Review 2: "CD4 T Cells and CD8α+ Lymphocytes are Necessary for Intravenous BCG-Induced Protection Against Tuberculosis in Macaques"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review: The authors have clearly shown that CD4 depletion has the greatest effect reducing IV BCG-induced protection, that CD8a depletion also has important effects, but that CD8b depletion has little effect. However, the authors interpretations that: 1) all 3 depletions are similarly effective, and 2) the susceptibility effects of the CD4 and CD8a depletions are due to the specific depleted populations and not indirect effects may not be totally correct.

Specific comments:

1. The authors have confirmed that all 3 populations (CD4, CD8a and CD8b expressing) are depleted by at least 90% by the appropriate antibody depletion regimens in animals that were either unvaccinated or vaccinated with high dose IV BCG. However, most of the data for efficacy of the depletions shown were collected prior to Mtb challenge. The only data shown for persistence of the numerical and functional depletions after infection were collected at necropsy from lung parenchyma (Fig 6). Confirmation of depletion in blood, LNs and airways during the infection period and necropsy would provide additional important information. 

2. The effects of CD4 and CD8 depletions are assumed to be due to the cells depleted rather than because of other indirect changes or relative functional effects on subsets of CD4 and CD8 T cells caused by these depletions. For example, both the CD8a and CD8b depletions led to markedly increased CD4 populations (Fig. 2A). The loss of these depleted CD8 populations certainly could be the reason for the loss of vaccine-induced protective immunity. Alternatively, differential expansion of distinct populations of CD4 subsets (eg-T regs, Th2 or other CD4 subsets) could be important for the increased susceptibility. Although less likely in this reviewer's opinion, in CD4 depleted populations, an increase in subsets of regulatory CD8 T cells filling the immunologic void could be contributing to the increased susceptibility. Similarly, selective effects on different subpopulations of the MAIT and gd T cell populations could have occurred. Further dissection of the subsets remaining after the depletions would help to discriminate between these possibilities. More discussion of these possibilities could be added.  

3. An important question with gd T cell populations and protective immunity in humans is what Vg/Vd chains are being expressed. Human and/or NHP gd T cells responding to Mtb infection vs BCG vaccination may be different. A considerable amount of data has been generated in support of the Vd2 populations developing potent Type 1 immune responses including Th1 cytokines and cytolytic granular effector mechanisms associated with inhibition of intracellular mycobacterial growth. Less data indicate that another major subset of gd T cells expressing Vd1 may be relevant for TB infection, as TB granulomas have been reported to contain monoclonal populations distinct from 1 granuloma to another. These latter results may be consistent with a different functional effect (eg-anti-inflammatory effects to prevent overexuberant responses). It would be important in future research to evaluate the TCR expressed by NHP associated with protection vs susceptibility, although this important question is outside the scope of this manuscript. However, this could be mentioned in the discussion to the current manuscript. 

4. The time points for studies done should be clarified in Fig. 2B and Fig. 3A&B.

5. Figure 2 would benefit from showing absolute numbers in addition to frequencies. 

6. Supplementary Figures & Tables are referred to in the text but this reviewer did not receive them to review.