New tools for immunologists: models of lymph node function from cells to tissues

Abstract

The lymph node is a highly structured organ that mediates the body's adaptive immune response to antigens and other foreign particles. Central to its function is the distinct spatial assortment of lymphocytes and stromal cells, as well as chemokines that drive the signaling cascades which underpin immune responses. Investigations of lymph node biology were historically explored in vivo in animal models, using technologies that were breakthroughs in their time such as immunofluorescence with monoclonal antibodies, genetic reporters, in vivo two-photon imaging, and, more recently spatial biology techniques. However, new approaches are needed to enable tests of cell behavior and spatiotemporal dynamics under well controlled experimental perturbation, particularly for human immunity. This review presents a suite of technologies, comprising in vitro, ex vivo and in silico models, developed to study the lymph node or its components. We discuss the use of these tools to model cell behaviors in increasing order of complexity, from cell motility, to cell-cell interactions, to organ-level functions such as vaccination. Next, we identify current challenges regarding cell sourcing and culture, real time measurements of lymph node behavior in vivo and tool development for analysis and control of engineered cultures. Finally, we propose new research directions and offer our perspective on the future of this rapidly growing field. We anticipate that this review will be especially beneficial to immunologists looking to expand their toolkit for probing lymph node structure and function.

Keywords: 3D culture; ex vivo model; human immunology; in silico model; in vitro model; lymphoid follicle; organ-on-chip (OoC); vaccination.

Figure 1

Schematic of the LN highlighting key aspects of lymph node organization and function. Each lobe shows one main feature: (i) spatial location of B and T lymphocytes, (ii) distribution of major stromal cell subsets; (iii) homing of naïve T cells into the node via high endothelial venules, and recognition of dendritic cells presenting cognate antigen by T cells, (iv) blood vessels within the lymph node and (v) germinal center formation. Figure created with BioRender.com.

Figure 2

Experimental and computational tools used in studying lymph node biology. Researchers have utilized in vitro models (cell culture in 2D wells, 3D constructs such as organoids and hydrogels, and microfluidic organ-on-chip devices), ex vivo culture of tissue slices alone or in combination with microfluidic chips (hybrid tissue-chips) and in vivo models in animals, to experimentally investigate lymph node structure and function. Computational ‘dry lab’ approaches, on the other hand, employ mathematical simulations to model biological environments. They have been used to complement experimental tools and can answer questions that are challenging to interrogate by current wet lab approaches. Figure created with BioRender.com.

Figure 3

Key immune events recapitulated in current and nascent models of the lymph node. Events being modeled include (A) chemotaxis, (B) cell motility, (C) cell-cell communication and (D) organ-level functions such as fluid flow, germinal center formation, and responses to vaccination. Figure created with BioRender.com.

Figure 4

Current approaches to modeling chemotaxis and cell-cell interactions. As an illustration, T cells are shown in green and DCs shown in yellow. (A) Chemotaxis and cell motility are readily modeled in microfluidic channels, including (i) gradient generator (system of parallel lanes) and (ii) ladder-style chip designs to ensure organized motion for easy imaging and quantification. Gradients of chemotactic factors or nutrients are established across the chip. (B) 3D culture of one or more cell types, with control over the biomaterials environment. (C) Trapping of cells in microwells ensures that individual cells or pairs of cells can be imaged over time, in high throughput. (D) Seeding a 2D monolayer of cells at the bottom of a flow chamber or microfluidic channel, with cell suspension flowed by above, enables study of cell-cell interactions under physiological and pathological flow conditions. Figure created with BioRender.com.

Figure 5

Examples of models of fluid flow and diffusion in the lymph node. (A) A computational fluid dynamics model, based on confocal microscopy images of the lymph node, predicted the advective fluid flow paths and velocities through the lymph node. The majority of the fluid flow passed around the edges of the node, in the sinus regions. Reproduced with permission from (132). Copyright 2015 Mary Ann Liebert, Inc. (B) Densely packed 3D cultures of reporter T cells predicted the distance over which secreted cytokines diffuse and elicit responses. Varying the proportion of IL-2 consuming T cells (100% vs 10% consumers) around IL-2 producing T cells indicated that IL-2 secretion was non-directional in this setup. Cells were stained with DAPI (blue) to identify nuclei, anti-IL-2Ra (green) to identify IL-2 consuming T cells and anti-pSTAT5 (red) to detect T cell response to IL-2. With greater consumption (100% consumers), IL-2 secretion remained more localized and had a smaller magnitude of signaling. Reproduced with permission from (135). Copyright 2017 Elsevier. (C) Schematic of a microfluidic chip developed to track diffusion of cytokines after delivery into live lymph node tissue slices through a port of entry. Reproduced with permission from (136). Copyright 2018 Elsevier.