Spanish HIV Experts Give Aid to ME/CFS

Joel (Snowathlete) talks with Dr Blanco, from IrsiCaixa, about the Spanish AIDS Research Institute‘s latest research on ME/CFS

T-lymphocyte (T-cell)
T-lymphocyte (T-cell)

Back in 2009 when XMRV hit the headlines a number of groups around the world took an interest in ME/CFS for the first time. One of these groups was the AIDS Research Institute IrsiCaixa, in Spain. Then XMRV tripped itself up and ME/CFS became invisible again…Except, it didn’t…

IrsiCaixa were still interested. Dr Blanco and his colleagues had taken a look at ME/CFS in 2010 and spotted a bunch of problems in the immune system. These guys really know their stuff when it comes to immunology and they knew that they were on to something. Here’s what Dr Blanco had to say about it:

“Our preliminary data showed alterations in all lymphocyte subsets analyzed (B, T and NK). However, these preliminary data were generated from patients recruited in one CFS unit. To avoid any bias, we expanded our study by recruiting patients from a different clinical center.

“Patients’ organizations partially funded the initial studies, with many patients contributing with small amounts to fund the research. These studies were aimed at identifying viral (XMRV) and immunological markers in CFS samples. Although the role of XMRV was rapidly ruled out, the researchers felt that the immunological studies were interesting enough to deserve further work. This additional effort was funded by the IrsiCaixa Foundation (a research institute funded by the Catalonian government and “La Caixa” foundation, a Catalonian bank).”

ME/CFS is well understood to have an element of immune dysfunction, and there are many papers on it. The immune system is complex, and contains many different cell types and subtypes which carry out different tasks. The cells most often studied are lymphocytes, the three major types of lymphocytes being T-, B- and natural killer-cells (NK). Within these three groups you have further division of phenotype, so two different T-cells, although related to each other, may possess characteristics that differentiate them, and disease may affect one of these phenotypes but not the other.

The studies that appear to shed the most light on ME/CFS – showing differences from healthy or disease controls – are those which look at the different phenotypes of T-, B- or NK-cells; either their count, or even better, their function. Despite interesting results, some of the immune abnormalities reported in ME/CFS over the years have been contradictory.

Back in 2011, researchers from Bond University in Australia published some very interesting findings in The Journal of Translational Medicine (Ekua W Brenu, et al) showing that the immune system of ME/CFS patients differed in phenotype and function, compared to controls. Now, new findings from Dr Blanco and his colleagues in Spain have been published in the same journal (Marta Curriu, et al) and report an assortment of immune abnormalities, some of which support the earlier findings from Australia.

From left to right: Marta Massanella, Julià Blanco, Jorge Carrillo, Cecilia Cabrera and Marta Curriu. (© IrsiCaixa. Raimon Solà).
From left to right: Marta Massanella, Julià Blanco, Jorge Carrillo, Cecilia Cabrera and Marta Curriu.
© IrsiCaixa. Raimon Solà

In short, what are they reporting?

They have reported that NK-cell phenotype is biased in ME/CFS and some T-cells have been found to be poorly responsive, and these abnormalities clearly identify ME/CFS affected individuals.

Phenotype count

Each cell in our body has one or more special surface molecules which carry out important functions. Each of these different molecules has a name but is also known by a number, called a cluster of differentiation (CD). These differences allow cells, such as lymphocytes, to be subdivided into different populations based on their relative expression of these surface markers.

You have likely heard of them before; they are talked about frequently in immune diseases, and you are probably aware of Rituximab – a potential treatment for ME/CFS – which depletes B-cells possessing the CD20 marker.

In this study Dr Blanco and his colleagues looked at these different markers on T-, B- and NK-cell lymphocytes and compared them to controls. In comparing the absolute count of these phenotypes, they found that some were no different from controls, but others were significantly different.

They found a slightly unbalanced composition of the overall T-cell subset, including higher representation of CD4 T-helper-cells in the CFS group. They also found a significantly lower frequency of cytotoxic T-cells with the CD56 marker, replicating the finding from Klimas, et al, in 2011, and Peterson, et al, in 2011.

cluster of diff
Cluster of differentiation tree showing some of the different markers that differentiate lymphocytes as reported in this study

This signature is also found to occur in Psoriasis, an autoimmune disease. I asked Dr Blanco whether he thought this might suggest ME/CFS is an autoimmune condition? 

