Saturday, 23 September 2017

How does Daclizumab work? Can animal studies tell us

Bhopale MK, Hilliard B, Constantinescu CS, Phillips SM, Rostami A. DAB389IL-2 recombinant fusion toxin effect on lymphocyte- and macrophage-producing cytokine subpopulation cells in experimentally induced demyelinating disease in mice. Immunopharmacol Immunotoxicol. 2017 :1-12

CONTEXT: We have reported previously that DAB389IL-2 recombinant fusion toxin targets IL-2R bearing CD4+ cells, and suppresses demyelinating disease in acute (A) - and chronic (C) - experimental autoimmune encephalomyelitis (EAE) animal models of multiple sclerosis.
OBJECTIVES: The present study was undertaken to investigate the effect of DAB389IL-2 treatment on various cytokine-secreting cell populations in A-EAE and C-EAE mice.
MATERIALS AND METHODS:The effects of DAB389IL-2 at doses of 200-, 800-, or 1600 kU administered i.v. on days 11-13 and 15 on the clinical score and cytokine-secreting cell populations were examined using flow cytometry.
RESULTS:C-EAE mice treated with 1600kU DAB389IL-2, but not A-EAE mice treated with 800 kU had significantly reduced disease. The CD3+CD25+ sub-population in spleens and spinal cords of A-EAE mice treated with 800 kU DAB389IL-2 a was increased, whereas in C-EAE mice treated with 1600 kU this population was increased. DAB389IL-2 treatment reduced CD3+CD4+, CD3+CD8+, CD4+CD8+, CD3+IL-2+, CD3+IFN-γ+ and CD3+TNF-α+ T cell subpopulations in the spinal cord in A-EAE, and C-EAE mice on day 16. CD11b+ macrophages that were IL-2-, IFN-γ-, and TNF-α- positive were reduced in A-EAE mice. DAB389IL-2 treatment reduced CD19+ B-cells positive for IL-2 or CD11b+ in the spinal cord in acute and chronic disease. DAB389IL-2 treatment also reduced lymph node CD3+CD8+, CD4+CD8+, CD3+CD25+ populations on day 16, and lymph node CD3+IL-10+ and peripheral blood CD3+CD25+ populations on day 24.
DISCUSSION AND CONCLUSIONS: Our study demonstrates that DAB389IL-2 fusion toxin suppresses EAE in a dose-dependent manner, and alters inflammatory cell sub-populations during disease development

One of the flavors of the decade in terms of disease mechanisms has been the identification of Fox3P, CD25+, CD4 T regulatory cells. You can't see a paper these days where this is not the mechanism of action of EAE inhibition.

This is all well and dandy, but in terms of MS there is one nasty fact. 

People ignore from their world view. 

This is daclizumab. 

This antibody blocks CD25 which is the high affinity interleukin2 (T cell growth factor) receptor. Block this and natural killer subsets increase and MS goes away, but a problem is so do the T reg cells as these are depleted.

I was always of a mind-set that that the inhibitory effect is not surprising because the action of daclizumab is because it is simply killing or blocking activated T cells and have more recently switched to thinking it is because it kills activated memory B cells. The NK story is a smoke screen. ProfG disagrees he thinks that this creates and better anti-viral response, due to more NK cells.

However T regs are dogma. They are there to stop autoimmunity developing, which I totally understand. However, one question I have always posed is why would we want to spend a lot of effort to generate an immune response, outside the control of T reg control to give you life long protection, only to have it reeled back in by T regs. 

Since T regs were discovered, most studies give the interleukin 2 blocker before disease induction and low and behold it blocks T reg function and autoimmunity gets worse.....Hurrah for dogma.

What happens when this is done after disease has become established?

I have often thought of doing this for a student project, we could by the antibody with their lab expenses and see what happens I predicted nothing or it makes EAE better because EAE is driven by T cells and the blocker blocks activated T cells. 

Now I have found a few examples where people block the CD25 molecule after disease has developed and disease gets hoorah for dogma again. 

However I have also seen studies where cladribine stops mouse EAE, however someone forgot to tell the authors that cladribine does not work properly on rodents (don't believe me ask Merck..I did and they should know) and is not T cell immunosuppressive. This could be deduced by reading the early cladribine studies that showed that rodent metabolise cladribine in a different way to humans. We knew this years ago (experiments done in 2004). 

