Oxygen levels at the time of activation determine T cell persistence and immunotherapeutic efficacy

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1. 

   Version published to 10.7554/elife.84280 on eLife

   May 11, 2023
2. 

   eLife

   Apr 16, 2023

   Author Response

   Reviewer #1 (Public Review):

   This study represents an important work in the field of (CAR)T-cell immunotherapy by analyzing the effect of different oxygen tension on the function and differentiation of T-cells (especially CD8+). Although it has been described that low oxygen levels can influence effector function/differentiation of T-cells, as nicely acknowledged by the authors in the introduction, a comprehensive analysis in the context of immunotherapy has been missing so far and this study adds significant findings that will be relevant for patient care in all fields applying (CAR)T-cell immunotherapy.

   The strength of the evidence is generally solid although there are some discrepancies between the different ways to induce HIF-1α (i.e. low O2, pharmacological inhibition, shRNA knockdown) that need to be clearly …

   Author Response

   Reviewer #1 (Public Review):

   This study represents an important work in the field of (CAR)T-cell immunotherapy by analyzing the effect of different oxygen tension on the function and differentiation of T-cells (especially CD8+). Although it has been described that low oxygen levels can influence effector function/differentiation of T-cells, as nicely acknowledged by the authors in the introduction, a comprehensive analysis in the context of immunotherapy has been missing so far and this study adds significant findings that will be relevant for patient care in all fields applying (CAR)T-cell immunotherapy.

   The strength of the evidence is generally solid although there are some discrepancies between the different ways to induce HIF-1α (i.e. low O2, pharmacological inhibition, shRNA knockdown) that need to be clearly stated and/or discussed.

   1. The first section of the results determines the impact of low oxygen and pharmacological HIF-1α stabilization on CD8+ T-cell activation/differentiation. Low oxygen diminishes cell growth but induces T-cell activation and effector cytokines, while HIF-1a stabilization mimics the effects on activation without alterations in expansion. Unfortunately, it remains unclear why effects upon low O2 are more pronounced although pharmacological HIF-1a stabilization is more efficient.

   1. As a next step, in vitro conditioned T-cells are transferred into a subcutaneous B16-OVA model. Although only the low O2 levels increase T-cell numbers in vivo after the transfer, the initial tumor burden was nicely decreased by both low O2 and HIF-1a stabilization. However, only the latter significantly improved survival and it remains unclear and uncommented why.

   1. Next, the authors address whether pre-conditioning of human CART-cells to induce HIF-1α either by pharmacological stabilization or by silencing of VHL shows similar effects. Surprisingly, both ways of HIF-1a stabilization resulted in different effects concerning differential gene expression and cytotoxic capacity of CART-cells. Accordingly, pharmacologically pre-conditioned CART-cells did not have a significant impact on survival in an in vivo model, while the VHL-silenced ones did significantly improve animal survival. This discrepancy between the two modes of HIF-1a stabilization remains uncommented. Unfortunately, it also remains unclear why the pharmacological HIF-1a stabilization significantly improved the survival in animals of the B16-OVA model and not in the human CART-cell model.

   1. After this, the researchers determine how the timing of hypoxic conditioning affects the (CAR)T-cells. Here it is convincingly shown that already a short period of hypoxic conditioning (1 day) with a subsequent expansion phase (additional 6 days) is sufficient to induce HIF-1a mediated alterations (e.g. metabolic changes, calcium flux, intracellular signaling). Although this section is coherent in itself, the switch between different times of hypoxic conditioning, expansion, and analysis is difficult to follow and might lead to confusion. The expression pattern of e.g. HIF-1a on day 1 and day 7 together with the nuclear amounts of NFAT and c-Myc might be misunderstood, like the other presented data as well.

   1. Last, short-term hypoxic conditioning of CART cells is tested in a solid tumour mouse model. The previously identified conditioning protocol also increases CART-cell function against solid tumours (as shown by enhanced cytotoxicity, reduced tumour burden, and prolonged survival). Unfortunately, although both HER2-CART-cells and CD19-CART-cells are shown to have superior cytotoxicity in vitro after the pre-conditioning, only HER2-CART-cells are demonstrated to be superior upon low O2 conditioning in an in vivo adoptive transfer mouse model and CD19-CART-cells remain an open question.

   Generally spoken, the limitations of the manuscript are:

   1. The occurring discrepancies of determining effects caused by the different modes of Hif-1a stabilization which certainly are caused by the complex nature of Hif-1a regulatory network, and;

   We now extend our observations and discuss these concerns more extensively in the manuscript.

