scRNA+TCR-seq Reveals the Proportion and Characteristics of Dual TCR Treg Cells in Mouse Lymphoid and Non-lymphoid Tissues

Reviewer #1 (Public review):

Summary:

This study presents findings on dual TCR regulatory T cells (Tregs) using previously published single-cell RNA and TCR sequencing datasets. The authors aimed to quantify dual TCR Tregs in different tissues and analyze their characteristics. Rather than perform the difficult experiments needed to ascertain the functional role of dual receptors, this study relies entirely on scRNA-VDJ-seq data published by two other groups. The findings primarily confirm prior work rather than provide new insights, and the methodology has significant weaknesses that limit the study's impact. We have concerns about the scientific integrity of this work.

Strengths:

(1) The use of single-cell RNA and TCR sequencing is appropriate for addressing potential relationships between gene expression and dual TCR.

(2) The data confirm the presence of dual TCR Tregs in various tissues, with proportions ranging from 10.1% to 21.4%, aligning with earlier observations in αβ T cells.

(3) Tissue-specific patterns of TCR gene usage are reported, which could be of interest to researchers studying T cell adaptation, although these were more rigorously analyzed in the original works.

Weaknesses

(1) Lack of Novelty: The primary findings do not substantially advance our understanding of dual TCR expression, as similar results have been reported previously in other contexts.

(2) Incomplete Evidence: The claims about tissue-specific differences lack sufficient controls (e.g., comparison with conventional T cells) and functional validation (e.g., cell surface expression of dual TCRs).

(3) Methodological Weaknesses: The diversity analysis does not account for sample size differences, and the clonal analysis conflates counts and clonotypes, leading to potential misinterpretation.

(4) Insufficient Transparency: The sequence analysis pipeline is inadequately described, and the study lacks reproducibility features such as shared code and data.

(5) Weak Gene Expression Analysis: No statistical validation is provided for differential gene expression, and the UMAP plots fail to reveal meaningful clustering patterns.

(6) A quick online search reveals that the same authors have repeated their approach of reanalysing other scientists' publicly available scRNA-VDJ-seq data in six other publications:

(1) Peng, Q., Xu, Y. & Yao, X. scRNA+ TCR-seq revealed dual TCR T cells antitumor response in the TME of NSCLC. J Immunother Cancer 12 (2024). https://doi.org:10.1136/jitc-2024-009376

(2) Wang, H., Li, J., Xu, Y. & Yao, X. scRNA + BCR-seq identifies proportions and characteristics of dual BCR B cells in the peritoneal cavity of mice and peripheral blood of healthy human donors across different ages. Immun Ageing 21, 90 (2024). https://doi.org:10.1186/s12979-024-00493-6

(3) Xu, Y. et al. scRNA+TCR-seq reveals the pivotal role of dual receptor T lymphocytes in the pathogenesis of Kawasaki disease and during IVIG treatment. Front Immunol 15, 1457687 (2024). https://doi.org:10.3389/fimmu.2024.1457687

(4) Yuanyuanxu, Qipeng, Qingqingma & Yao, X. scRNA + TCR-seq revealed the dual TCR pTh17 and Treg T cells involvement in autoimmune response in ankylosing spondylitis. Int Immunopharmacol 135, 112279 (2024). https://doi.org:10.1016/j.intimp.2024.112279

(5) Zhu, L. et al. scRNA-seq revealed the special TCR beta & alpha V(D)J allelic inclusion rearrangement and the high proportion dual (or more) TCR-expressing cells. Cell Death Dis 14, 487 (2023). https://doi.org:10.1038/s41419-023-06004-7

(6) Zhu, L., Peng, Q., Wu, Y. & Yao, X. scBCR-seq revealed a special and novel IG H&L V(D)J allelic inclusion rearrangement and the high proportion dual BCR expressing B cells. Cell Mol Life Sci 80, 319 (2023). https://doi.org:10.1007/s00018-023-04973-8

In other words, the approach used here seems to be focused on quick re-analyses of publicly available data without further validation and/or exploration

Appraisal of the Study's Aims and Conclusions:

The authors set out to analyze dual TCR Tregs across tissues, but the lack of robust controls, incomplete analyses, and insufficient novelty limit the study's ability to achieve its aims. The results confirm prior findings but do not provide compelling evidence to support the broader claims about the characteristics or significance of dual TCR Tregs.

Impact and Utility:

While the study provides a descriptive analysis of dual TCR Tregs, its limited novelty and methodological weaknesses reduce its likely impact on the field. The methods and data could have utility for researchers interested in tissue-specific TCR gene usage, but additional rigor is required to make the findings broadly applicable.

