Gene Expression
Nebulas
Gene Expression NebulasSummary: An unanticipated feature of the human neonatal CD4 T cell response is a robust capacity to produce CXCL8. However, this 'innate-like' function dissipates with age and is scarce in the adult. Here, we investigated the fate of CD4+CXCL8+ cells and their transition into conventional adaptive T cells. We show that CXCL8 is imprinted on immature thymocytes prior to TCR signalling and is maintained in T cell committed thymic progenitors and recent thymic emigrants (RTEs) of adults as well as neonates. Hence, rather than being unique to neonates, CXCL8-producing CD4+ T cells decrease with age in humans (and in humanised mice) owing to the decline in thymic output, coupled with the cells’ peripheral expansion. By cloning of CXCL8+CD4+ cells from cord blood, we were able to track effector function within daughter cells and demonstrate that these cells can convert to IFN-g producing cells. In sum, we provide direct evidence that ‘innate like’ CXCL8-producing CD4+ T cells emerge from the thymus and can transition into conventional adaptive Th1 cells
Overall Design: Examination of RNA-Seq count data from 96 single cells
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| Growth Protocol: | CD4 T cells from cord blood samples (n=2) were isolated by cell sorting on a FACS Aria (BD) after staining with antibodies for CD4 negative selection (Miltenyi). Samples were then activated for 2.5hrs with PMA and ionomycin (P/I) before washing and collection. |
| Treatment Protocol: | - |
| Extract Protocol: | The SMARTer kit (Clontech, Mountain View, CA, USA) was used for cDNA generation. 1μL of the ERCC spike-in mix (Life Technologies, Carlsbad, CA, USA) was included in the cell lysis mix at a dilution of 1:1000. cDNA quantity was measured using theQubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and a subset of cDNAs were checked for quality using the Agilent 2200 Tapestation (Agilent Technologies, Waldbronn, Germany). |
| Library Construction Protocol: | IL-8 qPCR: To determine expression levels of IL-8, real-time PCR was performed on all 104 single-cell cDNA samples using a sybr-green labelled primer/probe set Hs_CXCL8_1_SG QuantiTect Primer (Qiagen, Germantown, MD, USA). Two Taqman® endogenous control assays were used for normalisation purposes: GAPDH Hs99999905_m1 and EIF4A2 Hs00756996_g1 (Life Technologies). A standard curve of control cDNA from a sample known to express IL8 was run alongside the single-cell DNAs, and all samples were run in triplicate. Data was analysed using the ΔΔCt method. Library preparation and sequencing: A total of 96 single cells were taken forward for RNA-seq. Libraries were prepared using the Illumina Nextera XT Sample Preparation Kit (Illumina Inc., Cambridge, UK) with an input of 150pg of cDNA per sample. 75bp paired-end reads were generated for each library using the Illumina NextSeq® 500. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Unspecific |