Webinar

14 Ways to Measure Immune Cell Activation

Enjoy your complimentary webinar recording. Bookmark this URL to access the recording at any time.

Full Webinar Transcript

Kristen Haberthur
[00:00:13]

Hello, this is Kristen Haberthur, welcoming you to another Bitesize Bio webinar, which today is sponsored by Astarte Biologics. At Astarte Biologics, our mission is to help you get on with discovery. Whether you need extra help in the lab with custom research services and assay development, or are searching for hard-to-find immune cells and reagents, we can help you overcome your research roadblocks. If you’re looking for immune cell products, we’ve got you covered. Our extensive online inventory makes it easy to find the exact cells and donors you need for your experiments. From B cells to T cells and PBMC to stem cells, we have a variety of products from healthy and disease state donors, including antigen-specific T cells you can’t find anywhere else. Find it all Astarte Biologics.

So today’s presentation is titled 14 Ways to Measure Immune Cell Activation and it’s being presented by Anne Lodge, CEO of Astarte Biologics, a supplier of high-quality immune cells, reagents, and research services. She holds a Ph.D. in Cell and Molecular Biology from the University of Vermont and has an extensive background in cell-based therapeutics and immunology, including multiple patents.

As always, we will have a question and answer session after the presentation, so please type any questions that you have into the questions box, which appears to the right hand side of your screen and I’ll put them to Anne at the end. The recording of the webinar is going to be available at http://bit.ly/immunecellactivation. And again, it’s all one word, lower case, immunecellactivation. And I’ll be sure to add this to the chat section of the box to your right.

So, now, over to you, Anne, for the presentation.

Anne Lodge
[00:02:05]

Thank you for that introduction. And welcome to the webinar. A pleasure to speak with you all about immune cells and different assays we can use to measure immune cell activation. And, of course, the first question is, why would you want to measure immune cell activation? And the obvious one is immuno-oncology has become a very hot area of research. It’s bringing new treatments to bear on a variety of cancers. And the excitement in this area is spilling over to vaccine development, treatments for autoimmune and infectious disease as well.

The immune cells that we’ll be talking about today are primarily T cells, both CD4+ T cells that are usually helper T cells, and the CD8+ cytotoxic T cells. We’ll touch a little bit maybe on B cells, natural killer cells, and the myeloid cells. But less so, because I think with the focus these days is much more on the T cells. And we’ll leave the other players to another day.

[00:03:20]

To put all of those players in context, I borrowed this slide from a group called Geeky Medics. And I encourage you to check out their website. They’ve done a lovely job here of pulling together a number of different cell types to illustrate that they’ve been good about keeping a color coding intact. So if you have a pathogen, a virus, bacterium, what have you, these little red bits are kind of traced through the rest of the diagram quite nicely. The first interaction is with dendritic cells. There’s also activation of NK cells in many cases and neutrophils.

The dendritic cells are specialized in activating the adaptive arm of the immune response, presenting these little bits of the pathogen to helper T cells and also CD8 cells that are shown here with the unhappy antigen-presenting cells and getting both of these cells activated. And they further mature in the case of CD8s becoming killer cells. And in the case of CD4 helper cells, providing cytokines and other signals to B cells so that they can go on and make antibodies. And you can also see in the yellow boxes all of these soluble factors that are secreted. So there’s an awful lot going on. And biotechnology has brought us the ability to monitor a whole lot more than we ever used to. It’s helpful to kind of zero in on what might be great for screening or getting that initial handle on activation.

[00:05:22]

So this slide just kind of reiterates what’s in these previous one, that there’s an awful lot going on. There is a need for engagement of the antigen receptor, whether it’s a T or a B cell, a co-stimulatory receptor, and cytokines to support the growth of those cells. There’s a whole lot of genes upregulated and distinct phenotypes are developed as a result of the encounter with the antigen.

We’ll be talking about three categories of assays to measure immune cell activation. Proliferation is the first one and probably the most commonly used and we’ll spend a lot of time on that. Cytokine measurement is another great tool. Surface antigen expression is another option and can be useful and combined with other methods. And finally, cytotoxicity, which is often the endpoint that we want to get to when we’re doing therapeutics, interventions… is, “Hey, we want to have those cells either cytotoxic or not cytotoxic.” And so that’s a nice readout to be able to deploy.

[00:06:34]

So, first section is proliferation assays. And the choices that we have here are the traditional tritiated thymidine incorporation. The next generation bromodeoxyuridine incorporation. Tetrazolium dyes of various sorts and colors. CFSE dye reduction and CellTiter-Glo® are a measurement of ATP.