   1. The limitation of detected effects primarily on CD8+ T cells while CART-cells products usually are a mixture of CD4+ and CD8+ ones.

   Figure S6H now shows that the effects of shorter periods of low oxygen conditioning obtained with CAR-T cells generated from isolated CD8+ T cells are reproducible in CAR-T cells generated from PBMCs. We have found that a 24h incubation of PBMC-derived CAR-T cells in 1 %O2 increases cytotoxicity against target cell effector differentiation at day 7, when compared to the cytotoxic effects of cells cultured at 21% oxygen levels.

   Reviewer #3 (Public Review):

   In this study, Cunha et al. examined the role of different oxygen tensions (21%, 5%, and 1% O2) and HIF-1α stabilisation in regulating murine and human CD8+ T cell proliferation and function. The authors find that hypoxia (1% O2) and pharmacological PHD inhibition with FG-4592, enhance murine T cell activation but impair proliferation. Furthermore, adoptive cell transfer (ACT) therapy of CD8+ T cells from both conditions reduced tumour burden in a B16-OVA melanoma model. Short hypoxic conditioning (1% O2) of human CD8+ T cells for 1 day increased HIF-1α stabilisation, with increased activation, glycolysis, and mitochondrial function still observed following 6 days of normoxic cell culture. Short hypoxic conditioning of HER2 and CD19 CAR-T cells improved their activation and cytotoxicity in vitro, while HER2 CAR-T cell counts were increased in vivo, reducing tumour burden, and increasing survival when compared to 21% O2.

   Strengths:

   The paper convincingly demonstrates that short hypoxic conditioning in a defined window improves CAR-T cell function through in vitro cytotoxicity assays and following adoptive transfer in a preclinical HER2+-SKOV3+ positive tumour model. Thus, the major conclusion of the paper is mostly well supported by the data and could represent a novel strategy to improve CAR-T cell immunotherapy for solid tumours in the future.

   Weaknesses:

   The extent to which hypoxic conditioning-mediated improvement in CAR-T cell function is dependent on HIF-1-driven metabolic reprogramming is unclear and other potential mechanisms are not explored. 5FG-4592 and VHL silencing in HER2 CAR-T cells did not phenocopy each other faithfully. In addition, neither approach was as effective as short hypoxic conditioning with 1% O2 in improving CAR-T cell function in vitro or in vivo. Although the authors suggest the temporal dynamics of HIF-1α stabilisation is the key point, this is not convincingly proven, and no metabolic characterisation of these CAR-T cells was performed.

   The revised manuscript now includes live metabolic analyses in a Seahorse set up, using T cells following FG-4592 treatment or VHL silencing. We found exposure of human CD8+ T cells to FG-4592 leads to a suppression of their oxygen consumption rates, both at basal and maximal levels. This can underpin the observed reduced expression of effector molecules (PMID: 33398183). Treatment of human T cells with FG-4592 resulted in a dose-dependent reduction of in vitro cytotoxicity, similar to that observed with exposure to low oxygen (e.g., 7 day OT-I expansion in 1%O2 impairs antitumour function [Figure supplement 6L]).

   Regarding VHL silencing, we did not observe metabolic differences compared to controls. This might arise from the fact that shVHL vectors only caused an overall 30% reduction in VHL protein expression, and that the silencing occurred after T cells had been activated. As we show, the moment of activation is key for T cell differentiation and function, and this could explain the lack of metabolic differences between shNCT and shVHL-expressing cells. These points are now added to 5th paragraph of the Discussion section.

   It is unclear how changes elicited during short hypoxic conditioning are maintained following continued normoxic cell culture. Hypoxia is known to rapidly regulate histone methylation and chromatin structure in a HIF-independent manner (PMID: 30872525; PMID: 30872526). Are similar epigenetic changes observed in T cells, and if so, could these epigenetic changes underlie improved T cell activation?

   We thank the reviewer for the insightful comment on potential epigenetic changes observed in T cells cultured in hypoxia. We have now carried out an extensive analysis of histone methylation and acetylation (Figure 4H). Human CD8+ T cells cultured for 1 day in 1% and 6 days in 21% showed decreased acetylation of H3K9 and H3K27, reduced trimethylation of H3K4 and H3K27 and increased methylation of H3K9me2, as compared to the levels of cells continuously grown in ambient oxygen. These differences might underpin the altered differentiation and metabolic shifts of 1% cultured T cells and further indicate that the oxygen tensions during the first 24 hours of activation elicit permanent alterations in T cells. Future work will be dedicated to understanding the link between the observed alteration in histone post-translational modifications and T cell function in response to hypoxia.

