The lymph node (LN) is a pea-sized (0.1–2.5 cm long), bean-shaped organ of the lymphatic and immune system. A human possesses approximately 500–600 lymph nodes and collectively, lymph nodes enable timely detection, response, and clearance of harmful substances from the body. Each lymph node comprises of distinct substructures, which host a plethora of immune cell types working in tandem to coordinate complex innate and adaptive immune responses.
The conventional way to study lymph node functions is using animal models but this approach suffers from limitations including a lack of relevant immune system to humans, physiological differences, high costs, low reproducibility, and ethical considerations preventing eligibility for selected studies.
To overcome these challenges, researchers have, in recent years, created LN-mimicking models to recapitulate the architecture and function of healthy and diseased LN tissues without the limitations associated with animal models. These newer approaches can be classified into biomaterial-based models such as hydrogel, organoids, and 3D microfluidic organ-on-a-chip system. The advantages are that they can provide convenient and controllable platforms to investigate complex cell-cell/tissue interactions and for drug screening.
Here, we discuss recent progress in LN-mimicking models, and how they are being exploited for applications including chimeric antigen receptor T cell (CAR-T) expansion and disease modeling.
Biomaterials as synthetic bioreactors
Cell-based immunotherapy such as CAR-T therapy is a promising strategy to treat cancer. However, it is time consuming and costly to expand genetically modified CAR-T cells to achieve clinically relevant doses. The gold standard method to expand CAR-T cells is using superparamagnetic Dynabeads coated with anti-CD3 and anti-CD28 antibodies. However, beyond these stimulatory signals, the lymph node microenvironment also provides additional homing signals through cytokines. One example is CCL21 which interacts with naïve T cells to enhance CD4+ and CD8+ T cell proliferation.
To improve expansion of CAR-T cells, a group led by Judith Guasch, PhD, at the Institute of Materials Science of Barcelona, created a hydrogel made of polyethylene glycol and low molecular weight heparin loaded with CCL21 to mimic the extracellular matrix of lymph node (Pérez del Río et al., 2020). The group found that their material boosted CD4+ T cell proliferation by 30%. Interestingly, there was also a marked reduction in the percentage of naïve CD4+ T cells while that of central memory and effector memory phenotypes increased. The authors concluded that based on their findings, cytokine loaded hydrogels can alter the proportion of T cells and the use of chemical stimuli in hydrogels is a promising approach to manipulate T-cell expansion and differentiation pathways.
Yevgeny Brudno, PhD, and his group at the North Carolina State University recently designed a bioinstructive scaffolds for rapid manufacture of CAR-T cells in vivo (Agarwalla et al., 2022).
“Our biggest motivation in pursuing this work is the cost and difficulty of CAR-T cell manufacturing. Although CAR-T cells are potentially revolutionary, the manufacturing method is labor intensive (~4 weeks) and very costly (~$400,000). We simply won’t cure cancer with current production methods. We wanted to move to a point-of-care method where CAR-T cells could be made cheaply and quickly,” says Brudno.
The team named their technology Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER). To promote T-cell activation within the biomaterial, they conjugated anti-CD3 and anti-CD28 antibodies and although the conjugation was likely random, T cells expanded 10-fold after 18 hours, suggesting that the antibodies were functional. To further enhance T cell expansion within the scaffolds, IL-2 was also physically encapsulated in the hydrogel and could be sustainably released over five days.
“The major achievement of our research is that we were able to reduce CAR-T cell production times down to a few hours. This is compared to 2–4 weeks with current methods. We did this by creating a material we call MASTER that performs each of the different steps of CAR-T cell manufacturing inside the body. MASTER is seeded with a patient’s blood cells and immediately implanted and becomes a CAR-T cell factory releasing CAR-T cells to the patient,” Brudno adds.
When the team loaded peripheral blood mononuclear cells (PBMCs) and retrovirus encoding a CD19-specific CAR on MASTER, they found that there was CAR expression in 22% of the T cells and transduction predominantly occurred in T cells due to the selectivity of retroviruses in transfecting actively dividing cells. Importantly, less than 1% of the expanded cells showed any indications of exhaustion which is commonly observed in ex vivo expanded CAR-T cells. As MASTER has a well-connected macroporous structure or about 100–200 µm, CAR-T cells were able to migrate efficiently out of the scaffold.
A major concern using implantable technology for genetic engineering is transduction of surrounding host cells. The team found no evidence of transduction of neighboring fibroblasts or cells that might infiltrate the scaffolds which they attributed to the short stability half-life of 4–6 hours of viruses in vivo. Nevertheless, they acknowledge that this needs to be further studied, particularly for circulating CD4+ T cells which is prevalent in the circulatory system.
Finally, the team found that compared to conventionally produced CAR-T cells, implantation of MASTER was able to offer 2-fold better tumor-free survival. There was also stronger persistence in the latter as MASTER generated 30-fold higher absolute counts of CD45+CD3+CAR+ T cells in the bone marrow and spleen compared to conventionally generated CAR-T cells. The enhanced persistence was also confirmed with a tumor rechallenge study which showed that MASTER-treated mice were tumor-free up to 30 days after rechallenge while tumor growth was evident within two weeks in mice intravenously infused with conventionally produced CAR-T cells.
