This study is the first to provide an overview of cell function and receptor interactions, including assessing DC, neutrophil and monocyte function and receptors on T cells, BCRs and Bregs in moderate and severe CFS/ME patients. This is also the first study to examine lytic proteins in γδT cells, iNKT cells and Tregs in CFS/ME patients. Our results suggest that in some cases, functional immunological impairment may be related to differences in severity of CFS/ME patients, highlighting the variation in the illness. Significant alterations shown in moderate CFS/ME patients were often not present in severe CFS/ME patients and controls, revealing the possibility that moderate CFS/ME patients may have a different aetiology compared with the severely affected subgroup of CFS/ME patients.

Increased SLAM expression on total NK cells in moderate CFS/ME patients in this study may be a mechanistic response to the typically reduced NK cell cytotoxic activity in CFS/ME [10, 11, 36]. SLAM is a receptor expressed on the surface of T, B, NK and DC cells, functioning as an activating adaptor protein to amplify the recruitment of inflammatory cells, such as DCs, by activating IFN-γ [37-39]. SLAM receptors regulate NK cell activity via association with SLAM associated protein (SAP) family adapters, where binding receptors are then coupled to the Src kinase FynT to evoke protein tyrosine phosphorylation signals [40]. Heightened expression of SLAM in moderate CFS/ME may enhance the ability of NK cells to undergo cytotoxic activities [5, 7-11]. The activating receptor NKp46 is also typically involved in the recognition and lysis of target cells and was reduced in CD56brightCD16dim NK cells of severe CFS/ME patients [41]. NKp46 is highly expressed on the CD56brightCD16dim NK cell subset and although it is only weakly involved in cytotoxic activation, this reduction may contribute to the reduced NK cell cytotoxic activity prevalent in severe CFS/ME patients [5, 41, 42]. The expression of SLAM and NKp46 on NK cells significantly differed between moderate and severe CFS/ME patients, suggesting that perhaps these severity subgroups may vary in immunological presentation, as proposed by previous studies [5, 7, 13].

Effector memory and CD45RA effector memory cells demonstrate NK-like functions as they have the ability to detect abnormal major histocompatibility complex (MHC) expression and have a high capability for cytotoxic activities [43]. Our findings suggest that the number of CD45RA effector memory CD8+T cells of moderate CFS/ME patients may be enhanced due to sub-optimal function. In T cells, CD45RA effector memory cells are upregulated following cytokine directed proliferation, indicating that the subset is generated via homeostasis rather than antigen-dependent pathways [44, 45]. Moderate CFS/ME patients have an increased number of CD8+T CD45RA effector memory cells, potentially as a result of homeostasis where the cells may not be effectively undergoing degranulation and apoptosis [44, 45].

This research also found reductions in KLRG1 expression in total CD8+T and naïve CD4+T cells of moderate CFS/ME patients, which suggests that these cells may have a reduced ability to inhibit T cell function and activation. KLRG1 ligation inhibits the nuclear factor of activated T cells (NFAT) signalling pathway and downregulates CD95 mediated lysis to inhibit the activation of T cells [46]. Hence, blockades of inhibitory receptors tend to improve CD8+T cell responses by preventing inhibitory pathways [47]. It is therefore possible that reduced KLRG1 may be contributing to the pro-inflammatory response and T cell activation often found in CFS/ME patients [10, 48].

Increased KIR2DL5 on CD4+T cells in moderate CFS/ME patients may also be associated with alterations in KIR receptors in T cells in the same cohort [5, 7]. KIR2DL5 is an inhibitory KIR found in variable proportions of circulating T cells [49, 50] which is directly linked to a greater number of random combinations of KIR receptors expressed on these cells, which may be influencing optimal T cell functions in the illness [51]. Enhanced inhibitory signalling and modulation of immune responses are typical attributes of increased BTLA expression in T cells which may be present in moderate CFS/ME patients who have amplified expression of inhibitory receptor BTLA4 in central memory CD4+T cells [52]. Activation and function of CD4+T cells by NK cells is dependent on the engagement of the β 2 integrin LFA-1. LFA-1 adhesion is necessary for optimal cytotoxic activity by both NK cells and CD8+T cells, also mediating NK cell degranulation via synergy with NKG2D [53]. Decreased expression of LFA-1 on central memory CD8+T cells in moderate CFS/ME patients suggests that there may be a lack of LFA-1 adhesion in CFS/ME which is required for ideal cytotoxic activity by NK cells and CD8+T cells [53]. Similar to the pattern shown in SLAM and NKp46 receptors on NK cells, CD45RA effector memory CD8+T cells and CD4+T and CD8+T cell receptors significantly differed between moderate and severe CFS/ME patients.

Cellular adhesion may be important in CFS/ME as it is required for target cell contact and NK cell effector function. Regulation of adhesion molecules is necessary for integrin target cell ligand interactions as the release of adherence results in lymphocyte movement [54]. CD2 expression in CD56dimCD16− NK cells is reduced in moderate CFS/ME patients compared with severely affected patients, suggesting that these cells may have an impaired ability to adhere to target cells. This confirmed previous findings where CD2/CD18 co-expression was reduced in the same CD56dimCD16− NK cell phenotype in a cohort of moderate CFS/ME patients [24]. Increased CD2 is often associated with a higher cytotoxic ability [55] as CD2 acts as a contributor to induce NK cell activation [54, 55]. Higher expression of CD2 in severe CFS/ME patients potentially implies that more NK cells in these patients are in an active state and may have a greater ability than the moderate CFS/ME patients to induce NK cell activation and cytotoxic activities [55]. CD18+/CD2− CD56dimCD16− NK cells were also increased in the moderate CFS/ME patients, strengthening the theory that CFS/ME patients may have a weakened ability to activate NK cells as well as having impaired NK cell cytotoxic activity. Adhesion molecules CD18 and CD2 on CD56dimCD16− NK cells were also significantly altered in the moderate CFS/ME patient group compared with the severe CFS/ME patients, who appeared similar to the controls. In the case of CD18+CD11c− on the same CD56dimCD16− NK cell subset, however, increases in CD18+CD11c− increased in the moderate CFS/ME patients and significantly increased in the severe CFS/ME patients. Expression of the adhesion marker CD11c is heterogeneous and variable in NK cells although, typically activated NK cells are CD11c+ [55]. Increased CD18+CD11c− on CD56dimCD16− NK cells in severe CFS/ME patients indicates that these patients may have a reduced ability to adhere or that they may have a low number of activated NK cells. Therefore, differences in CD18+CD11c− adhesion molecules in severe CFS/ME patients may be associated with the reduced NK cell cytotoxic activity found in the illness [5, 10, 11].

CFS/ME symptom severity and presentation may be related to the immune dysregulation shown as the immune system interacts with physiological functioning via a number of body systems, including the central nervous system, digestive system and endocrine system [56]. Unrefreshing sleep and sleep disturbances are symptoms of CFS/ME and reports have indicated that NK cells are altered after sleep deprivation, demonstrating interactions between physiological symptoms and the immune system [57], particularly in CFS/ME patients. Similarly, it has previously been suggested that clinical severity status appears to be associated with reduced NK cell activity in CFS/ME patients [7, 13]. Although there are limited research findings for severe CFS/ME patients, the differences in NK cells, CD4+T and CD8+T cells between severity groups, found in this research, suggest that immune dysfunction in CFS/ME may be related to clinical symptoms and hence severity.