Single-cell transcriptional and proteomic profiling of tumor-infiltrating lymphocytes

To characterize the transcriptional environment of neoepitope-specific TILs, we performed multiplexed quantitative PCR analysis on tetramer-positive and -negative CD8+ T cell populations populations; by using an index sorting approach, we were able to simultaneously assay transcription and cell-surface phenotype at single-cell resolution. The 88 genes we selected for analysis accounted for: transcription factors shown to influence CD8+ T cell differentiation, chemokines and chemokine receptors, cytokines, activating and inhibitory co-receptors, and molecules associated with degranulation and cell survival. We observed a spectrum of dynamic transcriptional changes that take place during antigen-specific CD8+ T cell responses, specifically identifying three clusters with distinct transcriptional signatures across the sorted TIL populations, and these clusters differed in their functional status and in the expression of transcription factors, transcriptional regulators, signaling inhibitors, cytokines, cytotoxic granules, and trafficking molecules. Single-cell gene expression experiments were performed using Fluidigm’s DynamicArray microfluidic chips (Fluidigm) following manufacturer’s instructions (Two-Step Single-cell gene expression using EvaGreen Supermix on Biomark HD System). Single cells were sorted from three Pediatric leukemia patient samples using the Sony SY3200 into individual wells of 96-well plates that had been preloaded with 5 μL consisting of 1.2 μL 5X SuperScript VILO Reaction Mix (Thermo Fisher), 0.3 μL SUPERase-In (20 U/μL; Thermo Fisher), 0.25 μL 10% NP40 (Thermo Fisher), and nuclease free water (Teknova). Each plate contained two empty wells for use as non-template controls (NTC). After successful sorting, the plates were incubated at 65°C for 90 seconds, and chilled on ice for 5 minutes for RNA denaturation. The remaining components of the RT reaction were added to each well, including 0.15 μL 10X SuperScript Enzyme Mix (Thermo Fisher), 0.12 μL T4 Gene 32 Protein (NEB), and nuclease free water (Teknova). Thermal cycling conditions for the reverse transcription of single-cell mRNA into cDNA was 25°C for 5 min, 50°C for 30 min, 55°C for 25 min, 60°C for 5 min, 70°C for 10 min, and then hold at 4°C. After reverse transcription, the cDNA was amplified using 1.5 μL of a pool of the designed primers (STA mix; Fluidigm), 7.5 μL TaqMan PreAmp Master Mix (Thermo Fisher), and 0.075 μL 0.5M EDTA (Thermo Fisher). The thermal cycling conditions were 95°C for 10 min, 20 cycles of 96° for 5 sec and 60°C for 4 min, and then hold at 4°C. Amplified cDNA was then treated with Exonuclease I (NEB) to remove any unincorporated primers by adding 6 μL of 4 U/μL Exonuclease I (20 U/μL Exonuclease I diluted with water and 10X Exonuclease I Reaction buffer). Thermal cycling conditions were 37°C for 30 min, 80°C for 15 min, and then hold at 4°C. The cDNA was then diluted 5-fold for loading onto the chip. The sample mix to be loaded onto the chip included 2.5 μL 2X Sso Fast EvaGreen Supermix with low ROX (Bio-Rad), 0.25 μL 20S DNA Binding Dye Sample Loading Reagent (Fluidigm), and 2.25 μL of the prepared cDNA. The assay mix loaded onto the chip included 2.5 μL 2X Assay Loading Reagent (Fluidigm), 1X DNA Suspension Buffer (Teknova), and 100 μM of each forward and reverse primer mix (Fluidigm). Each chip was primed immediately before loading the 5 μL of each sample mix and 5 μL of each assay mix onto the chip. The chips were then loaded using the IFC Controller HX (Fluidigm) and transferred to the Biomark HD real-time PCR reader to analyze using the GE Fast 96x96 PCR+Melt v2 protocol.