So on first read of the manuscript I thought has this had been done
and it didn't block the early acute phase but blocked the chronic phase, but on re-read whilst writing this....I must say uuurgh,

In this study they used a toxin linked to interleukin2 so when it binds to the the CD25 it kills the target. Do this and Eh.....the number of T reg cells increases but T cells decrease and EAE is reduced so hooray dogma persists.  

Should I do that study project?


  1. I will be naive here.
    The different drugs that work good on MS do not seem to "agree" on which T cells do the harmfull job, but still seem to work somehow. The b-cell therapies have merged the treatment of most autoimmune diseases, but we have not concluded that all a.d. have the same initiation, and sometimes they are even inherited. Could it be that the problem is a balance problem rather than a memory problem? This is why the different drugs work? Because they re-balance T cells but not totally successfully and even still this is the way HSCT works, not by errasing the memory, but reborning all the types of T-cells?

    Based on the T cell exhaustion theory of EBV (

    1. "Based on the T cell exhaustion theory of EBV"

      22 years ago Tcells were used in children's leukemia..but it's just now being used in MS..11 patients treated in 3 years..typical..20 years for Ocrevus and 25 years for Cladribine to become available

      "To bridge the gap, Rooney and her team rescued the T cells removed from the donor marrow and separated out the ones that could specifically attack EBV. They then grew these ‘EBV killer T cells’ in the lab until they had sufficient numbers to inject into the children following their transplant."

      Why have none of these EBV experts ever guest posted on on EBV vaccine is being done in u.k.

      "The idea seemed simple – if gaps in the immune system were causing the cancer to develop, could we turn the tables and use the immune system to strike back at the cancers?"

    2. Very interesting article. The Atara Bio Ata 188 works somehow towards that direction.

      We are talking the rotten spare bones of cancer sold in the price of bon fillet. And the fact that autoimmune diseases are split between neurologists, cadriologists, pathologists etc is not helping at all. Whether it is caused by a virus or it transforms to a genetic deficit, we must find the theory that connects them. B cell therapies and HSCT just add to that direction.

    3. Have a read of this

      They are all interlinked.

  2. Because, in our work, the recovery of leuko-
    cyte and T cell subset absolute numbers was not associated
    with reemergence of inflammatory disease activity, other
    changes in the immune systems must be considered to ex-
    plain the observed disease relapse-free interval for the entire
    2-yr posttherapy follow-up.
    Our data show that a thymopoietic pathway of T cell
    regeneration is activated in MS patients treated with HSCT
    and leads to immune renewal. The evidence supporting this
    notion is as follows: (a) the increased frequency of pheno-
    typically naive CD4

    T cells; (b) the decreased frequency of
    central–memory T cells; (c) the expansion of phenotypically
    identified CD4

    RTEs; (d) the increased TREC levels
    in the CD4

    subset and the adequate recovery of CD8
    TRECs in spite of extensive peripheral division in this sub-
    set; (e) the overall improved clonal diversity of the TCR
    repertoire; and (f) the extensive clonotypic renewal show

    1. First question is did you look at memory B.
      In those with memory B was the expansion associated with EBV reinfection.

      Next again we are focused on t cells and the blood, they blood can be empty of cells but relapse occurs in arthritis.

      Studies generally fail to show or relation of blood cell levels and disease reactivation.

      Are the cells left in the brain important.

      Prof Coles aimed to get cells out of the thymus this was to stop secondary autoimmunity and it failed and perhaps made things worse.

    2. The default after t cell depletion is expansion of memory cells followed by thymic repopulation in hsct there are no memory t cells to repopulate so is it surprising that the normal t cell development pathway occurs?

