Saturday, 9 December 2017

Relapse without cell depletion in the blood

As you may be aware I have been banging on about the importance of B memory cells for most of the year, but it has been an uphill struggle to get this view accepted. 

However, response to therapy creates a powerful piece of insight that frankly can't be ignored. But I meet Ostriches ever day.




Ocrelizumab and rituximab have created a potential problem for the understanding of MS and other autoimmune diseases. 

Here you have a therapeutic antibody that depletes B cells, yet is very good at blocking relapsing MS.

Now you could take the view that B cells make toxic molecules and can do the damage, without having to evoke any T cell involvement.


Alternatively, you can take the view that it is the CD20 positive T cell population that does the trick. You put faith in this (flimsy) argument and accept that depletion of a few T cells is all important, whilst rejecting the fact that massive depletion,  
with CD4 monoclonal antibody, of T cells has had only marginal effect 

However, you can have the "love-in" view and say that you are blocking antigen-presenting cell function of B cells to T cells. 


This is the easy-option view that it is both cells and of course B cells probably wont exisit without some T cell involvement. However this is not the point. Many people are not ready to accept a different world-view. and this the stock answer when you ask "How does ocrelizumab work"

This is because people have spent their careers banging-on about T cells and why would they want to contemplate that "their life's work may not be that important".


For this reason, we have had immense difficulty publishing any alternative hypothesis.

However does the antigen presenting function really hold? We know B cells can present antigen, a Wellington Boot can present antigen if it expresses MHC....only joking but this feature was shown in the 1980s and people wasted years on it.


Staining of T and B cells and dendritic cells in human lymph nodes

Antigen presentation, typically occurs in lymph nodes. In tissues it will be via macrophages. In the figure above you can see that B cells line the outside of the lymph node.  This is called the cortex and they form follicles, which is where memory B cells are generated. There are a few T cells in the follicles to help them do this.

But most T cells are found in the paracortex. You can see them in the bottom left in the figure above (stained brown).  So when we look at where the B cells are (top left above), they aren't in the same areas as T cells (The blue is the nuclei of cells). So are memory B cells really important as antigen presenting cells?

The medulla is where the cells exit the lymph glands.

The important cells that present antigen to T cells are called dendritic cells which can be seen in the right on the figure above. They are common in the paracortex and have evolved to present antigen to T cells.  So why evolve B cells to do that part?
One referee said that we do "not prove our mechanism that memory B cells are important for disease activity in MS"...meaning reject the paper and the idea . 

My response to the Editor (that fell on deaf ears) is that the mechanism of virtually every human treatment is unproven. We are treating people...not mice and so it is not possible to prove that drug X works in any particular way.


The best case example could be natalizumab. 


We believe it works because it blocks CD49d on T cells from binding to VCAM-1 (CD105) on the blood vessel. It has a clear working mechanism, but does it work via the T cells, the B cell or something else?


Yes the latter can be the case, because when the action of natalizumab was first mentioned by Ted Yednock and Colleagues in Nature many years ago they showed that natalizumab blocked the binding of U937 cells to inflamed blood vessels. 


These cells are most like a monocyte (macrophage), so they showed an activity on the macrophage, not the T cell.


So back to the referee and they said that we "didn't show that memory B cell correlate with disease activity" so we haven't proven our mechanism. Yes this is true we had not shown this (yet), but was it going to happen?


It had been shown with both cladribine and alemtuzumab that disease activity does not correlate with T cell levels, so if MS was T cell mediated, this approach does not work. if this has to happpen to show which cells are important, then you conclude that MS is not a T cell problem


So should we say it can't be T cells.

The Editor was not bothered by this protestation and upheld the rejection. However, this case report (below) explains that problem.


Zecca C, Antozzi CG, Torri Clerici V, Ferrazzini M, Mantegazza RE, Rossi S, Gobbi C. Severe multiple sclerosis reactivation during prolonged lymphopenia after dimethyl fumarate discontinuation. Acta Neurol Scand. 2017. doi: 10.1111/ane.12882. [Epub ahead of print]

BACKGROUND: Delayed-release dimethyl fumarate (DMF) treatment can be associated with reduced lymphocyte and leucocyte counts, which might persist after DMF discontinuation.


