Distinct T Cell Receptor (TCR) gene segment usage and MHC-restriction between foetal and adult thymus

Reviewer #1 (Public Review):

Summary:
The manuscript by Rowell et al aims to identify differences in TCR recombination and selection between foetal and adult thymus in mice. Authors sequenced the unpaired bulk TCR repertoire in foetal and adult mice thymi and studied both TCRB and TCRa characteristics in the double positive (DP, CD4+CD8+) and single positive (SP4 CD4+CD8-CD3+ and SP8 CD4-CD8+CD3+) populations. They identified age-related differences in TCRa and TCRB segment usage, including a preferential bias toward 3'TRAV and 5' TRAJ rearrangements in foetal cells compared to adults who had a larger perveance for 5'TRAV segments. By depleting the thymocyte population in adult thymi using hydrocortisone, the authors demonstrated that the repertoire became more foetal like, they therefore argue that the preferential 5'TRAV rearrangements in adults may be resulting from prolonged/progressive TCRa rearrangements in the adult thymocytes. In line with previous studies, Authors demonstrate that the foetal TCR repertoire was less diverse, less evenly distributed and had fewer non-template insertions while containing more clonal expansions. In addition, the authors claim that changes in V-J usage and CDR1 and CDR2 in the DP vs SP repertoires indicated that positive selection of foetal thymocytes are less dependent on interactions with the MHC.

Strengths:
Overall, the manuscript provides an extensive analysis of the foetal and adult TCR repertoire in the thymus, resulting in new insights in T cell development in foetal and adult thymi.

Weaknesses:
Three major concerns arise: 1) the authors have analysed TCR repertoires of only 4 foetal and 4 adult mice, considering the high spread the study may have been underpowered. 2) Gating strategies are missing and 3) the manuscript is very technical and clearly aimed for a highly specialised audience with expertise in both thymocyte development and TCR analysis. Authors are recommended to provide schematics of the TCR rearrangements/their findings and include a summary conclusions/implications of their findings at the end of each results section rather than waiting till the discussion. This will help the reader to interpret their findings while reading the results.

Reviewer #2 (Public Review):

Summary:
The authors comprehensively assess differences in the TCRB and TCRA repertoires in the fetal and adult mouse thymus by deep sequencing of sorted cell populations. For TCRB and TCRA they observed biased gene segment usage and less diversity in fetal thymocytes. The TCRB repertoire was less evenly distributed and displayed more evidence of clonal expansions and repertoire sharing among individuals in fetal thymocytes. In both fetal and adult thymocytes they show skewing of V segment (CDR1-2) repertoires in CD4 and CD8 as compared to DP thymocytes, which they attribute to MHC-I vs MHC-II restriction during positive selection. However the authors assess these effects to be weaker in fetal thymocytes, suggesting weaker MHC-restriction. They conclude that in multiple respects fetal repertoires are distinct from and more innate-like than adult.

Strengths:
The analyses of the F18.5 and adult thymic repertoires are comprehensive with respect to the cell populations analyzed and the diversity of approaches used to characterize the repertoires. Because repertoires were analyzed in pre- and post-selection thymocyte subsets, the data offer the potential to assess repertoire selection at different developmental stages. The analysis of repertoire selection in fetal thymocytes may be unique.

