Association of T and NK Cell Phenotype With the Diagnosis of ME/CFS (Rivas et al., 2018)

[Wally]

This study was published on May 9, 2018. Not feeling well enough to process the details of the study, but I thought it might be of interest for others to review.

https://www.frontiersin.org/articles/10.3389/fimmu.2018.01028/full

 

[datadragon]

They were able to identify whether the people were ME/CFS patients or healthy and not sick in 70% of the cases using the observed differences in T and NK immune cells between patients and healthy controls. This can help in the diagnostic process of new ME/CFS patients, and help the recognition of the disease as it would have an objective measure. It would also help in the search of more specific treatments. They mention that more studies are needed to confim the findings and to contribute more info to establish the diagnosis criteria.

 

[Seven7]

I remember in one of the Klimas videos and Peterson was there. That they think twice to give Cfs diagnosed if NK was normal. Some of the old Cfs specialist have been using Nk as one of the markers for a while.

 

[AdamS]

This looks like a pretty good study, thanks for posting.

The study included 76 patients that fulfilled the revised Canadian Consensus Criteria (CCC 2010) for ME/CFS and 73 healthy controls, matched for age and gender

CCC...good start!

ME/CFS patients showed significantly lower values of T regulatory cells (CD4+CD25++(high)FOXP3+) and higher NKT-like cells (CD3+CD16+/−CD56+) than the healthy individuals.

Interesting. I’m currently in a bit of a crash so can’t do much analysis, interested to hear what people think though!

 

[TreePerson]

I tried looking at this but probably not well enough to understand it.

I wasn’t clear whether they found that CD4 were higher or lower in pwme. I think they start by saying that previous studies have found higher levels in patients but I am not clear whether this was confirmed. I’m interested because I know mine are quite low.

If anyone is well enough to explain the findings in very basic terms I would be really interested and grateful

Edited. I meant CD4 not T4

 

[Murph]

Certainly looks like they saw some real differences in some of the NK cells, as depcited in these charts.

Figure 3. Analysis of NK cell subsets in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients (n = 76) and healthy subjects (n = 73). (A,B) NK cells as CD16+/−CD56+(dim) and CD16+/−CD56++(high) were obtained in gated CD45+ lymphocytes. (C–H) Percentages of NKG2C, NKG2A, NKp46, NKCD69, NKCD57, and ILT2 NK cells were obtained after gating for CD16+/−CD56+ lymphocytes. Figures show median values (lines), interquartile ranges (boxes), and 10–90 percentile values (bars). p Values obtained by Wilcoxon non-parametric test.

 

[Murph]

Here's an interesting part of the discussion section

Previous studies have evaluated immune profiles in ME/CFS looking for potential biomarkers. Some have found a reduced NK cell cytotoxic activity (27, 28, 37–40) that has not been corroborated in other studies (30–33). Theorell et al. (33) argue that the cytotoxic activity could be reduced when using whole peripheral blood (41, 42) or PBMC immediately obtained (23, 43) but could appear normal when using frozen PBMC. Also other soluble factors like cytokines, catecholamines, and hormones would decrease according to the time lapse, since obtaining the sample that could explain the different results obtained in different studies. We used whole peripheral blood analyzed within 6 h of collection that may have allowed for detection of changes that could be lost when using a more processed sample.

We observed in the ME/CFS patients an increased NK CD56++(high) population (p = 0.0075), a small group of NK cells (maximum 10%) with greater cytokine secretion capacity, particularly IFNγ, and with low cytotoxic activity (44, 45). This would be in keeping with the observations by Tireli et al. (46), in a study with 40 patients and 35 healthy controls. Also, Hardcastle et al. (44, 45) observed increased NK CD56++(high) levels in patients with greater severity of the disease compared to the moderately affected patients. However, the same group had observed lower NK CD56++(high) cell numbers in two previous studies with 95 and 10 patients, respectively (23, 37), and in another study the reduced NK CD56++(high) cell numbers were related to the time course of the disease (28). NK CD56++(high) cells are more resistant to apoptosis than NK CD56+(dim) cells (47), so have a longer life span and can induce T cell proliferation that could lead to autoimmunity (48) and contribute to inflammation (49, 50).

