Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is characterised by significant impairments in physical activity as a consequence of severe fatigue and other flu like symptoms. CFS/ME remains an unexplained disorder with substantial physiological impairments where diagnosis is based on self-report measures. Fatigue is present in other disorders, including Multiple Sclerosis (MS), where 70% of patients are plagued with fatigue and this can be disabling in a minority of patients [
MS is a neurological disorder characterised by inflammatory demyelination in the CNS while CFS/ME patients may experience nervous system manifestations of lack of concentration and autonomic symptoms [
Expansion of autoreactive lymphocytes in MS results in inflammatory and active immune responses in the CNS as these lymphocytes are able to migrate to the CNS and induce damage [
It is apparent that CD8+ T cells are involved in the pathogenesis of CFS/ME and MS; hence, the aim of this study was to determine whether dysregulation in cytotoxic CD8+ T cells follows a similar pattern in CFS/ME and MS.
CFS/ME participants were defined according to the International Consensus Criteria (ICC) [
Whole blood (10 mL) was collected from all participants and analysed within 12 hours of collection. To identify subsets of CD8+ T cells at different stages of differentiation, samples were labelled with fluorochrome conjugated monoclonal antibodies, including CD3, CD8, CD27, and CD45RA (CD45 exon isoform 4). Cells were analysed on the Fortessa 2.0 (Becton Dickenson (BD) Biosciences, San Jose). For each CD8+ T cell assessment, forward and side scatter plots were used to determine the lymphocyte population. Cells of interest were identified from the lymphocyte population as cells expressing CD3+ and CD8+. The expression of cytokines, chemokine receptors, adhesion molecules, and migratory molecules on CD8+ T cells were also examined using the following markers: CCR5, CCR7, CXCR3, CD49d, CD29, CD18, CD11a, PSGL-1, and CD127. Glycoprotein, CD44, was also examined.
Inhibitory receptors were measured in whole blood cells stained with monoclonal antibodies including KLRG1, LAG3, CTLA4, and BTLA. The expression patterns of these inhibitory receptors were examined on the CD8+ T cell phenotypes. Coexpression of these receptors was also assessed on subsets of CD8+ T cells.
Statistical analyses were executed using SPSS (version 18.0, SPSS Inc., Chicago, USA) and Graph Pad Prism (version 6.0, Graph Pad Software, Inc., San Diego, USA). A test for normality was performed using the Kolmogorov-Smirnov tests. ANOVA was used to determine significance for normally distributed data while the independent sample Kruskal Wallis test was used as the nonparametric. Bonferroni analysis was used to assess significant parameter differences post hoc. Pearson chi square test was used to determine significant gender differences.
The characteristics of the participants recruited in the study are outlined in Table
Characteristics of participants and blood parameters.
CFS/ME | MS | Controls |
|
||||
---|---|---|---|---|---|---|---|
Overall | MS versus CFS/ME | MS versus control | CFS/ME versus control | ||||
Participants ( |
23 | 11 | 30 | ||||
Age (years) | 49.0 ± 2.5 | 56.0 ± 4.9 | 53.5 ± 2.2 | 0.72 | >0.99 | >0.99 | >0.99 |
Females, |
17 (73.9) | 10 (90.9) | 19 (63.3) | 0.44 | |||
Haemoglobin (g/L) | 135.0 ± 2.25 | 136.0 ± 2.90 | 139.0 ± 2.35 | 0.24 | >0.99 | 0.89 | 0.32 |
White cell count (×109/L) | 5.60 ± 0.32 | 6.80 ± 0.64 | 6.00 ± 0.25 |
|
|
0.52 | 0.28 |