CASE PRESENTATION: We report the case of a patient with severe disease reactivation despite prolonged lymphopenia after DMF discontinuation. We describe the frequency and impact of prolonged lymphopenia after DMF discontinuation at two tertiary MS centres. A 36-year-old female patient with multiple sclerosis was switched to DMF after 14 years of treatment with interferon beta-1a. DMF was suspended after 4 months because of persistent lymphopenia for 3 months. Six months later, the patient had a severe relapse with multiple enhancing brain lesions at MRI although lymphopenia was still persistent. Haematological assessment excluded other causes of lymphopenia, which was evaluated as a probable iatrogenic complication of DMF. The patient was treated with i.v. methylprednisolone 1 gr daily for 3 days with clinical recovery.


CONCLUSIONS: Prolonged lymphopenia after DMT discontinuation does not protect against disease reactivation. Starting a new immune therapy should be balanced against the option of a "wait and see." A different immunotherapeutic strategy such as an anti-B therapeutic approach could be considered.



In this person, there are low levels of lymphocytes due to the influence of dimethyl fumarate, yet they had a relapse.

So there are people developing MS attacks, whilst their blood is relatively empty of white blood cells. 

Now, it is probable that it is a just small population of cells that are causing the problems. However, the source of the problematic cells is unlikely to be from within the blood. 

There is only about 1-2% of the lymphocytes within the blood at any one time and the problem cells are likely to come from either primary (bone marrow) or secondary (lymphoid tissues) tissues or from within the CNS itself. So there is will be individuals that have few cells in the blood but can develop relapse. We have seen this with people developing arthritis relapse when there are few CD19+ B cells in the blood. However, there are other people with arthritis where B cell numbers in the blood act as a biomarker for disease activity.

Can this happen in MS?

Maybe, maybe not we just have to look and just have to look in the right way. Pharma probably has the data to give the first response. 

If there is biomarker activity as occurs in a number of other conditions this will be great, if not we have to home-in further to identify the disease-causing cells. 

On a practical point, it questions about waiting too long for cell numbers to return before switching to an alternative treatment after DMF, as there are a number of people who do not repopulate well after DMF. Maybe ProfG or DrK will tell you what we do.

25 comments:

  1. Ok. Playing devil's advocate. After alemtuzumab. Why doesn't the rapid repopulation of b cells reactivate ms disease. Given ther3 are no t reg cells to moderate the flammation? What if the disease is driven by b cells then given the life span of plasma cells. You will only need to give ocrelizumab tillnthose plasma cells are dead. Is this seen in practice. If so you conclusively prove your case. Regardless of opinion on t cells.

    ReplyDelete
  2. Nice post .good reasoning Md

    No doubt Memory b cell and anti cd20 theraphy surrely are part of the

    story but are they the full story ?

    A number of points of emphasis are highlighted in this
    case report. First, a secondarily progressive course developed
    during chronic B-cell depletion therapy with rituximab,
    an agent that appeared to have been effective in
    this patient during the relapsing phase of MS. Second,
    CSF analysis by flow cytometry demonstrated that levels
    of B cells were extremely low in the intrathecal compartment,
    an observation in agreement with earlier reports
    indicating that rituximab therapy can successfully deplete
    B cells from the CNS.4 Third, despite B-cell depletion,
    there was evidence for ongoing CNS inflammation: (1)
    OCB were present in CSF even after nearly 7 years of
    anti-CD20 therapy, indicating active robust intrathecal
    antibody synthesis, most likely by long-lived, CNS resident,
    plasma cells; (2) Clonally related B-cell populations
    were detected on both sides of the blood–brain barrier,
    where they might contribute to ongoing CNS damage; (3)

    doi: 10.1002/acn3.377

    Should this happen had he start Ritux earlier?

