Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Mucosal immunology

• Transcriptional control of regulatory T cell subsets

• Environment:Treg crosstalk

Main Findings

In this preprint, the authors investigate the interplay and regulation of three major colonic Treg subsets (RORc+ Tregs, Helios+ Tregs and double negative Tregs). They use 4 conditional knock out mouse lines of central transcriptional regulators in Tregs (Gata3, Izkf2, Rorc, Maf), gnotobiotic mouse models, multiple infection models, 16S rDNA amplicon sequencing, single cells RNA-sequencing and single cell ATAC sequencing to investigate the contribution and interplay of selective transcription factors on the development of colonic Treg subsets, the impact of specific commensal microbes on the heterogeneity of Treg subsets. They observe that deficiency in of either Gata3, Izkf2, Rorc or Maf has no major impact on the numerical abundance of Foxp3+ Tregs but leads to a change in the proportional abundance of the three major Treg subsets therein. The authors then analyse the gene expression and accessibility of genetic loci in the presence and absence of the above-listed transcription factors and identify unique accessibility of genes involved in the regulation of mucosal immunity. Moreover, the team’s ATAC-Seq analysis challenges previous observations on the role of other transcription factors (Tbet, Irf4, Blimp1) with selective effects on Treg subsets that warrant consideration and validation. These findings fall in line with their observations in a diverse spectrum of disease models and demonstrate a perturbed colonic, steady state immune landscape in the absence of selective Treg transcription factors and Treg subsets. Analysis of the microbiota in adult mice suggest that deficiency of Treg subsets has minimal to no impact on the composition of the microbiota implicating that Treg subsets may compensate for each other in the control of the microbiota. Using gnotobiotic mice, the authors then demonstrate that Treg-inducing microbes have no impact on the gene expression profile of the three major colonic Treg subsets but rather induce a change in their numerical abundance. Investigating T cell receptors sequences at the single cell level further reveals an even distribution and TCR sequences across Treg subsets that mirrors a comparable representation within the conventional T cell compartment. Collectively, this preprint addresses several open and central questions in the field of mucosal Treg biology.

Limitations

• The proportional changes in the colonic Treg subsets after deleting Rorc, Gata3, Helios or Maf are well documented. A complementation using a lenti- or retroviral overexpression system Tregs from the selective KO strains would be a great addition to their data and strengthen the conclusion.

• The authors suggest interactions of Gata3 and Helios in their models that could be confirmed by generating double conditional knock outs of both transcription factors in Tregs.

• The disease models used by the authors reveal unique roles for Tregs in shaping the disease outcome. It would be great to see if the elevated disease susceptibility or resistance is tied to any of the perturb immunocytes or Treg functions the authors describe. Depletion of cell types or neutralization cytokines could be used in their knock out mice to achieve this.

• It would great to pair their observations in mono-colonized GF mice with a more detailed analysis of factors that could affect Treg proliferation, recruitment or survival. An addition of known Treg-inducing microbes would strengthen this data set and allow for a more general interpretation. The selected 3 microbes, while useful to their study, may be a limitation. Would other microbes with similar features to those used in their experiments, phenocopy their observations?

• GF mice are colonized with single bacterial species at 4 weeks after birth. Would the abundance and gene expression across Treg populations differ if mice would have been born to GF parents that were mono-colonized with single bacterial species? This would be an interesting addition supporting the authors analysis of Treg subset ontogeny and address the impact of the early life environment on Treg abundance.

• While the manuscript is very well written and critical references cited, it would be great to see the experimental results done across all 4 conditional knock lines. Maf-deficient animals were not analysed, or results not displayed for some experiments.

Significance/Novelty

What is the novelty of the preprint for the field specific?

The authors show that central transcription factors control the abundance and function of distinct regulatory T cell subsets in the colon. They demonstrate that some of the transcription factors act systemically while others show a restricted action within the mucosa. They further show that these regulations are epigenetically made. Another important finding of their study is the role of the microbiota a sole “booster” of Treg subsets rather then selective effector of one Treg subset. Their findings suggest that Treg subset fate and function are regulated independently of the microbiota and the TCR, implicating tissue-specific niche factors for the maintenance and induction of Treg subsets.

How does the result of the preprint matter for general immunologists and/or patients?

This work extends our understanding of colonic Treg subsets as central regulators of disease outcomes and mucosal immune homeostasis. It assigns critical functions to transcriptional regulators to Treg subsets that are imprinted independently of the microbiota or the TCR-sequence. This suggests a conceptional change in how investigations in the microbiota:Treg crosstalk should be conducted in the future and are therefore critical for the broad scientific community.

Credit

Reviewed by Arthur Mortha (University of Toronto, Department of Immunology) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interest in relation to their involvement in the review.