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Issues with Lyosphere beads-background, especially CD8+

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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Aug 15, 2017 7:38 pm

Issues with Lyosphere beads-background, especially CD8+

Hi all,

This is going to be a long post. I also assembled example data into a Powerpoint file, but it's too big to post (stupid 2MB attachment limit....).

Therefore, you can find the PPT (or, broken down by Trial at this Google Drive: https://drive.google.com/drive/folders/ ... sp=sharing


We had some Lyospheres made by Biolyph from my standard CyTOF PBMC surface phenotyping cocktail. A few labs at Stanford (Nolan and Blish, in particular) had been successful with this, and I think some other labs outside of Stanford have as well. I have used this cocktail for 6 years now, and it's been reliable.

For historical reasons, I still use the DN3 polymer from Fluidigm. I labeled 4 preps each of the 33 antibodies in the cocktail, which was theoretically enough for 450 1-test Lyospheres. We used a particular excipient that was basically TBS, and had been successful for the Blish Lab. I sent the antibodies as 33 individual stock tubes (since we and others have had issues with background if the antibodies are mixed too far in advance). Biolyph returned to us ~375 1-test Lyosphere.

I had retained a fair amount of the original Liquid stocks (aliquots of which I had sent to Biolyph), so we could do head-to-head comparisons of Liquid vs Lyo when we got the shipment back. I used a healthy control PBMC donor that we have characterized well over the years, and used in the titration of the Liquid reagents for the Lyosphere calculations.

Not unexpectedly, one antibody did not retain activity after being included in the Lyosphere. However, a later experiment demonstrated that it could be spiked in as a liquid reagent, so it's not a big deal.

However: I had much higher background of the Lyosphere cocktail (which I did put through the 0.1um spin filter as always, and as I did the Liquid cocktail made fresh) of certain antibodies on certain cell types. TCRgd was one. So was CD56, but it was weird: the background was worse on NKT (CD3+ CD56+) than on NK cells (CD3-, then CD56mid CD16+).

In the 2nd trial, I tried doing extra washes, and larger volume washes. No effect. CD8+ cells are most affected by background.
In the 2nd trial, I also used a second PBMC donor that we have characterized. Oddly enough, this donor did *not* have any of the same background issues as the first donor (CD56 on NKT was appropriate, etc).

In the 3rd trial, I tried diluting the antibody to 0.5x, as well as blocking with low-MW PEG or heparin (as Rahman et al Cytometry A 2016, though it was intracellular background, there). Again, one donor consistently affected, one donor consistently unaffected. Blocking consisted of incubating the cells with the block in PBS, then adding the antibody (still in the presence of block). CD8+ cells are most affected by background.

In the 4th trial, I tried blocking (same way as above), using BD Human Fc block, total mouse IgG, and total human IgG (based on Andersen et al Cytometry A 2016, which was focused on background on monocytes). Again, same donor affected, other donor still unaffected by background. CD8+ cells are most affected by background.

In the 5th trial, I tried testing more donors, as well as replicate draws we had of the affected donor. This was to test both processing conditions (ie, maybe something had been screwed up in the processing of the affected donor), as well as donor variability. In short: about half the donors were affected, about half the donors weren't. In one case, two donors were processed by the same person, same media, same process, on back to back days; one was affected, one wasn't. I couldn't find a gender correlation, or age. I don't have CMV data on all the donors, but at least one affected donor was CMV- (the first affected donor was CMV+).

I showed this data to some other people; one person mentioned a weird CD16 polymorphism, and suggested that I look at CD16 expression on CD8+ cells. That *did* correlate reasonably well, though again, at least one donor that was affected had at least a decent number of CD16+ CD8+ (though much fewer than most affected donors). But the IgG or Fc blocks made no effect (trial 4 tested).


In summary:
1. Some donors affected in Lyosphere sample, some not. All are fine in the corresponding Liquid sample (always run on the same day, fresh, as the Lyo trials; same liquid master stock as was sent for Lyosphere synthesis).
2. CD8+ affected more than any other cell type.
3. Blocking with heparin, LMW PEG, BD human Fc block, total human or mouse IgG made no difference.
4. Roughly 50% of the donors tested were affected. No obvious correlation with processor, processing method, date of processing, nor with CMV. Some correlation with CD8+ CD16+, but not perfect.
5. Unlikely to be DN3 issue, as Liquid reagents also DN3. Similarly, some donors fine with Lyosphere cocktail.
6. CCR7 (affected on CD8+) and CD85j (the antibody that lost activity) were from R&D Systems, purchased as lyophilized antibody. Therefore, technically, Lyosphere synthesis would be their second lyophilization. However, CCR7 fine on some donors, and generally fine on CD4+ even on affected donors.
6. Additionally, TCRgd was liquid stock from Biolegend, and was affected.


I want to be clear: I don't blame Biolyph for this. They had been successful with several other groups, so there was no way to know this would happen to us. They offered to work with us on figuring this out, but we already sank $25-30K into this, and aren't overly thrilled with the idea of spending more only on the chance that we find a method that will work.

For anyone thinking of Lyospheres (or any other lyo/dry method), I will suggest:
1. Start small: maybe 100ug of each, for 50-150 beads. Biolyph said that they can go as low as ~20 beads per synthesis. Only if those work should you go ahead with a larger prep.
2. Test *multiple* donors. In some ways, I got lucky (?): the first donor I tested was Affected, otherwise I wouldn't have known until I did my first plate of samples.
3. Examine every cell type you can think of. I have no idea why CCR7 is more affected on CD8+ than on CD4+.

