That damned, elusive EBV

Will this study get the field behind the EBV hypothesis once and for all? Or will it be another bun fight?




PLoS One. 2018 Feb 2;13(2):e0192109. doi: 10.1371/journal.pone.0192109. eCollection 2018.

Epstein-Barr virus is present in the brain of most cases of multiple sclerosis and may engage more than just B cells.

Hassani A, Corboy JR, Al-Salam S, Khan G.

Abstract


Multiple sclerosis (MS) is a chronic neuroinflammatory condition of the central nervous system (CNS). It is a major cause of neurological disability in young adults, particularly women. What triggers the destruction of myelin sheaths covering nerve fibres is unknown. Both genetic and infectious agents have been implicated. Of the infectious agents, Epstein-Barr virus (EBV), a common herpesvirus, has the strongest epidemiological and serological evidence. However, the presence of EBV in the CNS and demonstration of the underlying mechanism(s) linking EBV to the pathogenesis of MS remain to be elucidated. We aimed at understanding the contribution of EBV infection in the pathology of MS. We examined 1055 specimens (440 DNA samples and 615 brain tissues) from 101 MS and 21 non-MS cases for the presence of EBV using PCR and EBER-in situ hybridization (EBER-ISH). EBV was detected by PCR and/or EBER-ISH in 91/101 (90%) of MS cases compared to only 5/21 (24%) of non-MS cases with other neuropathologies. None of the samples were PCR positive for other common herpesviruses (HSV-1, CMV, HHV-6). By quantitative PCR, EBV viral load in MS brain was mainly low to moderate in most cases. However, in 18/101 (18%) of MS cases, widespread but scattered presence of EBV infected cells was noted in the affected tissues by EBER-ISH. Immunohistochemical analysis of EBV gene expression in the 18 heavily infected cases, revealed that the EBV latent protein EBNA1, and to a lesser extent the early lytic protein BZLF1 were expressed. Furthermore, using double-staining we show for the first time that astrocytes and microglia, in addition to B-cells can also be infected. To the best of our knowledge, this is the most comprehensive study demonstrating that EBV is present and transcriptionally active in the brain of most cases of MS and supports a role for the virus in MS pathogenesis. Further studies are required to address the mechanism of EBV involvement in MS pathology.

"We seek him here, we seek him there...Is he in heaven - Is he in hell?"

-The Scarlett Pimpernel by E Orczy

No really! The cause of MS is as elusive as the Pimpernel was to the French. Despite mounting evidence, no research group has yet successfully managed to nail EBV to MS. This work by Hassani and colleagues is hope that it may yet happen!

Epstein-Barr virus (EBV) is a member of the Herpes family of viruses and is common in humans. It's target is B cells and can remain dormant or latent in memory B cells for their lifespan. A small percentage, however, do reactivate in what is called the lytic phase.

EBV is associated with more than one disorder, but is thought in MS at least it causes a virus-host immune system response imbalance, with the immune response to the virus to the virus itself being disrupted (Sundström P, Juto P, Wadell G, Hallmans G, Svenningsson A, Nyström L, et al. An altered immune response to Epstein-Barr virus in multiple sclerosis: a prospective study. Neurology. 2004;62: 2277–2282). Almost all PwMS demonstrate evidence of previous EBV infection, and those who have not been exposed (i.e. seronegative) have almost zero risk of developing MS. The spanner in the works is that ~95% of those without MS have also had previous EBV exposure (Ascherio A, Munch M. Epstein-Barr virus and multiple sclerosis. Epidemiology. 2000;11: 220–224).

The question then becomes can you place EBV successfully in the brain where it can cause MS? And that is what the authors have done. Hassani et al, examined the presence of EBV in 122 post-mortem MS brains and non-MS brains. Others have tried to do this in the past but not successfully for any number of reasons - techniques, sampling etc. They used a PCR technique and a highly sensitive and specific EBER- in situ hydridization (EBER-ISH) technique to localise EBV infected cells in brain tissues.

