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Purpose: mAbs are used to treat solid and hematologic malignancies and work in part through Fc receptors (FcRs) on natural killer cells (NK). However, FcR-mediated functions of NK cells from patients with cancer are significantly impaired. Identifying the mechanisms of this dysfunction and impaired response to mAb therapy could lead to combination therapies and enhance mAb therapy. Experimental Design: Cocultures of autologous NK cells and MDSC from patients with cancer were used to study the effect of myeloid-derived suppressor cells (MDSCs) on NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in vitro Mouse breast cancer models were utilized to study the effect of MDSCs on antibody therapy in vivo and test the efficacy of combination therapies including a mAb and an MDSC-targeting agent. Results: MDSCs from patients with cancer were found to significantly inhibit NK-cell FcR-mediated functions including antibody-dependent cellular cytotoxicity, cytokine production, and signal transduction in a contact-independent manner. In addition, adoptive transfer of MDSCs abolished the efficacy of mAb therapy in a mouse model of pancreatic cancer. Inhibition of iNOS restored NK-cell functions and signal transduction. Finally, nonspecific elimination of MDSCs or inhibition of iNOS in vivo significantly improved the efficacy of mAb therapy in a mouse model of breast cancer. Conclusions: MDSCs antagonize NK-cell FcR-mediated function and signal transduction leading to impaired response to mAb therapy in part through nitric oxide production. Thus, elimination of MDSCs or inhibition of nitric oxide production offers a strategy to improve mAb therapy. Clin Cancer Res; 24(8); 1891-904. ©2018 AACR ., (©2018 American Association for Cancer Research.)

CD20 is a widely validated, B cell-specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 Ab that prolongs survival of chronic lymphocytic leukemia (CLL) patients when combined with chemotherapy. Ofatumumab and GA101 (obinutuzumab) are CD20-directed Abs currently being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death, NK cell-mediated Ab-dependent cellular cytotoxicity, or complement-dependent cytotoxicity. Despite widespread study, ofatumumab and GA101 have not been compared with each other, nor studied for their interactions with monocytes and macrophages which are critical for the efficacy of anti-CD20 Abs in murine models. In CLL cells, we show that direct cell death and complement-dependent cytotoxicity are greatest with GA101 and ofatumumab, respectively. GA101 promotes enhanced NK cell activation and Ab-dependent cellular cytotoxicity at high Ab concentrations. Ofatumumab elicits superior Ab-dependent cellular phagocytosis with monocyte-derived macrophages. GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate that GA101 and ofatumumab are both superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 Abs in the treatment of CLL.

We recently reported that CD3- natural killer (NK) cells express the zeta chain of the T-cell receptor complex (zeta NK) in association with higher molecular weight structures whose expression differs between individual NK cell clones. Because NK cell cytolytic activity is known to be triggered by perturbation of the type III Fc gamma receptor (CD16), we sought to determine whether this activating molecule is included in the zeta NK molecular complex. Biochemical evidence for a physical association between CD16 and zeta NK was obtained by comparing immunoprecipitates formed using monoclonal antibodies reactive with each of these molecules by SDS/polyacrylamide gel electrophoresis, immunoblotting, and peptide mapping. In both clonal and polyclonal populations of CD3- NK cells, CD16 and zeta NK specifically associated with one another. Functional evidence for a specific association between CD16 and zeta NK in intact cells was obtained by demonstrating a coordinate down-modulation of both of these molecules induced by either phorbol 12-myristate 13-acetate or monoclonal antibodies reactive with CD16. Our results suggest that Fc gamma receptor type III (CD16) is included in the zeta NK complex and that this complex is likely to play an important role in NK cell activation.

Natural killer (NK) cells are large granular lymphocytes capable of killing tumour cells in a non-MHC restricted manner. NK cells do not express cell-surface CD3, or any known target recognition structure analogous to the T cell antigen receptor (TCR) heterodimers (alpha beta or gamma delta). Consistent with their lack of expression of a CD3-TCR complex, NK cells do not require prior sensitization or antigen presentation by accessory cells to specifically recognize their tumour targets. Although NK cells do not express CD3-TCR, they do express CD2, the target of an alternative activation pathway which is functional in both T cells and NK cells. In T cells, this alternative activation pathway utilizes some component of the CD3-TCR complex as a transducer molecule that is required for mitogenesis. The fact that NK cells are activated by this alternative pathway suggested that they might express a related subunit of the CD3-TCR complex capable of transducing the CD2-mediated signal. Here we show that human NK cells express the zeta-chain of the TCR complex in association with additional structures not included in CD3-TCR.

Natural killer (NK)3 cells are large granular lymphocytes that appear to play a significant role in the host's defense against viral infection. We performed an extensive phenotypic and functional characterization of NK cells on 41 patients with the chronic fatigue syndrome (CFS), or "chronic active Epstein-Barr virus infection" syndrome, and on 23 age- and sex-matched asymptomatic control subjects in an attempt to further characterize this illness. These studies demonstrated that a majority of patients with CFS have low numbers of NKH1+T3- lymphocytes, a population that represents the great majority of NK cells in normal individuals. CFS patients had normal numbers of NKH1+T3+ lymphocytes, a population that represents a relatively small fraction of NK cells in normal individuals. When tested for cytotoxicity against a variety of different target cells, patients with CFS consistently demonstrated low levels of killing. After activation of cytolytic activity with recombinant interleukin 2, patients were able to display increased killing against K562 but most patients remained unable to lyse Epstein-Barr virus-infected B cell targets. Additional cytotoxicity experiments were carried out utilizing anti-T3 monoclonal antibody to block killing by NKH1+T3+ cells. These experiments indicated that the NK cell that appears to be responsible for much of the functional activity remaining in patients with CFS belongs to the NKH1+T3+ subset, which under normal circumstances represents only approximately 20% of the NK cell population.