Project description:Checkpoint blockage has revolutionized cancer treatment. NKG2A is an inhibitory receptor expressed by cytotoxic lymphocytes, including NK cells. In contrast to other checkpoint inhibitory antibodies, anti-NKG2A antibodies have shown only limited success. Here, we designed a Cas9-based strategy to delete KLRC1 from human NK cells. Electroporation of KLRC1-targeting Cas9-RNP efficiently eliminated NKG2A expression from primary human NK cells. NKG2A-deficient NK cells showed normal proliferation, only minor transcriptional changes related to enhanced NK cell activation and maintained their phenotype and licensing status. Genetic deletion of NKG2A fully bypassed HLA-E inhibition and further enhanced NK cell activity against various tumor cell lines, thereby outperforming anti-NKG2A antibodies. In combination with antibody-coating of tumor cells to induce antibody-dependent cellular cytotoxicity, genetic deletion of NKG2A independently promoted cytotoxicity. Thus, Cas9-mediated targeting of NKG2A is an effective way to target this important inhibitory checkpoint. This technique is easily amenable to adoptive cell therapy in the clinical setting, where NKG2A deletion will promote anti-tumor responses and may help NK cells to better infiltrate and persist in an inhibitory tumor microenvironment.
Project description:A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells.
Project description:To identify possible transcriptional changes induced by NK cell education in this reductionistic model, we performed single cell RNA sequencing (scRNA-seq) on single positive Ly49A (NKG2A-Ly49G2-Ly49I-Ly49C-, denoted as sp-Ly49A, NK cells from Dd-/-, Dd+/- and Dd+/+ mice. To ensure minimum batch-effects, sorted sp-Ly49A cells from Dd-/-, Dd+/-, and Dd+/+ mice were hash-tagged with oligo-labelled CD45 antibodies, pooled and sequenced together.
Project description:NK cells are cytotoxic lymphocytes that play an important role in the innate immune response. The immune response of NK cells was shown under activated conditions. In order to identify such patterns, we performed RNA sequencing to confirm the gene expression profiles in NK cells expanded ex vivo compared to that of resting NK cells.
Project description:We report abnormal pregnancy outcomes in mice lacking NKG2A, including altered placental transcription. In humans, we find HLAB-21T snp to be associated with an increased risk of pre-eclampsia.
Project description:Clinical Natural Killer (NK) cell therapy for the treatment of leukemia is currently limited by the availability of larger numbers of functional NK cells. In vitro NK cell expansion can be used to generate higher numbers of NK cells and shows the potential to improve the cytotoxicity against tumor cells. A critical need for the clinical application of manufactured NK cells is their molecular and functional characterization. In order to characterize primary and expanded NK cells of all donors the cells were analyzed on the surfaceome level by using CSC technology in combination with total gene expression analysis using whole human genome oligo microarrays.
Project description:NK cells were isolated from the perpheral blood of healthy donors (n=6) and expanded ex vivo in the presence of feeder cells and IL-2. RNA from fresh and expanded cells was isolated, sequenced, and gene expression of the cell populations were compared.
Project description:Natural Killer cells (NK), a major constituent of innate immune system, have the ability to kill the transformed and infected cells without prior sensitization; can be put to immunotherapeutic use against various malignancies. NK cells discriminate between normal cells and transformed cells via a balance of inhibitory and activating signals induced by interactions between NK cell receptors and target cell ligands. Present study investigates whether expansion of NK cells could augment their anti-myeloma (MM) activity. For NK cell expansion, peripheral blood mononuclear cells from healthy donors and myeloma patients were co-cultured with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562-mb15-41BBL). A genome-wide profiling approach was performed to identify gene expression signature in expanded NK (ENK) cells and non-expanded NK cells isolated from healthy donors and myeloma patients. A specific set of genes involved in proliferation, migration, adhesion, cytotoxicity, and activation were up regulated post expansion, also confirmed by flow cytometry. Exp-NK cells killed both allogeneic and autologous primary MM cells more avidly than non-exp-NK cells in vitro. Multiple receptors, particularly NKG2D, natural cytotoxicity receptors, and DNAM-1 contributed to target lysis, via a perforin mediated mechanism. In summary, vigorous expansion and high anti-MM activity both in vitro and in vivo provide the rationale for testing exp-NK cells in a clinical trial for high risk MM. Differential gene expression profile in expanded natural killer (ENK) cells and non-expanded natural killer (NK) cells from healthy donors and myeloma patients Eight healthy donor and eight myeloma patients were used in the study. Non-expanded natural killer (NK) cells were isolated from PBMCs of healthy donors and myeloma patients. Expanded natural killer (ENK) cells were generated from same set of samples as mentioned in expansion protocol. All ENK and NK cells were used for gene expression profiling.