Surprisingly, they found that EBV was present in 90% of MS cases, but also found no presence of other viruses, such as HSV-1, CMV and HHV-6 that have been previously implicated in MS. Of note, they also found that not only did EBV target B cells, but also infected ~10-15% activated microglia and astrocytes (the innate immune system, see Figure). Therefore, the role of EBV in the inflammatory cascade needs to be looked at in greater detail.

Overall, a very interesting paper and others will now have to re-examine EBV in MS brains using the more sensitive techniques in order to identify scattered and low level EBV infection.

Figure: Double staining for EBV and cellular markers.
Double staining for EBV (EBER-in situ hybridization: dark blue staining) and different cellular markers (immunohistochemistry: brown) in the white matter of 3 different heavily infected MS cases. The pattern of double-staining seen in these 3 cases is representative of that seen in other double-positive cases. (A) EBV and CD20 (B-cell marker), (B) EBV and GFAP (astrocyte marker), (C) EBV and Iba1 (microglia marker). Double positive cells are indicated by the arrows.

The B cell tide has turned

Now the geneticists are getting involved with suggesting B cells are important in MS.

Was our paper on B cells in MS so ace and compelling?:-)

or are they on the anti-CD20 gravy train?

CD40 is a costimulatory protein found on antigen presenting cells and is required for their activation. 

The binding of CD154 (CD40L) on TH cells to CD40 activates antigen presenting cells and induces a variety of downstream effects.

The B cell can present antigens to helper T cells. If an activated T cell recognizes the peptide presented by the B cell, the CD40L on the T cell binds to the B cell's CD40 receptor, causing B cell activation. The T cell also produces IL-2, which directly influences B cells. As a result of this net stimulation, the B cell can undergo division, antibody isotype switching, and differentiation to plasma cells. The end-result is a B cell that is able to mass-produce specific antibodies against an antigenic target. 

Early evidence for these effects were that in CD40 or CD154 deficient mice, there is little class switching or germinal centre formation, and immune responses are severely inhibited.

CD40 is also expressed on B cell precursors in the bone marrow, and there is some evidence that CD40-CD154 interactions may play a role in the control of B cell (haematopoiesis) development.

This drove people to target CD40-CD40L to block B cell activity in autoimmunity. 

In Phase II clinical trials regarding multiple sclerosis and Crohn's disease, thromboembolisms occurred in at least three patients. A causal connection could not be proven, but since the same adverse effects were seen in trials with a similar antibody (hu5C8), the trials were halted.

CD86 and B7-2) is a protein expressed on antigen-presenting cells that provides costimulatory signals necessary for T cell activation and survival. It is the ligand for two different proteins on the T cell surface: CD28 (for autoregulation and intercellular association) and CTLA-4 (for attenuation of regulation and cellular disassociation). CD86 works in tandem with CD80 to prime T cells and is present on B cells

They found that carrying the risk allele rs4810485*T lowered the cell-surface expression of CD40 in all tested B cell subtypes. There was an increased proportion of alternative splice-forms leading to decoy receptors (P = 4.00 × 10-7) which would not signal. This CD40 allele was associated with decreased levels of interleukin-10.  This suggests that B cells might have an important antigen presentation and immunoregulatory role in the pathogenesis of multiple sclerosis

The risk allele rs9282641*G increased the expression of CD86..abit with this effect primarily seen in the naïve B cell subset. However I must say this failed the smack you in the eye test.

The authors suggest that "B cells might have an important antigen presentation and immunoregulatory role in the pathogenesis of multiple sclerosis". 

Indeed they may but it has to be said that dendritic cells, which are professional antigen presenting cells, express masses more CD40 and CD86 and so this could be where the true susceptibility association occurs. 

Also one wonders whether this reduced CD40 means that it is easier for the latent membrane protein 1 produced by EBV to mimic CD40 signalling to cause pathogenic B cells to be CD40L and T cell independent, meaning that it would be easier to activate the B cell .

