Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.12.22.423945

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Zöphel, S.·Schwär, G.·Nazarieh, M.·Konetzki, V.·Hoxha, C.·Meese, E.·Hoth, M.·Helms, V.·Hamed, M.·Schwarz, E. C.
View original source ↗Export metadata ↓

Abstract

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumour cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+-influx through store operated Ca2+ channels, formed by STIM-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. Here, we isolated total RNA from natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, this strategy led to the identification of KCNN4, RCAN3, CCR5 and BCL2 as potential candidates to regulate the efficiency of Ca2+-dependent target cell killing.

Source connections

Explore related subjects

immunologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Zöphel, S., Schwär, G., Nazarieh, M., Konetzki, V., Hoxha, C., Meese, E., Hoth, M., Helms, V., Hamed, M., Schwarz, E. C.. 2020-12-22. Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity. https://doi.org/10.1101/2020.12.22.423945

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Proteome-wide discovery of novel diagnostic antigens for coccidioidomycosis

Coccidioidomycosis, commonly called Valley Fever (VF), caused by Coccidioides spp., is an understudied fungal disease that accounts for a substantial fraction of pneumonias in endemic areas. Diagnosis remains challenging because the mainstay of diagnosis is serology, and current assays have modest sensitivity in early infection. Discovery of new antigens could improve serologic detection early in disease. Here, we use PepSeq, a DNA-programmable serology platform, to represent the complete proteome of Coccidioides within a library of 244,000 DNA-barcoded peptides. By profiling antibody reactivity at the protein and epitope levels in VF patients, naturally infected pigtail macaques, and healthy controls, we identify numerous previously-unrecognized antigens and epitopes, including some shared between humans and macaque responses. When translated into a pooled peptide ELISA, we show that a subset of identified antigens can discriminate VF cases from controls in an independent cohort with high accuracy (AUC = 0.91). These findings establish high-dimensional peptide serology as a scalable approach for fungal antigen discovery and identify new targets for improved VF diagnosis.

immunology↗
bioRxiv2026

Establishing an ex vivo platform for molecular profiling of skin structural cells using frozen human skin biopsies

Skin structural cells, including keratinocytes, melanocytes, endothelial cells, and fibroblasts, play critical roles in the development of chronic inflammatory skin disease. Inflammatory stimuli induce transcriptional reprogramming and epigenetic changes in skin structural cells, which contribute to disease pathogenesis. Although two-dimensional (2D) and three-dimensional (3D) skin culture models have been widely used to investigate inflammatory responses in skin structural cells, ex vivo systems that maintain skin structure and cellular composition remain limited. To address this gap, we explored using frozen skin biopsies as an experimental platform to study the responses of skin structural cells to inflammatory stimuli. Using a simple air-liquid interface culture method on permeable Transwell inserts, we showed that viable skin structural cells that retain cell-type-specific transcriptional profiles can be recovered from frozen skin biopsies after prolonged in vitro culture. Stimulation with IFN-{gamma} induced robust transcriptional and chromatin accessibility changes that were largely resolved after stimulus removal, with transcriptional responses to secondary stimulation comparable to the primary stimulation. Treatment of cultured skin biopsies with histone-modifying enzyme inhibitors effectively altered global histone modification levels, but did not substantially affect the transcriptional resolution of IFN-{gamma}-induced responses. Collectively, these findings establish cryopreserved human skin biopsies as a scalable ex vivo platform that enables mechanistic studies of inflammatory responses of multiple populations of skin structural cells.

immunology↗
bioRxiv2026

γδ T cell-derived IL-17A aggravates experimental epidermolysis bullosa acquisita in response to IL-1β

Epidermolysis bullosa acquisita (EBA) is a mucocutaneous autoimmune blistering disorder mediated by autoantibodies against type VII collagen and subsequent splitting of the dermal-epidermal junction. Interleukin (IL)-17A promotes the production of chemokines and cytokines involved in neutrophil recruitment and activation, suggesting that IL-17A may contribute to EBA pathogenesis. We aimed to clarify the role of IL-17A in experimental EBA. Using an antibody transfer-induced EBA mouse model, we demonstrate that IL-17A promotes disease development, as pharmacological blockade of IL-17A with a neutralizing antibody ameliorated skin lesions. Gamma-delta ({gamma}{delta}) T cells were identified as the predominant cellular source of IL-17A, supported by the attenuated disease phenotype observed in {gamma}{delta}TCR-deficient mice. In addition, CD4+ T cells and neutrophils contributed to IL-17A production in experimental EBA. Mechanistically, IL-1-beta, rather than IL-23, was identified as a critical mediator of IL-17A production by {gamma}{delta} T cells in vitro, consistent with our in vivo findings. Whereas EBA severity was not reduced in IL-23p19-deficient mice, disease was markedly attenuated in IL-1R-deficient animals. Together, our study provides the first evidence that {gamma}{delta} T cell-derived IL-17A plays a pivotal role in experimental EBA and identifies a previously unrecognized IL-1{beta}/{gamma}{delta} T cell/IL-17A axis that expands current understanding of EBA immunopathogenesis.

immunology↗