Platelets | 228.0 ± 13.34 | 264.0 ± 15.80 | 249.50 ± 10.69 | 0.76 | >0.99 | >0.99 | >0.99 |
Haematocrit (%) | 0.41 ± 0.01 | 0.40 ± 0.01 | 0.41 ± 0.01 | 0.35 | >0.99 | >0.99 | 0.49 |
Red cell count (×1012/L) | 4.49 ± 0.07 | 4.55 ± 0.13 | 4.61 ± 0.07 | 0.53 | >0.99 | >0.99 | 0.79 |
MCV (fL) | 89.17 ± 0.69 | 89.20 ± 0.85 | 89.53 ± 0.68 | 0.72 | >0.99 | >0.99 | >0.99 |
Neutrophils (×109/L) | 3.38 ± 0.25 | 4.20 ± 0.49 | 3.53 ± 0.17 | 0.39 | 0.53 | 0.82 | >0.99 |
Lymphocytes (×109/L) | 1.63 ± 0.09 | 2.30 ± 0.23 | 1.95 ± 0.10 |
|
|
0.13 | 0.11 |
Monocytes (×109/L) | 0.31 ± 0.02 | 0.45 ± 0.03 | 0.33 ± 0.02 |
|
|
|
>0.99 |
Eosinophils (×109/L) | 0.14 ± 0.015 | 0.21 ± 0.03 | 0.12 ± 0.01 |
|
0.13 |
|
0.91 |
Basophils (×109/L) | 0.02 ± 0.004 | 0.03 ± 0.004 | 0.02 ± 0.003 | 0.41 | 0.56 | >0.99 | >0.99 |
ESR (mm/Hr) | 10.50 ± 2.59 | 13.00 ± 5.29 | 10.00 ± 1.91 | 0.21 | >0.99 | 0.89 | 0.26 |
Data is represented as mean ± SEM, where
Clinical characteristics of CFS/ME and MS.
CFS/ME ( |
MS ( | |
---|---|---|
Dr. Bell’s Disability | 47.14% ± 2.20 | |
Expanded Disability Status Scale | 2.41 ± 0.79 | |
Multiple Sclerosis Severity Scale | 2.85 ± 0.89 | |
Courses | ||
(i) Relapsing-remitting |
|
|
(ii) Secondary-progressive |
|
|
(iii) Primary-progressive |
|
|
(iv) Clinically isolated syndrome |
|
|
Age of onset (years) | 35.28 ± 4.63 | 6.11 ± 2.45 |
Duration (years) | 14.96 ± 8.87 | 13.76 ± 3.83 |
Relapses rate | 2.4 ± 0.55 |
MS patients were not on any immunomodulatory therapies during this study, nor had they taken these previously. Of the 11 MS patients, there were relapsing-remitting (
In each group there were a large percentage of females in comparison to males but there was no significant difference in gender. Full blood count analyses were performed on all samples to determine the distribution of the different blood cells (Table
Distribution of total and subsets of CD8+ T cells in CFS/ME patients, MS patients, and nonfatigued controls.
CD8+ T cells (%) | CFS/ME | MS | Nonfatigued controls |
|
|||
---|---|---|---|---|---|---|---|
Overall | MS versus CFS/ME | CFS/ME versus control | MS versus control | ||||
Total |
13.25 ± 0.86 | 12.36 ± 1.89 | 13.24 ± 0.88 | 0.84 | >0.99 | >0.99 | >0.99 |
Naïve |
33.86 ± 3.89 | 36.76 ± 5.76 | 24.20 ± 2.19 | 0.07 | >0.99 | 0.12 | 0.29 |
CM |
31.88 ± 2.63 | 38.50 ± 2.36 | 32.93 ± 2.75 | 0.51 | 0.81 | >0.99 | 0.88 |
EM |
12.52 ± 2.15 | 15.83 ± 2.77 | 18.64 ± 2.13 | 0.07 | >0.99 | 0.08 | 0.51 |
EMRA |
10.18 ± 2.21 | 6.86 ± 2.22 | 13.38 ± 2.13 | 0.06 | 0.82 | 0.43 | 0.08 |
Surface expression of the following inhibitory receptors was examined on the CD8+ T cells, KLRG1, LAG3, CTLA4, and BTLA. These receptors were measured on total CD8+ T cells and subsets of CD8+ T cells at different stages of differentiation as previously described. Only BTLA was significantly elevated in the naïve and CM CD8+ T cells from the MS patients compared with the CFS/ME patients and the nonfatigued controls (Figure
Expression of BTLA on CD8+ T cells in CFS/ME, MS, and nonfatigued controls. BTLA was increased on naïve and CM CD8+ T cells in the MS compared with controls. Data is represented as median ± SEM, where
Expression of cytokine receptors including CCR7, CCR5, and CD127 was measured on total CD8+ T cells and subsets of CD8+ T cells at different stages of differentiation. CD49/CD29 was significantly reduced on the EM CD8+ T cells of the CFS/ME patients in comparison to the nonfatigued controls, while in the MS patients, CD49d/CD29 was significantly elevated in the naïve and EMRA CD8+ T cells in comparison to the nonfatigued controls (Figure
