Summary Document

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Dear all,
Thank you for all the replies. I have read them all and am very gratefull. I have brought this paper up because the implications of results are important for my work.
Personally, I have few comments.

I have often observed exactly same phenomenon with CD4 pos being higher in FoxP3 “background”as Clare (See Reply 4) and really did not know how to interpret the data. It was different from patient to patient and to donor, even within same run (stained, run same day). I usually include all single controls and adjust compensation for particular sample if necessary. If this is really a background then FMOs should resolve it.

In past, I have been doing a lot of leukemia stainings. We stained for CD45 PECy5/CD19 PE and kappa-Fitc and lambda Fitc. There was interesting connection there higher CD45 was MFI on pat cells more positivity I was getting for kappa and lambda, because only CD19 positive population would shift up and give positivity of kappa or lambda. As in this case it was not a compensation problem. It seemed that PE-Cy5 intensified the other channels. So compared to isotype it appeared positive. And because we did not do FMOs at the time we could not figure out the reason.

In this case however, I have to agree with Cindy (Reply 3) it really can be a compensation issue.

Like in some replies below, we will be doing some experiments validating the antibody ourselves. I will try to keep you informed.

Best regards,
Teona

P.S. If I have not posted your reply it means I have not received it or it got lost in the process. Please, if you would resend it I will be happy to add it to the letter.

Teona Roschupkina
Teona.Roschupkina@med.lu.se


Reply author:

Dear Teia,

Thank you for your questioning. I have forgotten to state that the FOXP3 in Fig3B was stained with 259D clone. Hopefully Blood will make that correction prior to printing. Please let me know if you have any further questions.

Sincerely,

Dat Q. Tran, M.D.
Laboratory of Immunology
NIAID, NIH
10 Center Drive
Blg. 10, Room 11N256 MSC 1892
Bethesda, MD 20892
phone: 301-496-3863
fax: 301-496-0222
email: dtran@niaid.nih.gov




Reply 1:

Hi Teia,

I think they used they 259D antibody in Fig.3b because they do get a Foxp3/negative/ population in the "CD45RA+ d5 +TGF-beta" cells. Regarding the PCR experiment: Their point seems to be that the Tcells with Foxp3 induced by activation in presence of TGF-beta have less Foxp3mRNA than the activated Treg and that this correlates with the lower percentage of positive foxp3 staining with 259D as shown in 3b (where NOT all cells are Foxp3+ in contrast to staining with PCH101). To me that alone would not be enough proof that the PCH101 is unspecific as correlation between mRNA and % positive cells is not necessarily good. But together with the other data it is quite convincing.

They mention that 2 populations were found with the PCH101. Couldn't that be positive and negative, with the negative just having a higher background than the isotype control? The did not really discuss that. They also did not discuss how the percentages of these 2 populations compare to the positive and negative population they get with the 259D. I have ordered the 259D and am going to compare my PCH101 results to that. I'll let you know how that goes.

Great you brought this paper up for discussion. Please let me know your thoughts and other replies you get as this is a quite important issue to my work.

Cheers,
Diana


Reply 2:

We have compared PCH101 with 259D and found no significant difference. However, we use the APC conjugate of PCH101, not the PE form used in the Shevak paper. Perhaps that accounts for some of the difference. Also, we work with G-CSF mobilized peripheral blood. The FoxP3 story and that of Tregs in general are still unfolding so many of the findings being made will be challenged going forward. Certainly, the story is going to get more interesting. My recommendation is to validate the antibodies yourself to your application. Relying in the literature or what the lab down the hall is doing can be costly; I’ve been misled in the past and learned the hard way the importance of confirming such details for myself.

Kevin Sheehan


Reply 3:

Dear Teia-

A friend who is on the mailing list forwarded me your email. I also use PCH101 and have read the Tran et al paper. Here is my take on Fig 3:

3A: First, I think there is a typo and the 'anti-TGFbeta' should say 'No TGFbeta'; at least according to the text and the legend. Second, I think the compensation for the stains with PCH101 looks improper. This is clearest on the isotype control plot. Third, to really compare two different Abs, they should have used Abs conjugated to the same flurochrome. Oddly, the compensation for the 259D Ab looks good. All this being said, I have seen some interesting things using PCH101 vs 206D (which Tran says gave similar results to 259D). Conjugated to Alexa 488, I see a greater % of FoxP3+ events in the CD25+ or CD25 Hi gate with PCH101 than 206D. So, there may still be a specificity issue with PCH101, but I think the cytometry presented in Fig 3A is not done with the best technique.

3B: I believe the staining is with PCH101. I was wondering why the 3rd and 4th plots look so different than their counterparts in 3A. I understand their graph labels to indicate that the 3rd plot should be the same as the 'anti-TGFbeta' PCH101 plot and the 4th plot should be the same as the '+ TGFbeta' PCH101 plot. Do you have any thoughts on this?

Thanks,
Cindy Zuleger
UW Madison


Reply 4:

Hi Teona - The shift of almost all CD4+ cells above background in Fig. 3A is something I have seen with clone PCH101 as well (I usually use the APC conjugate & I am staining samples of human peripheral blood, an average of 10 different patients/normals a week). Looking at the pictures I wonder if the brightest FOXP3+ group in the TGFB+ treated cells are the "truly" FOXP3+ cells and if the percentage there would also be around 60%, as seen w/ the other clone.

The shift I see is never quite that dramatic (I may get a half-log shift), and does not appear to be a titration issue, and generally the entire CD4 positive group shifts (highly suspicious in normals!). I have gotten certain lot numbers which seem to have more background than others. Since I do a lot of normal peripheral blood samples I am able to get a good sense about this shift being an artifact, or non-specific (call it what you will) - as no "normal" would have 30% of CD4+ cells being FoxP3+. And, more importantly, there is always a FOXP3 "bright" population out beyond the shift, which when gated upon has the expected phenotype in terms of CD25, CD62L, CD45RO/RA.

I am finding that if I use a 1/400 dilution of unlabelled msIgG in the Perm Buffer, & incubate my cells for at least 30 mins on ice in that as blocker before adding the FOXP3 AB (I add it directly to the well), I am getting much less shift relative to my isotype control. Also, after the incubation in FOXP3 is finished, I pellet the cells, aspirate sup, add the Perm Buffer again &let the cells it on ice for at least 10 mins before I re-pellet, and continue on. Maybe that helps, too.

I have mentioned this shift to our sales rep from eBiosciences, and his response has been that it doesn't make sense as they test it ll out w/ their reagents etc, and that maybe I'm not using the reagents properly, etc, etc, blah, blah, blah.

Our lab has been in contact with another well known TReg researcher (not Dr. Shevach) who has also noted this phenom. and is testing other clones.

I will be glad to ship back all of our PCH101 stock if the consensus is that it is unreliable.

Clare Rogers
University of Michigan
Department of Pediatrics