Tritiated thymidine is the use of radiolabeled thymidine, and that’s incorporated into the DNA as it’s replicated. So you can imagine all of the Ts here that designate thymidines becoming radioactive. And there’s lots of points to label in there. And you can then detect that using liquid scintillation. Since most immune cells aren’t dividing, they’re stuck in G0, G1 of the cell cycle. Any uptake of thymidine is an indication of proliferation and you have a good signal-to-noise ratio since there’s not any uptake to begin with.

This is an example of what that data can look like. In this case using mouse cells in a mixed lymphocyte reaction. Which is basically an in vitro measure of graft rejection. So if you mix cells from two unrelated individuals, it’s like a transplanted organ being rejected. And those T cells are going crazy because of those genetic differences. So down here in the bottom in green is the background negative control using spleen cells from a C57 black six mouse. If those are stimulated with cells from a BALB/c mouse, shown in orange here or in blue, even better here, you can see that there’s a good increase over that background level of thymidine from say around 10,000 and on these particular days up around 60,000 counts per minute. So, you know, six-fold increase quite believable.

It does, as you’ll notice, decline over time. And that’s one of the features of this type of assay is that you need to choose your time point because you’re adding thymidine for 18 hours and then looking at what’s incorporated into the cell. You want to get it during that time when assay is underway. And that isn’t going to come to an end, as shown here. You know, by day seven the action’s over. So an experiment like this is suggestive to, you know, within each laboratory, find the data that works best under your conditions.

The problem with this particular method is that tritium is radioactive and there’s been a lot of movement away from radioactive methods and finding other means of readout. And you need an instrument for detection of that signal. And having a devoted instrument just for that may not be desirable.

[00:10:22]

So kind of the next generation was the use of bromodeoxyuridine. If you look in here on the right hand side of this slide is the structure of thymidine with a little Bromine tagged in there. That is enough to generate monoclonal antibodies to that bromine derivative. And at the same time, it’s taken up and incorporated into replicating DNA the same way the tritiated version is. So you can then detect this after your labeling period either in an ELISA format or by fluorescent labeling.

So the ELISA outline we’re probably all familiar with. But in this case you’re adding bromodeoxyuridine to the cell culture overnight, usually. After that labeling period you remove the culture medium and fix the cells to adhere them to the bottom of the plate and also to permeabilize them to allow that antibody to penetrate into the nucleus where the label obviously is. You then add your bromodeoxyuridine and then usually a detection antibody labeled with peroxidase or your label of choice.

And you’ll get data that looks something like this. This is using human cells in this case rather than the mouse. And you have a measure of absorbance since it’s a colorimetric assay to indicate the number of cells that may have incorporated the bromodeoxyuridine. The control is relatively low in the presence of tetanus toxoid, which most people are immunized against, you can see that there’s an increase in the absorbance from point two up to about point six. Likewise, with PHA, this stimulates all T cells. So you’ll see an increase there. Lipopolysaccharide will actually stimulate B cell division and we’re picking up a little bit of that, although not as dramatic. And cytomegalovirus, many people are exposed to CMV or are serologically positive, and that will generate also a strong response.

Just another example here, again, showing the kinetics of the S-phase developing. You can see in the absence of any bromodeoxyuridine, there’s very little signal as in the absence of antigen, but the presence of bromodeoxyuridine still very low. And each colored bar represents a different day for labeling. And you can see that in the presence of this CMV antigen from day three increasing to day four, five, and six. But that clearly varies depending on the concentration of antigen and may even have some variation from lot to lot where you can see that this one definitely had its peak uptake of the label on day five and dropping off. So things that you need to maybe be aware of as you set up your experiments.

[00:13:58]

The other way to detect this that’s really distinct from something like titriated thymidine is that you can add your bromodeoxyuridine, incubate overnight. You could then collect the cells and stain for surface antigens, if you like, then fix and permeabilize and add a fluorescently labeled anti-bromodeoxyuridine. And then measure your proliferation using flow cytometry. And this gives you the advantage of seeing exactly what cells were incorporating the label during the experiment.

And this is some sample data, not of ours, but borrowed from somebody else where they were measuring not surface antigen but bromodeoxyuridine content and comparing to DNA content. Usually if you looked at this you’d say this is your normal complement of chromosomes and this is just before mitosis, when you have cells that have twice the number of  — twice the amount of DNA. And you can see that the bromodeoxyuridine is being incorporated and in between the two major populations here is where that S-phase is occurring. And there it is detected by the increase in bromodeoxyuridine.

