Glycolysis downregulation is a hallmark of HIV‐1 latency and sensitizes infected cells to oxidative stress

Abstract HIV‐1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV‐1 infection, but its role in the development and maintenance of HIV‐1 latency has not been elucidated. By combining transcriptomic, proteomic, and metabolomic analyses, we here show that transition to latent HIV‐1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, NADPH, fueling antioxidant pathways maintaining HIV‐1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV‐1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people living with HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV‐1 latency that can be exploited to target latently infected cells with eradication strategies.

Thank you again for submit ting your work to EMBO Molecular Medicine. One of the referees who init ially accept ed to review the manuscript finally dropped out . We have now heard back from the ot her two referees who evaluat ed your manuscript . As you will see from the report s below, the referees acknowledge the pot ent ial int erest of the st udy. However, they also raise subst ant ial concerns about your work, which should be convincingly addressed in a major revision of the present manuscript .
The referees' recommendations are rather clear and there is no need to reiterate their comments. Importantly, direct analysis of cell viability (infected vs. control) needs to be performed, the impact of latent and productive HIV-1 infections on the identified metabolic pathways needs to be assessed. Further, attention should be paid to the discrepancies with previous studies, and limitations with regard to the non-specific effect of metabolic intervention need to be discussed.
During our pre-decision cross-commenting process (in which the referees are given a chance to make additional comments, including on each other's reports), Referee #3 added "limitations with regard to the non-specific effect of metabolic intervention need to be discussed is the main concern for the conclusion and the relevance for this manuscript." Referee #2 said "I totally agree with all the requests and concerns raised by Referee #3. I think that all the issues raised by both referees have to be addressed by the authors before reconsidering the manuscript." We would welcome the submission of a revised version within three months for further consideration. Please note that EMBO Molecular Medicine strongly supports a single round of revision and that, as acceptance or rejection of the manuscript will depend on another round of review, your responses should be as complete as possible.
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You are also welcome to suggest a striking image or visual abstract to illustrate your article. If you do please provide a jpeg file 550 px-wide x 400-px high. Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress (EMM-2020-13901) The work presented by Iart Luca Shytaj et al., investigated the regulation of glycolytic metabolism during HIV-1 infection. The authors report that transition to latent HIV-1 infection downregulates glycolysis while viral reactivation inverts this effect. These observations are associated with downregulation of NAD+/NADH relaying on NADPH antioxidant maintaining HIV-1 latency. However, blocking NADPH downstream effectors favors HIV-1 reactivation from latently infected myeloid and lymphoid cell lines. This article brings new insights into glucose metabolism/redox system regulation during HIV-1 latency and might suggest new therapeutic strategies for HIV functional cure and reservoir eradication. However, discrepancies with previous studies should be further studied and discussed.
Major comments 1. Previous studies cited by the authors indicate that glycolysis and glucose transport are increased in HIV-1 infected cells. Moreover, two studies reported that glycolysis inhibition induces the death of infected CD4+ T cells and that HIV-1 latently infected macrophages metabolically carry no changes in glycolysis (Castellano P et al., Scientific Reports 2019, Valle-Casuso JC et al., Cell Met 2019. Thus, it's difficult to link these previous results to the conclusion obtained by the authors in the first part of their Results section (downregulation of the expression of glycolytic enzymes in HIV-1 productively and latently infected CD4+ T-cells as well as the absence of HC69 cell death following treatment with glycolysis inhibitor). 2. It is difficult to distinguish the experiments carried out by the authors and the results exploited to generate the RNA-seq or single-cell RNA-seq data presented in Figure 1. The experiments conducted in Figure 3 to test the ability of two drugs (auranofin and buthionine sulfoximine) to induce viral reactivation show the results obtained in cells from different origins (primary cells from PLWH and uninfected monocytes derived macrophages or lymphoid and myeloid cells lines) in the same graph. This kind of presentation is not suitable because it does not allow the reader to evaluate the efficacy of the drug depending on the cell type. In addition, it does not allow to see the results obtained on an appropriate number of experiments or patient cells. Similar comment for the cell viability following auranofin and buthionine sulfoximine treatment in Figure 3 B. 3. In Figure 3 C, the authors indicate that "direct and precise analysis of cell viability (infected vs. control) was not possible due to the low frequency of HIV-1-infected cells". However, these data are critical and have to be added to the manuscript since Figure 3 B shows an important effect on cell viability of the combination treatment auranofin+buthionine sulfoximine. The decrease in integrated proviral DNA could be linked to cell death. In addition, patients'characteristics from Figure  3C including viremia and transcriptional status have to be reported in the manuscript. 4. Importantly, the authors have to perform functional experiments to assess the impact of latent and productive HIV-1 infections on the most significant enriched pathways they identified such as the antioxidants metabolism or purine/pyrimidine pathways (glutamate metabolism).
Minor comments 1. In Figure 2B, the decrease in lactate level in cells infected with HIV-1JR-FL 3dpi is difficult to observe. 2. In Figure 2 F, glucose metabolism in productively-infected cells does not seem to be less impaired compared to latently-infected cells (Figure 2 E).

Referee #3 (Comments on Novelty/Model System for Author):
Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress Iart Luca Shytaj et al.
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation. Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference.
