Hi, folks.
I am a postdoc newly working at the Immunology field.
I am trying to establish a Treg suppression assay from the mouse. However, I have a weird issue unsolved yet. Here is the protocol.
Using cell sorter, I isolated mouse splenic CD4 naive T cells (mechanical separation, Live+CD4+D45RA+CD25-) and tumor-infiltrating Treg (Live+CD45+CD4+CD25+GITR+, following dead cell removal, CD4+CD25+ enrichment kit (STEMCELL kit)). Treg was verified after sorting by staining Foxp3 where more than 95% of isolated CD4+CD25+ were positive for foxp3. After cell sorting, I confirmed cell viability by quickly staining trypan blue and there's no major cell death.
I incubated CD4 T cells and Treg at different ratios (CD4 T cell number was fixed to 10^4 cells per well, and Treg number was every two-times diluted from 1:2 to 1:64 and no Treg per well) for 3 and 4 days. I added two times a higher number of CD3/CD28 activating Dynabeads (Invitrogen) compared to the CD4 T cell number. I did not add IL-2 because this cytokine could mask the suppressive function of Treg. I used RPMI 10% FBS supplemented with HEPES, P/S, pyruvate, and b-ME.
Unfortunately, under a microscope, I couldn't observe cell growth. However, there's a decreased CSFE (final 2.5 uM 20 min incubation at RT) intensity. It might be due to the lack of IL-2, a strong signal for T cell proliferation.
Basically, 1:2 should show the highest inhibition of T cell proliferation by Treg, and 1:64 should show the lowest inhibition. In my case, there's the opposite trend. 1:2 shows the highest proliferation of T cells and vice versa. I also stained Treg with an orange cell tracker, and there seemed to be a slight proliferation of Treg.
I performed three times of independent experiments by changing the number of T cell numbers, increased incubation time from three to four days. So far, I haven't explained my data perfectly with no precise mechanism or reason for my data. I double and triple checked the labeling between samples.
human T cell proliferation assay worked really vell without IL-2 addition, and there's a homogeneous peak of CSFE stained cells. However, I have always seen heterogeneous two peaks of CSFE stained mouse CD4 T cells isolated from the spleen. The cell suspension was vortexed before and after CSFE dye staining.
Could you help me to figure out these troubles?
Thank you.
Hi Dr Kim. In this system, you don't have APC. So you are relying instead on cd3/cd28 to stimulate.
So the non treg cells will make il-2
The treg may not be making il10 and tgf beta
But most important, you are lacking the effects of ctla4 stripping B7 because you lack apc in your coculture, and at the same time, you are delivering dominant signaling through anticd28.
So based on your protocol, lacking a source of b7 to engage ctla4, you have reduced treg function.
I
Dear Kim,
I have been working on mouse Tregs for many years and never faced such a problem . I disagree with Lora Benoit. Lacking APC will not cause Treg disfunction. Although one of the main mechanisms of action of Tregs is via CTLA4 but in your case you are mostly counting on anti-inflammatory cytokines such as IL-10, TGFb and sTNFR2.
Based on my experience there should be one of the following reasons:
1) You have used a high dose of activation beads. I suggest you to respect the protocol of Dynabeads.
2) Change your serum lot. Immunosuppression is strongly relate to FBS!!!
3) You are using very low ratios and starting numbers. I suggest you to increase your CD4 responder cells to 100K or 200K and decrease your Tregs accordingly. But use 1:1 ration to be sure that your Tregs are functional.
Moreover, do not count on Treg suppression in more than 1/8 or 1/10 ratios. Your Tregs are too diluted. I suggest you to use 1:1, 1:2, 1:4; 1:6: 1:8 and 1:10 ratios max.
4) Try to use another dye for your Tregs to see if they are proliferating too.
5) Do you remove CD25 population from your CD4 responder cells? you should use CFSE+CD4+CD25- T cells.
6) Try to activate (incubate Response cells and Beads at least for 30 min in 37°C before addition of Tregs.
Good luck
Sina
Sina Naserian 1) I already increased the concentration of beads by two times than invitrogen suggested. 2) This serum works when using human T cells, and we don't have another FBS, but it could be a good idea to change FBS possible. 3) Okay. I will increase the number of T cells. Meanwhile, I used U-shaped 96-well to concentrate T cells. In this case, I could use 48 well otherwise 96 well could be too crowded. 4) I will. 5) Yes. Niave CD4 T cells are negative of CD25. 6) I didn't try it. I added Treg followed by CD4 T cells and finally beads. Thank you for your valuable time and opinion.
