HEB collaborates with TCR signaling to upregulate Id3 and enable γδT17 cell maturation in the fetal thymus

Reviewer #1 (Public review):

The authors use Flow cytometry and scRNA seq to identify and characterize the defect in gdT17 cell development from HEB f/f, Vav-icre (HEB cKO), and Id3 germline-deficient mice. HEB cKO mice showed defects in the gdT17 program at an early stage, and failed to properly upregulate expression of Id3 along with other genes downstream of TCR signaling. Id3KO mice showed a later defect in maturation. The results together indicate HEB and Id3 act sequentially during gdT17 development. The authors further showed that HEB and TCR signaling synergize to upregulate Id3 expression in the Scid-adh DN3-like T cell line. Analysis of previously published Chi-seq data revealed binding of HEB (and Egr2) at overlapping regulatory regions near Id3 in DN3 cells.

The study provides insight into mechanisms by which HEB and Id3 act to mediate gdT17 specification and maturation. The work is well performed and clearly presented. We only have minor comments.

Reviewer #2 (Public review):

Summary:

The manuscript by Selvaratnam et al. defines how the transcription factor HEB integrates with TCR signaling to regulate Id3 expression in the context of gdT17 maturation in the fetal thymus. Using conditional HEB ablation driven by Vav Cre, flow cytometry, scRNA-seq, and reanalysis of ChIP-seq data the authors, provide evidence for a sequential model in which HEB and TCR-induced Egr2 cooperatively upregulate Id3, enabling gdT17 maturation and limiting diversion to the ab lineages. The work provides an important mechanistic insight into how the E/ID-protein axis coordinates gd T cell specification and effector maturation.

Strengths include:

(1) The proposed model that HEB primes, TCR induces, and Id3 stabilizes gdT17 cells in embryonal development is elegant and consistent with the findings.

(2) The choice of animal models and the study of a precise developmental window.

(3) The cross-validation of flow, scRNA-seq, and ChIP-seq reanalyses strengthens the conclusions.

(4) The study clarifies the dual role of Id3, first as an HEB-dependent maturation factor for gdT17 cells, and as a suppressor of diversion to the ab lineages.

Weaknesses:

(1) The ChIP-seq reanalysis indicates overlapping HEB, E2A, and Egr2 peaks ~60 kb upstream of Id3. Given that the Egr2 data are not generated using the same thymocyte subsets, some form of validation should be considered for the co-binding of HEB and Egr2, potentially ChIP-qPCR in sorted gdT17 progenitors.

(2) E2A expression is not affected in HEB-deficient cells, raising the question of partial compensation, a point that should be specifically discussed.

(3) All experiments are done at E18, when fetal gdT17 development predominates. The discussion could address whether these mechanisms extend to neonatal or adult gdT17 subsets.

Reviewer #3 (Public review):

Summary:

The authors of this manuscript have addressed a key concept in T cell development: how early thymus gd T cell subsets are specified and the elements that govern gd T17 versus other gd T cell subsets or ab T cell subsets are specified. They show that the transcriptional regulator HEB/Tcf12 plays a critical role in specifying the gd T17 lineage and, intriguingly, that it upregulates the inhibitor Id3, which is later required for further gd T17 maturation.

Strengths:

The conclusions drawn by the authors are amply supported by a detailed analysis of various stages of T cell maturation in WT and KO mouse strains at the single cell level, both phenotypically, by flow cytometry for various diagnostic surface markers, and transcriptionally, by single cell sequencing. Their conclusions are balanced and well supported by the data and citations of previous literature.

Weaknesses:

I actually found this work to be quite comprehensive. I have a few suggestions for additional analyses the authors could explore that are unrelated to the predominant conclusions of the manuscript, but I failed to find major flaws in the current work.

I note that HEB is expressed in many hematopoietic lineages from the earliest progenitors and throughout T cell development. It is also noteworthy that abortive gamma and delta TCR rearrangements have been observed in early NK cells and ILCs, suggesting that, particularly in early thymic development, specification of these lineages may have lower fidelity. It might prove interesting to see whether their single-cell sequencing or flow data reveal changes in the frequency of these other T-cell-related lineages. Is it possible that HEB is playing a role not only in the fidelity of gdT17 cell specification, but also perhaps in the separation of T cells from NK cells and ILCs or the frequency of DN1, DN2, and DN3 cells? Perhaps their single-cell sequencing data or flow analyses could examine the frequency of these cells? That minor caveat aside, I find this to be an extremely exciting body of work.