Expression of receptors in CFS/ME, MS, and nonfatigued controls. (a) CD49d/CD29 was reduced in EM subsets of CD8+ T cells in the CFS/ME patients but elevated in EMRA subsets of CD8+ T cells in MS patients compared to controls. (b) CD127 expression was reduced on naïve, EM, and EMRA subsets of CD8+ T cells in the CFS/ME patients but not CM subsets as CD127+ CD8+ T cells were evaluated in MS patients compared with controls. MS patients also demonstrated elevated naïve CD8 T cells compared with controls. Data is represented as median ± SEM, where
Expression of adhesion molecules on subsets of CD8+ T cells in CFS/ME, MS, and nonfatigued controls. Expression levels of PSGL-1 were reduced on EMRA CD8+ T cells in the CFS/ME patients compared with the nonfatigued controls. Data is represented as ± SEM, where
This preliminary study has identified significant impairments in subsets of CD8+ T cells in CFS/ME and MS patients. Overall the MS patients showed significant differences in the expression of receptors and adhesion molecules in comparison to the CFS/ME patients. These results demonstrate CD8+ T cells might play a role in the pathogenesis of MS compared with CFS/ME. PSGL-1 is elevated on CD4+ T cells in RR-MS patients and this may suggest an important role in the transmigration of lymphocytes to the CNS [
Previous research has indicated decreases in total CD8+ T cells in particularly EM and EMRA CD8+ T cells [
In summary, these preliminary findings provide new insight into the possibility of hyper activated inflammatory CD8+ T cell profile in untreated MS patients while CFS/ME patients may display an exhausted profile which permits viral prevalence and persistence. The above data may suggest that the differential expressions of receptors and adhesion molecules in MS patients are in response to imbalances in neuroimmune homeostasis. In comparison to CFS/ME patients, MS patients may have more severe immune dysregulation. Nevertheless it is likely that impairments in CD8+ T cells in CFS/ME patients relate to abnormal levels of adhesion and migratory molecules and these abnormalities may contribute to the persistent immune dysregulation observed and warrant further validation in a larger sample size.
Cluster of differentiation
Multiple Sclerosis
Chronic Fatigue Syndrome/Myalgic Encephalomyelitis
Central memory
Effector memory
Effector memory cells expressing CD45RA
B- and T-lymphocyte attenuator
Killer cell lectin-like receptor subfamily G member 1
Lymphocyte-activation protein 3
Cytotoxic T-lymphocyte-associated protein 4
Chemokine receptor type 7
Chemokine receptor type 5
Vascular cell adhesion protein 1
Interferon gamma
Tumor necrosis factor
Interleukin-7
Central Nervous System
Programmed cell death protein 1
Herpes virus entry mediator
Nuclear factor kappa-light-chain enhancer of activated B cells.
The authors alone are responsible for the content and writing of the paper.
The authors report no conflict of interests.
Ekua W. Brenu, Thao Nguyen, and Samantha Johnston wrote the paper, Simon Broadley provided the MS patients and reviewed the paper, and Sonya Marshall-Gradisnik and Don Staines provided supervision and assisted with the writing of the paper.
The authors would like to acknowledge the National Centre for Neuroimmunology and Emerging Diseases, Alison Hunter Memorial Foundation, Mason Foundation (Grant no. MA43120), and Queensland Government Science, Information Technology, Innovation and the Arts Smart Futures Fund (Grant no. 216702MRE) for their support.