RESUMEN
There are two known mechanisms by which natural killer (NK) cells recognize and kill diseased targets: (i) direct killing and (ii) antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated an indirect NK cell activation strategy for the enhancement of human NK cell killing function. We did this by leveraging the fact that toll-like receptor 9 (TLR9) agonism within pools of human peripheral blood mononuclear cells (PBMCs) results in a robust interferon signaling cascade that leads to NK cell activation. After TLR9 agonist stimulation, NK cells were enriched and incorporated into assays to assess their ability to kill tumor cell line targets. Notably, differential impacts of TLR9 agonism were observed-direct killing was enhanced while ADCC was not increased. To ensure that the observed differential effects were not attributable to differences between human donors, we recapitulated the observation using our Natural Killer-Simultaneous ADCC and Direct Killing Assay (NK-SADKA) that controls for human-to-human differences. Next, we observed a treatment-induced decrease in NK cell surface CD16-known to be shed by NK cells post-activation. Given the essential role of CD16 in ADCC, such shedding could account for the observed differential impact of TLR9 agonism on NK cell-mediated killing capacity.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Células Asesinas Naturales , Receptor Toll-Like 9 , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Receptores de IgG/metabolismo , Receptores de IgG/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacosRESUMEN
Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eµ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfocitos T , Microambiente Tumoral , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Microambiente Tumoral/inmunología , Microambiente Tumoral/efectos de los fármacos , Humanos , Animales , Ratones , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-alfa del Hepatocito/genética , Proliferación Celular/efectos de los fármacos , Proteínas que Contienen Bromodominio , ProteínasRESUMEN
Assays to quantify natural killer (NK) cell killing efficacy have traditionally focused on assessing either direct killing or antibody dependent cell-mediated cytotoxicity (ADCC) independently. Due to the probability that immunotherapeutic interventions affect NK cell-mediated direct killing and NK cell-mediated ADCC differently, we developed an assay with the capacity to measure NK cell-mediated direct killing and ADCC simultaneously with cells from the same human donor. Specifically, this design allows for a single NK cell population to be split into several experimental conditions (e.g., direct killing, ADCC), thus controlling for potential confounders associated with human-to-human variation when assessing immunotherapy impacts. Our Natural Killer cell Simultaneous ADCC and Direct Killing Assay (NK-SADKA) allows researchers to reproducibly quantify both direct killing and ADCC by human NK cells. Furthermore, this optimized experimental design allows for concurrent analysis of the NK cells via flow cytometric immunophenotyping of NK cell populations which will facilitate the identification of relationships between NK cell phenotype and the subsequent killing potential. This assay will be valuable for assessing the broader impact(s) of immunotherapy strategies on both modes of NK cell killing.