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  1. Author Response

    Reviewer 3 (Public Review:

    1. The overall research question and goal of this manuscript are unclear.

    The manuscript has been edited to improve clarity and emphasize the research goals.

    1. Many of the key experiments are executed in vitro, complicating results and making it hard to discern how some of the experiments translate to in vivo differentiation.

    As requested, we provide several new experiments showing CTL responses analyzed in vivo. Endogenous CTLs were analyzed using MHCI tetramers (Fig 2C, 2D and 5C), and transfers were used to compare mutant and wildtype CTLs in the same animals (Fig 1C) and for imaging flow cytometry (4B, 4C, 4E and 4F). Our in vivo and in vitro studies support similar conclusions.

    1. There is not a consistent approach of carefully examining Trm cells.

    We provide new figures showing IV staining (Fig 1C, 1D, 5C, 5D and supp Fig 1D). The data show that SMAD4 supports formation of KLRG1+ CTLs that localize in the vasculature. Many studies have shown that TGFb is involved in formation of TRM cells. To avoid duplication, we did not emphasize this subset.

    1. Many of the core findings in this manuscript have previously been reported either in the prior work from this group (J Immunol, 2015) or more recently by Wu et al. (Cell Moll Immunol 2020).

    This statement is not accurate. To our knowledge, only four papers have examined the regulatory functions of SMAD4 in peripheral CD8 T cells, using different promoters for gene-ablation. These studies had different objectives.

    i. Hu et al (2015) - distal Lck promoter. This paper did not examine gene expression, or utilize mice with multiple mutations. Our current paper is an extension of this work, with no duplication.

    ii. Cao et al. (2015) – proximal Lck promoter. This study did not examine gene expression, or utilize mice with multiple mutations. The cytokine response may have been altered by gene ablation during thymic development.

    iii. Wu et al (2021) – CD4-Cre was used to study regulation of CD103 by Ski/SMAD4. The role of SMAD4 during regulation of EOMES, CD62L, and KLRG1 was not analyzed. IV staining was not used.

    iv. Igalouzene et al (2022) - CD4-Cre was used to study autoimmune disease in mice that lack TGFbRII, with emphasis on cells the GI tract.

    Wu et al (2021) analyzed CD103 expression using S4TR2-DKO and TR2KO cells, while similar comparisons with S4KO and control cells were not shown. The authors concluded that “Smad4 is required to limit CD103 expression in CD8+ T cells through a mechanism that is downstream of TGFβR”. This statement is not supported by our work. While their study shows that Ski is negatively-regulated by TGFb, this is not the only mechanism that controls CD103 expression. We show that SMAD4 down-regulates the Itgae gene (CD103) and induces EOMES expression independently of TGFb. This novel observation has not been reported previously. Although this manuscript is an extension of our prior work, the data were generated using additional strains of geneticallymodified mice and experimental approaches that support novel conclusions.

    1. There is not a careful or consistent assessment of memory T cell populations in lymphoid or non-lymphoid compartments.

    To address this concern, new figures showing IV staining (Figs 1C, 1D, 5C and 5D, Supplemental Fig 1D).

    1. SMAD4 was required to maintain EOMES expression in activated CTLs. This data is fairly robust; however, could this be due to differences in cell states rather than a direct role for SMAD4 in sustaining EOMES expression?

    For this study, we analyzed EOMES expression in vitro and at multiple timepoints after infection (in vivo). The data show EOMES was consistently down-regulated in SMAD4-deficient CTLs at all time points regardless of phenotype (Figs 4A, 4C and 4E). Our prior work shows that cell-state impacts susceptibility to TGFb, since TCM cells did not upregulated CD103 during stimulation with TGFb (Suarez et al 2019) and KLRG1+ CTLs maintained EOMES expression during stimulation with TGFb (Fig 4F). This point is mentioned in the text. Our revised manuscript includes chip seq data showing that SMAD4 binds to the EOMES promoter (Fig 4G), indicating a direct role for SMAD4 during gene regulation.

    1. SMAD4 has multiple roles in regulating expression of CD103, including complimentary or independent roles of Ski (last two sentences of paragraph describing Fig 3 in the results section). There was no assessment of Ski in the results of this study. Additionally, despite many conclusions about the roles of SMAD proteins in controlling gene expression, there are no experiments to assess binding of these factors (e.g. ChIP-qPCR).
      The objective of our study was to examine the role SMAD4 during formation of TEFF and TCM cells. We did not study Ski expression, as interactions between SMAD4 and this molecule have been reported previously (Wu et al. 2020). We provide additional Chip-seq data showing that SMAD4 binds to the EOMES promoter (Fig 4G).

    We thank the reviewers for many suggestions that have greatly improved the quality of our manuscript.