There are some drawbacks to this method. I mean, it gets you away from the radioactivity, but you do need to fix and permeabilize the cells. And this can be, particularly in the plate-based assay, what we found is that it can take time. You really need to monitor that fixation and make sure it’s complete. And also because the cells are not normally adherent, they’re more of a suspension culture, you can lose some of your signal. Or you can get fixation where pieces of the cell pellet or cell layer will peel off and that will lose signal for you. It also takes time to develop since it is an ELISA type assay. Flow analysis can work better than doing that plate-based, but you do need the capability of doing flow analysis.

So these and other methods that are based on detecting S-phase have great signal-to-noise ratio and the tritiated thymidine read out in particular is very familiar to old immunologists like me. The downside is the use of radioactivity or in the case of bromodeoxyuridine, that need for fixation and permeabilization can be cumbersome.

[00:17:04]

So one of the other methods that has been tried is the use of tetrazolium dye. These are the things like MTT or XTT you may have seen in other publications, and they rely on the reduction of a substrate to a colored product by mitochondrial enzymes. With increased metabolic activity or increased cell numbers, you’re going to get increased conversion of the dye. But none of these are sensitive enough for what I would call a primary in vitro immune stimulation. You have to remember that there are only a few cells in a primary sample, whether from human or from mouse, only the small fraction of cells are actually responding to any given antigen.

So you’re looking for a signal in a whole lot of noise. With MTT or XTT or any variations on that, usually there is a large background because all of those cells are still alive, they’re just not proliferating. And when you have maybe 200,000 cells and you’re looking a few thousand to increase, you don’t get enough of a signal above that background. So I don’t have a good example data to show you for this particular method. So we’ll move on to the next one.

[00:18:44]

The dye reduction methods. This has become very popular in immunology circles where if you use a esterified fluorescent label, carboxyflourescein diacetate succinimidyll ester, or CFSE, which it’s usually called, and related compounds, will label surface proteins on cells. And with each cell’s division, the individual cell fluorescence is reduced by half. And this makes for a good readout as long as you’re, again, capable of running some flow cytometry.

We have this procedure on our website, but it’s summarized here. You basically prepare a cell suspension, add your fluorescent label, and allow that to link up with surface proteins, wash off any excess, and then culture those cells with whatever antigen or stimulus you want to provide. And we usually allow those cultures to run for anywhere between four and seven days, depending on the stimulus. After that you can collect the cells, counterstain with other cell markers or viability dyes. The viability dyes I would highly recommend. And then you can do some very elegant analyses depending on how capable you are with flow cytometry and how many colors are available to you.

So this is just a diagram showing the idea that you have an unlabeled cell that gets labeled with a fluorescent dye indicated by the blue dots. And that’s reduced with each cell division to fewer and fewer labels. And on a histogram plot of the number of cells versus fluorescence, you’ll start with the very bright cells and each generation will give you a separate peak. And there’s some very good software tools to help analyze this kind of data.

And here’s what that looks like in real life, not diagrammed out. In the top left here we have a control which is just cells that were labeled and allowed to incubate. There is some degree of background proliferation in this case. Not always as much as is shown here, but we can see that there’s sort of a trailing of cells that are dimmer than the original starting population. On this axis on the y is CD4 staining. So namely the CD4+ cells were proliferating in this case. When they’re cultured with tetanus toxoid, again, everybody’s immunized pretty much, you see more cells that have moved in that direction. Some of which are CD4, but some that are not CD4+, and these may well be B cells because they would encounter that antigen and also proliferate.

Cytomegalovirus is another example. It is a very strong stimulus to the immune system if you’ve been infected with it previously. And for variety, PHA, which stimulates all T cells. And we found, and others as well, have found that this is when you will see the characteristic multiple peaks. I think in the case of an antigen, everybody’s kind of synchronized and they just all move as a group. With things like PHA or Anti-CD3, they kind of go in cohorts. And so you have the cells that haven’t divided here, the next generation, and the next and the next, and the next. You can just barely make out that one. So they’re not synchronized, but kind of march off in cohorts, giving that characteristic peaks and allowing it to measure how far out each generation shifts.

We’ve also used this method to measure T regulatory cells, another one of those protocols that’s on our website under the Protocols page. When you take T cells and wiggle them and stimulate with Anti-CD3, you still have that remaining little peak of the original fluorescent level, but quite a bit of shift. And you can perhaps make out these green traces that are the software working out how many generations that’s gone on. When you add T regulatory cells, you can see that you have many more cells that are back here at that starting point. Still some proliferating, but far less than the control.

