New immunotherapies as well as vaccines are currently evaluated in animal models. However, this approach can lead to unpredicted toxicities or poor efficacy in clinical trials because of species-specific differences in immune responses.[1]

And while preclinical research can be conducted in vitro using human immune cells collected from blood, the results of such an approach often fail to predict patient responses.[2][3]

Understanding this issue, Ed Yong, a veteran science writer he won the Pulitzer Prize in explanatory journalism, very simple and yet extremely accurate, wrote in The Atlantic, “The immune system is very complicated.”*

In a series of articles published in The Atlantic, Young explained that as the COVID-19 pandemic had made abundantly clear, science still doesn’t fully understand the sophisticated defense mechanisms that protect us from microbe invaders. Why do some people show no symptoms when infected with SARS-CoV-2 while others suffer from severe fevers and body aches? Why do some succumb to cytokine storms of the body’s own making? We still lack exact answers to these questions.

Port Worthy

Very complicated, but now, it’s on a chip
One major reason why preclinical research using in vitro using human immune cells collected from blood fail, is that in vivo immune responses commonly occur within the highly specialized tissue microenvironment of lymphoid follicles (LFs) – structures that reside in lymph nodes and other parts of the human body which mediate immune responses. [2][3]

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However, scientists, have now a new tool to help them tease out the immune system’s mysteries, thanks to a group of researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University.

They cultured human B and T cells inside a microfluidic Organ Chip device and coaxed them to spontaneously form functional lymphoid follicles. They consist of different chambers that harbor “naïve” B cells and T cells, which together initiate the cascade of events that leads to a full immune response when they are exposed to a specific antigen.

This illustration demonstrates the structure of the LF Chip: B cells and T cells were cultured together in the extracellular matrix (ECM)-lined lower channel, and were “fed” via the consistent flow of nutrient-containing medium through the upper channel. This flow is also what appears to have caused the spontaneous assembly of the cells into lymphoid follicles. Image courtesy: Wyss Institute at Harvard University, Used with permission.

In addition to allowing researchers to probe the normal function of the immune system, these lymphoid follicle (LF) Chips can also be used to predict immune responses to various vaccines and help select the best performers, offering significant improvement over existing preclinical models like cells in a dish and non-human primates.

The achievement and results of this research, which was funded by the United States Defense Advanced Research Projects Agency (DARPA) were published in the March 14, 2022 edition of Advanced Science.[4]

“Animals have been the gold-standard research models for developing and testing new vaccines, but their immune systems differ significantly from our own and do not accurately predict how humans will respond to them. Our LF Chip offers a way to model the complex choreography of human immune responses to infection and vaccination, and could significantly speed up the pace and quality of vaccine creation in the future,” said first author Girija Goyal, Ph.D., a Senior Staff Scientist at the Wyss Institute.[4]

An accidental discovery
Like many great scientific discoveries, the LF Chip project is the result of serendipity in the lab. Goyal and other Wyss Institute scientists wanted to investigate how B and T cells circulating in the blood would change their behavior once they entered a tissue, so they obtained those cells from human blood samples and cultured them inside a microfluidic Organ Chip device to replicate the physical conditions they would experience when they encountered an organ.

When the cells were placed inside one of the two channels within the device, nothing remarkable happened — but when the researchers started the flow of culture medium through the other channel to feed the cells, they were surprised to see that the B and T cells started to spontaneously self-organize into 3D structures within the Organ Chip that appeared similar to “germinal centers” – structures within LFs where complex immune reactions take place. “It was so unexpected that we completely pivoted from the original experiment and focused on trying to figure out what they were,” Goyal explained.

When B cells and T cells were cultured in the LF Chip under flow conditions (left), they spontaneously formed 3D structures that were later identified as nascent lymphoid follicles. When the same cells were cultured in static conditions (right), no structures formed. Image Courtesy: Wyss Institute at Harvard University, Used with permission.

Imaris Snapshot
When B cells and T cells were cultured in the LF Chip under flow conditions (left), they spontaneously formed 3D structures that were later identified as nascent lymphoid follicles. When the same cells were cultured in static conditions (right), no structures formed. Credit: Wyss Institute at Harvard University

When the researchers started probing the mysterious structures that had formed inside the Organ Chip under flow conditions, they found that the cells were secreting a chemical called CXCL13. CXCL13 is a hallmark of LF formation, both within lymph nodes and in other parts of the body in response to chronic inflammation, such as in cancer and autoimmune conditions.

The team also found that B cells within the LFs that self-assembled on-chip also expressed an enzyme called activation-induced cytidine deaminase (AID), which is critical for activating B cells against specific antigens and is not present in B cells that are circulating in the blood. Neither CXCL13 nor AID were present in cells that were cultured in a standard 2D dish, suggesting that the scientists had indeed successfully created functional LFs from circulating blood cells.

In LFs in the human body, activated B cells mature and differentiate into multiple types of progeny cells including plasma cells, which secrete large amounts of antibodies against a specific pathogen. The team detected the presence of plasma cells in the LF Chips after they applied several stimuli used in the laboratory to activate B cells, such as the combination of the cytokine IL-4 and an anti-CD40 antibody, or dead bacteria. Remarkably, the plasma cells were concentrated in clusters within the LFs, as they would be in vivo.