“This is a very good question. The autoimmunity link to CFS was among our initial hypotheses. For this reason we deeply analyzed B cells. Contrasting with this hypothesis, we found no differences in B cell subsets between CFS and healthy individuals. In addition, we found (as others) a higher frequency of regulatory T cells in CFS. Considering that one of the main functions of T-regs is to control autoimmune responses, our data suggest that autoimmune responses are under a proper, if not enhanced, control in CFS. This paradox, that may be related to other functions of B cells, remains to be solved.”

Overall, B-cell phenotype count was found to be similar to controls.

When it came to NK cells, the researchers found that NK-cells expressing the markers CD69 and CD335 (NKp46) were significantly higher than controls and NK cells of the phenotype CD25 were significantly lower in number. A similarly altered NK-cell population has been reported in influenza infection or vaccination.

Phenotype function

Despite the differences found in NK-cell phenotypes, functional tests showed that their overall cytotoxic effect and sensitivity to ex-vivo cell death was similar to controls. This is a bit of a surprise, but Dr Blanco was kind enough to give us his thoughts on this.

“We were also surprised by the lack of differences in NK cell activity. Unfortunately, data from NK cell lytic activity was generated in a subset of patients, thus reducing the statistical power of these data. I think this is the main reason for the apparent lack of difference.”

Overall, as with absolute count, B-cell function was found to be comparable to controls.

The big differences are in the T-cells, where phenotype function was found to be distinctly different to controls. They report an increase in the proportion of CD4 T-cells expressing the PD-1 (CD279) marker, compared to controls. T-cells expressing the PD-1 marker have been reported to correlate with T-cell exhaustion in HIV-infected patients.

CD8 T-cells expressing the CD95 marker (another marker associated with T-cell exhaustion), were also found to be increased in the patient sample when compared to controls.

T-regulatory cells (T-reg) with particular markers (CD4+ CD25++ FOXP3+ or CD4+ CD25++ CD127-) were found to be significantly higher in the CFS sample, in line with a reduced number of CD4 T-cells expressing the Ki67 marker and supporting the earlier findings from Bond University in Australia.

CD8 T-cells expressing the CD5 marker (associated with impaired T-cell response as a result of chronic low level antigen exposure) were higher in the patient sample compared to controls.

And the patient sample demonstrated lower counts of the activation marker CD38 and memory CD8 T-cells displaying the marker CD45RO.

When they tested ex vivo proliferation of T-cells, the CD4 T-cell response was significantly lower in the patient sample. The CD8 proliferation response was not found to be significantly different, compared to controls, and the overall T-cell cell-death test matched controls.

These multiple differences in T-cell function point to an impaired T-cell response in patients with ME/CFS, which the authors describe as “a general hyporesponsiveness”.

How did they do it?

The study was designed in 2010 and so used Fukuda criteria rather than the later 2011 International Criteria that would have allowed selection of defined ME patients. In the paper, the authors show a good understanding and awareness of the potential difference between ME and CFS defined populations and/or subgroups within the disease(s). The majority of the ME/CFS patients in the study were classed as having a mild level of severity, with an approximate 50% reduction in pre-illness activity level, only three of the individuals being moderately severe. No severe cases were represented in the study.

Iriscaixa Lab
Iriscaixa Lab
© IrsiCaixa. Raimon Solà

They took blood samples from 22 ME/CFS patients with an absence of current identified infections, and 30 healthy controls, analyzing the portion of blood containing T-, B- and NK-cells.

Part of the blood drawn was analyzed to ascertain the absolute counts of the different lymphocyte phenotypes. This analysis is called immunophenotyping and was achieved via a process called flow cytometry. This process isn’t new, but it is clever. It involves the introduction of antibodies to the blood which attach to the surface proteins on the lymphocytes. As different phenotypes have different surface proteins, each antibody will only stick to specific cell phenotypes. This acts as a sort of label to identify each of the different cell phenotypes.

These labeled cells are then put through a machine called a flow cytometer, a very complex piece of kit, but the principle behind it is quite simple, it can read all these labeled cells extremely quickly, and give you a count of the different phenotypes.