A significative limitation of the study is that it focused only on glycolysis as the energy-producing pathway, while lipophagy is also at play and contribute to naïve and memory status, memory T cells in HIV infection. Loucif H,et al. . Lipophagy confers a key metabolic advantage that ensures protective CD8 T-cell responses against HIV-1. Autophagy. 2021 Jan 18:1-16. Cd4 T cell population is an heterogenous group with main Th 1, Th17, Th Follicular and Treg functions, which show plasticity and metabolism preference, and been regulated by mitochondrial activity. Loucif H, Plasticity in T-cell mitochondrial metabolism: A necessary peacekeeper during the troubled times of persistent HIV-1 infection. Cytokine Growth Factor Rev. 2020. Major limitation of the metabolic intervention is the non-specific effect, not targeting HIV infected cells but any cells. Arsenic trioxide already used in clinic for acute myeloid leukemia type 3 are relatively toxic and act on a specific type representing only 3 of AML. For HIV the low difference of latent infected CD4 cells vs. long-lived Th17 with stemness will not be very different on a metabolism basis. However, strategies propose based on study findings if can be specific for a cell subtype may affect elimination of latently infected cells while may boost of anti-HIV-1 cell mediated immunity, as reported by Loucif et al. 2020.
However, several issues have to be addressed: A possible approach to reach this goal is the investigation of the metabolic pathways exploited by the retrovirus to actively replicate and enter a latent state. This sentence is too vague, no clear that replication of virus (virions) lead to latent state, some infection in cell programmed to become long term memory with some stemness may drive the latency for the virus and not the virus inducing latency.
This individual had been treated with a combination of intensified ART and the NAD+ precursor nicotinamide, thus suggesting a possible contribution of glycolysis regulation in the therapeutic result obtained. Ref should be added Lebouché B, et al. . Impact of extended-release niacin on immune activation in HIV-infected immunological non-responders on effective antiretroviral therapy. HIV Res Clin Pract. 2021 Jan 6:1-9.
Data presented are complex and combine immunology with biochemistry the focus should be on CD4 T cells, not sure that myeloid cell analyses and their heterogenicity and tissue dependence may be better described in another paper.
Educative table should be added with glycolysis as the energy-producing pathway with NADH and the Krebs cycle to better understand study findings and their interactions.
Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress Iart Luca Shytaj et al.
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation. Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference.
To limit the study to T cells and remove myeloid cells To assess naive vs memory cells separated by flow to decipher subset of CD4 T cells according to glycolysis status clinical impart may be limited as intervention are non specific as apply to any human cells Referee #3 (Remarks for Author): Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress Iart Luca Shytaj et al.
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation.
Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference. A significative limitation of the study is that it focused only on glycolysis as the energy-producing pathway, while lipophagy is also at play and contribute to naïve and memory status, memory T cells in HIV infection. Loucif H,et al. . Lipophagy confers a key metabolic advantage that ensures protective CD8 T-cell responses against HIV-1. Autophagy. 2021 Jan 18:1-16. Cd4 T cell population is an heterogenous group with main Th 1, Th17, Th Follicular and Treg functions, which show plasticity and metabolism preference, and been regulated by mitochondrial activity. Loucif H, Plasticity in T-cell mitochondrial metabolism: A necessary peacekeeper during the troubled times of persistent HIV-1 infection. Cytokine Growth Factor Rev. 2020. Major limitation of the metabolic intervention is the non-specific effect, not targeting HIV infected cells but any cells. Arsenic trioxide already used in clinic for acute myeloid leukemia type 3 are relatively toxic and act on a specific type representing only 3 of AML. For HIV the low difference of latent infected CD4 cells vs. long-lived Th17 with stemness will not be very different on a metabolism basis. However, strategies propose based on study findings if can be specific for a cell subtype may affect elimination of latently infected cells while may boost of anti-HIV-1 cell mediated immunity, as reported by Loucif et al. 2020.
However, several issues have to be addressed: A possible approach to reach this goal is the investigation of the metabolic pathways exploited by the retrovirus to actively replicate and enter a latent state. This sentence is too vague, no clear that replication of virus (virions) lead to latent state, some infection in cell programmed to become long term memory with some stemness may drive the latency for the virus and not the virus inducing latency. Data presented are complex and combine immunology with biochemistry the focus should be on CD4 T cells, not sure that myeloid cell analyses and their heterogenicity and tissue dependence may be better described in another paper.
Educative table should be added with glycolysis as the energy-producing pathway with NADH and the Krebs cycle to better understand study findings and their interactions.
Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress Iart Luca Shytaj et al.
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation. Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference. A significative limitation of the study is that it focused only on glycolysis as the energy-producing pathway, while lipophagy is also at play and contribute to naïve and memory status, memory T cells in HIV infection. Loucif H,et al. . Lipophagy confers a key metabolic advantage that ensures protective CD8 T-cell responses against HIV-1. Autophagy. 2021 Jan 18:1-16. Cd4 T cell population is an heterogenous group with main Th 1, Th17, Th Follicular and Treg functions, which show plasticity and metabolism preference, and been regulated by mitochondrial activity. Loucif H, Plasticity in T-cell mitochondrial metabolism: A necessary peacekeeper during the troubled times of persistent HIV-1 infection. Cytokine Growth Factor Rev. 2020. Major limitation of the metabolic intervention is the non-specific effect, not targeting HIV infected cells but any cells. Arsenic trioxide already used in clinic for acute myeloid leukemia type 3 are relatively toxic and act on a specific type representing only 3 of AML. For HIV the low difference of latent infected CD4 cells vs. long-lived Th17 with stemness will not be very different on a metabolism basis. However, strategies propose based on study findings if can be specific for a cell subtype may affect elimination of latently infected cells while may boost of anti-HIV-1 cell mediated immunity, as reported by Loucif et al. 2020.
