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In Vitro Expanded Cord Blood γδ T Cells Acquired Unique Cell State Composition to Mediate Sustained Human AML Suppression [fb]

ABSTRACT: Single cell RNA-seq of cultured gamma-delta T-cells from cord blood were expanded over 2 weeks to identify cell states following in-vitro expansion. Cells were expanded in a feeder co-culture with the EBV-LCL feeder line. After 2 weeks, cells were challenged with the AML cell line K562. Single-cell RNA-sequencing was then performed to identify transcriptomic changes. RNA-seq was paired with single cell TCR sequencing to identify clonotypes mapping to specific cell states.

ORGANISM(S): Homo sapiens

PROVIDER: GSE214286 | GEO | 2023/09/08

REPOSITORIES: GEO

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Project description:We performed transcriptomic profiling of freshly isolated primary adult human alveolar epithelial type 2 cells (AEC2s), their isogenic cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). Distal lung preparations from adult donor lung explants from two individuals were cryopreserved using methods we have previously described. Primary AEC2s were purified using fluorescence activated cell sorting (FACS) to isolate cells co-expressing EPCAM and HTII-280. To establish cultures of primary AEC2s, these cells were combined with MRC5 fibroblasts at an 1:10 ratio on cell culture inserts in CK+DCI medium. iAEC2s were derived using our previously published lung directed differentiation protocol. We used the SPC2 human iPSC line and specifically the SPC2-ST-B2 (SFTPC tdT/WT ) clone containing a SFTPC tdTomato knock-in reporter. SFTPC tdTomato+ cells were sorted on day 41 of differentiation. iAEC2s were single-cell passaged in self-renewing feeder-free 3D cultures approximately every 2 weeks. We used the 10X Chromium System to generate single-cell transcriptomic data from 5 samples prepared in parallel and sequenced on the same day. We profiled (a) preculture primary AEC2s, (b) cultured primary AEC2s, (c) 3D feeder-free iAEC2s, (d) feeder-free iAEC2 cultured on cell culture inserts, and (e) iAEC2s cultured on cell inserts with MRC5 fibroblasts. The cultured primary AEC2 samples and the iAEC2 sample co-cultured with MRC5s were sorted for live EPCAM+ cells prior to encapsulation. All other samples were sorted for live cells. We find that although each population (fresh, primary cultured, and iPSC-derived AEC2s) occupies a distinct transcriptomic space, both cultured primary AEC2s and cultured feeder-free iAEC2s closely resemble their primary pre-cultured counterparts. We also find a continuum of progressively more proliferative states (from fresh primary to cultured primary to cultured iAEC2s) inversely associated with progressively less mature AEC2 states.

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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In Vitro Expanded Cord Blood γδ T Cells Acquired Unique Cell State Composition to Mediate Sustained Human AML Suppression [fb]

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