They used the remaining blood to analyze the function of these different phenotypes by a variety of assays.

Three of the CFS/ME sample group were later excluded from the findings: through the analysis of immune cells, one was found to have B-cell lymphocytosis, another was found to have IgA deficiency and a third was excluded due to sample unavailability. This left a sample size of 19 patients for results.

What does it mean?

We don’t yet know if these lymphocytes are directly involved in the pathophysiology of the disease, or whether they are a secondary effect. Specific NK-cell phenotype profiles have been associated with various viruses in the medical literature and the results may reflect the role of an unknown pathogen, but until such a pathogen is identified (if at all) in ME/CFS, this is merely speculation. Regarding the significant down regulation of T-cell mediated immunity; this characteristic may explain why people with ME/CFS often have various concurrent infections.

A biomarker

These findings may allow identification of ME/CFS patients as it is possible to look at their T-cell and NK-cell phenotype signatures, which should stand out when compared to a healthy signature. The authors evaluated the potential for identification of ME/CFS patients based on the reported abnormalities, and found that 8 of these T- and NK-cell phenotype markers, when used in a combination, provided a high sensitivity (100%) but with a moderate false positive rate 5/24 (specificity – 79%). Although the false-positive rate may be seen as problematic, when used in combination with current diagnostic criteria for ME/CFS it is likely that the specificity would be much higher.

Of course, this diagnostic model fits this dataset, and how it would perform against new, unseen data from new patients would be the true test of its efficacy.

This study does confirm some of the findings from Australia in 2011, and other prior studies, and that is fantastic news for us. It suggests that there is a good chance that someone looking at your phenotype profile would find the same signature reported in these two studies. It could be used to diagnose ME/CFS, and to prove what we already know – that we have dysfunctional immune systems, and these immune abnormalities may help focus in on the cause(s) of the illness.

In contrast, some results from this study do not support earlier findings reported by others, including Maes, et al. However the authors suggest that this apparent conflict may just be a result of prior in vitro stimulation which took place in the study by Maes, et al.

Is it a surprise that B-cells were not found to be abnormal?

The work by Mella and Fluge in Norway, using Rituximab, suggests that B-cells in ME/CFS patients may be abnormal and hints at the possibility of autoimmunity. But this new research from Spain found no significant differences in B-cell phenotype, or functions overall. Dr Blanco was good enough to give us his insights on this:

“As I mentioned above, our data suggest that autoimmunity is not a major player in CFS. However, data on Rituximab treatment suggesting a clinical improvement of CFS are clear. To solve this apparent paradox a deep analysis of B cells before and after Rituximab treatment would be very helpful. One possibility to bring together both observations is that the beneficial effect of Rituximab may be indirect, acting by removing B cells that produce cytokines that may contribute to CFS symptoms. This is obviously a hypothesis that requires a proper experimental demonstration.”

A recent study from Bansal, et al, with a sample of 33 patients, suggests a subtle deregulation of B-cells in ME/CFS patients. I asked Dr Blanco how these findings compared to his own observations of B-cells in ME/CFS:

“The paper by Bansal et al suggests abnormalities in B cells from CFS affected individuals. Interestingly some of these alterations seem to fit with our preliminary data. In fact we found a slightly reduced number of antibody producing cells and a low level of circulating IgG in some individuals. Unfortunately in our patients these alterations were not consistent and failed to be useful to identify CFS. However, we still believe that our data could be consistent with alterations in B cell phenotype or function that will be unveiled in larger studies.”

Additionally, as Dr Blanco and his colleagues point out in the paper itself, any B-cell dysfunction in ME/CFS could be present in the tissue rather than in the blood.

Possible causes

XMRV turned out to be contamination, but having taken a closer look at ME/CFS now, I asked Dr Blanco to speculate on what he thinks the potential cause of the illness might be: a retrovirus, virus, bacteria, or something else?

“It can be any of them. What I think is that some infections or other sources of stress may induce chronic unbalance in the inflammatory status. This may fit with some observations made by several authors. This unbalanced inflammatory status may help to explain not only immunological perturbations but also neurocognitive disorders reported in CFS.”

“What do you think of De Meirleir’s and Lombardi’s recent HERV/dendritic cell findings; do you think HERVs could play a role in this disease?”