   Complications may also arise when comparing different oxygen tensions given recent data that suggests standard cell culture conditions can lead to local hypoxia through a combination (https://www.biorxiv.org/content/10.1101/2022.11.29.516437v1) of cellular respiration and poor O2 diffusion. Although it is unclear how this will impact suspension T cells it does beg the question as to whether HIF-1α stability following T cell activation is (at least in part) mediated by pericellular O2 limitations in cell culture over time, even in presumed hyperoxic (21% O2) conditions? Or if T cells subsequently cultured at 21% O2 following short hypoxic conditioning (1% O2) still experience local hypoxia during the 6-day culturing protocol? It would be important to assess this in future work and at least discuss these potential weaknesses.

   Upon analysing HIF-1α accumulation on day 7, we only found substantial HIF levels in cells that had been in low oxygen tensions for the last 3 days of culture (Figure S4G). This suggests that cells were not experiencing hypoxia at the time of analysis on day 7, given that we did not observe substantial HIF accumulation. We have additionally designed an experiment where 21% and 1% 1 day T cells were cultured for 7 days with a single media change on day 4 (standard) or with 5 media changes (each media change performed on separate days to minimize local hypoxia in ambient oxygen). Regardless of the number of media changes, 1% 1d cultures showed increased effector differentiation and expression of effector molecules, relative to 21% cells (Figure S4H). We also did not observe any differences between control cells cultured with 1 or 5 media changes. As hypoxia elicits changes in T cell differentiation, this suggests cells do not experience local hypoxia during the phase of ambient oxygen expansion. Nevertheless, we very much agree that it is important to accurately assess oxygen concentrations in cell culture media.

   Read the original source
3. 

   eLife

   Apr 15, 2023

   eLife assessment

   In the current manuscript, the authors study the effects of hypoxia or genetic and pharmacologic modulation of the hypoxic pathways on T cells. The findings about T cells sense hypoxia and how hypoxia affects T cell (and CAR T cell) differentiation and function are significant and interesting for the field. The data supporting these findings are mostly robust, yet some questions remain open and some statements seem unsupported by evidence.

   Read the original source
4. 

   eLife

   Apr 15, 2023

   Reviewer #1 (Public Review):

   This study represents an important work in the field of (CAR)T-cell immunotherapy by analyzing the effect of different oxygen tension on the function and differentiation of T-cells (especially CD8+). Although it has been described that low oxygen levels can influence effector function/differentiation of T-cells, as nicely acknowledged by the authors in the introduction, a comprehensive analysis in the context of immunotherapy has been missing so far and this study adds significant findings that will be relevant for patient care in all fields applying (CAR)T-cell immunotherapy.

   The strength of the evidence is generally solid although there are some discrepancies between the different ways to induce HIF-1α (i.e. low O2, pharmacological inhibition, shRNA knockdown) that need to be clearly stated and/or discussed.

   Reviewer #1 (Public Review):

   This study represents an important work in the field of (CAR)T-cell immunotherapy by analyzing the effect of different oxygen tension on the function and differentiation of T-cells (especially CD8+). Although it has been described that low oxygen levels can influence effector function/differentiation of T-cells, as nicely acknowledged by the authors in the introduction, a comprehensive analysis in the context of immunotherapy has been missing so far and this study adds significant findings that will be relevant for patient care in all fields applying (CAR)T-cell immunotherapy.

   The strength of the evidence is generally solid although there are some discrepancies between the different ways to induce HIF-1α (i.e. low O2, pharmacological inhibition, shRNA knockdown) that need to be clearly stated and/or discussed.