Interestingly, we observed increased levels of NKT-like cells and lower NK CD56++(high) cells in the group of patients that had described an infection before the onset of the disease. These populations have a role in the regulation of the immune response through their cytokines. While NK CD56++(high) is relevant to regulate anti-viral and anti-intracellular infections, NKT cells can play a role in Th2 immune responses. However, higher levels of NK CD56++(high) cells in the “no infection” onset group could be due to the exposure to raised levels of catecholamines secondary to the chronic activation of the hypothalamic–pituitary–adrenal axis as described by Loebel et al. (51).

 

[Pyrrhus]

The author of this study, Jose Luis Rivas Delgado, made a video in Spanish explaining the results:

 

[Pyrrhus]

And here's the abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a pathological condition characterized by incapacitating fatigue and a combination of neurologic, immunologic, and endocrine symptoms. At present its diagnosis is based exclusively on clinical criteria.

Several studies have described altered immunologic profiles; therefore, we proposed to further examine the more significant differences, particularly T and NK cell subpopulations that could be conditioned by viral infections, to discern their utility in improving the diagnosis and characterization of the patients.

The study included 76 patients that fulfilled the revised Canadian Consensus Criteria (CCC 2010) for ME/CFS and 73 healthy controls, matched for age and gender. Immunophenotyping of different T cell and natural killer cell subpopulations in peripheral blood was determined by flow cytometry.

ME/CFS patients showed significantly lower values of T regulatory cells (CD4+CD25++(high)FOXP3+) and higher NKT-like cells (CD3+CD16+/−CD56+) than the healthy individuals. Regarding NK phenotypes, NKG2C was significantly lower and NKCD69 and NKCD56 bright were significantly higher in the patients group.

A classification model was generated using the more relevant cell phenotype differences (NKG2C and T regulatory cells) that was able to classify the individuals as ME/CFS patients or healthy in 70% of cases.

The observed differences in some of the subpopulations of T and NK cells between patients and healthy controls could define a distinct immunological profile that can help in the diagnostic process of ME/CFS patients, contribute to the recognition of the disease and to the search of more specific treatments. However, more studies are needed to corroborate these findings and to contribute to establish a consensus in diagnosis.

(emphasis and spacing added for readability)

 

[Pyrrhus]

The author also made a plain-English summary on Facebook of the study and its implications for patients:

Approach and objectives of the study
Study consisting of 76 patients with ME/CFS and 73 healthy controls, age and sex related. Both the sick and the healthy were double-diagnosed based on the 2010 ME/CFS Canadian Consensus Criteria. The consent for this study was granted by the BarnaClínic Hospital. The main objective of the study was to find differences in the immunophenotypic characterization -aspects- of specific T lymphocytes and Natural Killer (NK) subpopulations to improve the diagnosis of this disease, currently based almost exclusively on clinical criteria (unspecific symptoms). Other objectives were to observe if there were correlations between certain markers and symptom severity, if subgroups can be inferred, or if there is a correlation with Epstein Barr Virus (EBV) and human Cytomegalovirus (HCMV) viral serology.

Results
Out of the total populations under study in T and NK lymphocytes, 5 altered lymphocyte subpopulations are obtained vs. healthy controls: very low NKG2C values, NKCD69 high count, NK56 bright high count, T-regulators low count and NKT-like high count.

There are also significant differences in correlation of different parameters.

Predictive model and its utility for the patient
A predictive statistical model called WEKA is used, taking into account the different patient subpopulations, their "weight" within the set and the correlations between them. This model is able, through advanced statistical algorithms, to offer a veredict: patient or control. The system is able to diagnose more than 70% of those affected (approximately 3 out of 4) only using two subpopulations: NKG2C and T-regulators. This predictive system will be made available to our patients, in order to improve the diagnosis of the disease, referencing the interpretation of the results to this study.

Other results obtained
- There are no differences between IgG EBV nor HCMV serologies, so we thought that they should not be used to support the diagnosis of the disease.

