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FLAVIA2023-02-17T21:05:36Z
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Hello,
this is Flavia, and first of all, thank you for helping in the past in solving the issue of false positive spots in an IFN gamma ELISpot assay on mouse splenocytes.
This time I have a different problem.
I am using humanized mice that were injected with human breast cancer cells and treated with oncolytic adenovirus.  I am trying to test if the mice elicited a human-acquired immune response versus adenovirus and tumor peptides. I started with assessing the functionality of the test system and I plated 400k splenocytes. I used the ELISpot Pro: Human IFN-gamma (HRP). 340-2HST-02 kit. To stimulate the splenocytes I used the anti-CD3 provided in the kit (1:500 and 1:1000) and PMA/ionomycin (50ng/ml and 1uM) for 24 hours.
The issue I have this time is that in the wells treated with PMA/ionomycin, I have many small spots that are not well defined as in the assay I did a few years ago. In the well stimulated with anti-CD3, I have small spots at the edge of the well and very faint ones at the center of the well. In the wells with only media and no stimulus, the spots look like the wells treated with anti-CD-3, so I guess it is just background.
Now I am trying to understand if:
1. what I see in the PMA/ionomycin-treated wells are spots from stimulated NK or
2. the detection antibody 7-B6-1, HRP is non-specifically recognized in the PMA/ionomycin treated wells the mouse interferon-gamma secreted by NK.
Thank you in advance for any suggestions.
I am looking forward to hearing from you.
Regards,
Flavia
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Christian@mabtech.com2023-02-20T08:31:47Z

Admin

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Dear Flavia! Welcome back.

Looking at your results it actually looks to me like there is very little spot-formation at all. The small tiny things in PMA/ionomycin dont look like spots to me, more just artefacts from a massive number of cells added.

I have never worked with humanized mice but I presume that all their immune cells are "totally human"? Or can there be some chimerics going on? The antiCD3 antibody works on human cells but if something is "funny" with them, I guess there could be a situation where it simply don't work.

Overall, 400K splenocytes is rather high, especially for PMA+Ionomycin. For this stimuli I think you would only need around 10K cells/well. Also, PMA+ionomycin is light sensitive stimuli and needs to be handled with care otherwise it stops working. Light-exposure during actual experiment setup is of course ok but one should be aware.

Do you happen to have PHA available in your laboratory?

Your specific questions:

1. There might be spotformation in these wells but it is too many cells. I would titrate down from 400K and try much much fewer cells/well. Around 10K-30K.

2. If you take 400K cells per well and stimulate them with PMA+inomycion there is an inferno of protein secretion happening in these wells. Once you add detection antibody it will easily smudge against the membrane a littlebit. This non-specific binding is much much lower compared to actual IFNg, but there will always be some. By developing the plate for a long time with substrate you accentuate this result. Had there been real human IFNg secretion in these wells the membranes would have been totally darkened by the amount of IFNg secreted by the humanized cells. Since this is lacking I am suspicious of the fundamentals of the experiment: do humanized mice truly have natural "human" T-cells or is there some level of chimera going on?

Furthermore, how was the viability of the cells after splenocyte isolation? If viability is below 70% there can be a zero result. Was this fresh splenocytes or thawed cells?

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FLAVIA2023-02-21T18:12:56Z

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Hello Christian,

thank you for the reply.

I completely agree with what you said. If a real secretion of IFN gamma was present, the wells with 400k cells will be completely darkened by the strong signal. This is what happened in my old experiment when I was assessing the right number of cells to plate and then I went for 200k. I had a strong signal but it was clear those were positive spots.

Humanized mice should have human T cells. The mice are immunodeficient and engrafted with CD34 cells form cord blood. They sell only the mice at 4 months of age that  have a population of human CD45 positive cells bigger than 25. Within this population the CD3 cell are 1-3%. Maybe the stimulation with the tumor and virus didn't lead to a clonal expansion of T cells and this is why there is no signal at all.

To answer to your question. The viability of the splenocytes after thawing was more than 70%.

C

Christian@mabtech.com2023-02-22T10:37:22Z

Admin

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In humans, Merck did this landmark study around 2006 and found that cell viability needed to be above 84%, otherwise you dramatically lost antigen specific responses in ELISpot.

As a result, if viability is around 70-75% you can have very suboptimal results. In some donors, results can be decent with 75% viability but in others you can get zero result. All depends I guess on which cell population is actually dead or severely compromised (ie T-cells or not).