Myungchul Kim 96 well plates (U shape o V shape ) are perfect for this purpose.
However, you do not need to go to 48 well plates. keep the interaction t the maximum.
Regards
Kindly check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049949/
I have the same problem with Treg suppression assay, too. When I used 0:1, 1:1, 1:2, 1:4, and 1:8 Treg (CD4+CD25+):Teff (CD4+CD25-) ratio, 1:1 showed the highest proliferation rate than without Treg. And lower Treg ratio showed a lower proliferation rate.
Dear Dr Kim!
I`ve done these tests on murine cells, so I`ll tell about them.
I use irradiated syngeneic splenocytes as stimulator cells and aCD3 as a stimulus and T-regs worked very nicely. I`ve also tried to avoid the use of APCs using plate-bound antibodies instead, but there was no suppression at all or it was inconsistent. IMHO, this kind of tests is better to have APCs as CTLA4 is critical for it and if you avoid it you will block one of the critical suppression mechanisms. Otherwise you can try to optimize the stimulation protocol. Anyway, I`ve used irradiated splenocytes and succeeded :)
Although I was able to use even 10-20 thousands of responder CD8 T-cells for conventional proliferation tests, I use 50000 responders routinely as the results are much more consistent.
Another important issue is that I must say your CFSE staining isn`t good. There must be a clear single peak in the unstimulated samples and distinct peaks in the stimulated samples. Optimize your CFSE staining and consider CellTrace Violet (IMHO, cells tolerate it better than CFSE), but it doesn`t really matter. I think that probably you have troubles because of the incomplete removal of FBS at the staining step.
I wash cells with PBS or HBSS to remove any FBS before staining once or twice, stain them for 10-12 minutes at 37C in the dark in PBS or HBSS without any serum, then add FBS directly to 30-50% and wash once and it gives very nice staining. I also never vortex them, just pipette and invert the tube during the dye staining.
In some protocols they say to use RPMI and/or a small amount of FBS, but in my experience it makes staining awful (0.5% FBS in the staining buffer already rendered peaks very blurry later; more FBS - the situation is even worse).
Good luck!
Sina Naserian Dmytro Shytikov
Hi folks, I want to share my results again. Thank you for everybody who helps me here.
I increased the incubation time from 3 days to 5 days without IL-2, which worked well. As expected, compared to IL-2 treated responder cells, IL-2 negative cells proliferated less. However, there was clear proliferation of T cells in the absence of IL-2. In this time, each well contained 2.5*10^4 of responder cells (CD4+CD25-CD45RB+, CSFE+), 1*10^4 cells of Treg (CD4+CD25+CD45RB-), and 5*10^4 CD3/28 mouse activator Dynabeads. The CD4+CD25+CD45RB- showed 95% of Fopx3 intracellular staining.
Under microscope and based on the number of Tregs analyzed by flow cytometry, it’s likely that Treg in the absence of IL-2 did not proliferate. Unfortunately, in this time, Treg was found to not be stained by Far red dye.
Under flow cytometric analysis, responder cells that were co-cultured with Treg seemed to be less proliferate, because a total number of analyzed cells were lower in Treg coculture group than no Treg added group. However, CSFE staining showed that co-culture group seemed to be more proliferative than responder only group.
If there is IL-2 in the well, there was spontaneous proliferation of responder CD4 T cells, probably due to the role of IL-2 to activate T cells that had been stimulated by mechanical stimuli, such as smashing spleen.
For heterogeneous two peaks (CSFE positive control), I should have removed FBS more vigorously. However, I removed media and resuspended responder cells in 0.1% BSA PBS. For human CD4 cells, there was no this CSFE staining problem, showing clear one homogeneous peak of CSFE.
By the way, do you know why there are two populations of responder T cells in flow plot where one is higher FSC-H and the other is lower FSC-H? Based on IL-2 added flow plot, real lymphocytes seemed to be higher FSC-H group.
There are few options for me left. I have tumor-bearing mice that will be sacrificed soon. I am not sure that I can make it. I could change serum lot and activate responder cells and beads prior to addition of Treg. Since tumor-infiltrating Treg were extremely low in my case, I cannot increase the number of responders up to 100 to 200K.
Thank you.
Hi Dr Kim, to answer your question, the higher fsc is due to actvated, blasting lymphocytes
( aka lymphobladts) vs resting lymphocytes. Fsc is a function of cell size.
