Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies

Reviewer #1 (Public Review):

• A summary of what the authors were trying to achieve.

The authors cultured pre- and Post-vaccine PBMCs with overlapping peptides encoding S protein in the presence of IL-2, IL-7, and IL-15 for 10 days, and extensively analyzed the T cells expanded during the culture; by including scRNAseq, scTCRseq, and examination of reporter cell lines expressing the dominant TCRs. They were able to identify 78 S epitopes with HLA restrictions (by itself represents a major achievement) together with their subset, based on their transcriptional profiling. By comparing T cell clonotypes between pre- and post-vaccination samples, they showed that a majority of pre-existing S-reactive CD4+ T cell clones did not expand by vaccinations. Thus, the authors concluded that highly-responding S-reactive T cells were established by vaccination from rare clonotypes.

• An account of the major strengths and weaknesses of the methods and results.

Strengths
• Selection of 4 "Ab sustainers" and 4 "Ab decliners" from 43 subjects who received two shots of mRNA vaccinations.
• Identification of S epitopes of T cells together with their transcriptional profiling. This allowed the authors to compare the dominant subsets between sustainers and decliners.

Weaknesses
• Fig. 3 provides the epitopes, and the type of T cells, yet the composition of subsets per subject was not provided. It is possible that only one subject out of 4 sustainers expressed many Tfh clonotypes and explained the majority of Tfh clonotypes in the sustainer group. To exclude this possibility, the data on the composition of the T cell subset per subject (all 8 subjects) should be provided.
• S-specific T cells were obtained after a 10-day culture with peptides in the presence of multiple cytokines. This strategy tends to increase a background unrelated to S protein. Another shortcoming of this strategy is the selection of only T cells amenable to cell proliferation. This strategy will miss anergic or less-responsive T cells and thus create a bias in the assessment of S-reactive T cell subsets. This limitation should be described in the Discussion.
• Fig. 5 shows the epitopes and the type of T cells present at baseline. Do they react to HCoV-derived peptides? I guess not, as it is not clearly described. If the authors have the data, it should be provided.
• As the authors discussed (L172), pre-existing S-reactive T cells were of low affinity. The raw flow data, as shown in Fig. S3, for pre-existing T cells may help discuss this aspect.

Reviewer #2 (Public Review):

Summary: A short-term comparison of durability of S antibody levels after 2-dose vaccination, showing that better or more poorly sustained responses correlate with the presence of Tfh cells.

Strengths:
Novelty of approach in expanding, sequencing and expressing TCRs for functional studies from the implicated populations.

Weaknesses:
Somewhat outdated question, short timeline, small numbers, over-interpretation of sequence homology data.

Reviewer #3 (Public Review):

Summary: The paper aims to investigate the relationship between anti-S protein antibody titers with the phenotypes&clonotypes of S-protein-specific T cells, in people who receive SARS-CoV2 mRNA vaccines. To do this, the paper recruited a cohort of Covid-19 naive individuals who received the SARS-CoV2 mRNA vaccines and collected sera and PBMCs samples at different timepoints. Then they mainly generate three sets of data: 1). Anti-S protein antibody titers on all timepoints. 2) Single-cell RNAseq/TCRseq dataset for divided T cells after stimulation by S-protein for 10 days. 3) Corresponding epitopes for each expanded TCR clones. After analyzing these results, the paper reports two major findings & claims: A) Individuals having sustained anti-S protein antibody response also have more so-called Tfh cells in their single-cell dataset, which suggests Tfh-polarization of S-specific T cells can be a marker to predict the longevity of anti-S antibody. B). S-reactive T cells do exist before the vaccination, but they seem to be unable to respond to Covid-19 vaccination properly.

The paper's strength is it uses a very systemic and thorough strategy trying to dissect the relationship between antibody titers, T cell phenotypes, TCR clonotypes and corresponding epitopes, and indeed it reports several interesting findings about the relationship of Tfh/sustained antibody and about the S-reactive clones that exist before the vaccination. However, the main weakness is these interesting claims are not sufficiently supported by the evidence presented in this paper. I have the following major concerns:

1. The biggest claim of the paper, which is the acquisition of S-specific Tfh clonotypes is associated with the longevity of anti-S antibodies, should be based on proper statistical analysis rather than just a UMAP as in Fig2 C, E, F. The paper only shows the pooled result, but it looks like most of the so-called Tfh cells come from a single donor #27. If separating each of the 4 decliners and sustainers and presenting their Tfh% in total CD4+ T cells respectively, will it statistically have a significant difference between those decliners and sustainers? I want to emphasize that solid scientific conclusions need to be drawn based on proper sample size and statistical analysis.