   1. The first section of the results determines the impact of low oxygen and pharmacological HIF-1α stabilization on CD8+ T-cell activation/differentiation. Low oxygen diminishes cell growth but induces T-cell activation and effector cytokines, while HIF-1a stabilization mimics the effects on activation without alterations in expansion. Unfortunately, it remains unclear why effects upon low O2 are more pronounced although pharmacological HIF-1a stabilization is more efficient.
   2. As a next step, in vitro conditioned T-cells are transferred into a subcutaneous B16-OVA model. Although only the low O2 levels increase T-cell numbers in vivo after the transfer, the initial tumor burden was nicely decreased by both low O2 and HIF-1a stabilization. However, only the latter significantly improved survival and it remains unclear and uncommented why.
   3. Next, the authors address whether pre-conditioning of human CART-cells to induce HIF-1α either by pharmacological stabilization or by silencing of VHL shows similar effects. Surprisingly, both ways of HIF-1a stabilization resulted in different effects concerning differential gene expression and cytotoxic capacity of CART-cells. Accordingly, pharmacologically pre-conditioned CART-cells did not have a significant impact on survival in an in vivo model, while the VHL-silenced ones did significantly improve animal survival. This discrepancy between the two modes of HIF-1a stabilization remains uncommented. Unfortunately, it also remains unclear why the pharmacological HIF-1a stabilization significantly improved the survival in animals of the B16-OVA model and not in the human CART-cell model.
   4. After this, the researchers determine how the timing of hypoxic conditioning affects the (CAR)T-cells. Here it is convincingly shown that already a short period of hypoxic conditioning (1 day) with a subsequent expansion phase (additional 6 days) is sufficient to induce HIF-1a mediated alterations (e.g. metabolic changes, calcium flux, intracellular signaling). Although this section is coherent in itself, the switch between different times of hypoxic conditioning, expansion, and analysis is difficult to follow and might lead to confusion. The expression pattern of e.g. HIF-1a on day 1 and day 7 together with the nuclear amounts of NFAT and c-Myc might be misunderstood, like the other presented data as well.
   5. Last, short-term hypoxic conditioning of CART cells is tested in a solid tumor mouse model. The previously identified conditioning protocol also increases CART-cell function against solid tumors (as shown by enhanced cytotoxicity, reduced tumor burden, and prolonged survival). Unfortunately, although both HER2-CART-cells and CD19-CART-cells are shown to have superior cytotoxicity in vitro after the pre-conditioning, only HER2-CART-cells are demonstrated to be superior upon low O2 conditioning in an in vivo adoptive transfer mouse model and CD19-CART-cells remain an open question.

   Generally spoken, the limitations of the manuscript are:

   1. The occurring discrepancies of determining effects caused by the different modes of Hif-1a stabilization which certainly are caused by the complex nature of Hif-1a regulatory network, and;
   2. The limitation of detected effects primarily on CD8+ T cells while CART-cells products usually are a mixture of CD4+ and CD8+ ones.

   Read the original source
5. 

   eLife

   Apr 15, 2023

   Reviewer #2 (Public Review):

   In this work, Cunha et al provide an insightful and exhaustive analysis of the role of hypoxia and HIF-1a for T cell activation and function. The work contributes to the field by showing that transient hypoxia occurring simultaneously with T cell stimulation (antigen recognition) induces an effector program in T cells that results in increased cytotoxicity in vivo and in mouse models. Importantly, the induction of this effector phenotype is not necessarily linked with an increase in proliferation in vitro, and in vitro differences are mostly observed upon antigen re-challenging.

   The major strengths of the work are the use of different complementary methods to modulate HIF-1a (low oxygen conditions, inhibition of PDH by FG-4592, and deletion of VHL) and the combination of mouse and human models, especially …

   Reviewer #2 (Public Review):

   In this work, Cunha et al provide an insightful and exhaustive analysis of the role of hypoxia and HIF-1a for T cell activation and function. The work contributes to the field by showing that transient hypoxia occurring simultaneously with T cell stimulation (antigen recognition) induces an effector program in T cells that results in increased cytotoxicity in vivo and in mouse models. Importantly, the induction of this effector phenotype is not necessarily linked with an increase in proliferation in vitro, and in vitro differences are mostly observed upon antigen re-challenging.

   The major strengths of the work are the use of different complementary methods to modulate HIF-1a (low oxygen conditions, inhibition of PDH by FG-4592, and deletion of VHL) and the combination of mouse and human models, especially addressing how to implement the findings to the production of CAR-T cells. Besides, the authors not only evaluate T cell function but also dive into the pathways driving the responses observed, which provides mechanistic insight.

   While activation of HIF-1a through the different means mentioned before results in similar signatures in terms of T cell effector phenotype and animal response, there are some aspects that differ between the models. This is probably indicating that low levels of oxygen have other effects beyond the regulation of HIF, and that pharmacological modulation of HIF-1a might not be exactly equivalent to HIF-1a stabilization by real hypoxia.

   The work is useful to better understand the discrepancies in the field, where it has been previously shown that hypoxia can have both a pro-inflammatory effect and an immunosuppressive effect on T cells. The answer proposed by the authors is that it´s a matter of timing, and not so much the magnitude of the HIF-1a response. Despite this being relatively easy to control ex vivo, the challenge occurs when considering the role of hypoxia in vivo, which probably lasts longer than the transient hypoxia needed for beneficial effects on T cells, causing T cell exhaustion.