However, several issues have to be addressed: A possible approach to reach this goal is the investigation of the metabolic pathways exploited by the retrovirus to actively replicate and enter a latent state. This sentence is too vague, no clear that replication of virus (virions) lead to latent state, some infection in cell programmed to become long term memory with some stemness may drive the latency for the virus and not the virus inducing latency. This individual had been treated with a combination of intensified ART and the NAD+ precursor nicotinamide, thus suggesting a possible contribution of glycolysis regulation in the therapeutic result obtained. Ref should be added Lebouché B, et al. . Impact of extended-release niacin on immune activation in HIV-infected immunological non-responders on effective antiretroviral therapy. HIV Res Clin Pract. 2021 Jan 6:1-9.
Data presented are complex and combine immunology with biochemistry the focus should be on CD4 T cells, not sure that myeloid cell analyses and their heterogenicity and tissue dependence may be better described in another paper.
Educative table should be added with glycolysis as the energy-producing pathway with NADH and the Krebs cycle to better understand study findings and their interactions.

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress Iart Luca Shytaj et al.
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation. Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference. A significative limitation of the study is that it focused only on glycolysis as the energy-producing pathway, while lipophagy is also at play and contribute to naïve and memory status, memory T cells in HIV infection. Loucif H,et al. . Lipophagy confers a key metabolic advantage that ensures protective CD8 T-cell responses against HIV-1. Autophagy. 2021 Jan 18:1-16. Cd4 T cell population is an heterogenous group with main Th 1, Th17, Th Follicular and Treg functions, which show plasticity and metabolism preference, and been regulated by mitochondrial activity. Loucif H, Plasticity in T-cell mitochondrial metabolism: A necessary peacekeeper during the troubled times of persistent HIV-1 infection. Cytokine Growth Factor Rev. 2020. Major limitation of the metabolic intervention is the non-specific effect, not targeting HIV infected cells but any cells. Arsenic trioxide already used in clinic for acute myeloid leukemia type 3 are relatively toxic and act on a specific type representing only 3 of AML. For HIV the low difference of latent infected CD4 cells vs. long-lived Th17 with stemness will not be very different on a metabolism basis. However, strategies propose based on study findings if can be specific for a cell subtype may affect elimination of latently infected cells while may boost of anti-HIV-1 cell mediated immunity, as reported by Loucif et al. 2020.
However, several issues have to be addressed: A possible approach to reach this goal is the investigation of the metabolic pathways exploited by the retrovirus to actively replicate and enter a latent state. This sentence is too vague, no clear that replication of virus (virions) lead to latent state, some infection in cell programmed to become long term memory with some stemness may drive the latency for the virus and not the virus inducing latency. Data presented are complex and combine immunology with biochemistry the focus should be on CD4 T cells, not sure that myeloid cell analyses and their heterogenicity and tissue dependence may be better described in another paper.
Educative table should be added with glycolysis as the energy-producing pathway with NADH and the Krebs cycle to better understand study findings and their interactions.

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress Iart Luca Shytaj et al.
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation.
Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference. A significative limitation of the study is that it focused only on glycolysis as the energy-producing pathway, while lipophagy is also at play and contribute to naïve and memory status, memory T cells in HIV infection. Loucif H,et al. . Lipophagy confers a key metabolic advantage that ensures protective CD8 T-cell responses against HIV-1. Autophagy. 2021 Jan 18:1-16. Cd4 T cell population is an heterogenous group with main Th 1, Th17, Th Follicular and Treg functions, which show plasticity and metabolism preference, and been regulated by mitochondrial activity. Loucif H, Plasticity in T-cell mitochondrial metabolism: A necessary peacekeeper during the troubled times of persistent HIV-1 infection. Cytokine Growth Factor Rev. 2020. Major limitation of the metabolic intervention is the non-specific effect, not targeting HIV infected cells but any cells. Arsenic trioxide already used in clinic for acute myeloid leukemia type 3 are relatively toxic and act on a specific type representing only 3 of AML. For HIV the low difference of latent infected CD4 cells vs. long-lived Th17 with stemness will not be very different on a metabolism basis. However, strategies propose based on study findings if can be specific for a cell subtype may affect elimination of latently infected cells while may boost of anti-HIV-1 cell mediated immunity, as reported by Loucif et al. 2020.
However, several issues have to be addressed: A possible approach to reach this goal is the investigation of the metabolic pathways exploited by the retrovirus to actively replicate and enter a latent state. This sentence is too vague, no clear that replication of virus (virions) lead to latent state, some infection in cell programmed to become long term memory with some stemness may drive the latency for the virus and not the virus inducing latency. Data presented are complex and combine immunology with biochemistry the focus should be on CD4 T cells, not sure that myeloid cell analyses and their heterogenicity and tissue dependence may be better described in another paper.
