CFS/ME is a debilitating disorder hallmarked by unexplained debilitating fatigue accompanied by immune, neurological, musculoskeletal, cardiovascular and gastrointestinal symptoms . The diagnosis of CFS/ME is complex and relies on case definition for diagnostic criteria [1,2,3]. The underlying etiology of CFS/ME remains unknown; however, a significant reduction of NK cell cytotoxicity is a key and consistently reported feature of CFS/ME [4,5,6,7,8,9,10,11,12,13,14].
CFS/ME is believed to affect approximately 200,000 Australians  having a global prevalence of 0.2–6.4% . CFS/ME is reported more commonly in women than in men, with 75% of patients being female  and predominantly affecting 30- to 40-year-olds in developed countries . However, due to inconsistencies in CFS/ME case definitions the true prevalence is difficult to determine.
NK cells are effector lymphocytes of the innate immune system that eliminate pathogens and malignant cells, activate immune cells and provide cytokine producing functions . NK cells have tightly regulated cytotoxic activity against stress and antibody-coated cells [18,19,20,21,22]. The majority of human peripheral NK cells are CD56dim NK cell subset bearing the low-affinity Fc-γ-receptor CD16 . CD16 binds to the Fc portion of immunoglobulin (Ig) G and mediates antibody-dependent cellular cytotoxicity (ADCC) [23,24,25,26]. NK cell cytotoxicity (NKCC) involves numerous steps including adhesion to the target cell, activations of surface receptors, polarization of secretory granules and release of lytic proteins, including granzyme A and granzyme B, to induce apoptosis of the target cell [7, 21, 27].
NK cell activation is tightly regulated by activating receptors that recognise pathogen-derived, stress-induced and tumour specific ligands . CD16 plays a prominent role as an activating receptor for NK cells . NK cell activation initiates calcium (Ca2+)-dependent signal transduction through receptor cytoplasmic tails that contain immunoreceptor tyrosine-based activation motifs (ITAM) [11, 22, 29,30,31]. Ligation of ITAM-bearing receptor complexes results in the recruitment and activation of mitogen-activated protein kinase (MAPK) phosphorylation cascade . The phosphorylation of kinases induces the polarization of NK cell granules via the microtubule-organizing centre (MTOC) [32, 33]. Granule polarization ensures granule contents are released facing the target cell. NK cell cytotoxic granules are responsible for the storage and secretion of lytic proteins including perforin, a membrane-disrupting protein, and granzymes, a family of proteases [11, 27]. The secretion of perforin is suggested to create pores within the target cell membrane to facilitate endocytosis mechanisms, in which granzymes can enter the target cell to trigger apoptosis by cleaving pro-apoptotic caspases and influence nuclear damage [5, 7, 27, 34, 35].
Importantly, reduced NK cell function is the most consistently reported finding in both severe and moderate CFS/ME patients [4,5,6,7,8,9,10,11,12,13,14]. Impaired NKCC in CFS/ME patients is evident through delayed degranulation [7, 36, 37] and decreased lytic proteins, predominantly granzyme B [5, 11, 36, 38, 39]. The increase in NK cell degranulation in CFS/ME patients may suggest an inability to induce sufficient cytotoxic lysis or continued activation due to insufficient lytic proteins. Therefore, using flow cytometry to investigate expression of degranulation markers, CD107a and CD107b, and intracellular lytic proteins, granzyme A and granzyme B, is critical when investigating the cytotoxic activity of NK cells .
Rituximab (RTX), is a chimeric antibody that targets CD20 present on healthy and malignant B lymphocytes. RTX can trigger target cell death through three effector functions: 1) programmed cell death, 2) induction of complement-mediated cytotoxicity, and 3) ADCC mediated by Fc receptor-bearing immune cells such as NK cells . RTX works to opsonize the CD20 surface marker on B lymphocytes, this stimulates the recruitment of NK cells and ligation with CD16. The activation of CD16 to the Fc portion of RTX activates Ca2+-dependent elimination of B lymphocytes through ADCC.
Limited investigations have examined the potential role of therapeutic interventions in CFS/ME patients. We and others have reported elevated CD20+ B lymphocytes in CFS/ME patients [9, 42,43,44]. Moreover other investigators have employed anti-CD20 therapy as a possible therapeutic approach for the treatment of CFS/ME [45,46,47], where CFS/ME patients received two infusions at 500 mg/m2 of RTX two weeks apart. Clinical improvement was self-reported in sixty-four and 67 % of participants in these two separate studies [45, 47]; however this improvement was only maintained in 26.6% of patients twelve months post administration.
Importantly, a recent study conducted by Merkt et al. reported that in vitro treatment of NK cells with RTX at a concentration of 10 μg/ml resulted in significant inhibition of NK cell cytotoxic activity from healthy control donors . This investigation also reported a significant reduction of lytic proteins, predominantly granzyme B, and phenotypical and functional changes to CD16  in NK cells following RTX incubation from non-fatigued controls donors.
The effect of RTX on NK cells in malignancies and rheumatic diseases is documented [41, 48,49,50]. However, the possible role of RTX modulating NK cell cytotoxicity in CFS/ME patients is unknown as Lunde et al. only investigated NK cell subset numbers in CFS/ME patients receiving 500 mg/m2 of RTX . Therefore, this investigation aimed to examine NK cell cytotoxic activity, lytic proteins and degranulation following incubation of RTX at varying concentrations with NK cells from CFS/ME patients in a controlled and safe laboratory setting in vitro.