Unknown,Transcriptomics,Genomics,Proteomics

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Dynamics of Virus-specific CD8+ T cells in the Human Nasal Cavity

ABSTRACT: The goal of this experiment is to compare the transcriptomic profile of CD8+ T cells isolated from the nasal cavity and whole blood at two different state, baseline and CD3/28 activated. This will allow us to characterise the unique features of CD8+ T cells in the nasal cavity. We isolated CD8+CD45RO+ T cells from fresh nasal turbinate samples and PBMCs of four healthy donors (1 Females, 45 years old; 3 Males, 24, 40 and 41 years old). Single-cell RNA sequencing was performed on both nasal and blood CD8+ CD45RO+ T cells, either directly or following overnight TCR-mediated stimulation with anti-CD3/CD28 beads.

INSTRUMENT(S): FLOQSwab (Copan), Chromium Single Cell 5' Library & Gel Bead Kit v2, 10x Genomics Chromium Controller, Illumina NovaSeq 6000, Local workstation

ORGANISM(S): Homo sapiens

SUBMITTER: Qui Martin

PROVIDER: E-MTAB-15436 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gene expression profiling of cells isolated ex vivo is a unique tool to assess gene expression in vivo. Exemplified for CD4+CD45RO+ effector/memory T helper (T E/M) lymphocytes of human peripheral blood, we have analyzed different isolation procedures and storage conditions for the introduction of bias. Cells from clinical samples can be sorted to high purity by a flow cytometric sorter and assessed for their gene expression pattern but storage/shipping conditions and often necessary pre-enrichment due to low initial population frequencies bears the danger of introducing artificial changes. To test for changes introduced by different procedures, cells were enriched either by magnetic cell sorting (MACS) with Whole-Blood CD4 beads, density gradient centrifugation, alone or in combination with CD3 MACS, or by lysis of erythrocytes followed by MACS depletion of CD15+ cells prior to fluorescence-activated cell sorting (FACS) as CD3+CD4+CD45RO+ cells. A total of 7 different protocols were compared among each other and to T cells stimulated for 3 hours after isolation with Phorbol-12-myristate-13-acetate and Ionomycin (PMA/Iono). Preparation-induced de novo transcription was blocked in one of the protocols by addition of 2 M-BM-5g/ml Actinomycin D (ActD). To test for changes introduced by delays in sample preparation blood samples were drawn and either processed directly or kept at 4 M-BM-0C or room temperature (RT) for 2 or 6 hours before T E/M cells were isolated. Gene expression was compared among the various cell preparations and to T E/M cells isolated from 1 day old buffy coat fractions stored at RT. Sorted cells were lysed in TRIzol (Invitrogen) and frozen at -80M-BM-0C until used for RNA extraction. Total RNA was extracted using the miRNeasy kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary RNA was synthesized from 1 M-BM-5g total RNA using Message AmpII Biotin (Ambion, USA). Fifteen micrograms of fragmented cRNA of each sample were hybridized to 26 HG-U133A plus 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.4, both Affymetrix. All relevant GCOS data of quality checked microarrays were analyzed with High Performance Chip Data Analysis (HPCDA, unpublished), using the BioRetis database (www.bioretis-analysis.de), as described and validated previously.

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Dynamics of Virus-specific CD8+ T cells in the Human Nasal Cavity

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