Cytocentric Visionaries: Romain Vuillefroy de Silly
Part Two: What’s in a Word? If It Describes the Oxygen Level, Quite a Bit.
Dr. Romain Vuillefroy de Silly is a post-doc at the Ludwig Center for Cancer Research of the UNIL in Lausanne, Switzerland. He has published original research papers and several extensive reviews on the role of oxygen in T cell function. This is Part Two of a Two-part interview that Dr. Alicia Henn, Chief Scientific Officer of BioSpherix, conducted with him about his most recent review in Oncoimmunology.  This interview was edited for length.
Part One of our interview can be found here: Is Physiologic Oxygen be Better for CAR-T Cells In Vitro?
Alicia Henn: How do you feel about the newer terms “physioxia” and “physioxic” to describe physiologically relevant oxygen levels, rather than “hypoxia”, which can be either normal or pathologic?
Romain Vuillefroy de Silly: Nowadays, “normoxia” refers to the atmospheric oxygen tension that we routinely use in our cell incubators, while “hypoxia” can refer to either oxygen in physiological conditions (that are lower than in the atmosphere), or to pathologically low oxygen fractions found in the body. Recently, “physioxia” has been added in order to replace “hypoxia” when referring to physiological oxygen fractions.
Indeed, talking about hypoxia is not correct when referring to a physiological and non-pathological oxygen fraction, since hypoxia is a pathological state characterized by a deprivation of adequate oxygen supply.
I still do not feel comfortable using physioxia, as it means that we intentionally fail to recall what the term normoxia meant. Moreover, it adds complexity to the field (normoxia, physioxia, hypoxia) for non-specialist readers. That is why I believe that normoxia and hypoxia should be sufficient.
By definition, normoxia is adequate oxygen supply, while hypoxia refers to a pathological condition characterized by oxygen deprivation.
From my point of view, “adequate” refers to a physiological situation where oxygenation is as observed in vivo in non-pathological conditions. The oxygen content is neither below nor above physiological.
Normoxia should never refer to the atmospheric value of oxygen (i.e. 21%), which is wrong. I consider atmospheric oxygen fraction as “hyperoxic”.
I really think that the research community in this field should reach a consensus for these terms since it could lead to misinterpretations and unnecessary complexity.
AH: You are my hero for stating that comparisons of 1% to 21% oxygen are not physiologically relevant. Do you think that the tools available might be driving the experiments, rather than the real questions we need answered?
RV: Thank you very much! This concept is of special importance to me. I am not the first one stating that the 1% vs 21% comparison is not scientifically relevant to study hypoxia. Earlier papers from two main groups (Herzenberg and Kieda labs) lend credit to this concept.
Unfortunately, most of the hypoxia studies are using these conditions, leading me to wonder whether the effect they describe is the "steady state" physiological behavior of the cells instead of their response to hypoxia.
I am not sure that this common comparison is entirely the result of the available tools. From my point of view there are four main reasons.
First, working on hypoxia presents practical problems if it is to be scientifically rigorous. One needs to precondition buffers and media so the experiment starts with the expected oxygen fraction. Further, passaging cells needs to be done in a controlled atmosphere (workstation) so that cells do not get exposed to room air oxygen. These conditions (even if they are not always satisfied) considerably expand experimentation time and can be awkward. It is easier to compare 1% oxygen to room air to limit these practical issues to one condition (1%), since the room air is under the usual conditions. It renders experiments easier than if there was a physiological normoxic point.
Second, 1% to 21% oxygen means a twenty fold difference in oxygen presence, thus increasing the chance to observe an impact on the cell subtype studied. Since studies are mostly, but unfortunately, accepted for publication when showing differences, researchers will rather privilege this comparison instead of another, which leads to improved probability to see a difference.
Third, when we want to analyze something new, we follow the methodology of recognized publications, since they were validated by the scientific community. Since most, if not all, publications used 1% vs 21%, they follow.
Finally, since the experts in the hypoxia field did most of their work using this comparison, using another one could invalidate their previous findings. Therefore, it seems possible that they would not be prone to change the comparison parameters. Further, it is tempting to speculate that they might not be impartial while reviewing the work of other groups that tried to change the comparison concept. We actually experienced this when receiving reviews for our research paper, which ended with a rejection at the first round without any possibility to make improvements to our manuscript or to answer reviewers.
AH: Hal Broxmeyer’s group has published interesting data about HSC isolation in physioxia.  They found that 3% O2 was the critical level that affected HSC yields from bone marrow and cord blood. How important is it to consider the “oxygen history” of a cell for in vitro experiments?
RV: This is an interesting question that warrants to be investigated. I imagine it might be cell-and context- dependent. For example, we observed that when naive CD8+ T cells were primed in vitro at 5% O2 and reactivated at 21%, these cells secreted more IFNg as compared to those primed and reactivated under 21%.
These results would be in favor of an "oxygen history" hypothesis. However, this might not be the case for other cell types. Also, it is likely that, in this example, oxygen could have modified the TCR signaling and/or cytokine signaling in a non-specific manner. Therefore, whether it is an example of an "oxygen-specific history" is not clear to me.
AH: Can you take cells from a room air incubator and put them at 5% O2 and start your experiments right away as if the cells have been in vivo the whole time?
RV: I would rather adapt cells to 5% O2 before carrying out the experiment. From my point of view, the acute response to 5% O2 should not be monitored since it would still represent a "shock" for the cells to pass from a 21% atmosphere where they adapted to 5% O2. However, I cannot exclude that culturing cells at 21% modified them irreversibly.
AH: Do cells need to be in continuous physiologic oxygen from the isolation from the blood all the way through the experiments?
RV: Unfortunately, I cannot answer this question since I always carried out splenocyte extraction under atmospheric oxygen levels (before culture at 5% O2) and did not compare it to the same procedure at 5% O2, even if this would be the most scientifically rigorous methodology.
Thank you, Dr. Vuillefroy de Silly for your time and your expertise. We look forward to seeing what you do in the future!
1. Vuillefroy de Silly, R., P.Y. Dietrich, and P.R. Walker, Hypoxia and antitumor CD8+ T cells: An incompatible alliance? Oncoimmunology, 2016. 5(12): p. e1232236.
2. Mantel, C.R., et al., Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock. Cell, 2015. 161(7): p. 1553-65.