And with increasing numbers of T regulatory cells, you can push that proliferation just about to nothing. As a control, we included a different cell line that’s not a T regulatory and you can see that these histograms all look pretty much like the control, so nice to be able to trace that activity in vitro and be able to focus in on the cells that are being suppressed and screen out the ones that are providing the suppression.

So this is a really helpful technique because you can combine it with surface antigens. I didn’t show you a whole lot of colors there, but you can imagine you can tag all kinds of different receptors or cytokines — anything you can imagine that you could combine in. And you can also appreciate the number of cell divisions that are taking place. If you were only measuring, say S-phase, you wouldn’t necessarily see the effect of those T suppressor cells quite as clearly.

The downside is that it will take more cells than other methods because you need to have enough to run through the cytometer and get valid numbers. And at least in our situation, it’s not very high throughput. Maybe with other cytometers and kind of the latest and greatest instrumentation, sure. But it doesn’t tend to be a walkaway assay.

[00:25:28]

CellTiter-Glo measures the number of cells based on ATP content. It uses a firefly luciferase from Promega. It has a longer half-life so that you can measure that luminescence before it goes away and it is directly related to the number of cells present. It’s very sensitive to the cell number, so you don’t even need that many.

As illustrated in this slide, it has the same problem as the MTT and other tetrazolium dye methods in that it’s still hampered by the fact that you have a lot of primary response. You have a lot of cells that have ATP that are going to give you signal. So seeing the increase in cell number is still challenging and we haven’t been convinced by the increases, probably because we’re spoiled by our tritiated thymidine days.

So what you see in this slide is a primary immune response and different days — one, two, three, in this case. You can see some increase in the tetanus toxoid over control, but boy, it’s tough. You definitely see the increase in luminescence with PHA, if you’re stimulating everything in that case. Again, not very much to be seen in the presence of LPS or CMV. In some experiments you can see an increase over the control, but it’s just hard to convince yourself that that’s real, so to speak.

Where it is useful, and perhaps the MTT dyes are also useful in this case, is with purified antigen-specific cells as shown in this slide. We have the anti-tetanus T cells stimulated with increasing concentrations of the peptide they’re specific for. And we see an increase from no antigen to even a 30 ng/mL of peptide and continuing on. It’s a very quick method. It’s more sensitive than the tetrazolium dyes and certainly has its place.

[00:28:53]

Cytokine measurement is another very useful basket of methods because cytokines are produced in abundance following immune cell stimulation. You can either collect bulk medium from a culture after allowing time for stimulation to take place and measure either a single cytokine or now, with advances in technology, we can measure multiple cytokines in a relatively small volume of sample. We just need to keep in mind timing of that cytokine release to get the best data. The other method is the use of intracellular cytokine staining, which allows, again, as we saw with CFSE or bromodeoxyuridine staining, you can identify the cell population that’s involved in the response and quantitate that to some degree.

So here’s some sample data, again, from the antigen-specific T cells that respond to tetanus toxoid. So the cells were stimulated with antigen-pulsed dendritic cells and we measured 10 cytokines and the different colored bars indicate different concentrations of the antigen. What you notice right off, of course, is large output of interferon gamma. And this cell line also makes some IL-10 and IL-13. There’s a tiny bit of IL-5 and TNF alpha. Again, these are a bit swamped by the fact that you’ve got 8,000 picograms of interferon gamma.

There’s also an increase in IL-8 production. But what you’ll notice with IL-8 is that there’s 1,000 picograms to begin with, so this is something that has the highest background of any of these 10. And you’ll notice that these are just plain absent. IL-2, even though it’s thought of as being something that would be made, it tends to be consumed as fast as it’s made, so it’s not usually detected in any great quantity.

This is just similar data, not using a cell line but using peripheral blood mononuclear cells, PBMC, and looking at the kinetics of cytokine production over the first three days in culture. Again, the same 10 cytokines were looked at and we used either cytomegalovirus antigen or tetanus toxoid. And just to highlight, again, you’ll see that interferon gamma is made in abundance. There’s a little difference between the two antigen stimuli. You can see that there is IL-13 in response to tetanus toxoid and we can pick up some IL-2 there. Lots and lots of IL-8 and TNF alpha at lower levels assuming both our cases. So the difference here in picking up IL-2 would be the fact that these are PBMC, but it’s still sort of interesting that we can pick that up using tetanus toxoid, but we can’t really see it using CMV.