“This is again a very interesting question. Endogenous retroviruses may play a still undefined role in several human diseases. Interestingly, the expression of HERV genes is controlled or influenced by many inflammatory mediators that activate/repress transcription factors. Thus, the excellent study by De Meirleir and Lombardi on HERV provides an additional piece of information to have a complete picture of CFS.”

What next?

This study adds to the now significant (and still growing) body of evidence showing B-, T-, and NK-cell abnormalities in ME/CFS. This is a relatively small study. A bigger study is now needed to prove that the same signature is consistent in the wider ME/CFS population.

As well as replicating these findings in a larger sample, we also need to know how the ME/CFS profile compares to other diseases with similar symptomology. It may then be possible to diagnose patients by looking at their immune system profile, perhaps in combination with existing diagnosis criteria, allowing a more certain diagnosis of ME/CFS (assuming of course, that an extended study did not show a common profile in other comparable diseases).

One thing that may restrict the usefulness of this reported ME/CFS immune profile is co-infections. This study was careful to exclude patients with identifiable co-infections. This was a good thing, but people with co-infections may present a different profile. So there are still questions to be answered, but we have reason to be optimistic that this area of research will continue to be fruitful.

Having new researchers from different backgrounds working on our illness is exciting and full of promise, and I was delighted when Dr Blanco told us that he and his team plan to continue looking at ME/CFS.

“Our research team is an immunology group that works in collaboration with the HIV Clinical Unit in the Germans Trias I Pujol Hospital to understand immune damage caused by HIV infection.  Although we found the immunology of CFS is a new exciting area of research, our Hospital lacks a CFS Unit and we perform our work on CFS in collaboration with other Hospitals in Catalonia, limiting sample availability and processing and the design of large studies centralized in our laboratory.

“Despite this, I do think that this collaboration between research teams with a very different background yielded a strongly positive synergy that should be maintained. Therefore, our plans are to bring to the CFS field our ideas in collaboration with the leading research teams in CFS in Barcelona (which have the capacity to recruit larger cohorts) and to contribute to link immunological alterations with other clinical features of CFS.”

“Given your success in collaborating with CFS specialists in Spain, and with other ME/CFS specialists looking at lymphocytes as well, such as Dr Marshall and her colleagues in Australia, might there be an opportunity for your two groups to collaborate on research in this area?”

“It would be great to expand our contribution to CFS immunology in collaboration with leading laboratories worldwide. For instance, in HIV research the establishment of international cohorts has allowed new achievements on therapies and clinical guides. For CFS, the definition of well-defined international cohorts with standardized immunological/neurocognitive/endocrine follow-up and regular blood collection/biobanking might be a valuable tool for research. Again, clinicians that treat CFS should lead this initiative.”

“The ME/CFS community are grateful for your interest in the illness, and are keen to support your efforts. How can the ME/CFS community support your work?”

“The ME/CFS community has strongly supported our work with different fundraising projects. I know that national and international associations work to fund new projects. I would suggest that these projects involve both widely recognized ME/CFS research teams, and groups outside the ME/CFS field to favor synergies and new ideas coming into the field. Attracting new teams will increase the interest of the scientific community for CFS and a dynamic research may be the best weapon to fight the disease.

“I would like to thank you for your interest in our work and I hope that a collection of reliable biomarkers will soon be available for the diagnostic of CFS. This is important for patients, who will benefit from a faster diagnostic, and for health policy makers that will better evaluate the impact of the disease.”

A big thank you from the ME/CFS community to Dr Blanco and his colleagues for their interest in ME/CFS, and their excellent work.

Joel was diagnosed with ME/CFS in 2009 but struggled with the illness for some time prior to this. He loves to write, and hopes to regain enough health to return to the career he loved and have his work published. 

Marta Curriu, Jorge Carrillo, Marta Massanella, Josepa Rigau, José Alegre, Jordi Puig, Ana M Garcia-Quintana, Jesus Castro-Marrero, Eugènia Negredo, Bonaventura Clotet, Cecilia Cabrera, and Julià Blanco. Screening NK-, B- and T-cell phenotype and function in patients suffering from Chronic Fatigue Syndrome. J Transl Med. 2013; 11: 68.




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