“We are optimizing the technology to increase yield as well as cell numbers through studying in detail the molecular basis for how our scaffolds work. More importantly, we’re extending the concept to other applications, including solid tumors and HIV. First, as we see increased cell persistence and reduced cell differentiations with the scaffolds, we are testing these materials against solid tumors, including pancreatic and lung cancer. Second, we are working with the charity CaringCross to explore whether our scaffolds could be used for point-of-care CAR-T cell manufacturing as a cure for HIV. CaringCross is currently running a clinical trial of CAR-T cells against HIV, but they use traditional manufacturing techniques, which will not work in low- and middle-income countries,” says Brudno.
Due to the complexity of the lymphatic system, there has been limited literature to understand the effects of flow parameters and other stimuli on lymphatic vessels. A group led by Mandy Esch, PhD, at the National Institute of Standards and Technology, recapitulated the lymphatic vessel system using a microfluidic device with pulsatile flow pattern (Fathi et al., 2020). The team also tested different shear forces on the primary human lymphatic endothelial cells (HLECs), with low shear mimicking healthy conditions and no or high shear mimicking diseased states. They found that HLECs grew better in the presence of shear forces, and they also align with the direction of the flow. Interestingly, shear forces also reduced the production of IL-8 and tumor necrosis factor (TNF)-α which is supported by published literature. Overall, this study shows the advantage of integrating primary HLECs in 3D microfluidic devices to mimic the lymphatic system. The authors also suggested that their platform can be used to study interactions between cancer cells, T cells, and dendritic cells with the lymphatic endothelial cells that line lymphatic vessels.
Taking a step further, a group led by Donald Ingber, MD, PhD, professor, Harvard University, showed that using 3D microfluidics culture, they could induce primary blood-derived B and T lymphocytes to spontaneously self-assemble into ectopic lymphoid follicles that express Activation-Induced Cytidine Deaminase which mediates antibody class switching response within germinal centers in lymph nodes (Goyal et al., 2022). Importantly, the team also found that their microfluidic chips which contained autologous dendritic cells were able to show secretions of clinically relevant cytokines when stimulated with influenza vaccines and there was also immune-enhancing activity when the chip was tested with a squalene in water emulsion adjuvant.
“In this paper, we found that perfusion can drive the formation of lymphoid follicles from B and T cells, and that our lymphoid follicle chip is better at producing immunoglobin G (IgG) in response to seasonal influenza vaccine compared to traditional in vitro models. In unpublished data, we have recently shown that the lymphoid follicle chip also shows class switching to IgG in response to mRNA vaccines against a naive antigen, making it a valuable tool to test vaccines and adjuvants. We are also exploring the use of this model to understand tertiary lymphoid organogenesis in solid tumors,” says Girija Goyal, PhD, lead author of the paper, and senior scientist at Harvard University.
Immunological research has come a long way, and major findings including T cells and their antigen receptors and the functional importance of germinal centers for B cell development have come from studying inbred mice. Unfortunately, the mouse immune system is not identical to that of humans such as the affinity maturation and class switching and the effects of adjuvants. Attributing to a lack of accessible human organs to study immunity, many preclinical trials for vaccines that worked in animal models have failed in human trials.
Researchers have tried using slices of tissue explant cultures in vitro, but these tissues rarely survive beyond a week, and they typically represent a very distinct aspect of immunity and not its totality. To overcome these challenges, a group of researchers led by Mark Davis, PhD, professor, Stanford University, created tonsil organoids using discarded tonsil tissues (Wagar et al., 2021). They showed that the tonsil organoids recapitulated key germinal center features including somatic hypermutation and production of antigen-specific antibodies. Importantly, the organoids were also useful to assess humoral immune response to vaccines and different adjuvants. The team also found that similar strategy can be used to create lymph node organoids to study mechanisms underlying the human adaptive immunity.
Lymph nodes play a crucial role in adaptive immunity and there is significant value in generating models using biomaterials, microfluidics, and organoids to mimic them. Computational models can also be integrated with material designs to better understand the lymphoid tissue microstructure and microenvironment which current models and imaging methods cannot provide (Shou et al., 2022). A powerful approach would be to integrate both in vitro/vivo and in silico approaches integrated to strengthen basic patho-biological research, translational drug screening, and clinical personalized therapies involving the lymph nodes.
Agarwalla, P., et al. (2022). Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells. Nature Biotechnology. https://doi.org/10.1038/s41587-022-01245-x
Fathi, P., et al. (2020). Lymphatic Vessel on a Chip with Capability for Exposure to Cyclic Fluidic Flow. ACS Applied Bio Materials, 3(10), 6697–6707. https://doi.org/10.1021/acsabm.0c00609
Goyal, G., et al. (2022). Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ-on-a-Chip. Advanced Science, 9(14). https://doi.org/10.1002/advs.202103241
Pérez del Río, E., et al. (2020). CCL21-loaded 3D hydrogels for T cell expansion and differentiation. Biomaterials, 259. https://doi.org/10.1016/j.biomaterials.2020.120313
Shou, Y., et al. (2022). Integrative lymph node-mimicking models created with biomaterials and computational tools to study the immune system. In Materials Today Bio (Vol. 14). Elsevier B.V. https://doi.org/10.1016/j.mtbio.2022.100269
Wagar, L. E., et al. (2021). Modeling human adaptive immune responses with tonsil organoids. Nature Medicine, 27(1), 125–135. https://doi.org/10.1038/s41591-020-01145-0