We are using anti-CD19 antibodies from Bioxcell to deplete B-cells in mice. Strangely, when we check for depletion by staining cells from cheek blood of the mice, we see that there are no CD19+ cells but B220+ cells are still there.
This is weird as B220 is marker for B-cells, so we would expect no staining for either markers.
Does this mean that the depletion was unsucessful ?
1. If yes, then why do we not see CD19+ cells ?
2. The CD19 epitope is blocked by the depletion antibody and therefore is not stained in FACS ? (The clone for the depletion antibody and staining antibody are different)
Dear Khwab Sanghvi
I just briefly wanted to email you concerning your question. Besides the fact that B220 ain't 100% restricted to B cells and the fact that incomplete depletion has been reported for some anti-CD19 clones from Bioxcell, there's is the possibility that you have an exacerbated inflammatory response (for experimental or environmental reasons) in your mice that alters the pharmacokinetics of your depleting antibody. Please check the attached paper for details, where Laws and colleagues demonstrated that nicely for anti-CD20 depletion.
Article Inflammation Causes Resistance to Anti-CD20-Mediated B Cell Depletion
All the best,
Michael
Thanks a lot for the response. This paper is quite insightful.
In my setup, we are looking at the immunogenicity of a peptide vaccince in the presence and absence of B-cells (CD19 depletion to mimic this). We give a dose of 500ug of the anti-CD19 every 5 days (B6 mice). The total length of exp is 21 days.
We have had multiple problems:
1. The anti-CD19 based depletion of B-cells was successful in the beginning (as measured by CD19+ B-cells in cheek blood) but this depletion was lost after we do a boost vaccine (d10). As the paper suggests, it is possibly due to high inflammation (as we use TLR agonist as adjuvant) that leads to quick repopulation. But We thought that the mutiple dosing should be able to cope with this.
2. The other problem is that whether we see the depdletion of CD19+ cells or not, the B220+ cells are never reduced. I know they are not T-cells as I also stain for CD3. And the numbers are rather high for it to be another compartment. We have seen significant differences in certain experiements when comparing groups treated with anti-CD19 vs control, therefore I am hesistant to accept that the depdletion antibody is not doing much and its binding to CD19 is masking my FACS.
Dear Khwab Sanghvi
it does sound like a rather complicated experimental setup. I would suggest to use B cell knock out mice for your experiment to circumvent the issue of incomplete depletion entirely. Check the JAX strain
https://www.jax.org/strain/002288
It is also a nice negative control as these mice don't make any IgM (just in case antibodies and class switching is the readout for your vaccine efficiency). You might have to cross them out to B6 and do the experiments in littermates, but it would absolutely circumvent the flaws of your model (and save you the optimization of the depletion, which might be difficult when you wanna use a strong TLR agonist). I am pretty sure that you can obtain them rather easily from Hedda Wardemann (at the DKFZ) or Simon Fillatreau (at the DRFZ) or possibly Klaus Rajewsky himself (at the MDC), if you don't wanna buy them.
Think about it.
Cheers,
Michael
Dear Michael Förster ,
thanks again for the reply and the time.
Best
Khwab
Dear Khwab,
I'd like to know if you figured any solution for your issue. I have tried to use a combination of anti CD20 and CD19 to deplete B cells in mice (also using BioXcell antibodies - much lower doses than your - 100 ug of each Ab once a week). I have had the same issue, but additional markers confirmed that these cells are really B cells (they are B220+ CD20+ and light kappa IgG+; I also checked to define if they could possibly be DCs or NKs but they were negative for both markers) - not sure if these cells have any functional defects in the absence of CD19. If you have any updates, could you please share with me? I have heard that the clone SA271G2 is a good one to try B cells depletion. Have you tried other brands?
Best,
Mariana
Dear John,
I haven't used the SA271G2, but I will try it next. Just would like to know if any other person have experience with this. I saw this question similar to my issue with BioXcell antibody then I thought that maybe I could get some helpful info. The idea of using more clones at once is a good option.
Thank you!
Mariana
Dear John,
I will need your advice
I use a model of tumors that grow over a fortnight. Various results have led me to suspect a major role for plasma cells. To confirm this role, I would like to deplete these cells in mice (C57BL6). You will advise me to use anti C-19 and anti-B220 or only anti-CD19
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