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In contrast to responses against infectious challenge, T cell responses induced via adjuvanted subunit vaccination are dependent on interleukin-27 (IL-27). We show that subunit vaccine-elicited cellular responses are also dependent on IL-15, again in contrast to the infectious response. Early expression of interferon regulatory factor 4 (IRF4) was compromised in either IL-27- or IL-15-deficient environments after vaccination but not infection. Because IRF4 facilitates metabolic support of proliferating cells via aerobic glycolysis, we expected this form of metabolic activity to be reduced in the absence of IL-27 or IL-15 signaling after vaccination. Instead, metabolic flux analysis indicated that vaccine-elicited T cells used only mitochondrial function to support their clonal expansion. Loss of IL-27 or IL-15 signaling during vaccination resulted in a reduction in mitochondrial function, with no corresponding increase in aerobic glycolysis. Consistent with these observations, the T cell response to vaccination was unaffected by in vivo treatment with the glycolytic inhibitor 2-deoxyglucose, whereas the response to viral challenge was markedly lowered. Collectively, our data identify IL-27 and IL-15 as critical to vaccine-elicited T cell responses because of their capacity to fuel clonal expansion through a mitochondrial metabolic program previously thought only capable of supporting quiescent naïve and memory T cells.

RNA sequencing (RNA-seq) has become an important tool for examining the role of the transcriptome to biological processes. While RNA-seq has been widely adopted as a popular approach in many experimental designs, from gene discovery to mechanistic validation of targets, technical issues have largely limited the use of this technique to abundantly available sample sources. However, RNA-seq is becoming increasingly utilized for more specialized applications, such as flow cytometry-sorted cells and clinical specimens, due to protocol advances enabling the use of very low input material ranging from 10 pg to 10 ng of total RNA or 1-1000 intact cells. In this chapter, we present an optimized and detailed approach to RNA-seq for use with low abundance samples.

MiSeq, Illumina's integrated next generation sequencing instrument, uses reversible-terminator sequencing-by-synthesis technology to provide end-to-end sequencing solutions. The MiSeq instrument is one of the smallest benchtop sequencers that can perform onboard cluster generation, amplification, genomic DNA sequencing, and data analysis, including base calling, alignment and variant calling, in a single run. It performs both single- and paired-end runs with adjustable read lengths from 1 × 36 base pairs to 2 × 300 base pairs. A single run can produce output data of up to 15 Gb in as little as 4 h of runtime and can output up to 25 M single reads and 50 M paired-end reads. Thus, MiSeq provides an ideal platform for rapid turnaround time. MiSeq is also a cost-effective tool for various analyses focused on targeted gene sequencing (amplicon sequencing and target enrichment), metagenomics, and gene expression studies. For these reasons, MiSeq has become one of the most widely used next generation sequencing platforms. Here, we provide a protocol to prepare libraries for sequencing using the MiSeq instrument and basic guidelines for analysis of output data from the MiSeq sequencing run.