Your flow data is gorgeous. Good job on trouble shooting this assay. ; )
Dear Dr Kim
Good to hear!
I'd suggest to stain your cells with live/dead stain (FVS, PI or 7AAD, check them if interested) and exclude dead cells from your analysis. Although not critical, but it will make your CFSE profile better. And remove any protein from your cells before staining with CFSE. It really gives no benefits, I tried and saw only troubles.
See, FSC/SSC low cells are Resting cells, FSC hi / SSC moderate are proliferating cells, FSC low / SSC high - dead cells, FSC very low - debris.
And if you are not sure about the protocol, then try irradiated stimulator cells (25 Gy). Works for sure :)
And in the worst case just optimize your protocol and restart your experiment!
Good luck!
Dear Myungchul Kim ,
I have some fundamental issues with this new exp,
1) I still do not know and not convienced why do you use a double ratio of suggested activation Beads. You should normally use 2 µl/80K cells which gives you a 1:1 ratio. I think you are over activating your T cells! Big time.
2) 5 days is very long for an MLR test, especially with CFSE dye. You should perform the test 3 or max 4 days. After that, all cells are divided and you lose CFSE expression, therefore, you will find all cells on the left side.
3) If your Tregs are truly Tregs, you do not need IL-2 addition for keeping them 3 days in culture. This stimulates both Tregs and Activated T cells that are CD25high. Do not forget that CD25 is an activation marker that is highly expressed on your effector T cells too. That is why you eliminate them in the beginning. Even after 1 day of activation by Beads, the expression of CD25 (IL2R) will increase dramatically in effector T cells (CD25- cells). Your IL-2 addition will boost them even more.
In conclusion, I would say try:
a)100K Teffs (2 µl Beads only) + 100K Tregs 1:1 ratio
b) 100K Teffs (2 µl Beads only) + 50 K Tregs 2:1 ratio
c) 100K T cells alone + 2 µl Beads only
d)100 K T cells alone (no Beads)
Keep the co-culture 3 days only! no media changing!
Activate your responder cells separately 30 min in advance.
It will work! got no choice!
Your figure is not classically what we wait for. not even your controls.
Regards
Sina Naserian Thank you I do appreciate it. I will do an experiment tomorrow as you suggest and post it later again. How do you think about the FBS lot?
Thank you all!
Sina Naserian Please kindly note that we have the number issue when using tumor-infiltrating regulatory T cells. Tomorrow I may have a number of Tregs for establishing conditions, but it's really hard to obtain 100,000 Tregs per a single experiment. Do you happen to do an experiment using a small number of cells but with the same proportion of CD4 T cells and Tregs? Thank you
Dear Dr.Kim,
I do not think you have got FBS problem since you have enough proliferation!
Concerning the cell number, there is no problem keep the ratio of T cells (Teff and Tregs) and respectfully decrease the beads’ number.
Like a) 50K of each T cell in 1:1 ratio by adding only 1 ul of Beads
b) 50K of T eff and 25 K of Tregs in 1:2 ratio.
c) 50K Teff alone + 1 ul of Beads
d) 50K of Teff alone (no Beads)
It might get longer while recording the events by cytometer.
Hope it could help.
Just do not forget to vortex very well (30s atleast) the Beads prior using them!
Sina Naserian Unfortunately, my system did not work. I will use irradiated splenocytes and responder T cells with or without Treg.
People use "irradiated splenocytes" as APC. Do they deplete T cells after physical/enzymatic dissociation of mouse spleen?
Hi, Dr Kim!
Sad to hear! Actually, I`ve also tried to use plate-bound anti-CD3 as a method of stimulation for my in vitro suppression tests, but it also didn`t work(
Concerning your question, you can, but it`s not necessary. After irradiation with 25 Gy, there will be no single cell able to proliferate among the stimulator, so there is no need to deplete T-cells. I tried both options, non-irradiated T-cell-depeleted stimulators gave slightly better proliferation for the responders, but it didn`t really change the picture.
Good luck!
Dmytro Shytikov Hi, do you have a good paper that describes the dosage and duration of X-ray?
Myungchul Kim
Check this - doi:10.1007/978-1-61737-979-6_2
We use 25 Gy for irradiation, but lower dose must work fine as well. I guess, the use of such a high dose was critical for H3 assays, where survival of even small number of accessory cells might affect the results significantly.
You just need to tell the machine what dose you want to use and usually it determines the exposure time automatically.