   From the translational perspective, the study suggests strategies to improve CAR-T cell therapy but also has some limitations. Despite an improvement of cytotoxicity and survival observed in mouse models upon adoptive cell transfer or injection of CAR-T cells with previously increased HIF1a levels, these approaches do not result in curation and survival is still quite low in all groups. Interestingly, improved survival with HER2 CARs exposed ex vivo to low oxygen conditions for 1 day is clear and more promising.

   Read the original source
6. 

   eLife

   Apr 15, 2023

   Reviewer #3 (Public Review):

   In this study, Cunha et al. examined the role of different oxygen tensions (21%, 5%, and 1% O2) and HIF-1α stabilisation in regulating murine and human CD8+ T cell proliferation and function. The authors find that hypoxia (1% O2) and pharmacological PHD inhibition with FG-4592, enhance murine T cell activation but impair proliferation. Furthermore, adoptive cell transfer (ACT) therapy of CD8+ T cells from both conditions reduced tumour burden in a B16-OVA melanoma model. Short hypoxic conditioning (1% O2) of human CD8+ T cells for 1 day increased HIF-1α stabilisation, with increased activation, glycolysis, and mitochondrial function still observed following 6 days of normoxic cell culture. Short hypoxic conditioning of HER2 and CD19 CAR-T cells improved their activation and cytotoxicity in vitro, while HER2 …

   Reviewer #3 (Public Review):

   In this study, Cunha et al. examined the role of different oxygen tensions (21%, 5%, and 1% O2) and HIF-1α stabilisation in regulating murine and human CD8+ T cell proliferation and function. The authors find that hypoxia (1% O2) and pharmacological PHD inhibition with FG-4592, enhance murine T cell activation but impair proliferation. Furthermore, adoptive cell transfer (ACT) therapy of CD8+ T cells from both conditions reduced tumour burden in a B16-OVA melanoma model. Short hypoxic conditioning (1% O2) of human CD8+ T cells for 1 day increased HIF-1α stabilisation, with increased activation, glycolysis, and mitochondrial function still observed following 6 days of normoxic cell culture. Short hypoxic conditioning of HER2 and CD19 CAR-T cells improved their activation and cytotoxicity in vitro, while HER2 CAR-T cell counts were increased in vivo, reducing tumour burden, and increasing survival when compared to 21% O2.

   Strengths:
   The paper convincingly demonstrates that short hypoxic conditioning in a defined window improves CAR-T cell function through in vitro cytotoxicity assays and following adoptive transfer in a preclinical HER2+-SKOV3+ positive tumour model. Thus, the major conclusion of the paper is mostly well supported by the data and could represent a novel strategy to improve CAR-T cell immunotherapy for solid tumours in the future.

   Weaknesses:
   The extent to which hypoxic conditioning-mediated improvement in CAR-T cell function is dependent on HIF-1a-driven metabolic reprogramming is unclear and other potential mechanisms are not explored. 5FG-4592 and VHL silencing in HER2 CAR-T cells did not phenocopy each other faithfully. In addition, neither approach was as effective as short hypoxic conditioning with 1% O2 in improving CAR-T cell function in vitro or in vivo. Although the authors suggest the temporal dynamics of HIF-1α stabilisation is the key point, this is not convincingly proven, and no metabolic characterisation of these CAR-T cells was performed. It is unclear how changes elicited during short hypoxic conditioning are maintained following continued normoxic cell culture. Hypoxia is known to rapidly regulate histone methylation and chromatin structure in a HIF-independent manner (PMID: 30872525; PMID: 30872526). Are similar epigenetic changes observed in T cells, and if so, could these epigenetic changes underlie improved T cell activation?
   Complications may also arise when comparing different oxygen tensions given recent data that suggests standard cell culture conditions can lead to local hypoxia through a combination (https://www.biorxiv.org/content/10.1101/2022.11.29.516437v1) of cellular respiration and poor O2 diffusion. Although it is unclear how this will impact suspension T cells it does beg the question as to whether HIF-1α stability following T cell activation is (at least in part) mediated by pericellular O2 limitations in cell culture over time, even in presumed hyperoxic (21% O2) conditions? Or if T cells subsequently cultured at 21% O2 following short hypoxic conditioning (1% O2) still experience local hypoxia during the 6-day culturing protocol? It would be important to assess this in future work and at least discuss these potential weaknesses.

   Read the original source
7. 

   Version published to 10.1101/2022.11.25.517976v1 on bioRxiv

   Nov 27, 2022