Select microbiota species provides protection against food allergy via RORγt+ Tregs

It is now undoubtedly acknowledged that body's microbiota plays a decisive role in protection against allergy, including food allergy. But how exactly microbiota does it is less clear.

A new study in mice published in Nature Medicine suggests the certain microbiota species signal subset of FOXP3+ Tregs called RORγt+ Tregs via adaptor MyD88 to exert its protective role against food allergy.

The most of the experiments reported here were done in genetically modified mice called Il4ra^F709 that shows a predisposition to allergy due to an alteration in IL-4 signaling. Here, germ-free Il4ra^F709 mice were colonized with microbiota consortium differently enriched between non-allergic versus allergic infants. Out of those, defined mix of Clostridiales and Bacteriodales but not Proteobacteria could reduce allergic reaction in Il4ra^F709 mice. (Note, you can click the image to expand it to see it more accurately).

In a separate set of experiments the authors noticed that Il4ra^F709 mice or mice specifically deficient for RORγt+ Tregs subset displayed similar phenotype in response to allergic challenge. They thought there could be a connection.

Indeed, Il4ra^F709 mice deficient for RORγt+ Tregs subset lost an ability to resist allergic reaction when colonized with defined mix of Clostridiales and Bacteriodales.

Finally, the authors attributed the loss of protection to loss of MyD88 adaptor signaling in Tregs because Il4ra^F709 mice deficient for MyD88 signaling in Tregs also showed loss of protection against allergic reaction when colonized with defined mix of Clostridiales and Bacteriodales (Note, oral short chain fatty acid (SCFA) therapy failed to protect Il4ra^F709 mice against allergic response) .

In summary, we could conclude based on this and other studies that RORγt+ Tregs do play a decisive role in protection against unwanted inflammatory response (Note, however, that allergic sensitization protocol employed here is not exactly "translational" approach).

One major drawback of this study is that the authors failed to examine why it is that Clostridiales and Bacteriodales but not Proteobacteria or other species could signal via MyD88 to provide protection against allergic response. In my view it is not a difference in innate signaling molecules that distinguishes protective versus non-protective microbiota species but rather their antigenic composition that provides epitopes to RORγt+ Tregs to keep them active and in a good functioning condition (MyD88 could be just necessary to keep such antigen-specific Tregs active due to its role in metabolic pathways).

posted by David Usharauli

A neonatal temporal window for thymic epitope-specific Foxp3+ Treg formation

The thymus-derived Foxp3+ Tregs are indisputably the most important immune cell type. Surprisingly, little has been done to found out their antigen specificity. One reason for this lack of interest to study it has to do with the fact most scientists thought Tregs inhibited unwanted T cell responses antigen non-specific manner. So, they reasoned why to bother with TCR specificity. More recently however they started to pay close attention to antigen-specificity of Tregs since it became clear that antigen-specific Tregs showed superior, maybe even exclusive, therapeutic effect in animal models.  

So any study that advances our understanding of the formation of antigen-specific Tregs is immensely valuable. Below I will review one such research published in Nature Immunology from Eric Huseby's lab at the University of Massachusetts Medical School, Worcester, MA, USA.

In this study, they cloned several hundred TCRs from Foxp3+ GFP+ Tregs and screened their specificity in an in vitro IL-2 bioassay using standard hybridoma technology and library of ~1,750 unique self-peptides (it is astounding that so few labs have used this readily available approach). About 17 peptides showed a positive response. They chose to focus on 2 peptides derived from peptidyl arginine deiminase type IV (Padi4_92–105) and Adducin 2 (Add2_606–621). TCR specificity for Padi4_92–105 or Add2_606–621 was confirmed with respective KO mice.

Curiously, they noticed that thymic development of  Padi4_92–105 specific Tregs was time restricted and their formation rapidly went down after 3 weeks post birth.

More importantly, specific antigen expression was primarily responsible for both initial Treg formation and later its reduction.

However, even if there was initially an age-related decline in the frequency of Padi4_92–105 specific Tregs both in the thymus and periphery, their absolute numbers were maintained at a constant level in the periphery afterward. This is important to highlight.

Notably, this age-related antigen-dependent Treg reduction could be reversed in chimera where only thymic stromal but not bone-marrow derived cells expressed specific epitope. It could mean dose effect or specialized antigen-presentation pathway contributes to age-related decline in Padi4_92–105 specific Tregs formation.

Furthermore, out of several Padi4_92–105 specific TCRs with different antigen response potency, only moderate potency responders were enriched in Tregs in the periphery (the highest potency T cells were lost in the thymus and the lowest potency T cells ended up in Tconv spleen pool). In my view, this is conveniently too clean to my liking.

Also, the authors found Treg formation best correlated with the TCR:self-MHC half-life (t_1/2).

In summary, this study identified several self epitopes that drive mouse Treg formation in the thymus and this process is restricted to a few weeks post birth. It is not clear why or how the cessation of Treg formation is happening in the thymus here. As absolute numbers of such epitope-specific Tregs that seeded the periphery stayed constant it could indicate that a saturation feedback loop may exist between periphery and thymus that adjust Treg numbers. Additionally, the authors suggest that Treg formation could be predicted based solely on TCR:self-MHC dwell half-life (t_1/2). However, dwell time cannot explain their own observation about the age-related decline of Treg formation. What has changed in 8-week versus 3-week thymus to upend dwell time so dramatically? Besides, this paper did not address a mechanism of bifurcation that determines deletion versus Treg formation at the single thymocyte level that has been shown to occur independently of TCR affinity. 

Of note, these results could explain why some CD4+ TCR transgenic mice don't show thymic Foxp3+ Treg formation but still harbor them in the periphery, for example, marilyn CD4+ TCR transgenic mouse. As such mice are ordinarily examined when they are adults (>8 weeks) it will miss the thymic phase.  

posted by David Usharauli