Cytocentric Visionaries: Dr. David Lawrence, University at Albany
Inflammation and Oxidative Stress: Cell Conditions Matter, Even for CAR-T
Dr. David A Lawrence is Professor and Chair of Environmental Health Sciences, at the School of Public Health and the University at Albany in Albany, New York.
We recognize you as a cytocentric visionary because you have done ground breaking work in improving the conditions for cells in vitro and looking at the oxygen levels and how that changes toxicological assay results, particularly in lymphocytes. What made you want to start this?
Well I’ve always been interested in the biochemistry of the immune system and what influences the ability of the cells to respond. I did my immunopathology work at Scripps Clinic and Research Foundation in La Jolla. At that time, Mishell and Dutton had already shown that you had to reduce the oxygen partial pressure to 7% in a culture in order to get the mouse cells to make a good in vitro immune response . That raised our interest because we had some preliminary information that we didn’t see similar sensitivities with human cells. We began to question the difference between mouse and human and how that affects the biochemistry of the lymphocytes and the antigen-presenting cell.
Why were mouse cells so much more sensitive to a higher oxygen partial pressure?
My first graduate student, Randy Noelle, who is now a Professor of Microbiology and Immunology at Dartmouth Medical School, showed that the human cells are more resistant to the higher oxygen level than mouse cells. That’s partially due to the glutathione differences in the cells.
The mice, with less glutathione, were less capable of handling a higher oxygen partial pressure. This, in turn, caused more oxidation of proteins within the cell and on the cell surface, which was a greater inhibitory stress to the activation of the mouse immune cells.
So there’s a connection between their capacity to absorb reactive oxygen species in response to both exposure to supraphysiologic oxygen and other oxidative assault. With these differences between the different lymphocyte subsets, how important do you think it is to control supraphysiologic oxygen for CAR T cells during expansion?
I would think it’s very important because we’re also looking at the microenvironment in which the cells are responding. Some of the things that are probably bringing on cancers are inflammatory states. With inflammation there’s an increase in reactive oxygen species. They go hand and hand.
One of the interesting things about that is not only that there are the differences between B cells, CD8s and CD4s, but also in the activation of the different types of CD4 T cells. We haven’t really studied this carefully in humans, but in mouse studies, we’ve found that the T cells can respond well with an abundance of resting macrophages in - good T-cell activation and proliferation. However, when you put in activated macrophages, small numbers of them will inhibit the ability of the T-cells to become activated and proliferate.
We’ve shown that the critical differences are that activated antigen-presenting cells express less glutathione and expressing less glutathione activates a different type of T cell. So the antigen-presenting cells that activate Th1 cells express more glutathione than the antigen-presenting cells that activate Th2 cells.
And that fits the larger story of oxygen affecting T cell responses.
That’s somewhat logical, if you think about what an antigen-presenting cell is doing differently to activate a Th1 cell versus a Th2 and then what happens thereafter. When a Th1 gets activated and makes gamma interferon to activate macrophages, one of the things that macrophages need to do is to make nitric oxide, oxygen radicals to enhance killing. So if they’re going to be making those things they should have a higher glutathione level to protect themselves.
An antigen-presenting cell that is going to activate a Th2, is activating a Th2 preferentially for B cells to make antibodies and so there’s less need to worry about oxidative stress because that usually leads to more of an anti-inflammatory or antioxidant effect at least before immune complexes activate more inflammation So it makes some logic that activation by an antigen-presenting cell that activates a Th1 to make gamma interferon which is going to feed back and activate to a greater extent the macrophages making reactive oxygen species needs to be more protected and has more glutathione.
So APC would actually need less of their own capacity to detoxify ROS for a Th2 type response?
Right. For a Th2 response, you can see more skewing when antigen-presenting cell is expressing less glutathione. That’s the population that preferentially activates Th2s.
I’m thinking this may be important for not only basic research on what’s happening in vivo between different cells subtypes, but also be important for in vitro applications like antibody production.
Yeah. Most of the monoclonal antibodies are mouse monoclonal antibodies to start with, and you have to lower the oxygen partial pressure to get a good antibody response.
Have you seen much recognition of this in the broader community?