Reviewer #2 (Public review):

Summary:

The manuscript, "scRNA+TCR-seq Reveals the Proportion and Characteristics of Dual TCR Treg Cells in Mouse Lymphoid and Non-lymphoid Tissues" by Xu and Peng, et al. investigates whether co-expression of 2 T cell receptor (TCR) clonotypes can be detected in FoxP3+ regulatory CD4+ T cells (Tregs) and if it is associated with identifiable phenotypic effects. This paper presents data reanalyzing publicly available single-cell TCR sequencing and transcriptional analysis, convincingly demonstrating that dual TCR co-expression can be detected in Tregs, both in peripheral circulation as well as among Tregs in tissues. They then compare metrics of TCR diversity between single-TCR and dual TCR Tregs, as well as between Tregs in different anatomic compartments, finding the TCR repertoires to be generally similar though with dual TCR Tregs exhibiting a less diverse repertoire and some moderate differences in clonal expansion in different anatomic compartments. Finally, they examine the transcriptional profile of dual TCR Tregs in these datasets, finding some potential differences in the expression of key Treg genes such as Foxp3, CTLA4, Foxo3, Foxo1, CD27, IL2RA, and Ikzf2 associated with dual TCR-expressing Tregs, which the authors postulate implies a potential functional benefit for dual TCR expression in Tregs.

Strengths:

This report examines an interesting and potentially biologically significant question, given recent demonstrations that dual TCR co-expression is a much more common phenomenon than previously appreciated (approximately 15-20% of T cells) and that dual TCR co-expression has been associated with significant effects on the thymic development and antigenic reactivity of T cells. This investigation leverages large existing datasets of single-cell TCRseq/RNAseq to address dual TCR expression in Tregs. The identification and characterization of dual TCR Tregs is rigorously demonstrated and presented, providing convincing new evidence of their existence.

Weaknesses:

The existence of dual TCR expression by Tregs has previously been demonstrated in mice and humans (Reference #18 and Tuovinen. 2006. Blood. 108:4063; Schuldt. 2017. J Immunol. 199:33, both omitted from references). The presented results should be considered in the context of these prior important findings.

This demonstration of dual TCR Tregs is notable, though the authors do not compare the frequency of dual TCR co-expression by Tregs with non-Tregs. This limits interpreting the findings in the context of what is known about dual TCR co-expression in T cells.

Comparison of gene expression by single- and dual TCR Tregs is of interest, but as presented is difficult to interpret. Statistical analyses need to be performed to provide statistical confidence that the observed differences are true.

The interpretations of the gene expression analyses are somewhat simplistic, focusing on the single-gene expression of some genes known to have a function in Tregs. However, the investigators miss an opportunity to examine larger patterns of coordinated gene expression associated with developmental pathways and differential function in Tregs (Yang. 2015. Science. 348:589; Li. 2016. Nat Rev Immunol. Wyss. 2016. 16:220; Nat Immunol. 17:1093; Zenmour. 2018. Nat Immunol. 19:291).

Reviewer #3 (Public review):

Summary:

This study addressed the TCR pairing types and CDR3 characteristics of Treg cells. By analyzing scRNA and TCR-seq data, it claims that 10-20% of dual TCR Treg cells exist in mouse lymphoid and non-lymphoid tissues and suggests that dual TCR Treg cells in different tissues may play complex biological functions.

Strengths:

The study addresses an interesting question of how dual-TCR-expressing Treg cells play roles in tissues.

Weaknesses:

This study is inadequate, particularly regarding data interpretation, statistical rigor, and the discussion of the functional significance of Dual TCR Tregs.

Major Comments:

(1) Definition of Dual TCR and Validity of Doublet Removal
This study analyzes Treg cells with Dual TCR, but it is not clearly stated how the possibility of doublet cells was eliminated. The authors mention using DoubletFinder for detecting doublets in scRNA-seq data, but is this method alone sufficient?
We strongly recommend reporting the details of doublet removal and data quality assessment in the Supplementary Data.

(2) Inconsistency in the Proportion of Dual TCR T Cells in the Skin Across Figures
In Figure 3D, the proportion of Dual TCR T cells (A1+A2+B1+B2) in the skin is reported to be very high compared to other tissues. However, in Figure 4C, the proportion appears lower than in other tissues, which may be due to contamination by non-Tregs. The authors should clarify why it was necessary to include non-Tregs as a target for analysis in this study. Additionally, the sensitivity of scRNA-seq and TCR-seq may vary between tissues and may also be affected by RNA quality and sequencing depth in skin samples, so the impact of measurement bias should be assessed.

(3) Issue of Cell Contamination
In Figure 2A, the data suggest a high overlap between blood, kidney, and liver samples, likely due to contamination. Can the authors effectively remove this effect? If the dataset allows, distinguishing between blood-derived and tissue-resident Tregs would significantly enhance the reliability of the findings. Otherwise, it would be difficult to separate biological signals from contamination noise, making interpretation challenging.

(4) Inconsistency Between CDR3 Overlap and TCR Diversity
The manuscript states that Single TCR Tregs have a higher CDR3 overlap, but this contradicts the reported data that Dual TCR Tregs exhibit lower TCR diversity (higher 1/DS score). Typically, when TCR diversity is low (i.e., specific clones are concentrated), CDR3 overlap is expected to increase. The authors should carefully address this discrepancy and discuss possible explanations.

(5) Functional Evaluation of Dual TCR Tregs
This study indicates gene expression differences among tissue-resident Dual TCR T cells, but there is no experimental validation of their functional significance. Including functional assays, such as suppression assays or cytokine secretion analysis, would greatly enhance the study's impact.

(6) Appropriateness of Statistical Analysis
When discussing increases or decreases in gene expression and cell proportions (e.g., Figure 2D), the statistical methods used (e.g., t-test, Wilcoxon, FDR correction) should be explicitly described. They should provide detailed information on the statistical tests applied to each analysis.