When we consider our immune system encountering new microbes or foreign particles, we often picture a strong response, where adaptive immunity is generated so that next time you come across the microscopic invader it can be delt with quickly—usually with you none the wiser. We expect this kind of outcome from vaccines, for example, so that if you are later exposed to the corresponding infectious microbe, you won’t actually fall sick. Quite often, however, when your immune system encounters something new it will learn not to respond. This crucial component of immunity, termed immune tolerance, which allows us to interact with the continuous barrage of harmless microbes and antigens—both external and internal—without constant immune system overreaction. But what happens if you build tolerance to a vaccine? Variable responses to vaccines have long been observed in people, and recent evidence suggests that previous exposure to antigens which appear similar to vaccine antigens can result in decreased vaccine efficacy. If the pre-exposure comes from the gastrointestinal tract, such as from members of our gut microbiota, this is referred to as oral tolerance.
Regulatory T cells (Tregs)—specialized adaptive immune cells with important roles in keeping other immune cells in check by reducing inflammatory responses—are prevalent in the gastrointestinal tract and appear to be very important for establishing oral tolerance to vaccines. Specifically, a subset of Tregs that express the transcription factor RORt+ have been implicated in this phenomenon as they are particularly abundant in the gut and appear to be specific to microbial antigens. These RORt+ Tregs have also previously been shown to suppress type 2 immune responses, those which involve antibodies such like Immunoglobulin (Ig)E, IgG1, and cytokines such as Interleukin(IL)-4. It was these cells that were the main focus of the recent paper published in iScience by Dr. Nicole Potchen, who recently graduated from Jim Kublin’s lab in the Vaccine and Infectious Disease Division at Fred Hutch. Because RORt is a transcription factor, the research team hypothesized that its expression within Tregs would be important for regulating systemic responses to prior antigen exposure, possibly through impacting type 2 immune responses.
To address their hypotheses, they wanted to first test how prior antigen exposure impacted vaccine responses in their model. To do this, they used a well-characterized antigen called OVA, which they administered orally prior to an injection of an OVA-alum vaccine. Using OVA allows researchers to easily look for antigen-specific antibody responses, which is what they assessed here as well and noted that prior OVA exposure reduced OVA-specific IgG1 antibody levels following systemic OVA-vaccination. They also saw a reduction in IL-4 levels, noting that only type 2 immunity was suppressed in this system. After observing that there was in fact a suppression of systemic responses post prior exposure, the team investigated how the Treg cells responded to oral antigen exposure. There are a few different subtypes of Tregs, typically delineated by their transcription factor usage, which the research team characterized in their model; they observed that there was an increase in the RORt+ Tregs in the spleen after OVA exposure, but no major changes to other Treg subsets. They also saw a decrease in the proportion T helper 2 (Th2) CD4+ T cells, a non-regulatory-type of T cell which are involved in type 2 immune responses, consistent with their previous observation of IL-4 reduction.
To test whether these RORt+ Tregs would actually be important for oral tolerance responses, they generated a conditional knockout which lacked RORt expression specifically in the Treg cells. When they vaccinated these mice without any prior antigen exposure, there was no difference in vaccine immune responses compared to wildtype (WT) mice, importantly showing that loss of this subset of cells doesn’t impair normal vaccine responses. However, when they used their OVA pre-exposure model, they did not see any differences in vaccine response compared to the WT mice who were also pre-exposed. Since their hypothesis was that these cells would be crucial to oral tolerance, this result was not what the research team expected. Dr. Kublin commented that the results were so surprising “we were wondering, were the cages switched?” Dr. Potchen added that “Only after the 3rd time I saw it was I convinced”. This was especially convincing, they both explained, as the oral tolerance model is reliably reproducible, not something that is always common among in vivo studies.
To help further understand the potential importance of these cells, the team characterized how they accumulated in the gastrointestinal track during development. When they compared standard lab mice to germ-free mice, they saw that RORt+ Tregs increased concurrent to microbiota establishing residence after weaning, but were not as abundant in germ-free mice lacking these commensal microbes. This supported the hypothesis that these cells were likely reactive towards antigens present within the gastrointestinal tract, yet still, the loss of RORt did not alter the oral tolerance response. The research team also wanted to assess how changing the adjuvant component of the vaccine might impact RORt+ Tregs or oral tolerance. Adjuvants are molecules added to vaccines to increase the immunogenicity of the antigen and induce stronger vaccine responses. The adjuvant that they had been previously using, alum, skews the resulting immune response towards type 2 immunity, but other adjuvants can generate a stronger type 1 immune response -which is more associated with anti-viral immunity, for example. Whether these differences could alter outcomes on the development of tolerance or the potential contribution of RORt+ Tregs was the next question they asked. When they swapped out the adjuvant, alum, for ones that were more potent, they observed an increase in RORt+ Tregs after OVA vaccination consistent with previous reports. Yet, when they used their conditional knockout model, they still observed suppression of immune responses to subsequent vaccines. Altogether, their data supported a novel finding that RORt expression in Tregs was not crucial for oral tolerance.
The questions this paper elicits represent an exciting contribution to the field, as it may challenge the hypothesis that these cells, through RORt expression, are critical for oral tolerance to systemic responses. Dr. Kublin also emphasized the importance of studying this phenomenon, explaining that it likely represents a good way to study autoimmunity and other diseases. “Understanding how peripheral tolerance is maintained in the first place may shed light on how, under some circumstances, it is broken and results in disease,” Kublin noted. Despite their data showing a lack of a role for RORt expression in Tregs in this model, Dr. Potchen still believes that these cells do play a role in oral tolerance. “We knocked out a transcription factor”, she explained, “and it could just be that this particular transcription factor doesn’t matter, but that the cell type does. That’s what I think it is.” Therefore, RORt may constitute an identifiable marker for Tregs, but there could be other contributing factors—which aren’t yet characterized yet—that actually control and reflect the contribution Tregs have to oral tolerance. The results from this research also highlight an important way to approach scientific research, explained Dr. Kublin: “It’s a good example of pursuing science for the sake of a question and not prescribing it”. All in all, the study exemplifies the fact that just because a hypothesis seems obviously correct, the only way to know for sure is to actually test it.
This research was funded by the National Institutes of Health and the University of Washington.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Dr. James Kublin contributed to this work.
Potchen NB, Johnson AMF, Hager K, Graham J, Van P, Lyn-Kew KH, Warrier L, Talavera IC, Lund JM, Kublin JG. Oral tolerance to systemic vaccination remains intact without RORγt expression in regulatory T cells. iScience. 2023 Nov 22;26(12):108504. doi: 10.1016/j.isci.2023.108504. PMID: 38125026; PMCID: PMC10730369.