Well it’s been known for a long time. The problem of getting people to accept it is much broader than just immunology. Brain cells may be even more sensitive to the oxidative stresses. Some scientists take the easier route and haven’t really thought much about it.
For an example, as I mentioned in the late 60s, Mishell and Dutton made immunologists doing mouse immunology realize that in order to get good immune responses in vitro you needed to lower the oxygen partial pressure. But then in the 80s we found out that you can put in β-mercaptoethanol (BME) in the culture media, which helps. However, a lot of people assume what’s good for the mouse is good for humans. The problem there is activation of lymphocytes is a low level oxidative stress in and of itself, and human lymphocytes are less sensitive to oxygen partial pressure. Their glutathione level is already higher than in mouse lymphocytes.
So if you put BME in culture media with human lymphocytes, they don’t get activated as well because that is too much in reducing equivalents. They already have good glutathione levels and the ability to handle higher oxygen partial pressure in vitro.
So how much of our research today is based upon cell conditions that introduce artifact?
If you’re studying the role of drugs or toxicants, you need to keep the overall physical parameters as close to the natural physiology of the cell type that you are using. A substantial amount of my mouse research was looking at the effects of lead on the immune system. When you put lead in culture, that in and of itself skews the types of CD4 T cells to a Th2 response.
If you’re doing it at a higher oxygen partial pressure plus lead, you’ll get less of an immune response. Combinations of these things can be inhibitory, so if we put in antibiotics or drugs, any modifier, we have to consider the possibility that the compounds that you’re studying themselves are modified to a greater extent because they’re now in 20% oxygen.
Most of the cells in our body are not at atmospheric oxygen. So if you are interested in the immune system, that’s especially important because it’s a system that is there in part to make reactive oxygen species and nitrogen species for protecting us against pathogens. It utilizes oxygen to kill off pathogens.
The oxygen partial pressure is important to how cells in vitro responding to cancers, different cell types, activation of the T cells, activation of the phagocytic cells for killing. It’s all critical. In many pathologies, inflammation plays a critical role as well. In my mind inflammation and oxidative stress pretty much goes hand in hand.
Researchers have to think about so many factors for cell culture: serum lots, animal products, micro contaminations, reproducibility, cell line identity. How do you respond to people that just feel like they don’t want to have to think about oxygen too?
Certainly our human immune system is not bathed in fetal bovine proteins. But for me, oxygen levels are at the top of the list of things to control.
I guess it’s easier for people like me because when I was learning in the early 1970’s people made it clear to me that the in vitro conditions that you use should be as close to in vivo as possible. When I read the Mishell and Dutton paper, needing a lower oxygen partial pressure to get an immune response made a lot of sense. So I bought into it very early on.
Now we have been spending a lot of time trying to investigate the mechanisms involved in these processes. Keeping the oxygen partial pressures close to physiological is important if we are trying to understand how cells communicate in vivo.
That makes a lot of sense. So how about the practical considerations? How important is it for people to control conditions during cell handling?
Well it’s probably important. It’s probably a little less important than keeping them under high oxygen partial pressure for everything.
In trying to look at neuronal connectivity by SPR analyses, we grow brain regions on a chip in a low oxygen incubator but then have to take them out and bring them over to our SPR instrument. And that has been a failure. That has not worked for us at all.
My feeling is it’s probably because brain cells are even more sensitive to oxidative stresses than the immune system cells. We’ve been trying to do this with mouse brain. And we already knew that rat and human lymphocytes are probably more resistant to high oxygen than mouse. There’s not been much success at all with mouse cultures. I think that’s probably because of the oxygen partial pressure.
So what else do you want people to know about your work?
The redox chemistry of cells plays a major role in their functional reactivity and it’s very dependent upon the oxygen partial pressure. That’s the bottom line. And that there are differences, major differences, between different cell types.
Indeed. Well, thank you very much for your time, your work, and your perspectives, Dr. Lawrence. We look forward to seeing what you do in the future.
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 R.I. Mishell, R.W. Dutton, IMMUNIZATION OF DISSOCIATED SPLEEN CELL CULTURES FROM NORMAL MICE, The Journal of experimental medicine, 126 (1967) 423-442.
About the Author
Alicia D Henn, PhD, MBA
Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.