Webinar: Early Life Development of Thymic Microbiota-Specific T Cells

 

Gut microbiota is known to educate the immune system, balancing effector and regulatory T cells responses, but it remains unknown how bacteria-specific T cells develop. In this webinar, Dr. Daniel Zegarra-Ruiz of Memorial Sloan Kettering Cancer Center, recipient of the AAI 2020 Lefrançois-BioLegend Memorial Award, discusses how thymic T cell development is altered after colonization with model intestinal microbes. These findings include thymic expansion of bacteria-specific naive T cells, discovery of bacterial DNA in the thymus, enrichment of CX3CR1+ dendritic cells (CX3+ DCs), and the effect of depleting the gut microbiota with antibiotics. Thymic microbiota-specific T cells were found to differentiate into intestinal effector T cells, offering protection or mediating pathology. Together, Dr. Zegarra-Ruiz’s data suggests that trafficking of bacteria to the thymus by intestinal DCs drives T cell expansion of microbiota-specific T cells, shaping peripheral immunity against pathogens and the microbiota itself.

 

What you will learn:

 

• How to identify and characterize microbiota-specific T cells in the thymus
• How gut microbial depletion affects thymic populations
• How to study antigen-presenting cells traffic from the gut to the thymus
• How to assess thymic microbiota-specific T cells expansion and differentiation

Additional Q&A With Dr. Zegarra-Ruiz

Due to great interest in this talk, many submitted questions weren’t answered during the webinar. Dr. Zegarra-Ruiz graciously answered these questions, below.

 

1. Could you remind me: what is MLN?

 

MLN, or mesenteric lymph node is the lymph node that drains multiple sections of the gut and is critical for regulatory and effector immune responses.

 

2. If you were to re-introduce another microbe that is not native to B6 in the adult mice, would the processes seen in the young mice be recapitulated?

 

We don’t expect that to occur. As the presented data show, adult mice were not previously exposed to SFB or EC and colonization at this stage did not induce the effects seen in young mice. We hypothesize that this process stops as the microbiota matures and achieves a steady state. However, it would be interesting to assess in germ-free mice if this process occurs in any age.

 

3. Would a pathogenic bacteria introduced at weaning produce the same process?  What makes SFB different than another bacteria that doesn't colonize?

 

That’s an interesting question and it’s something we are soon going to try. We think that this process is common for commensals (even pathobionts if the host is not under any source of stress). However, we don’t think pathogens would ‘take the same route’ as commensals and thus would fail to induce the same effects.

 

4. You mentioned the impact of CCR5 blocking on reducing bacterial DNA in thymus. What about these CCR5+ cells?

 

We found that CCR5+ CX3CR1+ DCs were decreased as mice aged. Thus, blocking CCR5-dependent migration of CX3CR1+DCs in young mice was used to show the relevance of this chemokine receptor in this process.

 

5. Do you expect a different process between mice colonized at birth & at weaning?

 

Great question. The differences we find for these processes are found comparing mice with a developing microbiota (weaning) to those with an established microbiota (adult). Thus, we would hypothesize that a similar mechanism would occur in mice colonized at birth if the required immune components are developed at that point.

 

6. Very nice talk! Thank you. At what age do you start defining mice as adult?

 

For the purpose of this study, we are defining 12-week-old mice as adult.

 

7. What is the type of sorting for T cells and dendritic cells in the thymus?

 

T cells sorted for RNA-seq were Tetramer+ CD4+ CD8- CD25- CD73- CD3+ MHCII- DAPI-. We used an Aria Cell Sorter for Fluorescence activated cell sorting. Sorting of CX3CR1+ DCs in the thymus is ongoing.

 

8. Have you tried to find those DCs in the blood before they reach the thymus?

 

We have found that in young mice CX3CR1+DCs are the predominant APC population in the blood carrying stained-E. coli minutes after oral gavage. We have not tested this with other bacteria.

 

9. Very interesting presentation! I'm wondering what you think your findings say about the risk factors surrounding dysbiosis? Will young people be able to adapt and get off relatively unscathed thanks to this mechanism?

 

Really interesting question. As you see in our data this process occurs only in young mice as their microbiota matures. It would indeed be critical to assess if dysbiosis by any source (diet, infection, inflammation, etc.) could alter this process. However, we don’t think that drastic changes to this process early in development would go unnoticed and could have deleterious effects as mice age (we’re also testing this). Of course, assessing these processes in humans would be extremely informative but also quite challenging.

 

10. Do you have any idea what the microbiota-specific T cells are seeing? Did you consider single-cell sequencing to see any signatures on the TCR repertoire? Or spatial genomics to pinpoint the location of the bacteria-specific DCs in the thymus?

 

Pretty good points. Many more questions remain to be answered, but changes in the TCR repertoire is a critical point that we are working to elucidate. Similarly, we’re planning to do FISH to assess where and when is the bacterial DNA found in the thymus.

 

11. Can you propose a mechanism for the increase in CXC3R1+ DC levels induced by SFBs? Would other types of gut bacteria also induce these DCs?

 

We hypothesize that, in young mice, commensals are constantly being sampled by CX3CR1+ DCs and taken to the thymus since depletion of gut microbes leads to a decrease in thymic CX3CR1+ DCs. When we enrich with either SFB or E. coli, we are simply saturating this system to more clearly assess this mechanism.

 

12. Regarding tetramer products conjugated to fluorochromes: which vendor provided the products?

 

All tetramers were obtained from the NIH tetramer core.

 

13. Do you think there is a difference in the export mechanism between commensal vs pathogenic bacteria? Or is it a difference in the maturation (effector vs regulatory) of the bacteria specific T cells?

 

We think that this process is common for commensals (even pathobionts if the host is not under any source of stress). However, we don’t think pathogens would ‘take the same route’ as commensals and thus would fail to induce the same effects. But of course, we need to test this.

 

14. What do you think will happen if we start colonization earlier than 3 weeks?

 

Great question. The differences we find for these processes are found comparing mice with a developing microbiota (weaning) to those with an established microbiota (adult). Thus, we would hypothesize that a similar mechanism would occur in mice colonized earlier if the required immune components are properly developed.

 

15. Based on antibiotic experiments, it seems that bacterial antigens are playing a bigger role in positive selection than negative selection of T cells. Could you please comment?

 

Unfortunately, we haven’t studied in detail the microbiota effects in T cell selection. We are simply identifying expansion of microbiota-specific thymic T cells. Further studies are needed to assess their role in T cell selection.

 

16. Did you 16S sequence the plasma?

 

Unfortunately, we haven’t performed 16S of the plasma. However, in other assays we determined that bacteria had to be sampled by antigen presenting cells to be found in blood.

 

17. Did you try culturing the blood?

 

Yes, and multiple other tissues, but we were not able to detect growth aerobically (of E. coli).

 

18. Thank you for the great talk! I am wondering, how do you exclude the possibility of contamination when detecting bacterial DNA in the thymus? And how did you rule out cross-reactivity when identifying bacteria-specific T cells from the thymus? Thank you!

 

Great questions. Tissues were aseptically collected, and multiple controls were tested. In addition, we used standard curves to determine the limit of detection and get rid of contaminants. For the tetramers, we used multiple non-related tetramers (helicobacter, human peptides, etc.) to assess for proper detection.

 

19. Hi, nice talk. What would cause the dramatic drop in thymus cellularity after antibiotic treatment?

 

We hypothesize that this is due to loss of antigen source as the microbiota is depleted, because when antibiotics were given to germ-free mice there is no change in thymic numbers. However, further testing needs to be done to confirm this.