- No differences appear in the CD57 population in NK in % or in cell number, which have been associated with Lyme disease. Low CD57 values in NK could be associated with EBV.

- An inverse correlation has been found between high CD57 count in NK and low NKp46 count, which could be associated with HCMV; and low CD57 count in NK and high NKp46 count which could be associated with EBV. However, this association is also found in healthy controls and therefore lacks diagnostic value.

- High count of NKT-like (CD3 + CD16 + CD56 +) have been found in patients who report an infectious onset and high count of NKCD56 + Bright in patients who do not.

- There is a tendency (weak correlation) between symptom severity and high NKT-like count, and between greater fatigue vs. pain in low NKG2C count.

Possible hypotheses of how to frame these results (out of study)

- Group of 70% diagnosed with WEKA model.
It would correspond to low NKG2C and low Tregs count. It could be cause or consequence of EBV reactivations in a genetically predisposed population submitted to different stressors (chemical, labor stress, childbirth, multivaccination, other pathology or surgery), which could trigger a sequence of "no return" events, which in turn would affect different systems (ANS) and cause the pathology clinic. We think that the alteration of different "soluble substances" in serum (interleukins, catecholamines, hormones, autoantibodies) could affect healthy lymphoid subpopulations which could get sick in a pathological environment, hence the importance that future investigations be carried out in fresh blood. This sequence of events, initiated by an EBV reactivation, could end up causing autoimmunity and self-tolerance processes, thus chronifying the pathology.

- Group of the undiagnosed 30%.
One part could correspond to high NKG2C count, high NKT-like and high IgG HCMV - "separating" the said subgroup vs. the total population.

We think that high HCMV IgG levels can increase NKG2C count (this is known already) and also raise NKT-like (CD3 + CD16 + CD56 + / CD57 +). That is, it could be we could not diagnose this subgroup "on first instance" because NKG2C values were "masked" by the HCMV reactivation or by both (HCMV + EBV) at the same time, which in turn could initiate an etiopathogenic process similar to that described for EBV, even with a more marked clinical manifestation.

The fact that high NKT-like values are reported by those patients with an "infectious onset" could be due to a greater mononucleosis’ severity unleashed by HCMV rather than by EBV.

If so, we should try to find a similar predictive WEKA predictor model for this subgroup of patients.

What patients need
- This study should be replicated by other groups. This is the only way to get the international scientific and medical community to accept these results.

- It should be done under similar conditions to allow a comparison (equal lot size and fresh blood).

- With similar results, we could be able to:

· Improve the diagnosis or improve awareness, with all it implies: medical and social recognition, disability claims, pensions, etc; and new, effective and targeted treatments -not only symptomatic ones-.

· To start a process of diagnosis and "fine" characterization of patients in the current situation, and to influence the level of information and training of the medical community and patient association groups.

· Unlink all psychosomatic aspects and associated therapies (CBT) from ME / CFS.

- Continue with the research for much more specific biomarkers (eg. in the autoimmunity field, among others).

Our group is open to collaborate, share knowledge and join forces with all those national and international groups that wish to do so. It is all about influencing practical aspects that have a clear benefit in those affected by this disease: more research aimed at improving diagnosis, improving awareness and being pioneers in the advance of new pharmacological treatments that already work in ME/CFS and those used in other autoimmune pathologies which could be useful in subgroups of better characterized ME/CFS patients.

For this, it is necessary to have access to public financing and sufficient human and technical resources, to try to implement these advances in the patient groups ASAP.

“It is not about power, it is about love”, the saying goes. This is something we wanted and needed, but didn’t exist. I wanted it, we created it and here it is. Now, will all those researchers and excellent doctors out there want to give it continuity? There are so many who are awaiting for you!

José Luis Rivas. Researcher of ASSSEMBiomedics, pharmacist and ME/CFS patient. Casa del Mar. Barcelona, May 29th, 2018.

“It is not about power, it is about love”, the saying goes. This is something we wanted and needed, but didn’t exist. I wanted it, we created it and here it is. Now, will all those researchers and excellent doctors out there want to give it continuity? There are so many who are awaiting for you!