2. The paper does not provide any information to justify its cell annotation as presented in Fig 2B, 4A. Moreover, in my opinion, it is strange to see that there are two clusters of cells sit on both the left and right side of UMAP in Fig2B but both are annotated as CD4 Tcm and Tem. Also Tfh and Treg belong to a same cluster in Fig 2B but they should have very distinct transcriptomes and should be separated nicely. Therefore I believe the paper can be more convincing if it can present more information and discussion about the basis for its cell annotation.

3. Line 103-104, the paper claims that the Tfh cluster likely comes from cTfh cells. However considering the cells have been cultured/stimulated for 10 days, cTfh cells might lose all Tfh features after such culture. To my best knowledge there is no literature to support the notion that cTfh cells after stimulated in vitro for 10 days (also in the presence of IL2, IL7 and IL15), can still retain a Tfh phenotype after 10 days. It is possible that what actually happens is, instead of having more S-specific cTfh cells before the cell culture, the sustainers' PBMC can create an environment that favors the Tfh cell differentiation (such as express more pro-Tfh cytokines/co-stimulations). Thus after 10-days culture, there are more Tfh-like cells detected in the sustainers. The paper may need to include more evidence to support cTfh cells can retain Tfh features after 10-days' culture.

4. It is in my opinion inaccurate to use cell number in Fig4B to determine whether such clone expands or not, given that the cell number can be affected by many factors like the input number, the stimulation quality and the PBMC sample quality. A more proper analysis should be considered by calculating the relative abundance of each TCR clone in total CD4 T cells in each timepoint.

5. It is well-appreciated to express each TCR in cell line and to determine the epitopes. However, the author needs to make very sure that this analysis is performed correctly because a large body of conclusions of the paper are based on such epitope analysis. However, I notice something strange (maybe I am wrong) but for example, Table 4 donor #8 clonotype post_6 and _7, these two clonotypes have exactly the same TRAV5 and TRAJ5 usage. Because alpha chain don't have a D region, in theory these clonotypes, if have the same VJ usage, they should have the same alpha chain CDR3 sequences, however, in the table they have very different CDR3α aa sequences. I wish the author could double check their analysis and I apologize in advance if I raise such questions based on wrong knowledge.

Author Response

Reviewer #1 (Public Review):

• A summary of what the authors were trying to achieve.

The authors cultured pre- and Post-vaccine PBMCs with overlapping peptides encoding S protein in the presence of IL-2, IL-7, and IL-15 for 10 days, and extensively analyzed the T cells expanded during the culture; by including scRNAseq, scTCRseq, and examination of reporter cell lines expressing the dominant TCRs. They were able to identify 78 S epitopes with HLA restrictions (by itself represents a major achievement) together with their subset, based on their transcriptional profiling. By comparing T cell clonotypes between pre- and post-vaccination samples, they showed that a majority of pre-existing S-reactive CD4+ T cell clones did not expand by vaccinations. Thus, the authors concluded that highly-responding S-reactive T cells were established by vaccination from rare clonotypes.

• An account of the major strengths and weaknesses of the methods and results.

Strengths

• Selection of 4 "Ab sustainers" and 4 "Ab decliners" from 43 subjects who received two shots of mRNA vaccinations.

• Identification of S epitopes of T cells together with their transcriptional profiling. This allowed the authors to compare the dominant subsets between sustainers and decliners.

Weaknesses

• Fig. 3 provides the epitopes, and the type of T cells, yet the composition of subsets per subject was not provided. It is possible that only one subject out of 4 sustainers expressed many Tfh clonotypes and explained the majority of Tfh clonotypes in the sustainer group. To exclude this possibility, the data on the composition of the T cell subset per subject (all 8 subjects) should be provided.

We thank the reviewer for this comment. We will show the data in the revised manuscript.

• S-specific T cells were obtained after a 10-day culture with peptides in the presence of multiple cytokines. This strategy tends to increase a background unrelated to S protein. Another shortcoming of this strategy is the selection of only T cells amenable to cell proliferation. This strategy will miss anergic or less-responsive T cells and thus create a bias in the assessment of S-reactive T cell subsets. This limitation should be described in the Discussion.

We will describe the limitation and advantage of our strategy in the revised manuscript.

• Fig. 5 shows the epitopes and the type of T cells present at baseline. Do they react to HCoV-derived peptides? I guess not, as it is not clearly described. If the authors have the data, it should be provided.

We apologize for not mentioning it clearly. As we have confirmed the unresponsiveness using synthetic HCoV peptides, we will include these data in the revised manuscript.

• As the authors discussed (L172), pre-existing S-reactive T cells were of low affinity. The raw flow data, as shown in Fig. S3, for pre-existing T cells may help discuss this aspect.

We thank the reviewer for this helpful comment. We will add the discussion to the revised manuscript.