Educative table should be added with glycolysis as the energy-producing pathway with NADH and the Krebs cycle to better understand study findings and their interactions.
Please find our point-by-point response in red below each comment. To facilitate matching our comments to the new results, we have attached to this reply a copy of the revised figures/panels that are most important to support our answers.

General summary on the Referees´ comments:
The referees' recommendations are rather clear and there is no need to reiterate their comments. Importantly, direct analysis of cell viability (infected vs. control) needs to be performed, the impact of latent and productive HIV-1 infections on the identified metabolic pathways needs to be assessed. Further, attention should be paid to the discrepancies with previous studies, and limitations with regard to the non-specific effect of metabolic intervention need to be discussed.
During our pre-decision cross-commenting process (in which the referees are given a chance to make additional comments, including on each other's reports), Referee #3 added "limitations with regard to the non-specific effect of metabolic intervention need to be discussed is the main concern for the conclusion and the relevance for this manuscript." Referee #2 said "I totally agree with all the requests and concerns raised by Referee #3. I think that all the issues raised by both referees have to be addressed by the authors before reconsidering the manuscript." Regarding the direct analysis of cell viability, we now provide the data requested (Appendix 10B of the revised manuscript). These data are derived from the same experimental set up from which the data on proviral DNA were generated and demonstrate the general viability of the cells, i.e. that of the entire CD4 + T-cell culture comprising the patients' uninfected cells plus a small minority of infected cells. The whole cultures respond differently to auranofin and buthionine sulfoximine (BSO) in terms of cell viability than in terms of proviral DNA. Starting from the standpoint that we don't want to claim that auranofin has no impact on cell viability [we published a paper on this topic (Chirullo et al. Cell Death Disease 2013)], the data on the whole cell cultures show that the mortality is mainly driven by the prevously described effect of auranofin. There is no significant difference when the viability of the cultures treated with auranofin and the auranofin/BSO combination are compared. Instead, the effect of auranofin alone is minimal and non-significant on proviral DNA, and it is the addition of BSO to auranofin to be pivotal for HIV-1 DNA elimination. We believe that this is a strong argument in favor of a specific impact of the auranofin/BSO combination on proviral DNA. Moreover, we would like to clarify one point: cells from people living with HIV (PLWH) in which the proviral DNA was shown to be decreased by our strategy based on auranofin and BSO were sorted for cell viability before measuring integrated viral DNA. This measurement was normalized on the basis of the number of cells, calculated using the housekeeper gene CD3. We apologize for not being clearer in the former version of the manuscript on this topic. We have now included a figure detailing the sorting strategy used (Appendix 10A of the revised manuscript). In addition, the rationale of the experiment has now been more thoroughly described in the Results section. As mentioned in the previous paragraph, in patient-derived cells, latently infected cells are rare and mixed in a much larger population of surrounding uninfected cells. Therefore, while we did assess the viability of the whole cell population, it was not possible to specifically isolate the viability of those HIV-1 reservoir cells that harbor the virus from that of the surrounding cells of the same individual. For this reason, our strategy of combining cell sorting for viability and measurement of integrated HIV-1 DNA (normalized to the number of cells analyzed) is, in our view, the best possible setup to prove the preferential targeting of latently HIV-1 infected cells by auranofin and BSO in ex vivo samples of PLWH. On the other hand, in cellular models in which uninfected and infected cells could be clearly separated, we specifically compared cell viability in the presence or absence of auranofin/BSO, thus providing a further assessment of the preferential effect of the drug combination on latently infected cells ( Figure 5B and Figure EV5 in the revised manuscript). We therefore believe that what we propose is a step forward in the specific targeting of the viral reservoirs, although some off-target effects of course still remain. This limitation has been addressed in the new version of the Discussion.
As for the role of HIV-1 as a driver of metabolic changes, we now provide new data supporting the specific impact of the infection on the metabolic pathways identified. In two new sets of data, as 16th May 2021 1st Authors' Response to Reviewers detailed below, we now show that the HIV-1 accessory protein Tat, which is essential for viral transcription, determines, in the short term, an upregulation in markers of glycolysis and pyrimidine metabolism, which are necessary for retroviral replication. In the longer term, we show that Tat is fundamental for the upregulation of a key gene of the pentose phosphate cycle and the related antioxidant response (as requested by Referee 2). As for latent infection, we provide a new dataset, based on a homogeneously infected lymphoid cell line, showing that the latently infected cells maintain features acquired gradually during the late stages of productive infection and that some of these features are lost upon reactivation from latency. As these metabolic signatures are not present in uninfected cells or cells infected with Tat-deficient HIV-1 or in the same latently infected cell population after it subjected to reactivation from latency, we show that the late stages of HIV-1 replication induce key metabolic features favoring latency establishment. Of note, the use of a lymphoid cell line in the revised version of the manuscript, which is latently infected, but not quiescent, and is proliferating, contributes to exclude that the key metabolic changes observed in lymphocyte cultures undergoing HIV-1 latency are due only to reversal to a quiescent state. However, to complement our findings and acknowledge the possible contribution of cellular quiescence per se, we have now addressed this aspect specifically in the Discussion. We agree that the original version of the manuscript left some ambiguities when comparing our work to the previous literature. We believe that the revised version of the manuscript fully clarifies these seeming discrepancies and reconciles our results with the major findings of other groups. At the same time, we have tried to better explain and expand our new findings in the manuscript, which regards latency and latency reactivation.