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  2. Evaluation Summary:

    The authors set out to investigate the roles of SMAD4 and TGFbeta in regulating memory CD8 T cell differentiation during viral infection. To achieve their goal, the authors utilized a variety of available tools including, gene expression, mice that lack certain regulatory genes, and different tissue tissue culture approaches. Although the in vitro experiments yielded interesting results that will interest students of T cell immunology/biology, there is an absence of results from in vivo studies that would validate the in vitro observations.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    In this manuscript, Chandiran et.al explored the roles of Smad4 in regulating CTL differentiation during viral infection. By comparing the Smad4 or TgfbR2 deficient CD8 T cells during influenza infection, the authors noticed that Smad4 suppressed CD103 expression but promoted CD62L transcription. Further RNAseq experiments found that Smad4 deficient early effector CD8 T cells were endowed with tissue resident features. Among differential express genes, they observed significant changes in CD103, Eomes and CD62L expression. Using an in vitro culture system, the authors demonstrated that Smad4 could regulate Eomes mediated Itgae transcription. The authors also explored the Smad4/Eomes controlled CD62L expression during Tcm differentiation. Collectively, this manuscript stressed the reciprocal function of Smad4 and canonical TGFb pathways during the memory CD8 fate decision. While the gene expression regulation is of interest, the functional consequences of the regulation remains uncertain in the current study.

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  4. Reviewer #2 (Public Review):

    The work investigates the role of SMAD4 in the biology of circulating and tissue resident memory differentiation, and provides the surprising finding that SMAD4 does not seem to be involved in TGFb signaling, rather to be independent of it. The work potentially provides a novel signaling mechanism at the basis of memory T cell formation, but should be carefully revised before acceptance, especially in confirming in vivo findings that were obtained in vitro.

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  5. Reviewer #3 (Public Review):

    Strengths:
    • TGFB is a critical regulator of CD8 T cell fate and function; yet, the complex mechanisms of action and downstream regulatory factors remain unresolved. This works expands upon the role of TGFB in controlling expression of critical receptors and trafficking/retention molecules on CD8 T cells, such as CD127, KLRG1, CD103 and CD62L.
    • Downstream mediators of the TGFB signaling pathway are complex and at times operative counterintuitively. Therefore, carefully dissecting how TGFB and various SMAD factors impact T cell fate is an important and relevant research goal.
    • The authors should be commended for the wide range of tools and approaches they use to investigate the role of SMAD factors in CD8 T cell biology. This includes multiple strains of mice with perturbations in the TGFb/SMAD signaling pathway, diverse pathogens, RNAseq studies, in vitro and in vivo experiments, and manipulation of SMAD4 deficient cells through retroviral mediated genetic manipulations.
    • In vitro studies demonstrating that EOMES overexpression partially rescues the phenotype of SMAD4 deficiency in activated CD8 T cells are intriguing and generally well done.

    Weaknesses:
    • Despite some nicely designed and executed experiments, the overall research question and goal of this manuscript are unclear. The title/abstract/background seem to indicate the goal is to clarify the role of SMAD4 and TGFB in controlling CD8 T cell differentiation into circulating or tissue-resident memory populations. This is an important research question. However, many of the key experiments are executed in vitro, complicating results and making it hard to discern how some of the experiments translate to in vivo differentiation. Further, there is not a consistent approach of carefully examining Trm cells as the experiments tend to switch back and forth across circulating CD8 T cell populations, non-lymphoid localized CD8 T cells, and in vitro cells.
    • Related to the point above, the manuscript is a bit disjointed and at times difficult to follow. It's hard to tell if the goal is to understand how TGFBRII/SMAD4 regulates CD8 T cell differentiation during influenza virus infection vs discerning molecular mechanisms of SMAD-mediated control of CD103. If it's the former, much of the key in vivo experiments are lacking or the results have already been published. For the latter question, more molecular and genetic studies are necessary to understand this process (and the overall background and focus do not seem to fit with this research question).
    • Many of the core findings in this manuscript have previously been reported either in the prior work from this group (J Immunol, 2015) or more recently by Wu et al. (Cell Moll Immunol 2020). Key factors reported to be modulated by SMAD4 in this manuscript include CD103, KLRG1, and CD62L. It has already been shown these factors are modulated by loss of SMAD4, and the experiments outlined in this study do not dramatically expand on these findings. Intriguing results with dual TGBRII/SMAD4 dual KO cells have also been reported in similar experiments by Wu et al.
    • Many of the conclusions are not properly supported by the data. Based on the discussion, it appears the main conclusions are 1) TGFB and SMAD4 exert reciprocal functions in controlling circulating and tissue-resident memory formation (last sentence of first paragraph in discussion). As mentioned above there is not a careful or consistent assessment of memory T cell populations in lymphoid or non-lymphoid compartments. 2) SMAD4 was required to maintain EOMES expression in activated CTLs. This data is fairly robust; however, could this be due to differences in cell states rather than a direct role for SMAD4 in sustaining EOMES expression? 3) SMAD4 has multiple roles in regulating expression of CD103, including complimentary or independent roles of Ski (last two sentences of paragraph describing Fig 3 in the results section). There was no assessment of Ski in the results of this study. Additionally, despite many conclusions about the roles of SMAD proteins in controlling gene expression, there are no experiments to assess binding of these factors (e.g. ChIP-qPCR etc) to key genes.

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