Biology subjects

Pfefferle, A.

Publications and source records attributed to Pfefferle, A..

7 recordsLinked to original sources

bioRxiv2025

Toggling of NKG2A expression drives functional specialization of iPSC-derived CAR NK cells

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. NK cell function is dynamically regulated through education driven by inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). However, the acquisition of inhibitory receptors in iNK cells and their role during differentiation and education remains poorly defined. In this study, we monitored receptor repertoires, transcriptional states, and functional responses in a range of genetically engineered iNK cell lines. Transcriptional reference mapping placed iNK cells close to cytokine-activated NKG2A+ CD56dim peripheral blood (PB) NK cells. Despite their early differentiation stage, iNK cells displayed a well-developed cytotoxic effector program, which was also reflected in high protein expression of Eomes, granzyme B, and activating receptors DNAM-1 and NKG2D. Acquisition of NKG2A by iNK cells was associated with a more differentiated transcriptional state and superior functional responses against a broad range of targets, including those expressing low to moderate levels of HLA-E, suggesting attenuated inhibitory signaling through NKG2A in iNKs. CRISPR knockout of {beta}2-microglobulin (B2) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of educating interactions with HLA-E in cis or trans. Finally, CRISPR-mediated ablation of NKG2A led to a spontaneous compensatory surface expression of CD94/NKG2C heterodimers, associated with enhanced IFN-{gamma} production and cytotoxic activity against target cells with forced high expression of single-chain {beta}2m-HLA-E-peptide trimers. Our results indicate an education-independent functional maturation of iNK cells, characterized by potent effector programs coupled with a favorable early-stage transcriptional profile.

immunology↗

bioRxiv2023

Pan-cancer profiling of tumor-infiltrating natural killer cells through transcriptional reference mapping

The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic receptor-ligand interactions, and adaptive-like responses to viral infections. Here, we generated a single-cell transcriptional reference map of healthy human blood and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene regulator networks defining NK cell differentiation. Using transfer learning, transcriptomes of tumor-infiltrating NK cells from seven solid tumor types (427 patients), combined from 39 datasets, were incorporated into the reference map and interrogated for tumor microenvironment (TME)-induced perturbations. We identified six functionally distinct NK cellular states in healthy and malignant tissues, two of which were commonly enriched for across tumor types: a dysfunctional stressed CD56bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant effector CD56dim state. The ratio of stressed CD56bright and effector CD56dim was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of novel NK cell therapies and can be extended endlessly through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.

immunology↗

bioRxiv2020

The ratio of exhausted to resident infiltrating lymphocytes is prognostic for colorectal cancer patient outcome

Immunotherapy success in colorectal cancer (CRC) is mainly limited to patients whose tumours exhibit high microsatellite instability (MSI). However, there is variability in treatment outcomes within this group, which is in part driven by the frequency and characteristics of tumour infiltrating immune cells. Indeed, the presence of specific infiltrating immune cell subsets has been shown to correlate with immunotherapy responses and is in many cases prognostic of treatment outcome. Tumour-infiltrating lymphocytes (TILs) can undergo distinct differentiation programs, acquiring features of tissue-residency or exhaustion, a process during which T cells upregulate inhibitory receptors such as PD-1 and loose functionality. While residency and exhaustion programs of CD8 T cells are relatively well-studied, these programs have only recently been appreciated in CD4 T cells and remain largely unknown in tumour-infiltrating natural killer (NK) cells. In this study, we use single cell RNA-seq data to identify signatures of residency and exhaustion in CRC infiltrating lymphocytes, including CD8, CD4 and NK cells. We then test these signatures in independent single cell data from tumour and normal tissue infiltrating immune cells. Further, we use versions of these signatures designed for bulk RNA-seq data to explore tumour intrinsic mutations associated with residency and exhaustion from TCGA data. Finally, using two independent transcriptomic data sets from patients with colon adenocarcinoma, we show that combinations of these signatures, in particular combinations of NK activity signatures, together with tumour-associated signatures, such as TGF-{beta} signalling, are associated with distinct survival outcomes in colorectal cancer patients.

