Enhanced IL-15-mediated NK cell activation and proliferation by an ADAM17 function-blocking antibody involves CD16A, CD137, and accessory cells.

Background: NK cells are being extensively studied as a cell therapy for cancer. Their effector functions are induced by the recognition of ligands on tumor cells and by various cytokines. IL-15 is broadly used to stimulate endogenous and adoptively transferred NK cells in cancer patients. These stimuli activate the membrane protease ADAM17, which then cleaves assorted receptors on the surface of NK cells as a negative feedback loop to limit their activation and function. We have shown that ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo . In this study, we investigated the underlying mechanism of this process. Methods: PBMCs or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15 +/- an ADAM17 function-blocking antibody. Different versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab') 2 , and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A engagement on NK cells. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell anti-tumor activity. Results: The ADAM17 function-blocking mAb Medi-1 markedly increased initial NK cell activation by IL-15. Using different engineered versions of the antibody revealed that the activating Fcγ receptor CD16A, a well-described ADAM17 substrate, was critical for enhancing IL-15 stimulation. Hence, Medi-1 bound to ADAM17 on NK cells can be engaged by CD16A and block its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide, phagocytosis, or dysfunction. Synergistic activity by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A + NK cells and augmented their proliferation in the presence of PBMC accessory cells. Conclusions: Our data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the anti-tumor activity of NK cells in cancer patients. What is already known on this topic: NK cell therapies are being broadly investigated to treat cancer. NK cell stimulation by IL-15 prolongs their survival in cancer patients. Various stimuli including IL-15 activate ADAM17 in NK cells, a membrane protease that regulates the cell surface density of various receptors as a negative feedback mechanism. What this study adds: Treating NK cells with the ADAM17 function-blocking mAb Medi-1 markedly enhanced their activation and proliferation. Our study reveals that the Fc and Fab regions of Medi-1 function synergistically with IL-15 in NK cell activation. Medi-1 treatment augments the upregulation of CD137 by NK cells, which enhances their proliferation in the presence of PBMC accessory cells. How this study might affect research practice or policy: Our study is of translational importance as Medi-1 treatment in combination with IL-15 could potentially augment the proliferation and function of endogenous or adoptively transferred NK cells in cancer patients.

Paradoxical Effects of Endoplasmic Reticulum Aminopeptidase 1 Deficiency on HLA-B27 and Its Role as an Epistatic Modifier in Experimental Spondyloarthritis.

OBJECTIVE: We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 in experimental spondyloarthritis (SpA). METHODS: ERAP1-knockout rats were created using genome editing and bred with HLA-B27/human ß2 -microglobulin-transgenic (HLA-B27-Tg) rats and HLA-B7-Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology. RESULTS: ERAP1 deficiency increased the ratio of folded to unfolded (ß2 m-free) HLA-B27 heavy chains, while having the opposite effect on HLA-B7. Furthermore, in rats with ERAP1 deficiency, HLA-B27 misfolding was reduced, while free HLA-B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up-regulation of HLA-B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA-B27-Tg rats by two-thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA-B27, including reduced allogeneic T cell stimulation and loss of CD103-positive/major histocompatibility complex class II-positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up-regulation was mitigated. CONCLUSION: ERAP1 deficiency reduced HLA-B27 misfolding and improved folding while having opposing effects on HLA-B7. The finding that HLA-B27-Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss-of-function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA-B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.

A dominant function of LynB kinase in preventing autoimmunity.

Here, we report that the LynB splice variant of the Src-family kinase Lyn exerts a dominant immunosuppressive function in vivo, whereas the LynA isoform is uniquely required to restrain autoimmunity in female mice. We used CRISPR-Cas9 gene editing to constrain lyn splicing and expression, generating single-isoform LynA knockout (LynAKO) or LynBKO mice. Autoimmune disease in total LynKO mice is characterized by production of antinuclear antibodies, glomerulonephritis, impaired B cell development, and overabundance of activated B cells and proinflammatory myeloid cells. Expression of LynA or LynB alone uncoupled the developmental phenotype from the autoimmune disease: B cell transitional populations were restored, but myeloid cells and differentiated B cells were dysregulated. These changes were isoform-specific, sexually dimorphic, and distinct from the complete LynKO. Despite the apparent differences in disease etiology and penetrance, loss of either LynA or LynB had the potential to induce severe autoimmune disease with parallels to human systemic lupus erythematosus (SLE).