In particular, we have now stressed the main novelty of our work, i.e. the fact that we have focused our attention to the late stages of the infection, especially the development of latent HIV-1 infection.  (2019), we now show that our conclusions do not contradict their findings. In particular, our results, now confirmed by metabolomic analysis of another lymphoid cell model ( Figure 3 of the Revised the manuscript), show that the early steps of glycolysis, which are exclusively characteristic of this pathway, are always and specifically affected by HIV-1 latency. On the other hand, late glycolytic metabolites, which are shared with the intertwined pentose phosphate pathway, can be replenished by the latter, depending on the cell type considered. Indeed, Castellano et al. based their conclusion on the production of lactate, which is a late glycolytic product. In line with their finding, our revised results show that lactate can be increased in specific cell types upon latency, and this concept is now shown in Figure 3, Figure EV4 and Appendix S4 in the revised manuscript, and also clearly stated in the revised Discussion.
2. It is difficult to distinguish the experiments carried out by the authors and the results exploited to generate the RNA-seq or single-cell RNA-seq data presented in Figure 1. The experiments conducted in Figure 3 to test the ability of two drugs (auranofin and buthionine sulfoximine) to induce viral reactivation show the results obtained in cells from different origins (primary cells from PLWH and uninfected monocytes derived macrophages or lymphoid and myeloid cells lines) in the same graph. This kind of presentation is not suitable because it does not allow the reader to evaluate the efficacy of the drug depending on the cell type. In addition, it does not allow to see the results obtained on an appropriate number of experiments or patient cells. Similar comment for the cell viability following auranofin and buthionine sulfoximine treatment in Figure 3 B.
We have clarified the representation of the results in Figure 1 and Figure Figure 1G,H (which have been moved to Figure EV2 in the revised manuscript) are based on our own sc-RNA-Seq analysis of HC69 cells. We believe that combining datasets generated in our labs with datasets generated for other purposes in independent laboratories can increase the reproducibility of our observations. However, according to the suggestion, the origin of each dataset is now clarified in detail in the revised version of the manuscript. Moreover, according to this Referee's suggestion as well as to similar suggestions from Referee 3, previous Figure 3, now Figure 5 in the revised manuscript, only shows data derived from lymphoid cells and Figure EV5 of the revised manuscript shows the latency reactivation and viability effect separately for each cell type considered.
3. In Figure 3 C, the authors indicate that "direct and precise analysis of cell viability (infected vs. control) was not possible due to the low frequency of HIV-1-infected cells". However, these data are critical and have to be added to the manuscript since Figure 3 B shows an important effect on cell viability of the combination treatment auranofin+buthionine sulfoximine. The decrease in integrated proviral DNA could be linked to cell death. In addition, patients'characteristics from Figure 3C including viremia and transcriptional status have to be reported in the manuscript.
Please note the general answer to this question in the section "General summary on the Referees´ comments" above. Specifically, we apologize for the unclear description of the results in the previous version of the manuscript. The viability that could not be precisely measured was that of cells harboring latent HIV-1 DNA as compared to the surrounding uninfected cells in the same culture. We initially considered taking into account a FISH technique to address this issue. However, the precise quantification of viral DNA content using techniques based on fluorescence would be imprecise and could be still confounded by the presence of dead cells in the original population. The viability of the patients' whole cell population, instead, was taken into account, and is included in the figure below (Appendix S10B of the revised manuscript). These data are derived from the same experimental set up from which the data on proviral DNA were generated and demonstrate the general viability of the cells, i.e. that of the entire CD4 + T-cell culture comprising the patients' uninfected cells plus a small minority of infected cells. The whole cultures respond differently to auranofin and buthionine sulfoximine (BSO) in terms of cell viability than in terms of proviral DNA. As mentioned above, the impact of auranofin on lymphocyte viability had already been proven before (Chirullo et al. Cell Death and Disease 2013)]. Accordingly, the data on the whole cell cultures show that the mortality is mainly driven by the prevously described effect of auranofin. In line with this, there is no significant difference when the viability of the cultures treated with auranofin and the auranofin/BSO combination are compared (Appendix S10B of the revised manuscript). Instead, the effect of auranofin alone is minimal and non-significant on proviral DNA ( Figure 5C of the revised manuscript), and it is the addition of BSO to auranofin to be pivotal for HIV-1 DNA elimination. We believe that this is a strong argument in favor of a specific impact of the auranofin/BSO on proviral DNA.
Moreover, also the possible bias due to cell mortality was addressed by sorting of live cells before performing integrated HIV-1 DNA measurement, as detailed in the reply to the "General summary on the Referees´ comments" above and as shown below and in the (Appendix S10B of the revised manuscript). Finally, the description of the experimental rationale and results have been now rephrased at the end of the Results subchapter "Downstream inhibition of the glycolysis-alternative pentose phosphate pathway can induce a "shock and kill" effect in latently infected cells"

Appendix 10 of the revised manuscript. Gating strategy and viability of CD4 + T-cells of PLWH under ART left untreated or treated with auranofin (AF) and/or buthionine sulfoximine (BSO). CD4 + T-cells
were treated with auranofin AF (500 nM), BSO (250 μM) or a combination of the two, for 24 h. Viable CD4 + Tcells were sorted according to the gating strategy shown in Panel A. Viability data are shown as mean ± SD (Panel B). Data were analyzed by repeated measures one-way ANOVA followed by Tukey's post-test ** p< 0.01.