    Ritux. Also reduces not only b but t cells in the csf (go figure)

    An unexpected and intriguing finding was that CSF Tcells were reduced post-treatment in most
    subjects, with an overall decrease of 55%. CSF Tcells were reduced by more than 50% in 8/15
    (53%) subjects, decreased by lesser degrees in four, and increased in only three subjects,
    yielding an overall group decrease of more than 55% after treatment. The reason for this
    reduction is not known. One possibility is that B cells of CNS myelin specificity “home” to
    the CNS and in so doing alter the blood–brain barrier or secrete chemokines that recruit T cells
    as well. For example, B cell receptor triggering induces human B cell production of two T cell
    chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta) and MIP-1 alpha
    (Krzysiek et al., 1999)

    J Neuroimmunol. 2006 November ; 180(1-2): 63–70.

    AAN2017. B cells remain in the brain after rituximab treatment

    CNS autopsy verified severe multifocal brainstem and cerebral periventricular white matter plaque burden. Microscopically, several plaques manifested conspicuous perivascular lymphocytic cuffs, predominantly CD3+ T cells (mixed CD4/8) and macrophages, but individual CD20+ cells could still be identified within the parenchyma and perivascular cuffs.

    So preventing relapses is a good part of the story ,but does it

    prevent from progressive disease?

    Should Ritux treatment been given right after diagnosed impact, those

    plasma cell formation in the csf?


    Also those lessions are full of t cells shouldn´t we try to

    understand why?

    Obrigado

    Luis

    ReplyDelete
    Replies
    1. "They the full story"
      I doubt it..I don't think we have said it is the full story

      "doi: 10.1002/acn3.377"
      Yes we can build this into our ideas but at present I am talking about relapsing episodes.

      "Should this happen had he start Ritux earlier?"
      I don't know, but am hopeful that it will if we start early enough. This is the approach we should use.

      "Ritux. Also reduces not only b but t cells in the csf (go figure)"

      A you've fallen into the cause and effect trap. I'll be back

      Delete
    2. "Ritux. Also reduces not only b but t cells in the csf"
      J Neuroimmunol. 2006 November ; 180(1-2): 63–70

      You give anti CD20 mAb and T cell numbers drop, therefore anti-CD20 is depleting T cells. This paper was cited by a referee as to why MS was a T cell problem. Whilst this may be the case you cannot use this paper as evidence.
      If the antibody is doing to deplete cells is going to be when the antibody administered. However, the work in this paper looks 6 months after the antibody was administered. Therefore you are not seeing the cause of the antibody administration, but the consequence of the antibody administration. The T and B cells are present because of disease. The antibody gets rid of disease, so the cells will have disappeared. That T cells disappear is not a surprise and does not say that the antibody acts directly on the T cells. If we looked in these individuals there would be no-gadolinium enhancing lesions because disease has gone away, but if I said this shows that the antibody direct depletes gadolinium because it was binding to gadolinium you would say are you crazy? Gadolinium is an inert element that is a contrast agent designed to show leaking blood vessels and the antibody doesn’t cause production of gadolinium, it is simply that gadolinium was injected into a vein 6 months after the antibody was administered before you went in a scanner. But because there is no disease, there is no gadolinium in the CNS. Likewise there would be no T cells there.
      All too often we have this cause and effect, but it is a consequence. If were interested in looking at the presence of cytokine in the brain and it was not there because the cells that produce the cytokine are not there because there is no disease, because the drug works. But to say that the drug directly blocks the cytokine is a non-sense.

      "AAN2017. B cells remain in the brain after rituximab treatment"

      Rituximab does not get into the brain and so will not deplete the B cells there.

      "So preventing relapses is a good part of the story ,but does it
      prevent from progressive disease?"

      I am talking relapsing MS, we will create another scenario and mechanism for how progressive disease unfolds. However one thing at a time, but it is fair to say this will not be caused by CD4 T cells

      Should effective treatments be started at diagnosis? Yep

      "Those those lesions are full of t cells shouldn´t we try to
      understand why?"

