link: http://www.jov e.com/video/3960/.…”

“…To avoid contamination with saliva, remove the mandible and gently flush the nasal cavity from the posterior opening of the nose with ∼200 μL of PBS and collect nasal washes from the anterior openings of the nose (Kurono et al, 1999). Alternatively, holding the mouse vertically, carefully pipet 30 μL of sterile PBS into one nostril and collect the rinse from other nostril (Cisney et al, 2012). 9.…”