“These findings were especially exciting because they confirmed that we had a functional model that could be used to unravel some of the complexities of the human immune system, including its responses to multiple types of pathogens,” said Pranav Prabhala, a Technician at the Wyss Institute and second author of the paper.

Predicting vaccine efficacy on-a-chip
Now that the scientists had a functional LF model that could initiate an immune response, they explored whether their LF Chip could be used to replicate and study the human immune system’s response to vaccines.

In the human body, vaccination induces special cells called dendritic cells to take up the injected pathogen and migrate to lymph nodes, where they present fragments of them on their surface. There, these antigen-presenting cells activate the B cells with the assistance of local T cells in the LF, causing the B cells to differentiate into plasma cells that produce antibodies against the pathogen. To replicate this process, the researchers added dendritic cells to LF Chips along with B and T cells from four separate human donors. They then inoculated the chips with a vaccine against the H5N1 strain of influenza along with an adjuvant called SWE that is known to boost immune responses to the vaccine.

LF Chips that received the vaccine and the adjuvant produced significantly more plasma cells and anti-influenza antibodies than B and T cells grown in 2D cultures or LF Chips that received the vaccine but not the adjuvant.

The team then repeated the experiment with cells from eight different donors, this time using the commercially available influenza vaccine (FluzoneⓇ High-Dose Quadrivalent; Sanofi Pasteur) which protects against three different strains of the virus in humans. Once again, plasma cells and anti-influenza antibodies were present in significant numbers in the treated LF Chips. They also measured the levels of four cytokines in the vaccinated LF Chips that are known to be secreted by activated immune cells, and found that the levels of three of them (IFN-γ, IL-10, and IL-2) were similar to those found in the serum of humans who had been vaccinated with the commercially available influenza vaccine.

The Wyss researchers are now using their LF Chips to test various vaccines and adjuvants in collaboration with pharmaceutical companies and the Gates Foundation.

“The flurry of vaccine development efforts sparked by the COVID-19 pandemic were impressive for their speed, but the increased demand suddenly made traditional animal models scarce resources. The LF Chip offers a cheaper, faster, and more predictive model for studying human immune responses to both infections and vaccines, and we hope it will streamline and improve vaccine development against many diseases in the future,” said corresponding author Donald Ingber, M.D., Ph.D., who is the Founding Director of the Wyss Institute as well as the Judah Folkman Professor of Vascular Biology at Harvard Medical School (HMS) and Boston Children’s Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.


*Ed Yong (Edmund Soon-Weng Yong), a Malaysian-born British science journalist is a staff member at The Atlantic, which he joined in 2015. In 2021 he received a Pulitzer Prize for Explanatory Reporting for a series on the COVID-19 pandemic

Highlights of prescribing information
FluzoneⓇ High-Dose Quadrivalent; Sanofi Pasteur (Prescribing Information)

[1] Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV, Xu W, Richards DR, McDonald-Smith GP, Gao H, Hennessy L, Finnerty CC, López CM, Honari S, Moore EE, Minei JP, Cuschieri J, Bankey PE, Johnson JL, Sperry J, Nathens AB, Billiar TR, West MA, Jeschke MG, Klein MB, Gamelli RL, Gibran NS, Brownstein BH, Miller-Graziano C, Calvano SE, Mason PH, Cobb JP, Rahme LG, Lowry SF, Maier RV, Moldawer LL, Herndon DN, Davis RW, Xiao W, Tompkins RG; Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A. 2013 Feb 26;110(9):3507-12. doi: 10.1073/pnas.1222878110. Epub 2013 Feb 11. PMID: 23401516; PMCID: PMC3587220.
[2]  Eastwood D, Findlay L, Poole S, Bird C, Wadhwa M, Moore M, Burns C, Thorpe R, Stebbings R. Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells. Br J Pharmacol. 2010 Oct;161(3):512-26. doi: 10.1111/j.1476-5381.2010.00922.x. PMID: 20880392; PMCID: PMC2990151.
[3] Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA, Lockhart S, Shea JE, McClain JB, Hussey GD, Hanekom WA, Mahomed H, McShane H; MVA85A 020 Trial Study Team. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet. 2013 Mar 23;381(9871):1021-8. doi: 10.1016/S0140-6736(13)60177-4. PMID: 23391465; PMCID: PMC5424647.
[4] Goyal G, Prabhala P, Mahajan G, Bausk B, Gilboa T, Xie L, Zhai Y, Lazarovits R, Mansour A, Kim MS, Patil A, Curran D, Long JM, Sharma S, Junaid A, Cohen L, Ferrante TC, Levy O, Prantil-Baun R, Walt DR, Ingber DE. Ectopic Lymphoid Follicle Formation and Human Seasonal Influenza Vaccination Responses Recapitulated in an Organ-on-a-Chip. Adv Sci (Weinh). 2022 Mar 14:e2103241. doi: 10.1002/advs.202103241. Epub ahead of print. PMID: 35289122.

Featured image: Organ-on-a-Chip: Photo Courtesy: © 2022 Wyss Institute at Harvard University. Used with permission.


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