      If we look at an MS lesion the most prominent cells are macrophages, not T cells, remember MS is not EAE. Also CD4 is not necessarilty the promient cell type but in EAE it is. MS is not EAE

      Many of the cells in lesions are recruited there by the adhesion molecule profile they express and the chemokine receptors they express. they may be bystanders. If we transfer EAE with a T cell most of the cells in the lesion are not the pathogenic cells transferred.

      We know some of the cells in lesions are EBV directed CD8 T cells.

      I am sure T cells have their part to play but are they top of the hierarchy for treatment?

      Delete
    3. Thanks in advance for taking the time to reply

      Totally agree about cause-consequence scenarios

      But correct if i´m wrong here, but gadolinium will serve only to tell ,you have an disrupted bbb(immune cell trafficking between blood and cns)

      So this patients have had their b cell depleted and a close bbb 6 months after( not saying that ritux close the bbb)

      "If we look at an MS lesion the most prominent cells are macrophages, "

      "Interest in CD8+ T cells and B cells was initially inspired by observations in multiple sclerosis rather than in animal models: CD8+ T cells predominate in multiple sclerosis lesions,"
      http://www.thelancet.com/pdfs/journals/laneur/PIIS1474-4422(15)00313-0.pdf

      Abstract T cells are considered pivotal in the pathology
      of multiple sclerosis (MS), but their function and antigen
      specificity are unknown. To unravel the role of T cells in
      MS pathology, we performed a comprehensive analysis on T
      cells recovered from paired blood, cerebrospinal fluid (CSF),
      normal-appearing white matter (NAWM) and white matter
      lesions (WML) from 27 MS patients with advanced disease
      shortly after death. The differentiation status of T cells in
      these compartments was determined by ex vivo flow cytometry
      and immunohistochemistry. T-cell reactivity in shortterm
      T-cell lines (TCL), generated by non-specific stimulation
      of T cells recovered from the same compartments, was
      determined by intracellular cytokine flow cytometry. Central
      memory T cells predominated in CSF and effector memory
      T cells were enriched in NAWM and WML. WML-derived
      CD8+
      T cells represent chronically activated T cells expressing
      a cytotoxic effector phenotype (CD95L and granzyme....



      "Three
      main findings are reported. First, T cells in MS lesions
      are predominantly CD8+
      TEM
      cells expressing a cytotoxic
      effector phenotype indicative for local antigenic stimulation.
      Second, T cells cultured from WML in four of nine
      MS patients recognize autoBLCL. This reactivity was profound
      in TCL generated from AL and mAIL. Third, no
      substantial T-cell reactivity was observed towards seven
      human cMSAg in CSF- and brain tissue-derived TCL of
      MS patients expressing the major MS-associated HLA risk
      alleles HLA-A*03, -DRB1*15 and -DRB1*13"

      During the past decade, the focus on the role of CD4+
      T cells in MS pathology has shifted towards CD8+
      T
      cells; the most abundant T-cell subset identified in active
      WML of MS patients [12, 19]. The antigen specificity
      (e.g., autoantigens and/or EBV proteins) and the potential
      role of intra-lesional CD8+
      T cells (cytotoxic or regulatory)
      in MS pathology is still a matter of debate [3,
      22, 41, 54].

      (doi:10.1007/s00401-017-1744-4)

      Mind you,i am not a t cell taliban...Loolllllllllllllll

      :)

      Cheers

      Obrigado
      Luis

      Delete
    4. So if you are arguing for CD8 you are already arguing that the CD4 idea is wrong and the EAE mafia is out of touch.

      "Interest in CD8+ T cells and B cells was initially inspired by observations in multiple sclerosis rather than in animal models: CD8+ T cells predominate in multiple sclerosis lesions,"


      Yet we are still putting so much effort in to CD4 Th17 cells as a mechanism.

      autologous EBV-transformed B-cells

      AL and mAIL. Active lesion & mixed active/inactive lesions (mIAL),
      MS-associated autoantigens (cMSAg)

      Delete
    5. But correct if i´m wrong here, but gadolinium will serve only to tell ,you have an disrupted bbb(immune cell trafficking between blood and cns)

      Yes you are right but I only selected gadolinium to show how we make the direct cause and effect connection when there is not one.