[00:32:37]

Another way to look at cytokine production is by intracellular cytokine staining. And this is an outline of the method used for the data I’m about to show where we used antigen-presenting cells incubated with peptide overnight. T cells were added to those and then that co-culture was continued overnight. For the last four hours of culture, Brefeldin A was added to just prevent secretion of cytokines. The cells were then collected from culture. The dead cells were stained to exclude them from analysis and CD8 was used to look for that particular cell type. And then the whole prep was fixed and stained for the presence of interferon gamma. And half a percent saponin is used to permeabilize the cells during that staining step so that the antibody can gain access to the inside of the cell.

It was kind of a neat experiment. The red histograms here are  co-cultures with an irrelevant peptide included and the black histograms were with the peptide that the T cells actually recognized. Using the CD8 we were able to analyze the antigen-specific T cells specifically and show that the fluorescence had shifted to the right when stained with interferon gamma. If you do the same thing and ignore the T cells but focus on the antigen-presenting cells in the culture, you see that the background fluorescence and the fluorescence in the presence of relevant peptide are completely overlapping, showing that there was no specific interferon gamma signal in those cells and demonstrating for colleagues that, “No, these are the cells that are making interferon gamma in the culture.”

So the nice thing about cytokine assays is that they are a sensitive measure, both for primary cell assays and purified antigen-specific cultures. There is a need for an initial workup to understand the best timing when your cytokine might be made and allowing best detection. And unless you do the intracellular staining, you can’t really identify which cells are producing the cytokine. So I kind of glossed over it a bit there, but you can imagine in a culture of PBMC, monocytes will produce a fair amount of cytokines such as TNF alpha or IL-6 and that may complicate analysis or interpretation of your data.

[00:35:40]

So in the time remaining, I have a couple more areas to touch on. One of those is activation markers. You can look at the expression of surface antigens that are upregulated upon activation. And what are commonly used is CD69, CD25, which is the IL-2 receptor. CD44 is also used, as is KI67, which is a nuclear antigen. The surface antigens, I would say, are easier to work with than something like KI67, but since KI67 is used across cell biology, we need to consider it.

You can combine any of these with cell surface markers to give detail into what is participating, whether it’s B cell, T cell, CD4 or CD8, memory, naïve — whatever population you want to kind of tease out, you can do that. I don’t have any good data to show you, but trust me, you would have some of the same issues I think as with any of the methods determining what the best time is for capturing that data and combining your markers in an effective way to pick out the relevant population.

[00:37:09]

The last method I want to touch on is cytotoxicity. As I mentioned, this is a nice readout to have in your back pocket. It’s how do we measure the ability of natural killer cells or T cells to kill a target cells. Whether that’s tumor cells or cells that might be infected with a virus or bacterium. Traditional methods label target cells, both Chromium 51, and that is reduced on entry into the cell, retaining within the cell and reducing the leak back out into the culture medium. So when those target cells are then washed, you can measure release of the label upon lysis. But again, that’s the good old days of radioactivity and not many of us really want to deal with radiolabels.

The non-radioactive methods that are available tend to have a higher background. There are some fluorescent labels that can be used or even intracellular proteins or enzymes that have been used such as LDH, lactate dehydrogenase. But these tend to have higher backgrounds and they don’t allow distinguishing signal that might be coming from the target cell versus from the cytotoxic T cells in the culture. And there is a need for better methods.

[00:38:48]

One that we’ve had success with, we took the K562 cell line, which is a target cell used to measure NK activity. The K562 cells lack major histocompatibility complex antigens, MHC antigens on the cell surface, so there are readily lysed by NK cells. We transfected those with luciferase, and you can use those to measure release of luciferase into the medium as the cells lyse. So you collect the culture supranate and you measure that for luminescence, or you can look at the loss of luciferase. This example over here on the right is showing the release of luciferase into the medium with higher release when more NK cells are added. And targeted alone, there’s not much released at all.

[00:39:58]

The other option, if you don’t want to go through making a stable cell line, is to label your targets with CFSE. As we talked about earlier, that’s going to give you a nice, bright label on the cell surface. You can incubate with those with NK cells and we found that this method worked best if you increase your incubation time to overnight. And then you can label cells with a viability dye, 7-AAD, to detect dead cells. So then you’re going to be looking for labeled target cells that are now taking up the dead cell dye. And that’s illustrated up here. These are target cells alone on the y-axis is your CSFE, so they’re all up here. And the NK cells would be shown down here, but we’re not even showing them, it just makes analysis easier. 7-AAD on the x-axis gives a little bit of background depth on the uptake of the label.

As you add in NK cells, you see an increase in the number of dead cells present and that continues to increase as the ratio of the NK cells-to-target cells increases. So this is a helpful technique that gives you great confidence that you’re truly reading out the death of the relevant cell population. It doesn’t require flow cytometry. Again, means that the throughput is not that great. There are other methods coming online that measure cytolysis using luminescent labels or even non-labels, but detecting release of antigen or release of internal proteins. And these may allow better readout of this important function of T cells.