Many of the 200  or so identified susceptibility MS genes are immune-associated and many are thought to be involved in T cell biology such as the IL-2 receptor and the IL-7 receptor. 

However, these are both expressed by B cell lineage cells, so more evidence for the  B club;-).

Is the problem of MS due to EBV in the B cells?

What genes are active in MS? 

In this study they looked at lesions and surrounding white matter and found a load of CD8 T cells, a lot of CD20 B cells and a few CD4 T cells and plasma cells (antibody making cells).

The B cells contained EBV 


In this study they take brain tissue and then cut it into very thin slices (eg. a 100th of a mm) and then you look to see if you can find a lesion. You use a laser to cut out the cells from the section (laser capture) and then you remove the nucleus to see what proteins the cells were making. 

There were CD4 T cells in all the lesions, CD8 T cells in all of the lesions and CD19 B cells in all the lesions. Not many CD4 T cells and only 1-3% lesions contained IL-17/IL-22 (Th17 cytokines). If if there is more IL-17 in the blood (see beleow) how come there 

So are these few cells causing autoimmunity as the EAEers would have us believe There were 70% of lesions with gamma interferon, which could be Th1 derived or could come from CD8.

There was about 70-95% of lesions with clear B cell growth and differentiation factors like IL-6, IL-10 (yes it is a B cell factor and IL-10 does not have to be a regulatory cytokine), BAFF, lymphotoxin etc etc.

There are CXCR3 and CXCL12 chemokines in most lesions ,which recruit B cells 

However the paper is open access and they do vastly different association for these products 

One or more EBV genes were detected in immune infiltrates from 9 of 11 MS cases and in 41.3% of the samples. 

EBV genes were detected more frequently in meningeal than in WM perivascular infiltrates (55.6 vs 28% of the samples) and genes expressed during viral latency were detected more frequently (i.e. present in memory cells) than genes associated with the viral lytic which is where the virus kills the cell and releases the cells contents which is live virus). 

LMP1 and LMP2A are viral genes, deliver surrogate B cell survival and differentiation signals (CD40 and B cell receptor, LMP1 was detected more commonly than LMP2 suggest that the B cells may be more T cell-independent .

The authors suggests that "EBV could be the main antigenic trigger of an immunopathological, CD8+ T cell-mediated response that damages the brain/spinal cord in MS. This model is consistent with the notion that CD8+ T cells are the main drivers of bystander tissue damage in EBV-associated immunopathologic diseases"

So are CD8 cells in the brain to destroy EBV infected B cells? 

The authors suggest that "the use of drugs that, by directly targeting the virus and its cellular reservoir, could be more effective in normalizing an altered EBV-host interaction in MS. For example, B cell-depleting therapies could lower EBV load and hence the burden of EBV-induced immunopathology in MS more efficiently than other drugs". 

Is this what we are doing?

Veroni C, Serafini B, Rosicarelli B, Fagnani C, Aloisi F. Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis. J Neuroinflammation. 2018;15:18.

BACKGROUND:It is debated whether multiple sclerosis (MS) might result from an immunopathological response toward an active Epstein-Barr virus (EBV) infection brought into the central nervous system (CNS) by immigrating B cells. Based on this model, a relationship should exist between the local immune milieu and EBV infection status in the MS brain. To test this hypothesis, we analyzed expression of viral and cellular genes in brain-infiltrating immune cells.

METHODS:Twenty-three postmortem snap-frozen brain tissue blocks from 11 patients with progressive MS were selected based on good RNA quality and prominent immune cell infiltration. White matter perivascular and intrameningeal immune infiltrates, including B cell follicle-like structures, were isolated from brain sections using laser capture microdissection. Enhanced PCR-based methods were used to investigate expression of 75 immune-related genes and 6 EBV genes associated with latent and lytic infection. Data were analyzed using univariate and multivariate statistical methods.

