Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC).

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.

Inclusion of family members without ME/CFS in research studies promotes discovery of biomarkers specific for ME/CFS.

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.

Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) in Familial Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

BACKGROUND: Chronic fatigue syndrome (CFS) is an illness of unknown origin that may have familial risks. Low natural killer (NK) lymphocyte activity was proposed as a risk for familial CFS in 1998. Since then, there have been many studies of NK lymphocytes in CFS in general populations but few in familial CFS. Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK lymphocytes helps control viral infections. ADCC is affected by variant CD16A receptors for antibody that are genetically encoded by FCGR3A. METHODS: This report characterizes ADCC effector NK cell numbers, ADCC activities, and FCGR3A variants of five families each with 2-5 CFS patients, their family members without CFS and unrelated controls. The patients met the Fukuda diagnostic criteria. We determined: CD16Apositive blood NK cell counts; EC50s for NK cell recognition of antibody; ADCC lytic capacity; FCGR3A alleles encoding CD16A variants, ROC tests for biomarkers, and synergistic risks. RESULTS: CFS patients and their family members had fewer CD16Apositive NK cells, required more antibody, and had ADCC that was lower than the unrelated controls. CFS family members were predominantly genetically CD16A F/F s for the variant with low affinity for antibodies. ROC tests indicated unsuitability of ADCC as a biomarker for CFS because of the low ADCC of family members without CFS. Familial synergistic risk vs. controls was evident for the combination of CD16Apositive NK cell counts with ADCC capacity. CONCLUSIONS: low ADCC may be a risk factor for familial CFS. Furthermore, characterization of familial CFS represents an opportunity to identify pathogenic mechanisms of CFS.

An improved method to quantify human NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) per IgG FcR-positive NK cell without purification of NK cells.

Natural killer (NK) lymphocyte ADCC supports anti-viral protection and monoclonal antibody (mAb) anti-tumor therapies. To predict in vivo ADCC therapeutic responses of different individuals, measurement of both ADCC cellular lytic capacity and their NK cellular receptor recognition of antibodies on 'target' cells are needed, using clinically available amounts of blood. Twenty ml of blood provides sufficient peripheral blood mononuclear cells (PBMCs) for the new assay for lytic capacity described here and for an antibody EC50 assay for Fc-receptor recognition. For the lytic capacity assay, we employed flow cytometry to quantify the CD16A IgG Fc-receptor positive NK effector cells from PBMCs to avoid loss of NKs during isolation. Targets were 51Cr-labeled Daudi B cells pretreated with excess obinutuzumab type 2 anti-CD20 mAb and washed; remaining free mAb was insufficient to convert B cells in the PBMCs into 'targets'. We calculated: the percentage Daudis killed at a 1:1 ratio of CD16A-positive NK cells to Daudis (CX1:1); lytic slopes; and ADCC50 lytic units. Among 27 donors, we detected wide ranges in CX1:1 (16-73% targets killed) and in lytic slopes. Slope variations prevented application of lytic units. We recommend CX1:1 to compare individuals' ADCC capacity. CX1:1 was similar for purified NK cells vs. PBMCs and independent of CD16A V & F genotypes and antibody EC50s. With high mAb bound onto targets and the high affinity of obinutuzumab Fc for CD16A, CX1:1 measurements discern ADCC lytic capacity rather than antibody recognition. This assay allows ADCC to be quantified without NK cell isolation and avoids distortion associated with lytic units.