[00:42:06]

So in conclusion, there are many ways to measure immunity, and selecting a method really depends on what you have available for instrumentation, how many samples you plan on analyzing, whether you’re measuring a primary recall response or a polyclonal stimulation of every T cell that’s there, one’s going to take a much greater signal-to-noise ratio. And you need to identify the cells involved. If you’re going to need to identify which cell is doing what, then you’re probably going to need to access a flow cytometer. In any case, almost any method is going to require some initial workup to understand in your particular laboratory, with your culture media and cells, at what point is the ideal time point to measure?

And with that, I will open up the section to questions and I thank you all for taking the time to call in and listen.

[00:43:32]

Kristen Haberthur

Thank you for that, Anne. That was a really excellent presentation. We actually have a few questions from the audience. And if anyone else has a question while we’re going through this section, please feel free to post it in the questions box that appears to the right of your screen.

So, Anne, our first question comes from Jay. And they ask, “Are there in vitro assays that incorporate the innate and adaptive immune response?

Anne Lodge

That look at both innate and adaptive-

Kristen Haberthur

Yeah, I think that’s what they mean, is if both at the same time.

Anne Lodge

Well, you know, you can utilize cytokine secretion in that way, I suppose. Because you would have activation of those adaptive cells to present antigen to — activation of the innate that then go on to stimulate the adaptive portion and you could measure that with either intracellular cytokines or cytokine in bulk. So that might be a way to go for that.

Kristen Haberthur

I agree. And then they also ask, “Does interferon gamma measure both CD4 and CD8 activation?

Anne Lodge

Yes. At least in my experience it does. So the intracellular cytokine staining I showed was using CD8+ T cells, but the bulk was using a tetanus toxoid-specific T cell line that is CD4+. So either cell type can secrete interferon gamma.

[00:45:16]

Kristen Haberthur

Alright, great. Lolita asks, “Are there any ways to identify exhausted T cells?”

Anne Lodge

That’s an excellent question.

Kristen Haberthur

I thought you’d like that one.

Anne Lodge

It’s an excellent question because certainly you kind of want to identify them, but they’re exhausted, so they’re really not doing much. You can’t measure activity. But there are some — it’s one of those things that we’ve been pondering here. Is there a way that we could develop an exhausted cell that we could look at? PD-1 expression is the first one that comes to mind. So if you can stain for PD-1, that would probably label either recently activated or exhausted T cells.

Kristen Haberthur

Would you ever stain it in conjunction with CD69?

Anne Lodge

Yeah, that might be a useful way to go. Because I would expect the recently activated would express both 69 and PD-1. Because it’s normally upregulated immediately after antigen encounter. But with the exhausted cells, they just continue to express PD-1.

Kristen Haberthur

That makes complete sense. Jay is back, they have another question. “Do you have a recommendation for a good review on markers of T cell activation?”

Anne Lodge

You know, I don’t really have a good review in mind, because you can kind of —it doesn’t take much of a look through the literature to find papers that use CD69 or CD25. You have to go back further I think to see the use of KI67.

Kristen Haberthur

Are there any ones — I just remember when I was in school, I really had a penchant for nature immunology reviews. But that’s usually a pretty strong go-to.

Anne Lodge

It is a strong go-to to search through that. Thank God for the internet, it’s easier to find that now. Just showing my age. But I know that I read something recently on a — people were proposing a new one. And boy, if you see that, you see somebody touting their new marker that might be even better, they naturally have reviewed other methods and you can kind of dig back into that.

[00:48:07]

Kristen Haberthur

I’ve got a lot of questions here, this is wonderful. So the next question is, “What do you think are the best or key flow markers or cytokines for T cells that are the most universal?”

Anne Lodge

You know, I go for interferon gamma just because I’ve been watching that thing pile up in cells or in culture media. That’s a great one to measure. It’s not as easy as, say, slapping CD69 on there, but boy, close to universal.

Kristen Haberthur

If you were running a flow panel, what would be your standard marker setup?

Anne Lodge

So it depends on if you want to go basic or you have lots of colors available. I’ve seen some proposed panels for teasing out, you know, central memory versus effector memory, all of those that are really helpful in understanding primary cells. We do pretty basic immuno phenotyping of our cells, which for PBMC is CD3 and 19 to pick out the Ts and Bs, CD14 to label the monocytes, and then CD16 and CD56 to get the NKs. That leaves a lot to the imagination as to what else is in there.