      Delete
    6. Eae mafia
      Lollllllllllllllllllllllllllllllllllllllk

      Delete
  3. Dear Devil

    Please read
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474520/

    You have fallen into the T cell MSologist trap.

    The CD19 population of B cells is what you are referring to and yes they are back to normal within 3-6 months and abnormal for 6-12months and yes you are right there are no T regs to control the B cells...thats why you get autoimmunity after alemtuzumab.

    However when you see that the response is a composite of B cells you will see that the memory B cells are depleted for the whole two years and more. The cells repopulating are immature and naive B cells not the memory B cells

    Please read

    June 12
    http://multiple-sclerosis-research.blogspot.com/2017/06/newsflash-how-alemtuzumab-works-and-why.html

    June 13
    http://multiple-sclerosis-research.blogspot.com/2017/06/alemtuzumab-kills-b-cells-not-really.html

    June 15
    http://multiple-sclerosis-research.blogspot.com/2017/06/why-does-alemtuzumab-cause-b-cell.html

    "What if the disease is mediated by B cells then given the life span of plasma cells you only need to give ocrelizumab till they are dead"

    Problem is that ocrelizumab kills CD20+ cells and plasma cells do not kill plasma cells. They are long-lived and essentially can last a life-time this is what immunity is..so do you have to take it for ever.

    Next problem is these plasma cells may be in a niche in the brain and not targeted by orcrelizumab in the blood. Therefore, antibody responses can persist after ocrelizumab. However some antibody responses are reduces so a grey area as usual.

    ReplyDelete
  4. Thanks MD. I'm fan of your b cell theory. But good to ask questions. Never good to follow any idea in blind faith by deferring to someone else's intellect or spirituality. Depending on the context. You have my thumbs. Err for its worth.

    ReplyDelete
  5. Re: prolonged lymphopaenia after dmf short course discontinuation.
    Yet another report of this! Pity no trial data showing what happens when dmf discontinued. Pretty rubbish drug if it leaves people with total lymphocytes <0.5 for years after and ineligible for treatment with anything else. So what's going on here? Merck anything to say? I happen to have n=1 data to show it is CD8 subset that are almost completely depleted. Suspect I am not alone. For a moderately effective drug, ie only moderate memory B cell deletion, to be left with next to no CD8 T cells for (the rest of your life?) and ineligible for any further treatment is a hefty price to pay.

    ReplyDelete
    Replies
    1. Merck anything to say?

      I suspect they would say why use this drug use mavenclad:-).
      Remember DMF is a Biogen Drug not a Merck drug.

      If CD8 is near zero and people relapsing still, it would say something about CD8s. You are correct that CD8 depletion is a well noted effect of DMF

      Delete
    2. Oops sorry meant Biogen ;-)
      Merck would doubtless say have Mavenclad :) even if eligible not much chance of lymphocytes ever returning to the 0.8 required to start :(

      Delete
  6. In the interest of balance read this

    Phenotype and genotype of interfollicular large B cells, a subpopulation of lymphocytes often with dendritic morphology Teresa Marafioti, Margaret Jones, Fabio Facchetti, Tim C. Diss, Ming-Qing Du, Peter G. Isaacson, Michela Pozzobon, Stefano A. Pileri, Amanda J. Strickson, Soo-Yong Tan, Fiona Watkins and David Y. Mason Blood 2003 102:2868-2876; doi: https://doi.org/10.1182/blood-2003-03-0692

    ReplyDelete
  7. I feel your struggle to publish your work. Hopefully another editor will see the light.

    However: Why the skepticism against B cells as APCs? I buy all you arguments for why memory B cells are probable mediators of pathology, but why not also look at this in light of HLA-DRB1*15:01 (and other) association?

    Memory B cells have been described to substantially upregulate their HLA-DR expression upon BCR stimulation, and hence been postulated to require BCR stimulation in order to work as APCs. I'm personally not entirely convinced that BCR stimulation is the only way B cells may function as effective APCs. More work needs to be done on how B cells endo-lysosomal pathways work and are regulated in human cells upon various inflammatory stimuli.