    3. As far was i know ebv reactivacion in Hsct patients is linked with Ptld
      And you have to have preempting theraphy if you test positive with rituxan
      You are at risk of you do Allogenic transplant and use a T cell depleter longer
      Anyway i think in the most ablative setting is a rare condition
      Thanks for reply

    4. Post-transplant lymphoproliferative issues has been more evident in people with MS.

    5. "Post-transplant lymphoproliferative issues has been more evident in people with MS"

      With all do respect Dr:Baker but thats not correct
      Allogenic hsct for any conditions ( ms included) are more at risk solid organ transplant (Heart, kidney lung) and if you use continuos immusupression like atg

      Epstein-Barr Virus Reactivation and Lymphoproliferative Diseases

      The risk of EBV reactivation is increased in patients receiving high doses of ATG in the conditioning regimen [242], and may lead to potentially life-threatening lymphoproliferative disorders [242]. Currently, this complication can be prevented through close and regular monitoring of EBV reactivation and use of pre-emptive therapy with rituximab. A low dose of anti-CD20 antibody (rituximab) administered on day +5 after HSCT may also prevent this complication [243]. The risk of EBV reactivation after HSCT should be appreciated, and prevented or treated appropriately.

      Risk factors for EBV-PTLD
      Risk factors for developing EBV-PTLD can be considered
      as existing pre-20,24,33-35 or developing post-transplant7,34-37
      (Table 3). Importantly, assessing the risk of EBV-PTLD isdependent on the HSCT context with potentially complex
      interactions between the primary hematological malignancy,
      HSCT procedure, source, and other factors. Given
      that the risk of EBV-PTLD is predominantly related to the
      degree of T-cell depletion or impairment, this should be
      regarded as the principal risk factor (AIIu). Strategies that
      deplete T cells from the graft increase the risk of EBVPTLD.
      CBT confers an intrinsic risk for EBV-PTLD because of
      T-cell naïvety related to the HSC source. A high incidence
      of EBV-PTLD in both pediatric and adult patients after
      CBT, following reduced intensity conditioning regimens
      using anti-thymocyte globulin (ATG) or alemtuzumab
      (anti-CD52), has also been reported.28,39 This likely reflects
      both the delayed recovery of EBV-specific CTLs after such
      transplants, alongside the persistence of recipient-derived
      B cells. The use of alemtuzumab during conditioning in
      other types of HSCT can also be regarded as a risk factor
      for EBV-PTLD.27,29 There appears to be a dose-dependent
      risk with the in vivo use of ATG in children,40 which is
      probable also in adults. Current data do not suggest any
      significant differences between children and adults with
      respect to epidemiology and risk factors.
      Patients undergoing HSCT can be classified for the risk
      for EBV-PTLD as low risk (auto-HSCT), standard risk
      (MFD allo-HSCT without risk factors, haplo-PTCy-
      HSCT), and high risk (MFD with at least one risk factor,
      MUD/MMUD, alternative donors including CBT).

      If you go to any pubmed ,or blood ,search for Autologuos hsct

      for ms its very very dificult to find any paper regarding ptld and ebv reactivation
      Have talk to 2 major hematologists here in my country and in their career have had no single case of ptld
      Also an Indian hematologist Dr Rahul Bhargava that does Hsct for ms and other conditions as this to say:
      After doing 500 transplants including haploidentical and mud transplants we are yet to see a ebv reactivation in even a single pt.

    6. Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell
      transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative
      disease following T-cell–depleted SCT

      BLOOD, 15 AUGUST 2001 z VOLUME 98, NUMBER 4

      The only 3 case i know for Ptld of hsct for ms and sse are this:
      Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) occur with
      congenital or acquired immunodeficiency disorders but are most often seen in
      immunosuppressed patients after solid organ or allogeneic hematopoietic stem cell
      transplantation (HSCT). A lymphoproliferative disorder occurs when there is a failure to
      control proliferation of EBV-infected B cells because of impaired T-cell immunity. EBVassociated
      posttransplantation lymphoproliferative disorder (PTLD) is an infrequent
      complication after high-dose chemoradiotherapy and autologous HSCT for malignant diseases.
      In 1 single-center study, it was reported that there were 4 (0.7%) cases among 612 patients
      reviewed [1]. There have been other infrequent case reports of EBV-PTLD after autologous
      HSCT, but none to date have been reported after HDIT for patients with severe autoimmune
      diseases [2–8]. As a result of the modifications to the high-dose chemoradiotherapy, including
      lymphocyte depletion by CD34 selection and the addition of ATG before and after HSCT,
      patients may be profoundly immunosuppressed. We report here 2 cases of EBV-PTLD after
      autologous HSCT that were associated with an increased degree of immunosuppression after
      rabbit ATG was substituted for equine ATG in the HDIT regimen for patients with a positive
      skin test to the equine product

      Biol Blood Marrow Transplant. 2003 September ; 9(9): 583–591.