RESULTS: Genes related to T cell activation, cytotoxic cell-mediated (or type 1) immunity, B cell growth and differentiation, pathogen recognition, myeloid cell function, type I interferon pathway activation, and leukocyte recruitment were found expressed at different levels in most or all MS brain immune infiltrates. EBV genes were detected in brain samples from 9 of 11 MS patients with expression patterns suggestive of in situ activation of latent infection and, less frequently, entry into the lytic cycle. Comparison of data obtained in meningeal and white matter infiltrates revealed higher expression of genes related to interferonγ production, B cell differentiation, cell proliferation, lipid antigen presentation, and T cell and myeloid cell recruitment, as well as more widespread EBV infection in the meningeal samples. Multivariate analysis grouped genes expressed in meningeal and white matter immune infiltrates into artificial factors that were characterized primarily by genes involved in type 1 immunity effector mechanisms and type I interferon pathway activation.

CONCLUSION: These results confirm profound in situ EBV deregulation and suggest orchestration of local antiviral function in the MS brain, lending support to a model of MS pathogenesis that involves EBV as possible antigenic stimulus of the persistent immune response in the central nervous system.

However, without wanting to upset the authors too much, they are the group that have proposed that B cell follicles ares associating with, and implicated in, the cause of progression. However as in their own words

"several groups have reported absence or paucity of EBV in postmortem MS brain samples"....whilst....

"we have repeatedly shown not only presence of EBVinfected B-lineage cells but also EBV latency disruption and reactivation in the MS brain"

"RNA/protein nor deregulated EBV infection was detected in brain tissues from patients with other infectious and non-infectious neuroinflammatory diseases"..."ruling out the possibility that an active EBV infection in the CNS is the general consequence of immune cell invasion and local activation."

Therefore not all people agree on this and so requires replication by other groups who do not have a vested interest in this story.

What do others find?

Trenova AG, Slavov GS, Draganova-Filipova MN, Mateva NG, Manova MG, Miteva LD, Stanilova SA. Circulating levels of interleukin-17A, tumor necrosis factor-alpha, interleukin-18, interleukin-10, and cognitive performance of patients with relapsing-remitting multiple sclerosis. Neurol Res. 2018 Jan 3:1-7. doi: 10.1080/01616412.2017.1420522. [Epub ahead of print]

Multiple sclerosis (MS) is associated with cytokine imbalance and high rate (40-70%) of cognitive impairment. The objective of this study is to investigate the relationship between serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-17A, IL-18, IL-10, and cognitive performance in patients with relapsing-remitting MS (RRMS). 
Methods The study comprised 159 patients with RRMS (mean age 40.08 ± 8.48 years) in remission phase and 86 age-, gender-, and education-matched healthy controls. Paced Auditory Serial Addition Test (PASAT), Symbol Digit Modalities test (SDMT), and Isaacs test were used for assessment of working memory, attention, visuo-perceptual abilities, information processing speed, and executive functions. Serum cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). 

Results Patients had significantly increased serum concentrations of TNF-alpha and IL-17A and decreased levels of IL-10 compared to the controls (p < 0.05). Negative correlation was found between serum TNF-alpha and SDMT score in patients with disease evolution longer than 10 years (rxy = -0.258 p = 0.033); PASAT and SDMT scores were in negative correlation with concentration of IL-17A (rxy = -0.229 p = 0.004; rxy = -0.166 p = 0.041). Cognitive impairment was established in 46.5% (n = 74) of the patients. Cognitively impaired patients had significantly higher serum IL-17A than cognitively preserved individuals (p = 0.007). Multiple linear regression analysis revealed IL-17A as a significant predictor of cognitive performance in RRMS patients. 

Conclusion The results from this study suggest that pro-inflammatory cytokines IL-17A and TNF-alpha simultaneously with decreased IL-10 are involved in cognitive deterioration in RRMS.

When I was rreally interested in cytokines, we looked for cytokines like TNF in the blood and didn't find any. Now it could be sensitivity of the assay.Alternatively it mean what was produced was used, so that if excess is found, it suggests more is produced than needed. 

However it had been reported that TNF levels in blood in MS, correlated with disease activity and the the results appeared to good. Indeed when the person writing the paper was asked to repeat the analysis with coded and blinded samples, so they wouldn't know what was what...They ran a mile never to be heard off again. 

