Gene expression profiles of primary human NK cells before and after expansion on CSTX002 feeder cells, with and without IL-21 stimulation
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ABSTRACT:
PROVIDER: PRJNA529864 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8812370_1.fastq.gz | Fastqsanger.gz | |||
| SRR8812370_2.fastq.gz | Fastqsanger.gz | |||
| SRR8812380_1.fastq.gz | Fastqsanger.gz | |||
| SRR8812380_2.fastq.gz | Fastqsanger.gz | |||
| SRR8812371_1.fastq.gz | Fastqsanger.gz |
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Project description:NK cell development, maturation, and activation by cytokines is driven by alterations in gene expression mediated by activation and repression or transcriptional programs. In particular, we have extensively studied the role of STAT3 in human NK cells. This was based in part on a method we developed for in vitro expansion of large numbers of highly active NK cells using a genetically-modified feeder cell expressing 4-1BBL and membrane-bound IL-21. To dissect the various gene expression profiles induced by IL-21 from the various other signals received from the feeder cell, we purified peripheral NK cells from 4 healthy subjects (naïve, N), expanded NK cells for 14 days using CSTX002 feeder cells (expanded, E), and extracted RNA from the cells without (Neg) or after (Pos) the cells were activated with IL-21. We then performed RNA sequencing on each sample.
2019-03-30 | GSE129044 | GEO
Project description:RNA-seq analysis of feeder cells for NK cells expansion
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Project description:Ex vivo activation and expansion of natural killer (NK) cells is a strategy to produce sufficient numbers of these effector cells for adoptive immunotherapy. Therefore we aimed at the development of an automated, clinical scale NK cell expansion process and compared NK cells after manual and automated expansion.We found only a small subset of 124 genes that significantly varied between both sample groups. These genes were associated with movement of leukocytes were identified including a group of genes for NK cell movement (CMKLR1, CX3CR1, S1PR5, GNLY and CXCR1) which were slightly higher expressed after automated compared to manual expansion. Nevertheless, the expansion profiles of NK cells after automated or manual expansion were rather similar in comparison to the remarkable difference between primary and expanded NK cells in general represented by the vast majority of regulated genes between the analyzed sample groups. Pathway analysis revealed that most of regulated genes upon expansion were associated with cell cycle regulation, DNA replication, DNA recombination and DNA repair, regulation of apoptosis and cell survival as well as cytokine signaling. Furthermore, the expression of many NK cell relevant markers was changed upon expansion with the strongest effects on up regulation of TRAIL and FASL, inhibitory TIGIT and chemokine receptors CCR2, CCR5 and CXCR6. In addition, granzyme M was down regulated but other important effector molecules like TNFa, perforin and granzymes A, B and K were up regulated. In summary we could show that manual and automatically expanded NK cells show a similar expansion profile while the differ significantly from primary NK cells. Up regulation of many NK cell relevant markers indicate for an enhanced NK cell functionaility after ex vivo activation and expansion. 24 samples were analyzed in total. 6 biological replicates represented by cells from different donors. 4 different samples per donor: freshly isolated NK cells (d0) and NK cells expanded for 14 days under 3 different conditions: in co-culture with EBV-LCL feeder cells and 500 U/mL IL2 either cultivated manually in T75 flasks (T) or automatically using the CliniMACS Prodigy system (P); NK cells cultured with 500 U/ml IL2 without feeder cells (I)
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Project description:NK cell expansion on uAPC helper cells
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Project description:Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A- and HLA-B- K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by greater than 900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.
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Project description:PBMCs were stimulated with irradiated K562-mIL21 and 221-mIL21 feeder cells, respectively, as well as supplemented with IL-2 and IL-15. NK cells were purified from expanded cells using flow cytometry on day 7 and day 14 for RNA sequencing (RNA-Seq).