    7. "That's not correct".....Maybe ProfG can comment on this and the London experience

  3. With regard to CD4þCD25þ Foxp3þ Treg cells, it
    has been reported that the number of this lymphocyte
    subset increased in SLE patients who achieved complete
    remission after AHSCT. In post-transplant patients, both
    CD4þCD25þ Foxp3þ Treg cells and an unusual CD8þ
    Foxp3þ Treg cell subset have been shown to return to
    levels seen in normal subjects, accompanied by almost
    complete inhibition of pathogenic T-cell response to crucial
    peptide autoantigens from histones in nucleosomes,
    the major lupus autoantigen from apoptotic cells (18).
    Scand J Rheumatol 2012;41:110–115

    1. After hsct the cells are gone so expansion of t regs to stop further autoimmunity makes sense.

      Nice to see you have CD8 in the mix

  4. Despite a rapidly accumulating clinical experience with autologous stem cell transplantation (ASCT) as a treatment for severe refractory autoimmune disease, data on the mechanisms by which ASCT induces immune tolerance are still very scarce. In this study it is shown that ASCT restores immunologic self-tolerance in juvenile idiopathic arthritis (JIA) via 2 mechanisms. First, ASCT induces a restoration of the frequency of FoxP3 expressing CD4+CD25bright regulatory T cells (Tregs) from severely reduced numbers before ASCT to normal levels after ASCT. This recovery is due to a preferential homeostatic expansion of CD4+CD25+ Tregs during the lymphopenic phase of immunereconstitution, as measured by Ki67 and CD44 expression, and to a renewed thymopoiesis of naive mRNA FoxP3 expressing CD4+CD25+ Tregs after ASCT. Second, using artificial antigen-presenting cells to specifically isolate self-reactive T cells, we demonstrate that ASCT induces autoimmune cells to deviate from a proinflammatory phenotype (mRNA interferon-γ [IFN-γ] and T-bet high) to a tolerant phenotype (mRNA interleukin-10 [IL-10] and GATA-3 high). These data are the first to demonstrate the qualitative immunologic changes that are responsible for the induction of immune tolerance by ASCT for JIA: the restoration of the CD4+CD25+ immune regulatory network and reprogramming of autoreactive T cells.

  5. Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients
    with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the
    immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive
    conditioning regimen, and re-infusing the autologous haematopoietic cell product. ‘Non-myeloablative’ conditioning regimens
    to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial
    with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory
    stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the
    changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained
    from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years.
    We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multicolour
    fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution posttransplantation
    we observed significant though transient increases in the proportion of CD4+FoxP3+ T cells and CD56high
    natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+CD57+ cytotoxic T cells
    were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a
    CD161high proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization

    Division of Brain Sciences, Department of Medicine, Imperial College, London, UK

    Non-myeloablative autologous haematopoietic
    stem cell transplantation expands regulatory cells
    and depletes IL-17 producing mucosal-associated
    invariant T cells in multiple sclerosis

    1. During immune reconstitution posttransplantation
      we observed significant though transient increases in the proportion of CD4+FoxP3+ T cells and CD56high
      natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+CD57+ cytotoxic T cells
      were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a
      CD161high proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization

      Division of Brain Sciences, Department of Medicine, Imperial College, London, UK

      Non-myeloablative autologous haematopoietic
      stem cell transplantation expands regulatory cells
      and depletes IL-17 producing mucosal-associated
      invariant T cells in multiple sclerosis


    2. Are we doing experiments to prove Treg function in MS or should be be doing experiments in a way to try and disproove the Treg hypothesis

  6. I think we have become too obsessed with EBV, that we lose the bigger picture. It might seem like a short-cut to just observe and fight the results of EBV, and there sure is a memory matter in all that but is it the primarily problem? Because we are kinda making circles with the same state of mind.
    In the first paper the issue is the matter of exhaustion not the exhaustion in EBV necessarily.