Reviewer #3 (Public Review):

Summary: The paper aims to investigate the relationship between anti-S protein antibody titers with the phenotypes&clonotypes of S-protein-specific T cells, in people who receive SARS-CoV2 mRNA vaccines. To do this, the paper recruited a cohort of Covid-19 naive individuals who received the SARS-CoV2 mRNA vaccines and collected sera and PBMCs samples at different timepoints. Then they mainly generate three sets of data: 1). Anti-S protein antibody titers on all timepoints. 2) Single-cell RNAseq/TCRseq dataset for divided T cells after stimulation by S-protein for 10 days. 3) Corresponding epitopes for each expanded TCR clones. After analyzing these results, the paper reports two major findings & claims: A) Individuals having sustained anti-S protein antibody response also have more so-called Tfh cells in their single-cell dataset, which suggests Tfh-polarization of S-specific T cells can be a marker to predict the longevity of anti-S antibody. B). S-reactive T cells do exist before the vaccination, but they seem to be unable to respond to Covid-19 vaccination properly.

The paper's strength is it uses a very systemic and thorough strategy trying to dissect the relationship between antibody titers, T cell phenotypes, TCR clonotypes and corresponding epitopes, and indeed it reports several interesting findings about the relationship of Tfh/sustained antibody and about the S-reactive clones that exist before the vaccination. However, the main weakness is these interesting claims are not sufficiently supported by the evidence presented in this paper. I have the following major concerns:

1. The biggest claim of the paper, which is the acquisition of S-specific Tfh clonotypes is associated with the longevity of anti-S antibodies, should be based on proper statistical analysis rather than just a UMAP as in Fig2 C, E, F. The paper only shows the pooled result, but it looks like most of the so-called Tfh cells come from a single donor #27. If separating each of the 4 decliners and sustainers and presenting their Tfh% in total CD4+ T cells respectively, will it statistically have a significant difference between those decliners and sustainers? I want to emphasize that solid scientific conclusions need to be drawn based on proper sample size and statistical analysis.

We will carefully describe the interpretation of the data with statistical analysis in the revised manuscript.

1. The paper does not provide any information to justify its cell annotation as presented in Fig 2B, 4A. Moreover, in my opinion, it is strange to see that there are two clusters of cells sit on both the left and right side of UMAP in Fig2B but both are annotated as CD4 Tcm and Tem. Also Tfh and Treg belong to a same cluster in Fig 2B but they should have very distinct transcriptomes and should be separated nicely. Therefore I believe the paper can be more convincing if it can present more information and discussion about the basis for its cell annotation.

We apologize for the insufficient explanation and will describe how we performed cell annotation in the revised manuscript.

1. Line 103-104, the paper claims that the Tfh cluster likely comes from cTfh cells. However considering the cells have been cultured/stimulated for 10 days, cTfh cells might lose all Tfh features after such culture. To my best knowledge there is no literature to support the notion that cTfh cells after stimulated in vitro for 10 days (also in the presence of IL2, IL7 and IL15), can still retain a Tfh phenotype after 10 days. It is possible that what actually happens is, instead of having more S-specific cTfh cells before the cell culture, the sustainers' PBMC can create an environment that favors the Tfh cell differentiation (such as express more pro-Tfh cytokines/co-stimulations). Thus after 10-days culture, there are more Tfh-like cells detected in the sustainers. The paper may need to include more evidence to support cTfh cells can retain Tfh features after 10-days' culture.

We thank the reviewer for raising this important point. We will describe the limitation of the strategy. In addition, we will include some data in accordance with the reviewer’s recommendation.

1. It is in my opinion inaccurate to use cell number in Fig4B to determine whether such clone expands or not, given that the cell number can be affected by many factors like the input number, the stimulation quality and the PBMC sample quality. A more proper analysis should be considered by calculating the relative abundance of each TCR clone in total CD4 T cells in each timepoint.

We will also show the proportion of clonotypes in the revised manuscript.

1. It is well-appreciated to express each TCR in cell line and to determine the epitopes. However, the author needs to make very sure that this analysis is performed correctly because a large body of conclusions of the paper are based on such epitope analysis. However, I notice something strange (maybe I am wrong) but for example, Table 4 donor #8 clonotype post_6 and _7, these two clonotypes have exactly the same TRAV5 and TRAJ5 usage. Because alpha chain don't have a D region, in theory these clonotypes, if have the same VJ usage, they should have the same alpha chain CDR3 sequences, however, in the table they have very different CDR3α aa sequences. I wish the author could double check their analysis and I apologize in advance if I raise such questions based on wrong knowledge.

We thank the reviewer for carefully reading our manuscript. Although the two clonotypes, donor #8 clonotype post_6 and _7, have exactly the same TRAV5 and TRAJ5 usage, they have different CDR3a aa sequences due to random nucleotide addition in rearrangement. Likewise, donor #27 clonotype post_1 and donor #13 clonotype post_15 had the same TRAV9-2 and TRAJ17 usage but different CDR3a.