But, you know, every once and a while we will run CD4 and CD8 just to see what the ratios are because there are people who have different ratios of fours and eights. And it is interesting to dig in a little bit further and get those memory versus naïve, central versus effector. And so if you have the colors available to you and you’re clever enough to put them together right, you can have a really interesting panel.

[00:50:27]

Kristen Haberthur

Definitely. Timur asks, “What would be the best surface markers to measure the activation of murine APCs following some kind of antigen take up?”

Anne Lodge

That’s a good question. So when we’ve looked at activation of dendritic cells, we’ve often measured the CD80, CD83, and even CD54, which is not negative, but you can see a good shift, an increase in expression upon activation. Those are always a good step. As is class II, so you can just stain for HLA-DR and you’ll see that go up upon activation. So, a number to choose from there.

Kristen Haberthur

“What is the best timing to measure higher cytokine production in culture?”

Anne Lodge

I would go with later than what you typically see. We always measure our recall antigen responses on day four, because for the most part, stuff accumulates in the culture medium and some cytokines are just getting going after 48 hours. So in order to pick up, to increase our sensitivity, we go out to four days. I think some, though, are more quickly released and maybe actually decline. That would be things like TNF alpha, maybe even IL-2, would be actually dropping on day four rather than coming up.

Kristen Haberthur

That makes total sense. We have another one. This is different. The question is, “How do the ATP release-based assays, such as CellTiter-Glo, differentiate between cellular metabolism versus replication?”

Anne Lodge

Well, they don’t. And that’s one of the catches. They’re just measuring ATP. So if the cell is more metabolically active, you’re going to see an increase in that. But the increase of additional cells seems to be greater, in at least from what I’ve seen. Because I’ve certainly done activation of things like macrophage and you don’t see the activated macrophages — which I’m sure are more metabolically active — you don’t see a great increase in ATP activity with those. Maybe some marginal.

But it’s one of those things to keep in mind, because… And that’s part of why it doesn’t necessarily work well for a primary response is that you’ve got a lot of cells just sitting there with ATP in them and you get that big background.

[00:53:50]

Kristen Haberthur

Jay has another question. They ask, “What set of flow panels would you suggest to measure from early to killing?” I want to say that they’re asking from beginning activation to killing. If I may, I think that goes back to your initial question where we’re talking about your basic panels that you would include things about, like, IL-2, interferon gamma, CD95, CD28, to knock out the naïve versus all the essential and effector memories.

Anne Lodge

But there are people looking at surface expression for killing. Like annexin V. Seeing the apoptosis of your target cells. There’s one that’s on the tip of my tongue that I won’t remember that’s expressed on the effector cells when they’re undergoing lysis, or when they’re active. So they’re busy killing and spitting out granzymes and things like that.

[00:55:08]

Kristen Haberthur

Okay. “Would you expect certain levels of T cell proliferation isolated from a stimulated (say, vaccinated) host?”

Anne Lodge

Well, I’d certainly expect to see something. If the vaccination is effective, you should be able to detect it in vitro. But boy, what the level’s going be is hard to judge.

Kristen Haberthur

I would imagine it depends on when the vaccination occurred.

Anne Lodge

Sure. When the vaccination occurred, what the antigen is, how old is your donor? You know? You would have some people, you know, we have a normal donor who’s certainly healthy. And I remember we managed to get samples from him shortly after a tetanus booster and shortly after the booster, his tetanus response was great, you know? And I’m talking about a month after his boost, but he’s back down to pretty weak.

So, you know, he’s not that old, but maybe old enough that he’s not maintaining his immune response as he should.

Kristen Haberthur

Oh, absolutely. Aging….

Anne Lodge

Aging does have an effect.

[00:56:44]

Kristen Haberthur

It does have an effect, yes. This is a longer question, but I think it’s a good one. So Nick asks, “We have been investigating NK cell and granulocyte degranulation using CD107a degranulating marker. But the big question is, if this is actually showing degranulation or cell death? Do you have any idea on better degranulation measurements of any degranulating cell types such as NK cells and/or other granulocytes?”

Anne Lodge

Well, I guess the way to distinguish that is to look at the life of your NK cells. If you’re looking at CD107a, which is the marker I couldn’t come up with, but I’ve seen it used. If you’re looking at that, you could certainly look at how live those cells are. Whether you’re using annexin V and see that they’re on their way, or 7-AAD and see that they’re really dead.

I’ve seen it and I kind of go, “Yeah, I guess that sort of works.” But it’s so much more satisfying to see a target cell die, you know?

Kristen Haberthur

Yes. I agree. So making sure to maybe stain with a live/dead stain.

