The manuscript by Mielle et al., entitled “Glutaminolysis enhances IL-10 secretion from B lymphocytes via GSK3 inhibition,” aims to provide insight into the mechanistic understanding of the ontogeny of IL-10 producing Breg cells using an in vitro CpG induced human B cell culture system. The research group carefully addresses the regulatory role of glutaminolysis in IL-10 production of B cells and suggests a possible mechanism; ASCT2 mediated uptake of glutamine is necessary to activate mTORC1 via a-KG, which inactivates GSK3 and then mediate IL-10 production in CpG activated human B cells These findings might provide insight into the links between metabolic regulation and B cell-mediated immune tolerance. However, there are several issues to be discussed or addressed to give a more precise conclusion. I believe this manuscript deserves to be published in the European Journal of Immunology, provided that the authors address the concerns (especially #3) listed below.

1) Statistic comparison between Glc(-)Gln(+) and Glc(+)Gln(-) in Fig 1D is necessary. Also, Fig. S2B showed variances in the % of B10 in each group. So, it would be beneficial to present data with statistical comparisons and pair each experiment, similar to Fig 2B. Statistics are missing in all of Fig S2 data.
2) The authors used Gln deprivation and BPTES treatment to evaluate the importance of glutaminolysis in Breg generation and cytokine production. However, BPTES was less potent than Gln deprivation in the inhibition of Breg generation (Fig S2B vs. Fig 2B) and the production of cytokines (Fig 1E vs. Fig 2C). In addition, the production of all examined cytokines (IL-10, IL-6, TNF-a) was affected in the absence of Gln, while only IL-10 production was affected in BPTES treatment. Moreover, Figs. 2G, H depict the importance of mTORC1 activity and rapamycin treatment phenocopies the deprivation of Gln. These results suggest that the glutaminolysis pathway influenced by BPTES partly contributes to mTORC1 activity and the presence of additional pathways to activate mTORC1 fully in this system, such as CpG stimulation. Therefore, it is uncertain whether GSL1 mediated glutaminolysis is a major contributor to mTORC1 activity in this system. This point requires a resolution.
3) Interestingly, GSK3 inactivation itself was enough to generate IL-10 producing Bregs, and GSK3 appears to be a key mediator of Breg generation. Although authors suggest GKS3 inactivation is downstream of glutaminolysis mediated mTORC1 activation, it is still possible that CpG also contributes to GSK3 inactivation via PI3K pathway and acts upstream of glutaminolysis, mTORC1 activity, or both, resulting in the observations presented in Fig 1, 2. This possibility could be addressed by testing whether SB216 or CHIR99 treatment can induce ASCT2 expression. Also desired is examining whether Gln deprivation, BPTES, or rapamycin treatment inhibit pharmacological GSK3 inactivation mediated Breg generation and IL-10 production without CpG stimulation.
4) Statistics are missing in several data.

The authors carefully revised the previous manuscript and I believe current manuscript is deserved to be published in journal.