So you can guess what I thought of the data. This report may suggest the neuro was correct. They find what you expect if you follow dogma...an increase in pro-inflammatory cytokines and a decrease in regulatory cytokines. 

The interpretation of results can depend on the way you look at things.

Look at this picture. What do you see?

Do you see an old woment (you see a nose) or a young women (you see a jaw)?

Now Look at this. What do you see?

Don't know what this is?.

An educational comment for pwMS and researchers

It is interleukin 10.

It maybe doesn't mean much to you, but it may help you understand papers a bit better and questions whether you should accept dogma without thought.


Say this to a T cell immunologist and they see an immunoregulatory cytokine.

"Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatorycytokine"

In humans, IL-10 is primarily produced by monocytes and, to a lesser extent, lymphocytes, namely type 2 T helper cells (T_H2), mast cells, CD4⁺CD25⁺Foxp3⁺ regulatory T cells, and in a certain subset of activated T cells and B cells. 

IL-10 is a cytokine with multiple, pleiotropic, effects in immunoregulation and inflammation. It downregulates the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages.

Now show this to a B cell immunologist and they see something different 

IL-10 is a cytokine that enhances B cell survival, proliferation, and antibody production and could be produced to help make antibody producing cells

How about Interleukin 4.

T cell immunologist says "IL-4 decreases the production of Th1 cells, macrophages, IFN-gamma, and dendritic cell IL-12."

or a  B cell immunologist says "IL-4 stimulates activated B-cell proliferation, and the differentiation of B cells into plasma cells. It is a key regulator in humoral  immunity. IL-4 induces B-cell class switching and up-regulates MHC class II production to potentially act as an antigen presenting cell.

How about interleukin 6?

It is viewed as being pro-inflammatory?, 

However, It can be anti-Inflammatory but it can also supports the growth of B cells .

So lets looks in MS and see what has been have found

Guerrier T, Labalette M, Launay D, Lee-Chang C, Outteryck O, Lefèvre G, Vermersch P, Dubucquoi S, Zéphir H.Proinflammatory B-cell profile in the early phases of MS predicts an active disease. Neurol Neuroimmunol Neuroinflamm. 2017; 5(2):e431. 

OBJECTIVE:To assess whether any alteration of B-cell subset distribution and/or the cytokine production capacities of B cells could be associated with any stage of MS and could be predictive of MS evolution.
METHODS:We prospectively enrolled radiologically isolated syndrome (RIS), clinically isolated syndrome (CIS), naive patients with relapsing remitting MS (RRMS) of any disease modifying drug, and healthy controls (HCs). Peripheral blood B-cell subset distributions and the interleukin (IL)-6/IL-10-producing B-cell ratio were assessed by flow cytometry to evaluate their proinflammatory and anti-inflammatory functional properties.
RESULTS:Twelve RIS, 46 CIS, 31 RRMS patients, and 36 HCs were enrolled. We observed that a high IL-6/IL-10-producing B-cell ratio in patients with RIS/CIS was associated with the evolution of the disease in the short term (6 months). This imbalance in cytokine production was mainly explained by an alteration of the production of IL-10 by B cells, especially for the transitional B-cell subset. In addition, a significant increase in IgD-/CD27- memory B cells was detected in patients with CIS and RRMS compared with Hs (p = 0.01). Apart from this increase in exhausted B cells, no other variation in B-cell subsets was observed.
CONCLUSIONS:The association between a high IL-6(pro-inflammatory)/IL-10(anti-inflammatory) -producing B-cell ratio and the evolution of patients with RIS/CIS suggest a skew of B cells toward pro-inflammatory properties that might be implicated in the early phases of MS disease.

So as you can see the B cell production of IL-6 is viewed as pro-inflammatory and IL-10 is antiinflammatory, but are these simply B cell producing agents? 

I don't have the answer which is which, but use this to illustrate that many things you get told you may have different explanations

End of Year Revision

As the year draws to a close, it has been a productive year for papers and ideas. 

