No abstract

BACKGROUND:The search for a biomarker specific for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) has been long, arduous and, to date, unsuccessful. Researchers need to consider their expenditures on each new candidate biomarker. In a previous study of antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer lymphocytes, we found lower ADCC for ME/CFS patients vs. unrelated donors but ruled against low ADCC as a biomarker because of similar ADCC for patients vs. their family members without ME/CFS. OBJECTIVE: We applied inclusion of family members without ME/CFS, from families with multiple CFS patients, as a second non-ME/CFS control group in order to re-examine inflammation in ME/CFS. METHOD: Total and CD16A-positive 'non-classical' anti-inflammatory monocytes were monitored. RESULTS: Non-classical monocytes were elevated for patients vs. unrelated healthy donors but these differences were insignificant between patients vs. unaffected family members. CONCLUSIONS: Inclusion of family members ruled against biomarker considerations for the monocytes characterized. These pilot findings for the non-classical monocytes are novel in the field of ME/CFS. We recommend that occupational therapists advocate and explain to family members without ME/CFS the need for the family members' participation as a second set of controls in pilot studies to rapidly eliminate false biomarkers, optimize patient participation, and save researchers' labor.

No abstract

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

Serial killers are more desirable than single-hit attackers when it comes to treating cancer. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK lymphocytes is a significant component of many monoclonal antibody anti-cancer immunotherapies. However, there is a significant problem associated with ADCC: the NK cells lose their CD16A Fc-receptors and can no longer recognize target cells coated with antibodies, due to the action of NK cell metalloproteases. In this report, we searched for evidence for serial ADCC before the effector cells lose their receptors. Human ADCC measured by 51Cr-release at multiple NK effector to tumor cell ratios (E:Ts) was largely finished after 4 hours when the targets were Daudi B leukemia cells coated with non-fucosylated obinutuzumab anti-CD20. We reasoned that serial killing is evident when the number of dead targets exceeds the number of initial CD16A NK effectors. We assessed the dead Daudi per NK effector (D/E) at an E:T of 1:4 to detect serial killers given an excess of targets. For 28 healthy human subjects, the total ratio of dead targets per CD16Apos NK effector cell ranged from 0.6 to 2.2, with a median of 1.5 and average of 1.5 +/− 0.4. Four donors (14%) had D/Es >2. The D/E values were similar for CD16A AA158 F/F donors compared to F/V & V/V donors, even though the V-positive donors had greater initial NK cellular levels of CD16A. We conclude that this gross measurement (D/E) can detect serial ADCC even without the information provided by time-lapse cinematography to indicate the actual fraction of effectors that are serial killers. This simple measurement may be useful to screen for promotion of serial killing by reagents that reduce proteolytic loss of NK cell CD16A.