4. Importantly, the authors have to perform functional experiments to assess the impact of latent and productive HIV-1 infections on the most significant enriched pathways they identified such as the antioxidants metabolism or purine/pyrimidine pathways (glutamate metabolism).
To address this question, we have added a set of experiments isolating the effect of HIV-1 transcription (modulated through either exogenous administration of the Tat protein or genetic deletion of the Tat gene) on the expression of key genes of glycolysis, the pentose phosphate pathway, the antioxidant pathways thioredoxin/glutathione and glutamine metabolism. These newly included results show that: 1) In U1 cells, in which the Tat/TAR axis is not functional, incubation with exogenous Tat concomitantly increases expression of HIV-1 (as expected) and of GLUT-1 (please see below Appendix 6 of the revised manuscript). This result is in line with one main finding of the manuscript, i.e. that HIV-1 reactivation is accompanied by glycolysis upregulation. Moreover, in the same experimental setup, Tat induced upregulation of Carbamoyl-Phosphate Synthetase 2 (CAD), which is required for pyrimidine synthesis from L-glutamine and aspartate. Interestingly, upregulation of CAD, but not of GLUT-1, was also observed in uninfected U937 cells, suggesting that Tat per se could play a direct role on pyrimidine metabolism. Finally, Tat administration led to an increase, albeit not significant, in the expression of G6PD, which is the enzyme initiating the pentose pathway leading to NADPH production. As we had previously shown that expression of G6PD and of other antioxidant factors is increased during late productive and latent HIV-1 infection (Shytaj et al. EMBO Journal 2020) we further analyzed their modulation in primary CD4 + T-cells infected with wild type and Tatdeficient HIV-1 during early and late productive infection.
2) Experiments in CD4 + T-cells showed that infection with Tat-deficient HIV-1 led to impaired viral transcription (as expected) and, unlike infection with wild type HIV-1, was not associated with upregulation of G6PD (please see Figure 4 of the revised manuscript below). We then considered the role of Tat-deficiency on the expression of the antioxidant genes thioredoxin reductase (TrxR) and glutamate cysteine ligase (GCLC), which encode for the downstream antioxidant targets of NADPH that are inhibited by AF and BSO. Interestingly, infection with Tat-deficient HIV-1 was not associated with an increase in the expression of each of the two genes (panels C,D).
Overall, these new experiments show that increasing HIV-1 expression can per se upregulate the glycolytic potential as well as the expression of key genes of the pentose phosphate pathway and of glutamine metabolism. Please also see the new results subchapter "HIV-1 transcription is a specific driver of metabolic changes" and our reply to the "General summary on the Referees´ comments" above.  infected with wild type HIV-1 pNL4-3 . Panels B-D) relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B), and of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells. Data were first normalized using 18S as housekeeping control and then expressed as Log 2 fold mRNA expression in wild type vs Tat-deficient infection (panel A) or in infected vs mock infected cells (panels B-D), which were calculated using the 2-ΔΔCT method (Livak & Schmittgen, 2001). GCLC= Glutamate-cysteine ligase; TrxR1= thioredoxin reductase 1.
Minor comments 1. In Figure 2B, the decrease in lactate level in cells infected with HIV-1JR-FL 3dpi is difficult to observe.
In light of this comment and of our new data showing that lactate, as other late glycolytic products, can be replenished by intertwined pathways in some cell types, this Figure has now been moved to the supplementary information (Appendix S4) and the role of pathways other than glycolysis in determining the levels of late glycolytic products is now addressed in detail in the Results and Discussion of the revised manuscript (in particular please see subchapter "Decreased initial glycolytic metabolism during latent HIV-1 infection" of the Results).

In Figure 2 F, glucose metabolism in productively-infected cells does not seem to be less impaired compared to latently-infected cells (Figure 2 E).
We agree with the Referee on the lack of clarity in our previous data presentation. The difference is now much more evident after providing a direct comparison between latently infected cells and cells in which HIV-1 was reactivated ( Figure 2C, Figure EV3C of the revised manuscript). However, it needs to be highlighted that the development of latency has emerged as a gradual process. Thus, we agree with this Reviewer that the differences between these two states should not be overinterpreted and we have amended our text accordingly throughout the manuscript.

