Project description:Adoptive transfer of anti-tumor cytotoxic T cells is a novel form of cancer immunotherapy, and a key challenge is to ensure the survival and function of the transferred T cells. Immune cell survival requires adaptation to different micro-environments, and particularly to the hypoxic milieu of solid tumors. The hypoxia-inducible factor (HIF) transcription factors are an essential aspect of this adaptation, and we undertook experiments to define structural determinants of HIF that would potentiate anti-tumor efficacy in cytotoxic T cells. We created retroviral vectors to deliver ectopic expression of HIF-1ɑ and HIF-2ɑ in mouse CD8+ T cells, together or individually, and with or without sensitivity to their oxygen-dependent inhibitors Von Hippel-Lindau (VHL) and Factor Inhibiting HIF (FIH). We found that HIF-2ɑ, but not HIF-1ɑ, drives broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. We further found that a specific mutation replacing the hydroxyl group acceptor site for FIH in the HIF-2ɑ isoform gives rise to the most effective anti-tumor T cells after adoptive transfer in vivo. Lastly, we show that co-delivering an FIH-insensitive form of HIF-2ɑ with an anti-CD19 chimeric antigen receptor greatly enhances cytolytic function of human CD8+ T cells against lymphoma cells both in vitro and in a xenograft adoptive transfer model. These experiments provide a means to increase the anti-tumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor. Method: OT-I CD8+ T cells were activated with 100 ng/ml OVA for 24 hours before retroviral transducton with with mouse HIF1 or mouse HIF2-expressing vectors, expanded in IL-2 for 5 days before being sorted on Thy-1.1 surface expression on a BD FACSAria Fusion cell sorter (BD Biosciences) directly into RLT Plus lysis buffer (Qiagen, #1053393). Total RNA was subjected to quality control with Agilent Tapestation according to the manufacturer’s instructions. To construct libraries suitable for Illumina sequencing the Illumina TruSeq Stranded mRNA Sample preparation protocol, which includes cDNA synthesis, ligation of adapters and amplification of indexed libraries, was used (Illumina, #20020594). The yield and quality of the amplified libraries were analysed using Qubit by Thermo Fisher and the Agilent Tapestation. The indexed cDNA libraries were normalised and combined and the pools were sequenced on the Nextseq 550 for a 50-cycle v2.5 sequencing run generating 75 bp single-end reads. Basecalling and demultiplexing was performed using CASAVA software with default settings generating Fastq files for further downstream mapping and analysis.

Project description:Purpose: In addition to developing antivirals targeting specific SARS-CoV-2 viral proteins, there has been a growing effort in developing therapeutics targeting host proteins exploited by SARS-CoV-2 for their replication or implicated in viral pathogenesis. As such therapeutics can impair normal functioning of host proteins, a clear understanding of their impact on cellular mechanisms is critically important. Here, we characterized the interactions of a host-targeted anti-SARS-CoV-2 therapeutic, SNX-5422, with the cellular machinery using a physiologically relevant ex vivo human primary tracheobronchial epithelial (TBE) cell model. Methods: Fully differentiated human TBE cells (EpiAirwayTM) from 3 independent donors with no reported respiratory disease or smoking history were obtained from MatTek (Ashland, MA). The cells were cultured at the air-liquid interface in 1 ml of AIR-100-MM culture medium (MatTek) in 6 well plates at 37°C in 5% CO2. Upon receipt of cells, the cultures were acclimated for 16-24hr prior to the start of experiments. Human TBE cells from each donor were treated with 1µM SNX-5422 or 0.1% DMSO added to the media on the basolateral side of the culture, in three biological replicates. After 48hrs, cells were resuspended in TRIzol reagent (Thermo Fisher) and total RNA from the cells was extracted by phase separation with chloroform and subsequently using the RNeasy Mini Kit (Qiagen). RNA-Seq libraries were prepared using TruSeq RNA library Prep Kit v2 (Illumina, Inc. USA). Before pooling and sequencing, fragment length distribution and library quality were assessed on a TapeStation 2200 (Agilent Technologies), and the libraries were validated by Qubit Fluorometers (Thermo Fisher). All libraries were then pooled in a concentration at 4nM and sequenced in quadruplets on a NextSeq 500 Illumina sequencing platform system using NextSeq 500/550 High Output Kit v2.0 (150 cycles) (Illumina, Inc. USA). Conclusion: This study identified differentially expressed genes between DMSO-treated and SNX-5422-treated human TBE cells.

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Lactate regulation of activation in CD8+ T cells

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