Anne Lodge

A live/dead stain makes perfect sense.

[00:58:20]

Kristen Haberthur

Okay. I have lots of questions coming in. Alright. “So just wondering if there is any comment on artificial activation chemistries, i.e. soluble antibodies versus beads versus APCs?”

Anne Lodge

Oh, interesting. Just as ways of activating T cells?

Kristen Haberthur

Yeah. Maybe like there’s one preferred over the other depending on the experiment at hand.

Anne Lodge

I would say that we’ve been using… we do use Anti-CD3, Anti-CD28. It’s not really bead-based. I think they’ve just combined the length of the antibodies together. It’s a product from Stemcell Technologies. But of course, there’s also bead-based Anti-CD3, Anti-CD28 out there. And I’ve heard people say, “Oh, it’s more physiologic.” And it’s like, “Well, yeah, sort of.” Not really, because…

Kristen Haberthur

It’s really concentrated.

Anne Lodge

Yeah. And you can see the difference on that CFSE. You activate everything, for one thing. And you see that they march off in cohorts. Which has always intrigued me why that is. But it’s distinct from the antigen-specific stimuli that we looked at. So it’s useful. It gives a great signal. It expands a bunch of cells. But let’s not kid ourselves that it’s physiologic. Because, it just can’t be. I suppose it’s better than using PMA or…

[01:00:06]

Kristen Haberthur

Fair enough. “So what is your experience with neutrophil function assays? Stimulation effector function using commercial kits or facts?”

Anne Lodge

I haven’t used any commercial kits. I think somebody just pointed one out to me the other day and I was like, “Oh, that’s interesting.” Because we do produce neutrophils, and they’re tricky little buggers to work with, we’ve been able to see phagocytosis. It took us a while to demonstrate oxidative burst using fluorescent dyes that shift upon reduction of those free radicals. So that’s kind of cool, but boy, I wouldn’t want to do it in a big way. You know, it was just sort of to demonstrate that they can do it type of thing.

So phagocytosis, reactive oxygen — a few people have asked me about migration assays, and I would expect that they would migrate. The hard thing is that, at least in my experience, anytime you throw serum in — and we’re all used to using fetal calf serum — you do that and you activate the neutrophils and everything becomes one big sticky gob. So that’s the trick. Keep them away from serum, and you might be able to get them to do most anything.

[01:01:46]

Kristen Haberthur

That’s a good idea. “Is there any way to measure anergic T cells?”

Anne Lodge

Oh, that’s kind of similar to the exhausted T cell question. So there’s expression of those checkpoint inhibitors, which kind of mark them out as being probably anergic. And there’s ways to make them unanergic, especially now that there’s anti-PD antibodies or anti-LAG-3 antibodies or whatever you want to throw in there to kind of revive them. But first you’ve got to find them and then you’ve got to see if you can get them to do something. Because that’s sort of the definition.

[01:02:38]

Kristen Haberthur

Alright, just a couple more questions. First is, “What is the best cytotoxicity method which can distinguish between CD T cells and target cells?”

Anne Lodge

Oh. I like the one that I presented in here with the labeled target followed up with 7-AAD. Because, boy, there’s no doubt who’s dying there. You can see it by flow and, you can label your effector cells and just fake them right out. You have no doubt that you have death in your target. And at least when I’ve run it, you don’t see death in the effector cell population. And hey, if you want to move to something that’s a little more  — that doesn’t tie up the flow cytometer, you can always do that preliminary experiment to show that that’s what’s happening and then expand out to something more.

[01:03:47]

Kristen Haberthur

Yeah. That’s a good point. Last question, “How do you induce T cell anergy?”

Anne Lodge

Well, there’s more than one way to do that. You know, way back in the day you could induce energy by presenting in the absence of the co-stimulatory signal. And there was a lab that did a whole bunch of experiments on that lines. Kind of a light fixation of your antigen-presenting cell, you know? Get an antigen and then follow up with fixative so that they won’t express the co-stim antigen. And there you go. You’ve got them in an anergic state at that point.

Kristen Haberthur

Perfect. Awesome. Thank you so much, Anne. That was the last question. That was a fantastic discussion. I’d say a very helpful presentation. So thank you again to Anne, and thank you to our sponsor, Astarte Biologics. Finally, thank you to all of the audience for taking the time to attend and to listen in and to engage in our discussion. If you’ve enjoyed the seminar and would like to view the recording, the recording should be up on our webinar page on bitesizebio.com within the next 24 hours. And you can visit it using the link that I provided in our chat box, which again is http://bit.ly/immunecellactivation. All one word, all lower case.