The authors state "we investigated three mechanistic questions that
cannot be addressed in humans".
- First, we established that a
fraction of mature B cells in the spleen is resistant to anti-CD20.
- Second, we
determined that, after cessation of treatment, splenic and bone-marrow B cells
reconstitute in parallel, substantially preceding B cell reappearance in
- Third, we observed that, in a model involving activated B cells,
the post–anti-CD20 B cell pool contained an elevated frequency of
differentiated, myelin-reactive B cells. Together, our findings reveal
mechanisms by which pathogenic B cells may persist in anti-CD20 treatment”.
So to me this rather says that B cell therapy should not work in
humans........But it does! Or maybe they are saying CD20 depletion should not work in mice.
So perhaps All I should say is “Read the review above and perhaps one can see
that you can actually address these questions in humans”
However, you know me too well. So off we go....
In this study they deplete with CD20-depleting antibody and then look at the cells repopulating and
they see an increase in CD27+ cells in both myelin oligodendrocyte glycoprotein (MOG) protein and MOG peptide induced
But as MOG-induced disease is largely monophasic (a single attack) I
would say “So what! What does it tell us?"
Not much, as disease activity will not resume in the
C57BL/6 mouse model no matter what happens to the cells, so we can’t determine the consequences of the changes.
The kinetics of B cell repopulation in the blood of humans is known.
There we see a stereotyped
repletion response with immature and then mature B cells showing repopulation,
probably from the bone marrow. There is a marked depletion of memory B cells in
virtually every study (see above). The mature (naïve) B cells is not the
In addition, in this study they then looked at the B cell repertoire and
found there was a reduced diversity of repertoire, suggesting that the B cells
are initially repopulated from surviving B cells (I say....Surprise, Surprise?) but the
repertoire that survives is different. This is seen in MS (There are a number of studies on this but they were not cited in the discussion..Therefore so much for putting your work into the context of what is known).
In this mouse study, the B cells that return after MOG protein have more
antigen presenting function and they suggest that there are more autoreactive
cells. Surely this is opposite of what you would think you would want. Therefore what are these studies actually telling us.....I suppose that mouse studies are not going to give us a useful answer?
However does it tell us anything? In MS there is long-term benefit from a
short-term treatment. In mice there is no long-term benefit. So is the rapid repopulation by pathogenic cells the reason why there is no long-term treatment
effect in mice?
Maybe, but I suspect not. In the C57BL/6 mice used, the attack causes nerve loss and this
does not recover. Hence you get the the flat-lining of the clinical disease. The data tells us
that the net result of a short term treatment of mice with CD20-depleting
antibody, whether it is MOG protein or MOG peptide means nothing in the long-term (See below).
However, first things first. This paper sets out to show “the unknown” like so
many other papers.
The referee/editor buys into this and impact factor ten paper
here we come.
However, I have to say that "I hope the authors do not do their animal
experiments/pathology like their literature reviewsJ"
Simply read the paper above to see this.
You can indeed and do know, albeit it is only to some extent and not as
controlled as the animal experiments, what goes on in human lymph nodes/tonsils,
spleen, blood and bone marrow following rituximab.
For example the spleens are removed as a treatment aim in thrombocytic
purpura and this has occurred after rituximab, as have lymph node and bone
marrow biopsies. So the discussion of the paper is rather lacking to say the
"I'm just being constructive here".
Next, also with a bit of reading it would become evident that human and
mouse B cell biology is not the same, perhaps because human B cell biology is
heavily influenced by Epstein Barr Virus, which does not infect rodents.
In humans memory cells can conveniently be detected by their expression
of CD27, but this appears not to be the case in mice, seemingly ignored by the
authors of this paper, who imply they are detecting antigen-activated (i.e. memory)
B cells by detecting CD27. They use CD138 to detect plasma cells, which I am not going to gripe about..
However, it has been reported by Xio et al. 2004 that “It (CD27) is not a marker for somatically mutated B cells and is
present at very low frequency on memory B cells”. Indeed memory B cells in mice are very complex and consist of multiple subtypes. Read Bergmann et al. 2013).
In this current study the inference is that the CD27 positive B cell
population go up and as disease is inhibited then the memory cell idea surely
If you accept, which I don’t (see above), that CD27 is a type of memory cell
marker in mouse.
