IgG1 Immune Complexes Inhibit Naïve T Cell Proliferation but Stimulate a Subset of Their Progeny

We examined the effects of well-defined, soluble IgG1 immune complexes on the phenotypes of human peripheral naive CD8+ T cells. We demonstrate that IgG1-ICs inhibit the proliferation and differentiation of one subset of naïve T cells but stimulate the division of another naïve-like subset. We utilize RNA-Seq to characterize the inhibited and stimulated T cell subsets. (1) Well-defined immune complexes (ICs) were formed using trinitrophenol-conjugated bovine serum albumin (TNP-BSA; 25-35 TNP-moieties/BSA molecule) as the antigen and monoclonal anti-trinitrophenol IgG1 antibodies to form TNP-BSA-anti-TNP IgG1 ICs. Additionally, we generated ICs (Fc5 IgG1 ICs) comprising an engineered aglycosylated IgG1 Fc (Fc5 IgG1) that selectively binds FcγRI with high affinity but presents no binding to all other FcγRs. (2) Proliferation experiments were set up for three unique human donors (017, 050, and 054) as described in the article methods section (Preparing T cells for In Vitro Functional Assays: Activation, IC-Incubation, and Culture). Briefly, CellTrace-labelled naïve CD8+ T cells were activated and incubated with 50μg/mL IgG1-ICs (017, 050, and 054; n=3) or Fc5 IgG1-ICs (017 and 054; n=2) in technical duplicates. Dynabeads were removed at t= 60 hrs. After 5.5 days in culture, IgG1-IC-treated T cells were sorted based on CellTrace fluorescence into two fractions: Inhibited T cells (InhT) and Stimulated T cells (StimT). Fc5-IgG1-IC-treated T cells (Fc5T) were not sorted and served as controls. (3) Total RNA was extracted from InhT, StimT, and Fc5T samples (N=16). RNA samples were then forward processed by the Genomic Sequencing and Analysis Facility (GSAF) at UT Austin. Briefly, mRNA was isolated (poly-A capture) and stranded libraries were created (dUTP Method; NEB NGS kit) for paired-end sequencing (PE75, NextSeq 500). If any replicate for a particular donor failed to create a quality library, GSAF equally amplified all samples from that donor (6 PCR cycles) and re-attempted library creation. (4) Bioinformatically, adapters were trimmed from the reads using Cutadapt and then aligned using HISAT to the human genome version hg38. Read counts were generated by FeatureCounts using parameters for fractional counting.