I predict that during 2018, we will get even more clarity on the story.

But do you believe the academic minnow or the whale?

As we have said many times before, people do not all agree on the driving force of MS.

Most people are still in the T cell camp or midway into the T cell camp. Even those in the B cell camp are not convinced and so do not provide any clarity into what is going on. 

Is it Chewbacca Science, like the chewbacca defence...you throw a load of facts in the air and it simply confuses every one, so every one agrees with you but non-one knows what's really going on. 

You are never wrong because you suggest every possibility. Everyone is happy.

However, it allows you to lack focus and so waste time by not doing the important things.

 Chewbacca Science-Lovely and Cuddly

Greenfield AL, Hauser SL.B Cell Therapy for Multiple Sclerosis: Entering an Era. Ann Neurol. 2017. doi: 10.1002/ana.25119.

Monoclonal antibodies that target CD20 expressing B cells represent an important new treatment option for patients with multiple sclerosis (MS). B cell depleting therapy is highly effective against relapsing forms of the disease and is also the first treatment approach proven to protect against disability worsening in primary progressive MS. Moreover, evolving clinical experience with B cell therapy, combined with a more sophisticated understanding of humoral immunity in preclinical models and in patients with MS, have led to major progress in deciphering the immune pathogenesis of MS. Here, we review the nuanced roles of B cells in MS autoimmunity, the clinical data supporting the use of ocrelizumab and other anti-CD20 therapies in the treatment of MS, as well as safety and practical considerations for prescribing. Lastly, we summarize remaining unanswered questions regarding the proper role of anti-CD20 therapy in MS, its limitations, and the future landscape of B cell-based approaches to treatment. 

In my opinion, this paper seems to be little more than an advert for ocrelizumab, particularly as it states the manufactures provide "writing assistance for CD20-related presentations"

Anyway, it says:

"Although most disease-modifying therapies for MS have traditionally been conceptualized as functioning via T cell-based mechanisms, a growing body of data indicates that all have demonstrable effects on B cells as well".

"Common themes include 

• Promoting naive rather than memory or plasmablast (alemtuzumab); 
• Shifting B cell cytokines towards an anti-inflammatory tone (beta interferon, glatiramer acetate, fingolimod);
• Increasing B-regs (beta interferon, glateriamer acetate, fingolimod and dimethyl fumarate); 
• Decreasing class II MHC and constimulatory molecules on B cells required for antigen presentation (beta interferon and dimethyl fumarate); 
• Sequestering B cells in lymphoid organs (fingolimod); 
• Blocking VLA-4 mediated B cell trafficking to the CNS (natalizumab); or
• Direct cytolysis of B cells (alemtuzumab, teriflunomide, mitoxantrone)" 

As for ocrelizumab

• Potentially pathogenic memory B cells remain at reduced levels
• Fewer GM-CSF producing B cells and higher levels of IL-10
• Fewer pro-inflammatory Th1 & Th17 cells
• Large number of CD25+FOXP3 regulatory T cells

So as the year draws to a close, 

I ask you to remember what you have learnt this year and ask you to consider this ....

Do you want to believe the Jumble of Conflicting and Inconsistent Ideas? as all the above are possible. It means the MS treatment landscape is difficult to comprehend and navigate or

Is it simpler that MS treatments all physically or functionally deplete memory B cells (maybe to stop them becoming plasmablasts) to prevent them entering the CNS.

This gives a unifying idea on aetiology, pathology and response to treatment?

It makes treatment choices easier.

Baker D, Marta M, Pryce G, Giovannoni G, Schmierer K. Memory B Cells are Major Targets for Effective Immunotherapy in Relapsing Multiple Sclerosis. EBioMedicine. 2017;16:41-50.

Which fits the MS reality?

Let's put it to the test and see what happens in 2018.

I predict that more people will fall off the fence

Or maybe I'll eat humble pie.

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.

A BAFFling tale: how does fingolimod affect B cells?

