O'Brien, L. | bioRxiv Science

bioRxiv2021

Evaluation of the SARS-CoV-2 inactivation efficacy associated with buffers from three kits used on high-throughput RNA extraction platforms

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QiaCube HT and the ThermoFisher Kingfisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type; SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer from the MagMax Pathogen RNA/DNA kit (ThermoFisher), used on the Kingfisher Flex, which included guanidinium isothiocycnate (GITC), a detergent, and isopropanol demonstrated a minimum inactivation efficacy of 1 x 105 TCID50/ml. An alternative lysis buffer from the MagMax Viral/Pathogen Nucleic Acid kit (Thermofisher) also used on the Kingfisher Flex and the lysis buffer from QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QiaCube HT (both of which contained GITC and a detergent) reduced titres by 1 x 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 minutes, 68 {degrees}C) did not completely inactivate the virus, demonstrating a reduction of 1 x 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

microbiology↗

bioRxiv2021

Non-canonical Hedgehog signaling through L-type voltage gated Ca2+ channels controls CD8+ T cell killing

Cytotoxic CD8+ T lymphocytes (CTLs) are critical to the immune response against intracellular pathogens and cancer and act by eliminating infected and malignant cells through targeted secretion of cytotoxic granules. Hedgehog (Hh) signaling has been shown to be critical for CTL killing. Interestingly, Hh signaling in CD8+ T cells is not induced by extracellular Hh ligands but is initiated upon T cell receptor (TCR) engagement. How the TCR induces the Hh pathway independently of extracellular Hh ligands is unknown. Here we show that the Hh transcription factor Gli1 is essential for efficient CTL function and is induced downstream of the TCR by an extracellular Ca2+ influx selectively controlled by L-type voltage gated Ca2+ channels localized at the plasma membrane. We demonstrate that this novel mode of Hh signaling induction is independent of the canonical Hh pathway and represents the primary mechanism of Gli1 induction in naive CD8+ T cells, while CTLs can also activate Gli1 via MAP Kinase signaling. Importantly, we show that this L-type voltage gated Ca2+ channel-controlled Gli1 induction is functionally required for CTL killing in mice and humans. Gli inhibitors are currently in clinical trials against various cancers and our observations indicate that they likely inhibit the anti-tumor response. Significance statementCytotoxic CD8+ T cells (CTLs) kill infected and malignant cells by targeted secretion of cytotoxic granules. Hedgehog signaling is critical for effective CTL killing and is activated by the T cell receptor (TCR) independently of exogenous Hedgehog ligands. This study shows that Hedgehog transcription factor Gli1 is required for CTL killing and identifies L-type voltage gated Ca2+ channels (Cav1) as essential regulators of CTL killing in mouse and human, by virtue of their ability to activate Gli1 downstream of the TCR. This Cav1-Gli1 axis operates independently of canonical Hedgehog signaling. Our work suggests that caution is required when using Gli inhibitors, currently in trials as anti-cancer therapeutics, since they may dampen the anti-tumor response.

immunology↗