Below are the detailed responses to Referee 1 submitted to Journal during the resubmission of our manuscript:

Minor Comments:
1. “Some of the Figure panels are so small that it is below the level of resolution, and the reader is unable to efficiently evaluate the author’s conclusions. In particular, the values for FACs plots on Fig 6-9 are illegible.”
We apologize to both Reviewers for the small font size. We have modified the figures and increased the font size of the text Figures 6-9 to improve legibility.

2. “Fig 1C (i-vi): Please provide the percentages for of each population with respect to either CD45 or CD3 positive leukocytes.”
We have now included the percentages for each of the immune cell populations with respect to CD45 positive leukocytes for Figure 1C (i-vi), as suggested by this reviewer (See Results section page 8). We have also included the methodology for calculating the percentage of each cell population in the legend for Fig. 1 (Page 32) and in Materials and Methods Section (see Page 21). Briefly, the percentages were calculated based on the total number of CD45+ immune cells in the tumor. For example, to calculate the percentage of mDC in the tumor, the total number of gated mDC (CD11c+ MHCII+ CD45+) was determined and their percentage was calculated by dividing the total number of mDC by the total number of CD45+ cells in the tumor.

3. “Fig 1 and associated text: The authors state that GBM GL26 tumors are “densely infiltrated with immune cells”. Could the authors comment on the apparent preferential staining of immune cells on the tumor edge (see Fig 1B).”
We thank the reviewer for the careful examination of our data. We apologize for the selection and inclusion of low power images that were not representative of the majority of our samples. We have included new images that more accurately represent typical infiltration of immune cells into the tumors. Confocal images in figure 1 B exhibit profuse infiltration of CD45+ cells and F4/80+ cells throughout the tumor mass and also in the border of the tumors (See new Figure 1 C and Results section page 8).

4. “Fig 2C: This figure was not adequately explained in the Results. Authors should clarify that tumors were engrafted in Treg deficient nu/nu immune deficient mice.”
We have modified the Materials and Methods (Page 19) and Results (Page 9) sections of our revised manuscript to clarified that the tumors were engrafted in Treg deficient nu/nu mice (see references 25; 35). We also expanded the explanation of this figure in the Results section (Page 9-10).

5. “Authors need to clearly indicate the cell populations gated for calculating the % values reported (For instance Fig 3 & 5).”
We thank the reviewer for this suggestion. We have now included the methodology for calculating the percentage of each cell population in the Materials and Methods section (Page 21) and in all the figure legends (Figs 3-9). Briefly, the percentages were calculated based on the total number of CD45+ immune cells in the tumor. For example, to calculate the percentage of mDC in the tumor, the total number of gated mDC (CD11c+ MHCII+ CD45+) were counted and their percentage was determined by dividing the total number of mDC by the total number of CD45+ cells in the tumor.

6. “P12: Statement that “PC61 depleted all CD25+ cells” is an over-statement. PC61 appears to deplete approximately 60% of the CD4+ CD25+ cells in the spleen and the decrease for the CD4- population in the spleen was not quantified.”
We apologize for our over-statement and we thank the reviewer for the suggestion. We have modified the text in our revised manuscript to include a more cautions description of the population of cells that PC61 decreases. We found that administration of PC61 reduces the population of CD25+CD4+ cells by 60% in the spleen (Fig. 7 A). Please see the corrected statement in Result Section, Page 12.

General Question:
7. “How do the authors propose that PC61 Treg depletion improves survival rates without generating an anti-tumor response (i.e. no DTH response; Fig 3)?”
We thank the reviewer for this comment and have now included an enhanced discussion of the potential reasons why PC61 Treg depletion improves survival rates without generating an anti-tumor response (see Discussion Section, pages 14-15). Treg depletion induced brain tumor regression and increased the survival of tumor bearing mice; only if depletion was done when the tumors were very small, i.e., 0.45mm3. These findings are in agreement with other reports that show an anti-tumor effect of T reg depletion in mouse models of neuroblastoma (see reference 40), leukemia, fibrosarcoma and myeloma (see reference 34). We observed that the antitumor effects of Treg depletion were not mediated by a T cell dependent anti-tumor immune response. Our results show that Treg depletion did not generate tumor-specific T cells as assed by ELISPOT assay (Fig. 6 D), nor did it induce anti-tumor immunological memory. These data suggest that the innate immune system could play a role in the anti-tumor effect elicited by Treg depletion in this model. In fact, T regs have been shown to inhibit the activity of the innate immune system by directly suppressing the cytotoxic effect of NK cells (see reference 41). This inhibitory mechanism is dependent on TGF-β and cell-cell contacts (see references 41-44). Since we found that NK cells comprise ~20% of the immune cells that infiltrate GL26 intracranial tumor (Fig. 1 C), the cytotoxic activity of these cells could be involved in the antitumor effects observed after T reg depletion (pages 14-15).


8. Mis-types
a. “Page 9: Depletion of Tregs 15 days after tumor regression significantly increased the number of long term survivors. I think the authors meant the engraftment not regression?”
We apologize for this mistake and have changed the revised manuscript to read “Depletion of Tregs 15 days after tumor cell implantation significantly increased the number of long term survivors” (Page 10).

b. “Page 10: “injection of Tregs” I think the authors meant depletion of Tregs or injection of PC61?”
We apologize for this error and have changed the revised manuscript to read “injection of PC61” (Page 10).

c. “Page 21: Vermes reference is not formatted.”
We are sorry for this mistake; we have correctly formatted the above mentioned reference (Page 30, ref 48).