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Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

The diversity of T cell subpopulations demands a growing number of biomarkers to be studied from small samples. To bridge the gap between multicolor flow cytometry or classical qPCR and RNA-Seq, we applied highly multiplexed whole transcriptome AmpliSeq-RNA with 20,803 genes to perform sensitive and specific gene expression profiling (GEP). This study analyzed untouched primary human naïve and memory T cells and compared results to microarrays. Method 8 T cell subsets were isolated by FACS from 4 healthy donors without restimulation: CD4+ or CD8+ naïve (CCR7+CD45RA+), central memory (CCR7+CD45RA−), effector memory TEM (CCR7−CD45RA−) and TEMRA (CCR7−CD45RA+). AmpliSeq transcriptome human gene expression kit (Thermo Fisher) generated barcoded libraries from total RNA. 8–10 samples multiplexed per Ion 540 chip were sequenced on an Ion S5 System. Samples were also analyzed by Affymetrix HG-U133 Plus 2 genechips. Results The resulting Excel matrix of normalized reads [RPM = reads per million reads] had a dynamic range of more than 5 log units. Biological replicates of T cell subsets showed high correlation with r ≥ 0.925. Technical replicates (same libraries) demonstrated high reproducibility with r ≥ 0.995. A high concordance with genechip data were found for expression pattern of analyzed markers. For several very high or low abundant genes the AmpliSeq-RNA data were more sensitive and more specific than the hybridization-based genechip data. Conclusion AmpliSeq-RNA (counting PCR products by NGS) is an interesting alternative to perform straightforward GEP. The analysis of phenotypically well-defined T cell subsets without artificial in vitro stimulation allows more precise determination of in vivo relevant functions.