Weaknesses:
(1) Problematic experimental design and some lack of familiarity with prior work have resulted in highly problematic interpretations of the data, particularly for TCRA repertoire development.
The authors note fetal but not adult thymocytes to be biased towards usage of 3' V segments and 5'J segments. It should be noted that these basic observations were made 20 years ago using PCR approaches (Pasqual et al., J.Exp.Med. 196:1163 (2002)), and even earlier by others. The authors also note that in fetal thymus this bias persists after positive selection, and it can be reproduced in adults during recovery from hydrocortisone treatment. The authors conclude that there are fewer rounds of sequential TCRA rearrangements in the fetal thymus, perhaps due to less time spent in the DP compartment in fetus versus adult. However, the repertoire difference noted by the authors does not require such an explanation. What the authors are analyzing in the fetus is the leading edge of a synchronous wave of TCRA rearrangements, whereas what they are analyzing in adults is the unsynchronized steady state distribution. It is certainly true, as has been shown previously, that the earliest TCRA rearrangements use 3' TRAV and 5'TRAJ segments. But analysis of adult thymocytes has shown that the progression from use of 3' TRAV and 5' TRAJ to use of 5' TRAV and 3' TRAJ takes several days (Carico et al., Cell Rep. 19:2157 (2017)). The same kinetics, imposed on fetal development, would put development of a more complete TCRA repertoire at or shortly after birth. In fact, Pasqual showed exactly this type of progression from F18 through D1 after birth, and could reproduce the progression by placing F16 thymic lobes in FTOC. It is not appropriate to compare a single snapshot of a synchronized process in early fetal thymocytes to the unsynchronized steady state situation in adults. In fact, the authors' own data support this contention, because when they synchronize adult thymocytes by using hydroxycortisone, they can replicate the fetal distribution. Along these lines, the fact that positive selection of fetal thymocytes using 3' TRAV and 5' TRAJ segments occurs within 2 days of thymocyte entry into the DP compartment does not mean that DP development in the fetus is intrinsically rapid and restricted to 2 days. It simply means that thymocytes bearing an early rearranging TCR can be positively selected shortly after TCR expression. The expectation would be that those DP thymocytes that had not undergone early positive selection using a 3' TRAV and a 5' TRAJ would remain longer in the DP compartment and continue the progression of TCRA rearrangements, with the potential for selection several days later using more 5'TRAV and 3'TRAJ.
(2) The authors note 3' V and 5'J biases for TCRB in fetal thymocytes. The previously outlined concerns about interpreting TCRA repertoire development do not directly apply here. But it would be appropriate to note that by deep sequencing, Sethna (PNAS 114:2253 (2017)) identified skewed usage of some of the same TRBV gene segments in fetal versus adult. It should also be noted that Sethna did not detect significantly skewed usage of TRBJ segments. Regardless, one might question whether the skewed usage of TRBJ segments detected here should be characterized as relating to chromosomal location. There are two logical ways one can think about chromosomal location of TRBJ segments - one being TRBJ1 cluster vs TRBJ2 cluster, the other being 5' to 3' within each cluster. The variation reported here does not obviously fit either pattern. Is there a statistically significant difference in aggregate use of the two clusters? There is certainly no clear pattern of use 5' to 3' across each cluster.
(3) The authors show that biases in TCRA and TCRB V and J gene usage between fetal and adult thymocytes are mostly conserved between pre- and post-selection thymocytes (Fig 2). In striking contrast, TCRA and TCRB combinatorial repertoires show strong biases pre-selection that are largely erased in post-selection thymocytes (Fig 3). This apparent discrepancy is not addressed, but interpretation is challenging.
(4) The observation that there is a higher proportion of nonproductive TCRB rearrangements in fetal thymus compared to adult is challenging to interpret, given that the results are based upon RNA sequencing so are unlikely to reflect the ratio in genomic DNA due to processes like NMD.
(5) An intriguing and paradoxical finding is that fetal DP, CD4 and CD8 thymocytes all display greater sharing of TCRB CDR3 sequences among individuals than do adults (Fig 5DE), whereas DP and CD8 thymocytes are shown to display greater CDR3 amino acid triplet motif sharing in adults (with a similar trend in CD4). The authors attribute high amino acid triplet sharing to the result of selection of recurrent motifs by contact with pMHC during positive selection. But this interpretation seems highly problematic because the difference between fetal and adult thymocytes is dramatic even in unfractionated DP thymocytes, the vast majority of which have not yet undergone positive selection. How then to explain the differences in CDR3 sharing visualized by the different approaches?
(6) The authors conclude that there is less MHC restriction in fetal thymocytes, based on measures of repertoire divergence from DP to CD4 and CD8 populations (Fig. 6). But the authors point to no evidence of this in analysis of TRBV usage, either by PC or heatmap analyses (A,B,D). The argument seems to rest on PC analysis of TRAV usage (Fig S6), despite the fact that dramatic differences in the SP4 and SP8 repertoires are readily apparent in the fetal thymocyte heatmaps. The data do not appear to be robust enough to provide strong support for the authors' conclusion.

Reviewer #3 (Public Review):

Summary:
This study provides a comparison of TCR gene segment usage between foetal and adult thymus.

Strengths:
Interesting computational analyses was performed to find interesting differences in TCR gene usage within unpaired TCRa and TCRb chains between foetal and adult thymus.

Weaknesses:
This study was significantly lacking insight and interpretation into what the data analysed actually means for the biology. The dataset discussed in the paper is from only two experiments. One comparing foetal and adult thymi from 4 mice per group and another which involved hydrocortisone treatment. The paper uses TCR sequencing methodology that sequences each TCR alpha and beta chains in an unpaired way, meaning that the true identity of the TCR heterodimer is lost. This also has the added problem of overestimating clonality, and underestimating diversity.
Limited detail in the methods sections also limits the ability for readers to properly interpret the dataset. What sex of mice were used? Are there any sex differences? What were the animal ethics approvals for the study?