2020-08-07 | GSE155812 | GEO
Project description:We have developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells (iNKs) derived from induced pluripotent stem cells (iPSCs). We used GeneChip microarrays to compare the whole transcriptome of iNK cells to the transcriptomes of primary T cell and NK cell populations, K562 feeder cell-expanded primary NK cells, fibroblasts, iPSCs and iCD34+ cells.
2020-11-11 | GSE157327 | GEO
Project description:NK cells are frequently expanded for the clinic using irradiated engineered K562 feeder cells expressing a core transgene set of membrane-bound mb IL-15 and/or mbIL-21 together with 41BBL. Prior comparisons of mbIL-15 to mbIL-21 for NK expansion lack comparisons of key attributes of the resulting NK cells including their high dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL-15- and mbIL-21-based feeder cells are absent. We addressed these gaps and found that using mbIL-15 versus mbIL-21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL-15 alone drove superior functionality by nearly all measures, while those expressing mbIL-21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL-21, whereas in sequence NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine highlighting the plasticity of NK cell differentiation. The sequence mbIL-21 followed by mbIL-15 was especially advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL-15 alone in 2 of 3 xenograft models. Our findings define the impact of mbIL-15 versus mbIL-21 during NK expansion and reveal a previously under appreciated tradeoff between NK yield and function for which sequential use of mbIL-21- followed by mbIL-15-based feeder cells may be the optimal approach in many settings.
2023-09-30 | GSE234384 | GEO
Project description:NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that limiting its feasibility and clinical efficacy. One difficulty is the paucity of clinical grade manufacturing platforms to support the large scale expansion of highly active NK cells. We created an NK cell feeder cell line termed 'NKF' through overexpressing membrane bound IL-21 that is capable of inducing robust and sustained proliferation (>10,000-fold expansion at 5 weeks) of highly cytotoxic NK cells. The expanded NK cells exhibit increased cytotoxic function against a panel of blood cancer and solid tumor cells as compared to IL-2-activated non-expanded NK cells. The NKF-expanded NK cells also demonstrate efficacy in mouse models of human sarcoma and T cell leukemia. Mechanistic studies revealed that membrane-bound IL-21 leads to an activation of a STAT3/c-Myc pathway and increased NK cell metabolism with a shift towards aerobic glycolysis. The NKF feeder cell line is a promising new platform that enables the large scale proliferation of highly active NK cells in support of large scale third party NK cell clinical studies that have been recently intiatied. These results also provide mechanistic insights into how membrane-bound IL-21 regulates NK cell expansion.
| S-EPMC6797802 | biostudies-literature
Project description:Ex vivo activation and expansion of natural killer (NK) cells is a strategy to produce sufficient numbers of these effector cells for adoptive immunotherapy. Therefore we aimed at the development of an automated, clinical scale NK cell expansion process and compared NK cells after manual and automated expansion.We found only a small subset of 124 genes that significantly varied between both sample groups. These genes were associated with movement of leukocytes were identified including a group of genes for NK cell movement (CMKLR1, CX3CR1, S1PR5, GNLY and CXCR1) which were slightly higher expressed after automated compared to manual expansion. Nevertheless, the expansion profiles of NK cells after automated or manual expansion were rather similar in comparison to the remarkable difference between primary and expanded NK cells in general represented by the vast majority of regulated genes between the analyzed sample groups. Pathway analysis revealed that most of regulated genes upon expansion were associated with cell cycle regulation, DNA replication, DNA recombination and DNA repair, regulation of apoptosis and cell survival as well as cytokine signaling. Furthermore, the expression of many NK cell relevant markers was changed upon expansion with the strongest effects on up regulation of TRAIL and FASL, inhibitory TIGIT and chemokine receptors CCR2, CCR5 and CXCR6. In addition, granzyme M was down regulated but other important effector molecules like TNFa, perforin and granzymes A, B and K were up regulated. In summary we could show that manual and automatically expanded NK cells show a similar expansion profile while the differ significantly from primary NK cells. Up regulation of many NK cell relevant markers indicate for an enhanced NK cell functionaility after ex vivo activation and expansion.
2015-04-23 | GSE62654 | GEO