The study shows that they are resistant to CD20 depletion . However, the work shows that certain B cell subsets are depleted and therefore
the percentage of the other stuff goes up, but without looking at the absolute
numbers of cells, we have no idea if this is a indeed a real increase in
numbers. They may be staying the same or going down. However, if we accept that the data
suggests that a population of B cells is less depleted by CD20 depletion.
Now let’s look at the therapy data. I am sorry to say it hardly passes the Smack
you in the Eye Test. The control group in one experiment looks like the Test group
in the other experiment and vice versa. The effects last a few days and suggests that the data is all within “experimental wobble” (biological variation),
meaning there is really no or very limited biological impact on the mice.
This is what our data showed and also found by others, indeed a poster Boschert U et al by Merck (ISNI 2018 Brisbane) showed that CD20 antibody-B cell depletion had no impact on MOG-induced EAE in C57BL/6 mice. (How did the Bruton tyrosine kinase inhibitor work…it’s a macrophage blocker...but that’s another story)
In this study MOG protein induced disease, claimed to be B cell dependent, is inhibted by CD20 antibody depletion and MOG peptide induced disease claimed to be B cell independent is augmented by CD20 antibody depletion (see below)
Would you say that the animals are protected or do they get disease?
The therapeutic effect in my eyes is marginal. Anti-CD20 depletion before immunization makes things worse in MOG35-55 group. OK I have seen this before, but let's look at the data, the main difference is where the control group is. Also if a few animals (we don't know as it is not in the main paper) don't get disease the line is skewed making it very difficult to interpret.
However, if the models (MOG peptide) are B cell independent, then why does anti-CD20 have a significant effect?
This is interesting as most importantly the authors seem to have forgotten about their own data, published previously in Molnarfi N, Schulze-Topphoff U, Weber MS, Patarroyo JC, Prod'homme T, Varrin-Doyer M, Shetty A, Linington C, Slavin AJ, Hidalgo J, Jenne DE, Wekerle H, Sobel RA, Bernard CC, Shlomchik MJ, Zamvil SS. MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies. J Exp Med. 2013 210(13):2921-37.
Where they show and state "Thus, only rhMOG-induced (recombinant human = rh) EAE is considered B cell dependent (Lyons et al., 1999; Oliver et al., 2003; Marta et al., 2005).
If it is only Human MOG that is B cell dependent, whereas mouse MOG and MOG35-55 are B cell independent. Then the effects seen here and previously (Weber et al. Anal Neurol 2010: 68:369) are surprising as an effect of B cell depletion is claimed in mouse MOG induced disease. If you look at the methods (Supplementary data) section they apparently use mouse MOG protein to induce EAE, so there should be no effect.
So if the experiment is reproducible and the literature is correct, then results of the protein and peptide should have been the same, so maybe experimental wobble is indeed at work.....Oooops.
Adding the two experiments together then suggests that there is no biological effect of depleting CD20 antibody in mice, which I think is probably the case.
Importantly, in this paper, the authors imply that CD20 depletion in humans is associated with increases in proliferating memory cells similar to this study in mouse, perhaps implying it is a good thing. However, if you read the references cited to justify this point it is clear that the human study implicates the occurrence of memory B cells to be associated with a worse prognosis and disease (re) activation, just as we have been banging on about.
So back to the paper. So we have CD20 depletion causing major depletion
of CD27 B cells and inhibition of relapse in humans, however if you actually look at the data there is no/minor impact (increase) on CD27
B cells and no/minor impact on clinical EAE. So actually it all fits. The memory B cells may remain to
be an important subtype of cell. QED.
So we are still right. Is this crazy logic?
P.S. It doesn’t seem to bother the people putting square pegs into round
COI: I have none, but editor of paper is working in same place as an
author….Em.....Rant over :-(
P.S. If this type of clinical data is indeed experimental wobble then it suggests that the conclusions of a large number of EAE papers that show similar types of clinical effects become meaningless and suggests that there will be little translational value of many EAE studies in predicting efficacy/biology in humans.
But we know this, Don't we?
P.S.S. If you are a scientist, this is surely a good Journal Club paper as it should teach you a lot on the need to read around papers to understand them and also teach you about data analysis (stats tests used in paper are all wrong as well:-0) and interpretation.
Maybe I have it all wrong