In their manuscript, the authors used their famous birth cohort study to analyze the correlation of Treg numbers and different postnatal factors, at birth, 6 and 12 months after birth. They have found a correlation between naïve Treg and activated Treg with household size. This has been correlated with decreased allergic outcome in those children later in life.
The manuscript is very descriptive and lacks any functional analysis to back the authors findings. They have correlated the numbers of Treg cells to allergic outcomes without any functional evidence.

Major Comments
- The authors showed the results of 3-4 or over 4. Is there any reason for that? With a 3 household, that means the child is a lone-child and with 4, a sibling is there. Or did the authors mean it 3 or 4 + the new child? Please explain and re-analyze for 3 alone and 4 alone and +4. Does it make a difference?
- The comments on breastfeeding are scarce. What happened to the children over all? The authors mentioned 1 third will continue for 6 months? What about the rest?
- Many studies showed a clear positive correlation between blood Treg numbers and breastfeeding. In this study there was no correlation. Can the authors explain why? Did the data collected not verified about the breastfeeding timing?
- The authors kept mentioned activated Tregs compared to naïve Tregs. Can they please comment on induced Tregs (iTregs)? Those Tregs are very important in the disease and tolerance building.
- Have the authors checked Helios staining as part of functional analysis of Tregs. I would strongly suggest so.
- Was the activity of the Treg changed? The authors claimed that aTregs are higher, were their suppressive activity affected as well? I would suggest doing that.
- In figure 1, please show gating strategy.
- The authors kept discussing about microbial external factors in the discussion, but the results are written very shortly and lacks consistency. I have missed the whole microbial part! I didn’t see any microbiome analysis, neither to the mother’s milk, the household nor the feces of the parents and children.
- The biggest concern remains that this is only an observational study and no evidence backing their claims on Treg and household and less allergic outcome. More functional analysis should be conducted.

In their manuscript Ponsonby et al. tried to link household size with regulatory T-cell numbers with development of early allergic disease. This is a resubmission of the old manuscript. I couldn’t track the changes done in the first submission. As I was one of the reviewers reviewing this manuscript before, I still have multiple open points that I didn’t see them answered! I haven’t seen answers to my previous questions as well.
After reviewing this manuscript, I have the following points

Major comments:
1- Are the activated Tregs same as induced?
2- What is the Helios expression?
3- Did the authors check CD25 markers and CD127 low cells? Many T-cells acquire a low foxp3 expression upon stimulation.
4- My old question about large and small household still exists. I don’t see it clear. What are the criteria?
5- I’m still missing the microbial factors in the methods and results, it comes suddenly in the figures.
6- The authors use words like “markedly” in a way to convince us. The data doesn’t look very convincing, it is merely a correlation.
7- Their IgE analysis via skin-prick test is invalid they need to measure titers. I asked for that in my earlier comments (If I am not wrong) to be able to conclude an appropriate conclusion.
Minor comments:
1- The gating in figure 1 is not very convincing and seems wrong, why would any T-reg cells have low foxp3? How was this being defined?
2- Is Foxp3 locus for the suppressive activity same as the CNS locus? It needs to be cleared.