Slightly off topic, but the top tier journal reviews actually often do not take me as long as badly written papers for lower impact journals. Of course the worst combo is badly written and top tier journal.

ok, thanks. This tells me that your overloading was perhaps a bit extreme. We are overloading 2-fold currently, and were thinking about doing it a bit more.

You aimed for a final loaded cell number of 30k-50k cells in 10x, meaning you sorted something like 75k-125k cells? We typically have a loss of 2.5fold. You never saw clogging? We also overload, but not quite as much as you seem to have done.

Great work! I already liked the preprint.

Fantastic, congrats!

Understanding this helps interpret T-cell states across different experimental contexts.

Thanks to @katharinakocher.bsky.social & Felix Drost for leading this work, and looking forward to discussion with the single-cell and TCR communities.

(5/5)

💡 A central idea is an “observer effect” in immunology:
the very process used to detect antigen reactivity also reshapes the phenotype we record — similar to the Hawthorne effect in psychology or classical measurement effects in physics.

(4/5)

This study builds on the concept of reverse phenotyping we originally introduced here:
🔗 www.nature.com/articles/s41...
(Fischer et al., Nat Comm 2021)

We now applied the analytical approach to a more standardized model of human T cell biology, i.e. vaccination.

(3/5)

By longitudinal sampling, single-cell RNA/TCR-seq, DNA-barcoded class II dextramers, and TCR replacement, we asked: What do spike-reactive CD4⁺ T cells look like before we activate them?
• At rest: a Th-neutral EM phenotype
• After ex-vivo restimulation: a Th1-like transcriptional signatures

(2/5)

...I would have thought that T cell singlets could be more protected from overstimulation and would preserve more functionality? Also, may I ask why you used the name "cluster" instead of "doublet" or "multiplet"? Looking forward to your response!

Congrats, fantastic work! I'm honestly a bit surprised you see that APC/tumor-interacting cells are more functional when looking at the same TCR, like you do in Fig. 1i. It's intuitive that clusters enrich for TCRs with tumor antigen reactivity (Fig. 4), but if you control the antigen...

What I find most remarkable is the phenotypic stability you show (e.g. Fig. 4E-F). Sure, no sign of exhaustion, but in CD8 responses to vaccination analyzed in peripheral blood one sees massive phenotypic dynamics within the course of 1yr. Tfh are different.