Most unfortunately, this research group was disbanded in 2018 due to lack of funding.

 

[Pyrrhus]

By the way, this study is a follow-up to a pilot study by other Spanish researchers:

Screening NK-, B- and T-cell phenotype and function in patients suffering from CFS. (Curriu et al., 2013)
https://forums.phoenixrising.me/thr...s-suffering-from-chronic-fatigue-syndr.22476/


And here are other discussions about lymphocytes such as NK cells or T cells:

Biomarkers in CFS: Natural Killer function (Fletcher/Klimas 2010)
https://forums.phoenixrising.me/thr...l-killer-function-fletcher-klimas-2010.11051/

Cytotoxic lymphocyte microRNAs as prospective biomarkers for CFS/ME (Brenu, Staines, Marshall-Gradisnik et al., 2012)
https://forums.phoenixrising.me/threads/new-phanu-study-on-microrna-markers.17324/

Longitudinal investigation of natural killer cells and cytokines in CFS/ME (Brenu, Staines, Marshall-Gradisnik et al., 2012)
https://forums.phoenixrising.me/thr...l-killer-cells-and-cytokines-in-cfs-me.16125/

Increased expression of activation antigens on CD8+ T lymphocytes in ME/CFS: inverse associations with lowered CD19+ expression and CD4+/CD8+ ratio, but no associations with (auto)immune, leaky gut, oxidative and nitrosative stress biomarkers (Maes et al., 2015)
https://forums.phoenixrising.me/thr...mphocytes-in-myalgic-encephalomyelitis.42118/

Unperturbed cytotoxic lymphocyte phenotype and function in ME/CFS patients (Theorell et al., 2017)
https://forums.phoenixrising.me/thr...ocyte-phenotype-and-function-in-me-cfs.52458/

Longitudinal associations of lymphocyte subsets with clinical outcomes in CFS (Mehalick et al., 2018)
https://forums.phoenixrising.me/thr...-subsets-with-clinical-outcomes-in-cfs.57597/

Decreased Expression of the CD57 Molecule in T Lymphocytes of Patients with Chronic Fatigue Syndrome. (Espinosa, 2019)
https://forums.phoenixrising.me/thr...chronic-fatigue-syndrome-espinosa-2019.77241/

ME/CFS patients exhibit altered T cell metabolism and cytokine associations (Hanson et al. 2020)
https://forums.phoenixrising.me/thr...ytokine-associations-hanson-et-al-2020.79143/

And a recent review paper about NK cells:
A systematic review of natural killer cells profile and cytotoxic function in ME/CFS (Eaton-Fitch et al. 2019)
https://systematicreviewsjournal.biomedcentral.com/articles/10.1186/s13643-019-1202-6

...As well as this oldie-but-goodie:
Comparative Analysis of Lymphocytes in Lymph Nodes and Peripheral Blood of Patients with Chronic Fatigue Syndrome (Fletcher et al., 1999)
https://forums.phoenixrising.me/thr...analysis-of-lymphocytes-in-lymph-nodes.10697/
(only accessible to Phoenix Rising members with at least 100 posts)

 

[pattismith]

ME/CFS patients showed significantly lower values of T regulatory cells (CD4+CD25++(high)FOXP3+) and higher NKT-like cells (CD3+CD16+/−CD56+) than the healthy individuals. Regarding NK phenotypes, NKG2C was significantly lower and NKCD69 and NKCD56 bright were significantly higher in the patients group.

T regulatory cells have been found to be lower in autoimmune conditions, such as systemic lupus erythematosus (96) and active RA (97), as well as inverted Th17/T regulatory cells ratio, with an elevated proinflammatory response (98).

In RA, treatment with low dose of methotrexate (MTX) increase levels of T regulatory cells and in immune thrombocytopenic purpura (99) with low doses of rituximab (RTX) together with steroids vs steroids only improve clinical symptoms. In a pilot study with three ME/CFS patients, symptoms improved after treatment with MTX and RTX (100), and in a larger study (101) improvement were seen after treatment with RTX only.

If our Treg are low, MTX might help us.

The probem is to manage the mental side effects of low dose MTX