The MAIT cell response to controlled oral enterotoxigenic E. coli challenge

Mucosa-associated invariant T (MAIT) cells recognize conserved microbial antigens presented by the non-polymorphic MR1 molecules and play important roles in barrier immunity. Enterotoxigenic E. coli (ETEC) is a major cause of diarrheal disease especially among children in lower-income countries. Here we investigate the potential role of MAIT cells in ETEC infection using samples from a human experimental ETEC challenge study. MAIT cells were activated with elevated function and proliferation in blood on day 7 after challenge, reflected both at protein and transcript levels, a pattern most evident among individuals who developed mild to severe diarrhea (MSD). The MSD-positive group had elevated expression of CCR9 and α4β7 on MAIT cells and experienced expansion of the peripheral MAIT cell pool at day 28 after challenge. Finally, the size of the MAIT cell pool correlated with MSD disease severity score. These findings indicate that MAIT cells respond to ETEC infection in a way associated with the development of symptomatic disease. Peripheral blood MAIT cells were purified by sorting (2,304 - 88,172 total cells) using a FACS Aria SORP (BD Biosciences), pelleted, and overlaid with 250 μl of RNAlater (ThermoFisher) and frozen at -20°C. RNA was extracted using the RNeasy Mini Kit (Qiagen), and RNA quality and concentration were assessed with the Agilent 2100 Bioanalyzer Pico Chip. RNA-seq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) according to the manufacturer’s instructions. Amplified material was purified using Agencourt AMPure XP beads (Beckman). cDNA quantity was assessed on a Qubit 3.0 (ThermoFisher) and fragment size was evaluated on a 2100 BioAnalyzer (Agilent). The PCR products were next indexed using the Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Briefly, products were tagmented using the Amplicon tagment mix containing Tn5 transposase, and indexed using Nextera index 1 (i7) and index 2 (i5) primers. The libraries were again cleaned-up with Agencourt AMPure XP beads, quantified, pooled, and sequenced across 75 base pairs (bp) using a single-end strategy with a 75-cycle high output flow cell on a NextSeq 500 (Illumina). Fourteen biological replicates were sequenced per experiment, with two donor-matched time points corresponding to pre and 7-days post ETEC challenge. Median reads per sample was 22.9 million reads. The Unix based program Spliced Transcripts Alignment to a Reference (STAR) v.2.6.1 with human genome hg38 was used for alignment (36), and transcription mapping was performed using RNA-seq by Expectation Maximization (RSEM) v.1.3.1(37). The FeatureCounts program was used for counting mapped reads (38), and RUVSeq v1.12 was utilized to remove unwanted variation (39). A differentially expressed gene list was subsequently generated by edgeR v3.20. R package (40), and the GSEA method (31) was employed to find statistically significant pathways with 5917 gene sets of GO(Gene Ontology) in Molecular Signatures Database (MSigDB) issued by the Broad Institute.