Referee #3 (Comments on Novelty/Model System for Author):
Neuro-immunometabolism is the new frontier in HIV cure research. It is well established that CD4 T cells are preferentially infected when glycolytic pathway is activated when cell respond to an immune response or in the context of non-specific inflammation, often assessed by the expression of the glucose transporter GLUT-1. The glycolysis/redox state during HIV-1 infection influence autophagy that regulates cell fate. Investigators used elegant technology with transcriptomic, proteomic, and metabolomic datasets, with single cell analyses to show as expected that less energy is required for latency evidenced by the downplay of glycolysis and re-increases after activation. Such slowdown of glycolysis is accompanied with a higher reliance on the antioxidant thioredoxin (Trx) and glutathione (GSH) systems linked to stemness. Results point to exploit cell specific glycolytic effect to reduce HIV reservoir and mitochondria to be the great regulator. However, Hormesis could make stress a more complex issue as little stress might be better that no stress or too much stress limiting some experiment interpretation. Zimmermann A, et al.. When less is more: hormesis against stress and disease. Microb Cell. 2014 We agree that GLUT1 is a crucial factor for glucose influx and glycolysis regulation. In this regard, we have now included an experiment specifically isolating the role of HIV-1 transcription on GLUT1 expression (please see the reply to question 4 of Reviewer 2 and Appendix Figure 6 in the revised manuscript). We also agree that defining the hormetic dose zone of AF and BSO (or of similar compounds) might be crucial to achieve enough selectivity of the treatment and target with higher preference latently infected cells. While we are currently working in that direction, and we have preliminary data suggesting that significantly lower BSO concentrations might be sufficient to target infected cells, our experiments in the paper are a proof-of-concept and thus are conducted using well established drug concentrations equal to in vitro equivalents of standard doses administered in clinical trials or animal experiments with AF and BSO. Moreover, the aim of the present study is not only translational but also targeted at pathway discovery. Therefore, we needed to adhere to the AF and BSO dosages reported in the literature to inhibit their molecular targets efficiently. In this regard, we have now acknowledged at the end of the revised Discussion the need for future studies to identify the hormetic dose interval in order to achieve optimal selectivity with this strategy and we have accordingly quoted the paper suggested by the Referee.
Investigations are timely and novel but highly dependent of the model used and have Lymphoid and myeloid analyses make the reading of the text and context difficult and long.
In light of this comment and of comment 3 of Referee 2, the main figures of the revised manuscript now only contain data derived from lymphoid cells. In particular, we have included a novel metabolomic data set of lymphoid Jurkat T-cells (please see below Figure 2 of the revised manuscript) which consolidates our finding that glycolytic metabolites are downregulated during latent infection, except those that can be replenished by intertwined pathways (such as the pentose phosphate pathway). All data derived from other cell types have been moved to the Extended View or Appendix Figures. We think it appropriate to keep the other cell types as supplementary information as HIV-1 reservoirs are known to be highly heterogeneous. Therefore, the possibility to compare lymphoid cells with other cell types allows to identify those metabolic pathways that are broadly conserved markers of latency (i.e. downregulation of early glycolytic metabolites and reliance on pentose phosphate activity) from those that are cell-type dependent (e.g. late glycolytic metabolites).  (Xia et al, 2009). Panel C) Heatmaps of glycolytic metabolites in latently infected Jurkat T-cells as compared to their uninfected counterparts or Jurkat T-cells with HIV-1 reactivated by TNF as compared to latently infected cells. Data are displayed as Log 2 fold change expression. Adjusted P values (q values) were calculated by the Benjamini-Hochberg false discovery rate. Panels D,E) Relative ratios of NADH/NAD + (D) and ATP/ADP (E) in latently infected and reactivated cells. Data were normalized using the matching uninfected control.
A significative limitation of the study is that it focused only on glycolysis as the energy-producing pathway, while lipophagy is also at play and contribute to naïve and memory status, memory T cells in HIV infection. Loucif H,et al. . Lipophagy confers a key metabolic advantage that ensures protective CD8 T-cell responses against HIV-1. Autophagy. 2021 Jan 18:1-16.
We wish to thank this Referee for the interesting suggestion. We have expanded our analysis in order to take into account this alternative pathway of energy production. Our metabolomic data, including the new metabolomic analysis performed in Jurkat T-cells (see our reply to the first point raised by this Referee and Figure 3 of the revised manuscript), shows that lipid metabolism is downregulated in latently infected cells, and that this downregulation is in line with the less efficient tricarboxylic acid cycle observed in lymphoid cells. Interestingly, we observed an increase in the number (but not density) of lipid droplets in infected cells (please see the Appendix Figure 5 of the revised manuscript below) consistent with a downregulation of lipid catabolism. We interpret these new data as consistent with a switch off, during latent infection, of those pathways more active in the presence of efficient immunity such as that described by Loucif et al. (2021). We have now discussed this concept in the revised version of the Discussion. Figure EV1 of the revised manuscript. Single-cell transcriptional modulation of glycolytic enzymes in CD4 + T-cell subsets. The scRNA-Seq expression of the entire glycolytic pathway was analyzed in primary CD4 + T-cells infected in vitro with VSVG-HIV-1-GFP and sorted for viral expression as detailed in (Golumbeanu et al, 2018). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 (low latency reactivation potential) and 2 (high latency reactivation potential) were identified by principal component analysis as described in (Golumbeanu et al, 2018). T cell subtypes were identified using the SingleR R package and allocated to each cluster irrespective of the in vitro treatment. The expression level of the HUMAN-GLYCOLYSIS pathway was calculated as the average expression of genes comprising the gene list.
Major limitation of the metabolic intervention is the non-specific effect, not targeting HIV infected cells but any cells. Arsenic trioxide already used in clinic for acute myeloid leukemia type 3 are relatively toxic and act on a specific type representing only 3 of AML.