    ReplyDelete
    Replies
    1. Happy to accept B cells are antigen presenting, but we need to think about it.

      Have you asked whether the DR B1 variant is better at allowing EBV B cell infection along with CD21?

      Delete
    2. I will do a post on folicular T cells and if we look at the memory B they have co-stimulatory molecules required for antigen presenting cells so there will be cross talk, I'm sure. The anatomy of the spleen is of interest too.

      Delete
  8. Dr Hauser
    "We believe the relapsing phase is related to an autoimmune attack mediated by the adaptive immune system, namely T cells and B cells, whereas the cause of the progressive phase of MS is less clear, but may be due to some combination of an adaptive immune response similar to relapsing MS but locked behind a closed blood-brain barrier, plus activation of innate immunity, cells like microglia and activated macrophages that may secrete product toxic to both myelin producing cells and nerve cells."

    https://jamanetwork.com/journals/jama/fullarticle/2666528?utm_source=silverchair&utm_medium=email&utm_campaign=article_alert-jama&utm_content=olf&utm_term=121317

    Obrigado

    ReplyDelete
    Replies
    1. similar to relapsing MS but locked behind a closed blood-brain barrier... really...surely relapsing MS is all about unlocking the BBB so how is it similar?

      Obviously didn't read the smoldering lesion papers:-(

      Delete
  9. Ostriches, HSCT. Yeah, just can't relate. End sarcasm.

    ReplyDelete
    Replies
    1. Stuck record, boring, yeah, Why end sarcasm:-)

      Delete
    2. My broken record is no more broken than the scientists and neurologists who hang on to an ineffective, inadequate status quo. It's so sad, so hurtful and damaging and above all, unnecessary.

      Use me as 'entertainment' and the hsct clown all you want. Its highly effective, way more than dmd's, cheaper than dmd's and a disgrace the way people like you fight against it for self preservation purposes.

      Delete
  10. Maybe CD8+ T cells are more than one category. Maybe this is why DMFs long term depletion does not affect the evolution of MS. Because wrong CD8+?

    How do immune cells remember an infection or a vaccination so that they can spring into action decades later?

    When someone gets a vaccine or is exposed to a new infectious agent, cells that recognize the invader but had never have been called into action before - called naive cells - respond by dividing like crazy and developing infection-fighting functions. This creates a large pool of so-called memory cells, named for their ability to remember the specific infectious agent and respond effectively to repeat threats later. Over time, the large pool shrinks to a small number of long-term memory cells, which are primed to provide late protection. But scientists have debated how these memory cells are maintained and ready to strike for so long after the initial exposure.

    This study found that one way the pool is maintained for years after vaccination is through the development of several unique features. On the surface and through the actions of their genes, they look like cells that have never been exposed to an infection, but on their DNA the researchers found a fingerprint, called a methylation pattern, that identifies them as having been through battle as an infection-fighting cell, which are called effector cells.

    "These cells are like veteran soldiers, camped in the blood and tissues where they fight their battles, waiting for yellow fever to show up," said Hellerstein. "They are resting quietly and they wear the clothes of untested new recruits, but they are deeply experienced, ready to spring into action and primed to expand wildly and attack aggressively if invaders return."

    The extremely long life-span of the surviving memory cells allows them to specialize over time into a unique, previously unrecognized type of T cell. The long-term memory cells have some molecular markers that make them look like naive cells that have never activated, including a gene expression profile that looks like that in naive cells, yet have other molecular markers on their DNA of having gone through battle as effector cells.

    "These results make it clear that true long-term memory cells were once effector cells that have become quiescent," Hellerstein said. "This apparently keeps them poised to respond rapidly as new effector cells upon re-exposure to the pathogen."


    Read more at: https://phys.org/news/2017-12-cells-infections-decades.html#jCp

    ReplyDelete
    Replies
    1. Absolutely right CD8 are not a single population

      Delete

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