    1. All I say is that is a phaenomenon that deserves further investigation. You had posed yourself important questions when you had posted this article that need to be addressed, and not necessarily only around EBV. The EBV theory leaves many holes unaswered about the a.d. and their heredity. Are the memory cells passed through the blood to the child? I think this is unlikely.
      We suspect from the Lemtrada research that when B cells proliferate before the Ts create autoimmunity, but we have no certain answer to what is the defect of the T cells in general that can cause this imbalance. So, a wild guess is exhaustion (from any cause). And then maybe imbalanced proliferation of other parts of the white cells. The great variery of expression of a.d. (especially through heredity cases) need a wide theory with the element of "luck". Also, the fact that what is helpful to one a.d. is harmful to another -which is often the case- is not adequately answered too.
      As I said wild guesses to my unanswered questions. Thanks for your reply.

    2. Can I continue my blabing? Its just a post in a blog, no animals get harm :/
      What are the lessons we learn from HSCT? It is that is works good on new patients with RRMS but not that good at SPMS. There is a dark factor that always despays at the papers that says "Fewer prior DMTs", to have good results.
      The fact that immune regulation is needed at any point of the disease is explained by profG, so I don't need to. What long term effects can DMTs can have on the immune system is reported at the article about DMF.
      There are two separate things. One is the the immune system activity and the other one is the damage caused by it.
      So why it gets complicated so much at HSCT? Why the advanced patients cannot sustain stability whereas the new ones can?
      Is it because the immune system (and not only the CNS) is beyond repair? Is it because it is unable to sustain the "exhausted" parts of the immune system long enough even if the memory cells are removed?
      Another reason could be that the stem cells that are re-imported are damaged at that point (there is an article about the stem cells quality after chronic inflammatory disease), so a trial with just chemotherapy and long term isolation would worth the try. Thanks.

    3. "So why it gets complicated so much at HSCT? Why the advanced patients cannot sustain stability whereas the new ones can?"
      A cpuple of reasons, firstly in "advanced" pwMS the spare capacity of the nervous system has been used up, so every new neuron that dies is significant and those that are left may have to work harder to maintain function. Also HSCT is not going to alleviate the problem of the population of activated microglia in the brain and spinal cord, which continue to do their stealthy damage for a considerable time after new lesions have been prevented. We need to find a way of turning these "hot" microglia off to try and restore balance.
      There are ideas on how to do this.

    4. That is certaintly a reason (the toxicity nerve damage), but wouldn't it be apparent soon enough and not around the 4 year of stability (which is often the case) and even without the appearance of improved EDSS? And why others with very high EDSS have success after the 5 year (a time that marks sustained results)? Sure it is individualised but we need the reasons.

      "Also HSCT is not going to alleviate the problem of the population of activated microglia in the brain and spinal cord,"

      "Disproof of inflammation in the CNS
      If we can’t prove that the lymphocytes attacking the brain and spinal cord are gone and aren’t satisfied with the artificial situation of studies on
      ex-vivo lymphocyte cultures, are there other ways to investigate inflammation in vivo?
      The interstitial fluid of the brain is in direct connection with the CSF through the glymphatic system,
      a recently defined brain-
      wide paravascular pathway that facilitates efficient clearance of so lutes and waste from the brain.
      Thus soluble mediators of inflammation in the CSF mirror local inflammation in the brain. A disproof of inflammation in the CNS should require demonstration of normalization in the levels of such soluble factors.
      Measurement of antibodies and cytokines should be ideal for this purpose. (...) No study demonstrating absence of inflammation in the CSF after HSCT has been made so far (...)"
      So yes and no. Still unproved.

    5. Can I continue my blabbing....No thanks. Move the conversation to unrelated blogger comments.

    6. I refer you to MouseDoctor's comment below.

  7. Luis if you see this give us your lights!

  8. This post is about daclizumab and not hsct. If you have something useful to say about daclizumab if not move your conversation to unrelated blogger comments please.

    1. I apologize. It wasn't supposed to be about HSCT, rather than Tregs idea in general. Thanks.


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