cancer biology↗

bioRxiv2019

A Temporal Transcriptional Map of Human Natural Killer Cell Differentiation

Natural killer cell repertoires are functionally diversified as a result of differentiation, homeostatic receptor-ligand interactions and adaptive responses to viral infections. However, the regulatory gene-circuits that define the manifold cell states and drive NK cell differentiation have not been clearly resolved. Here, we performed single-cell RNA sequencing of 26,506 cells derived from sorted phenotypically-defined human NK cell subsets to delineate a tightly coordinated differentiation process from a small population of CD56bright precursors to adaptive NKG2C+ CD56dim NK cells. RNA velocity analysis identified a clear directionality in the transition from CD56bright to CD56dim NK cells, which was dominated by genes involved in transcription and translation as well as acquisition of NK cell effector function. Gene expression trends mapped to pseudotime, defined by increasing entropy, identified three distinct transcriptional checkpoints, reflecting important changes in regulatory gene-circuits. The CD56bright NK cell population dominated pseudotime with two distinct checkpoints separating precursors from intermediate states that gradually took on transcriptional signatures similar to CD56dim NK cells. The final checkpoint occurred during late terminal differentiation of CD56dim NK cells and was associated with unique divergent gene-expression trends. Furthermore, we utilized this single-cell RNA sequencing resource to decipher the regulation of genes involved in lysosomal biogenesis and found a coordinated gradual increase in the RAB4 and BLOC1S gene families with differentiation into CD56dim NK cells. These results identify important gene programs driving functional diversification and specialization during NK cell differentiation and hold potential to guide new strategies for NK cell-based cancer immunotherapy.

immunology↗

bioRxiv2019

Intra-lineage Plasticity and Functional Reprogramming Maintain Natural Killer Cell Repertoire Diversity

Natural killer (NK) cell repertoires are made up of a vast number of phenotypically distinct subsets with different functional properties. The molecular programs involved in maintaining NK cell repertoire diversity under homeostatic conditions remains elusive. Here we show that subset-specific NK cell proliferation kinetics correlate with mTOR activation, and that global repertoire diversity is maintained through a high degree of intra-lineage subset plasticity during IL-15-driven homeostatic proliferation in vitro. High-resolution flow cytometry and single cell RNA sequencing revealed that slowly cycling sorted KIR+CD56dim NK cells with an induced CD57 phenotype display increased functional potential associated with inhibitory MHC interactions and activating DAP12 signaling. In contrast, rapidly cycling cells upregulate NKG2A and display a general loss of functionality associated with a transcriptional increase in RNA-binding metabolic enzymes and cytokine signaling pathways. These results shed new light on the role of intra-lineage plasticity during NK cell homeostasis and suggest that the functional fate of the cell is tightly linked to the acquired phenotype and determined by transcriptional reprogramming.\n\nOne Sentence SummaryHigh-resolution flow cytometry combined with single-cell RNA sequencing reveal a role for intra-lineage plasticity and functional reprogramming in maintaining phenotypically and functionally diverse NK cell repertoires during IL-15-driven homeostatic proliferation.

immunology↗

bioRxiv2018

Induction of the BIMS Splice Variant Sensitizes Proliferating NK Cells to IL-15 Withdrawal

Adoptive transfer of allogeneic NK cells holds great promise for cancer immunotherapy. There is a variety of protocols to expand NK cells in vitro, most of which are based on stimulation with cytokines alone or in combination with feeder cells. Although IL-15 is essential for NK cell homeostasis in vivo, it is commonly used at supra-physiological levels to induce NK cell proliferation in vitro. As a result, adoptive transfer of such IL-15 addicted NK cells is associated with cellular stress due to sudden cytokine withdrawal. Here, we describe a dose-dependent addiction to IL-15 during in vitro expansion, leading to caspase-3 activation and profound cell death upon IL-15 withdrawal. NK cell addiction to IL-15 was tightly linked to the BCL-2/BIM ratio, which rapidly dropped during IL-15 withdrawal. Furthermore, we observed a proliferation-dependent induction of BIM short (BIM S), a highly pro-apoptotic splice variant of BIM, in IL-15 activated NK cells. These findings shed new light on the molecular mechanisms involved in NK cell apoptosis following cytokine withdrawal and may guide future NK cell priming strategies in a cell therapy setting.

immunology↗

bioRxiv2018

TRPML1-mediated Modulation of Dense-core Granules Tunes Functional Potential in NK Cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

immunology↗