If anyone has any ideas, we would greatly appreciate suggestions: as I said, we have $25-30K sunk into this, and would like to use the remaining ~300 Lyospheres sitting in our cold room...

Thanks,
Mike
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kunicki

Contributor

Posts: 38

Joined: Thu Apr 13, 2017 8:46 pm

Post Tue Aug 15, 2017 10:54 pm

Re: Issues with Lyosphere beads-background, especially CD8+

Hello Mike,

Would you consider characterizing the two donors for various fatty acid/scavenger receptors to explain the higher background, such as CD36? Perhaps Biolyph has done some of this work already? I am not so familiar with their technique, but I imagine their lyosphere leaves some added material in the Ab cocktail, no?

Best,
Matthew
Last edited by kunicki on Wed Aug 16, 2017 5:52 pm, edited 1 time in total.
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Chowduck

Contributor

Posts: 29

Joined: Wed Nov 19, 2014 4:39 pm

Post Wed Aug 16, 2017 4:11 am

Re: Issues with Lyosphere beads-background, especially CD8+

Hi Mike, I noticed in the pptx you commented about not attempting to buffer exchanging because addressing the reagent would not explain the donor variability but this seems to be 2 simultaneous issues (reagent and donor) causing the background that need to occur concurrently. Eliminate one would solve your problem and the reagent side of things would be the best place to start. Please forgive me if any of this seems naïve, I’m brainstorming in the hope that something helpful might come of this.

Reagent:
May I suggest buffer exchanging a reconstituted lyosphere with a 30kDa or 50kDa centrifugal filter with 3 washes to eliminate the possibility that there are chemical components present in the lyosphere that are altering staining. On a small scale you would lose a substantial amount of antibody through binding to the filter, but it would help to identify if this is a factor. In the event it is, and the contaminants are hydrophobic another less costly method might be to add lipidex beads to the reconstituted lyosphere to soak up hydrophobic components then pellet the lipidex out before staining (assuming the DN3 or X8 do not bind to the lipidex). If buffer exchanging has no impact it has to be something structural with the antibody-DN3 complex. NMR and x-ray are out since they are a mixture of proteins. Perhaps HPLC, or a low density non-reducing SDS-PAGE / native PAGE to see if you are getting aggregates that are still small enough to pass through the 0.1um filter? Or re-submit pure anti-CD8 only to Biolyph to see if you can repeat the defect then have the structure of the post-lyosphere anti-CD8 compared to the liquid state to gain some insight into what to try next. Also relevant: "Infrared Spectroscopic Studies of Lyophilization‐ and Temperature‐Induced Protein Aggregation" (doi:10.1002/jps.2600840407)


Donor:
With regards to the donor variability, that’s the really perplexing part! Especially the one donor who switched groups on sequential days. It might be insightful to collect dietary data such as: time since their last meal and what they ate in terms of high fat/sugar content to see if there is any correlation. A very brief search indicates CD8+ T cell function can be altered by modulating either cholesterol or glucose metabolism (doi:10.1038/nature17412 and doi: 10.1172/JCI69589) potentially on a time scale where a differences might be detected if a donor were to say: skip breakfast and donate prior to eating lunch. Hypothetically protein(s) expressed only on specific cell types as a result of dietary factors could be binding specific antibody aggregates that are forming because of the lyophilization that are not bound by other cells. Going out on a limb here which is why I would focus more on the reagent.

Our lab is in talks with Biolyph to prepare similar lyosphere and a DIY pilot is in the pipe. We use exclusively the X8 so I will be certain to report back if we experience similar issues to help narrow down what's going on here.

Regards,
-Greg
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Aug 16, 2017 2:57 pm

Re: Issues with Lyosphere beads-background, especially CD8+

Hi Greg,

I'm not sure what you mean by "Especially the one donor who switched groups on sequential days."

Were you referring to my statement "In one case, two donors were processed by the same person, same media, same process, on back to back days; one was affected, one wasn't."? If so, I think there's been some confusion: I was referring to Trial 5, where the 1910 and 2427 donors were separate donors, but processed by the same person using the same protocol, one the day after the first (2427 on 11/15/16, 1910 on 11/16/16). To me, this was the strongest evidence that it's a donor-specific issue, as this would largely remove any processing differences.


Re donor information: some of what you suggesting looking at, we have no control over, and have no way to get the information. The donors I investigated were "healthy donors" donating at Stanford Blood Center; we got LRS chambers from the donation. Similarly, we never have that sort of information from our customer samples.

To Matt's point: I guess we could try looking at CD36 expression. But I'm not sure how useful that would be: from a purely practical experimental POV, it wouldn't be something like age/gender/CMV status that we could stratify *prior to* the experiment, and only use the Lyospheres on the "CD36- donors" or whatever. That's the real issue: so far, to find the "weird" donors, we have to run the experiment. If we do, and then find out that it's a "weird" donor, then we've wasted time, reagent, and sample.

In short, we either need to find a correlate we can test for ahead of time, without something as fine-scale as dietary info; or we need to find a way to block or clean up the reagent to avoid the problem in the first place.


Mike
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Chowduck

Contributor

Posts: 29

Joined: Wed Nov 19, 2014 4:39 pm

Post Wed Aug 16, 2017 4:00 pm

Re: Issues with Lyosphere beads-background, especially CD8+

Hi Mike,

Thanks for clarifying; I misunderstood thinking it was the same donor producing opposite results.

Protein aggregates in the reconstituted lyosphere is where I would check next, size exclusion chromatography would likely be simplest.

-Greg

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