We agree with this limitation. As mentioned in the reply to question 1 of this Referee our drug treatment in this study was designed as a proof of concept based on dosages previously administered in clinical trials or animal experiments of AF and BSO. However, the selectivity range of these and other drugs with a similar mechanism of action might be improved when targeting specifically the HIV-1 latent reservoir. This point is now mentioned in the revised Discussion. We would also like to add that, while a better selectivity for latent cells might be achievable, the temporary elimination of part of the uninfected cells, if well tolerated, might be both desirable and necessary to achieve a sustained HIV-1 remission without antiretrovirals (functional cure), due to generation of effective immunity following temporary lymphorestriction (the so called "Zitvogel effect", reviewed in Benhar et al. J Clin Invest 2016).
However, several issues have to be addressed: A possible approach to reach this goal is the investigation of the metabolic pathways exploited by the retrovirus to actively replicate and enter a latent state. This sentence is too vague, no clear that replication of virus (virions) lead to latent state, some infection in cell programmed to become long term memory with some stemness may drive the latency for the virus and not the virus inducing latency.
In the revised version of the manuscript, we have included data that strongly support a direct role of HIV-1 transcriptional state in determining the metabolic state of the host cell (please see reply to question n4 of Reviewer 2). However, we agree that metabolic changes of the cell itself, for example due to the reversal to a quiescent state, might also be responsible for the development of latency and this complementary scenario is now explicitly mentioned in the Discussion. We thank this Referee for pointing out this reference, which has now been added in the description of the different types of metabolic approaches to an HIV cure.
Data presented are complex and combine immunology with biochemistry the focus should be on CD4 T cells, not sure that myeloid cell analyses and their heterogenicity and tissue dependence may be better described in another paper.
In line with this comment and with question n2 of the Referee, the main figures in the revised manuscript now only contain data derived from lymphoid cells. All data derived from other cell types are now presented in the Expanded View or Appendix material. In this regard, please note that the metabolomic data, previously available only for myeloid cells, have been now substituted with an entirely new dataset derived from lymphoid Jurkat T-cells, which allowed us to reach very similar conclusions and rendered the main message of the paper, i.e. the role of downregulated glycolysis as a specific marker of HIV-1 latency, more cohesive (please see Figure 2 of the revised manuscript above).
Educative table should be added with glycolysis as the pathway with NADH and the Krebs cycle to better understand study findings and their interactions.
In the introduction other metabolic pathway are also under investigation for HIV cure and should be shortly discussed with new reference.
A specific paragraph has now been added to the Introduction.
To limit the study to T cells and remove myeloid cells As mentioned in the replies to questions n2 and n7, the main figures in this revised version now only contain data derived from lymphoid cells. Data from other cell types has been moved to the Expanded View or Appendix material, where we think it can be useful to pinpoint those metabolic changes that are conserved across latently infected cells of different lineages (i.e. glycolysis downregulation and compensatory activity of the pentose phosphate pathway).

To assess naive vs memory cells separated by flow to decipher subset of CD4 T cells according to glycolysis status
Unfortunately, the separation of naïve and memory T-cells by flow cytometry can hardly be done in our model, because cells are first activated to allow HIV-1 infection. Once this occurs, cells typically lose the naïve phenotype. Moreover, measuring the whole glycolytic pathway by flow cytometry would be challenging. However, we agree with this Referee that, from the data that we presented, one might ascribe the different expression of glycolytic genes observed in infected and uninfected cells to a variable contribution of CD4 + T-cell subsets. To address this question, and the partially overlapping question n4 of the same Referee, we have now provided evidence that downregulation of the glycolytic pathway is a marker of latency independently of the cell subset considered (please see above Figure EV1 of the revised manuscript). 1st Revision -Editorial Decision 14th Jun 2021 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from the two referees who were asked to re-assess it. As you will see below, the referees are now supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following amendments: 1. In the main manuscript file, please do the following: -remove the red color font.  Table needs to be called out as Appendix Table S1 -NOT Appendix S1.
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The upregulation of glycolysis in activated cells favors HIV-1 infection and initial viral replication. This study discovers that, to transit into a latent form, which can shield the virus from immunity and antiretroviral drugs, HIV-1 needs to downregulate glycolysis.
• Restoration of glycolytic activity is required for HIV-1 reactivation from latency.
• Latently infected cells rely on pentose phosphate metabolism and its downstream effectors, i.e., the antioxidant glutathione and thioredoxin pathways, for their survival.
• Preferential targeting latently infected cells with drugs inhibiting thioredoxin and glutathione pathways leads to both HIV-1 reactivation from latency and death of infected cells.
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This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript.

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.
definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
The typical sample size for biological in vitro assay was commonly adopted (n =3). For some in silico analysis (e.g. microarray) due to cost considerations and to the availability of partially overlapping data sets a n=2 was selected. For experiments on CD4+ T-cells derived from patients´cells, and aimed at a main outcome (i.e., success of the therapeutic in-vitro effect), we considered an effect size of 33% (difference between means), an SD of 20% of the mean, an alpha error of 0.05. The required calculated sample size was 7 ( https://clincalc.com/Stats/SampleSize.aspx ) and we chose a sample size of 8 for our experiments graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Yes
Parametric testing was adopted when normality could be hypothesized (sample size ≤ 3) or restored through an appropriate transformation. Data sets characterized by sample size > 3 which did not pass the normality tests (D´Agostino & Pearson or Shapiro-Wilk) and for which a transformation was not applicable were analyzed by non-parametric tests. For both parametric and non-parametric tests, post-test comparisons were used to compare specific groups as described in the