Fingolimod is an effective DMT which binds to the sphingosine-1-phosphate receptor on white blood cells. We don’t fully understand why it works in MS, but the most popular theory is that it traps lymphocytes inside the lymph nodes, preventing them from entering the blood and therefore from penetrating into the CNS. It may also have direct anti-inflammatory effects, protect the integrity of the blood-brain barrier, and (possibly) act directly in the CNS as a neuroprotective agent.

After people receive fingolimod, the number of B cells in the peripheral blood is suppressed. In addition, the B cells which remain in the blood are immature, so-called ‘transitional’ B cells. It is not fully understood how fingolimod does this, or whether this is relevant to the mechanism of benefit in MS.

BAFF is a signalling molecule involved in regulating the survival and proliferation of B cells. BAFF has received lots of attention in MS and other autoimmune diseaseas for a few reasons...

1.  A rare variant in the gene for BAFF is associated with increased risk of MS and SLE, another autoimmune disease.
2. BAFF levels go up after B cell depletion with drugs like ocrelizumab and rituximab.
3. Atacicept, a BAFF inhibitor, made MS worse in a clinical trial.
4. BAFF is an important regulator of B cells, and there is lots of emerging evidence we’ve blogged about elsewhere that B cells are central players in MS.

A new study investigated the relationship between BAFFlevels and B cell counts in response to fingolimod therapy. To do this, they first measured BAFF levels in the blood of 30 pwMS treated with fingolimod, 32 pwMS on no DMTs, and 25 healthy controls. Most of the people in the untreated group had never had DMTs (27/32) whereas 5 had had beta-interferon. The fingolimod-treated group had higher BAFF levels than both other groups. This suggested that fingolimod might increase BAFF levels in the blood. In support of a causal link, 3 people who had blood taken before and after fingolimod showed an increased in blood BAFF levels after starting treatment.

The authors then asked whether this increase in BAFF levels is related to changes in the numbers and types of B cells found in the blood after fingolimod treatment. Interestingly, BAFF levels were correlated with the total number of B cells. When these cells were looked at in a bit more detail, the authors found that increasing BAFF levels were associated with a shift in the relative make-up of these B cells: more BAFF was related to a higher proportion of transitional, immature B cells, and a lower proportion of memory B cells.

Here is a picture of the key graphs which I’ve rehashed:  

This study, for me, provides another important piece of evidence that BAFF is a master regulator of B cell pools in health, disease, and following administration of drugs, like fingolimod, which affect B cell numbers in the blood.

The emerging picture is that the body senses a decrease in the circulating B cell pool, aims to counteract this by producing more BAFF, and this subsequently leads to the release of transitional, immature B cells from the bone marrow, as we seen after B cell depletion therapy. The really interesting questions for me are

1) What is the sensor?

2) What is the key cell type (or types) responsible for producing BAFF in the body?

A better understanding of the relationship between BAFF and B cell pools will almost certainly improve our understanding of MS and other autoimmune diseases. As the atacicept nightmare tale shows us, hitting the BAFF axis can have a dramatic impact on disease activity. Knowing more about this axis will hopefully allow us to target BAFF in a way that benefits pwMS.  

***

Abstract

Patients with multiple sclerosis (MS) who are treated with fingolimod have an increased proportion of transitional B cells in the circulation, but the underlying mechanism is not known. We hypothesized that B cell-activating factor of the tumor necrosis factor family (BAFF) is involved in the process. Compared with healthy controls and untreated MS patients, fingolimod-treated MS patients had significantly higher serum concentrations of BAFF, which positively correlated with the proportions and the absolute numbers of transitional B cells in blood. Despite the elevated concentrations of BAFF in fingolimod-treated MS patients, serum levels of soluble transmembrane activator and calcium-modulating cyclophilin ligand interactor, and B cell maturation antigen were not elevated. Our results show that fingolimod induces BAFF in the circulation and expands transitional B cells, but does not activate memory B cells or plasma cells in MS, which is favorable for the treatment of this disease.