Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Round 1

Reviewer 1 Report

The manuscript is sound, but it is sometimes difficult to see the novelty. And there are some discrepancies with published data. Here are my comments:

- Part of the claimed interest of the manuscript is the short-term polyclonal stimulation procedure to obtain Tregs in vitro. It should be described sufficiently in the main text, especially in order to be able to understand how it differs from previous strategies to expand Tregs (in the current version only describes the presence of stimulating additives line 199). Looking at the methods, the procedure seems actually fairly classic, relying mostly on IL-2 and anti-CD3 and anti-CD28 coated beads and TGF-bеtа. Either there is a trick, and the reader would like to know what it is. Or it’s just the classic way, and then there should be no misleading mention of a specific protocol (line 77).

Otherwise, if the culture and differentiation of Tregs is similar or identical to what has been described and validated before, the figures 1 and 2 and even figure 3 do not make much sense as they do not show any new data or findings

- In figure 1 cells steadily expand and remain highly viable till day 9. By contrast, in most plots of fig. 2 there are clearly more cells at day 7 (and sometimes day 1). Can the author explain this discrepancy?

- The results of figure 3 should be discussed in the context of reference 7 (Epigenetic Control of the foxp3 Locus in Regulatory T Cells), which states that TGF-beta induced Tregs in vitro show a much lower degree of demethylation compared to naturally occurring peripheral CD25+CD4+ Tregs. Ref 7 should also be discussed in the context of figure 2, as it states that in vitro generated Tregs loose Foxp3 Expression upon restimulation

- Results about the splice variant of IKAROS are essentially descriptive and provide little information Treg biology as stated in 328. Could the author show any data, or at least explain a bit more why the changes they observe in the population of splice variants of IKAROS contribute to Treg development? Same comment applies for the data on cell cycle regulation in figure 6.

- Line 427 and beyond: the text says figure 6 instead of 7.

- Figure 7. The figure legends states that it shows a representative experiment of a total of 5. It is understandable for A and C, but much less for B and D, which show ratios. All the 5 experiments should be pooled here, and the stat test should be performed on the means of all 5 experiments.

- Results of figure 7 seem to be in contradiction with reference 7 (where the loss of Foxp2 upon restimulation in absence of TGF-beta leads to a low suppression of proliferation) . Could the authors explain why briefly?

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

In this report, Blinova et al. had described a novel method for the quick expansion of Tregs from donor naïve human CD4+ T cells. They performed an extensive and detailed assessment of the characteristics and function of these expanded Tregs (eTregs), including the suppressive function.

In general, this is a nice work and data are presented with high quality. I only have a couple of minor experimental requests:

• Protein expression of Helios, Ikaros and other transcription factors should be assessed in eTregs.
• In addition to proliferation, cytokine production of suppressed vs non-suppressed effector T cells could be shown.

Author Response

Please see the attachment

Author Response File: Author Response.docx