In this manuscript, Huisman et al. describe in detail a reexamination of the classical concept of Public T-Cell Receptors (TCRs). The manuscript is focused on a particular Public TRC and related and highly-similar TCRs, restricted by HLA-A2 and specific for a peptide (FLY) derived from the EBV LMP2 protein. The authors elegantly address the subject with an appropriate set of in vitro experiments (in vitro mutagenesis, transfections, cytometry, and functional studies), that arose from previous works and findings from the same team.
Overall, the work is of interest and findings have not been reported before. The text is clear and well written, without an excess of information. The figures are well done and are useful for the general interpretation of the paper (figure 2 could be easily improved). The supplementary figures are adequate, specially Suppl. Fig. 3.

Major recommendation:

The main aim of the article is to experimentally demonstrate the novel concept of Highly-similar TCRs, as an update of the classical Public TCRs. They have selected a Public TCR, namely HLA-A02:01-restricted EBC-LMP2FLY TCR as a proof-of-concept. They have focused on position 5 of the CDR3β loop of this particular TCR and highly-related TCRs that recognize the same ligand. The authors nicely showed that this particular, and highly variable position of the Vβ chain (that is encoded in the germline Vβ gene segment TRBV6-5) could be widely changed for every amino-acid without a substantial decline in its performance. This is shown by using in vitro mutated TCRs at this particular 5th residue with amino-acids that have been already found in healthy individuals (in blue throughout the paper) or even amino-acids that were never found (9 in orange throughout the figures). The cells used for transfection and further experiments are primary T cells (either CD8+ or CD8- T cells) and the Jurkat cell line. The results are consistent in all the experimental approaches. The main exception to this rule is Glycine (G) and only under special circumstances, the lack of the proper co-receptor, CD8. Surprisingly, the rest of the amino-acids in the assay functioned very well, even Cysteine (C), irrespective of their size, hydrophobicity, or charge. Once the technical and experimental data have been satisfactory established, one important point remains to be addressed. What is the importance of the fifth amino-acid in the CDR3β of these highly-similar TCRs in the fine recognition of the ligand, HLA-A02:01FLYALALLL?
In order to further clarify the relevance of their findings, I strongly recommend including modeling of the Public TCR interaction with its ligand, HLA-A*02:01FLYALALLL. This could allow viewing the location of the fifth CDR3β amino-acid in the HV3 loop and its possible interaction with either the peptide or the HLA-A. This will not be a formal demonstration, but it could easily be performed with some computer tools like TCRmodel (https://doi.org/10.1093/nar/gky432).

Minor alterations:

• Wild-type or wildtype are both used through the text, unify

• Wildtype tyrosine is used several times referring to the Public TCR, but all the found variants in different individuals (10 amino-acids plus the Tyr) are formally wildtype. I agree that WT for Tyr is useful for the reading, but it should be note early in the text that Tyr is germline encoded in the TRBV6-5-gene

• Line 143: TRAJ1-2 should be corrected to TRBJ1-2

• Line 232: CD8-transduced Jurkat E6 ΔTCR. Probably means CD8alpha alone transduced Jurkat, but it’s worth mentioning if it is CD8 alpha o CD8 alphabeta.

• FIGURE 2: In Figure 2, the colors follow those of Figure 1 and the rest of the Figures. Red for the WT, Blue for the ex vivo found residues and Orange for the rest. Whereas, there is a logical sequence in that, the figs 2B and 2D are difficult to see. I suggest to change the color in these two figures, more than a striped lane.

• Bibliography (Reference #16): Change from bioRxiv 2021 to Front Immunol 2022 Mar 24;13:851868. doi: 10.3389/fimmu.2022.851868.

• Bibliography (Reference #2): Consider to change the classical reference Padovan et al. Science 1993, for a more general and updated Review on TCR. Suggestion (Bradley P. & Thomas P.G. Annu Rev Immunol. 2019 Apr 26;37:547-570.)

Thanks a lot for this nice job. I honestly think that your article has improved with these corrections.