Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity.

Reproduction is a functional outcome that relies on complex cellular, tissue, and organ interactions that span the developmental period to adulthood. Thus, the assessment of its disruption by environmental chemicals would benefit significantly from scalable and innovative approaches to testing using functionally comparable reproductive models such as the nematode C. elegans. We adapted a previously described low-throughput in vivo chromosome segregation assay using C. elegans predictive of reproductive toxicity and leveraged available public data sources (ToxCast, ICE) to screen and characterize 133 physiologically-relevant chemicals in a high-throughput manner. The screening outcome was further validated in a second, independent in vivo assay assessing embryonic viability. In total, 13 chemicals were classified as reproductive toxicants with the two most active chemicals belonging to the large family of Quaternary Ammonium Compounds (QACs) commonly used as disinfectants but with limited available reproductive toxicity data. We compared the results from the C. elegans assay with ToxCast in vitro data compiled from 700+ cell response assays and 300+ signaling pathways-based assays. We did not observe a difference in the bioactivity or in the average potency (AC50) between the top and bottom chemicals. However, the intended target categories were significantly different between the classified chemicals with, in particular, an over-representation of steroid hormone targets for the high Z-score chemicals. Taken together, these results point to the value of in vivo models that scale to high-throughput level for reproductive toxicity assessment and to the need to prioritize the assessment of QACs impacts on reproduction.

Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity.

Reproduction is a functional outcome that relies on complex cellular, tissue, and organ interactions that span the developmental period to adulthood. Thus, the assessment of its disruption by environmental chemicals is remarkably painstaking in conventional toxicological animal models and does not scale up to the number of chemicals present in our environment and requiring testing. We adapted a previously described low-throughput in vivo chromosome segregation assay using C. elegans predictive of reproductive toxicity and leveraged available public data sources (ToxCast, ICE) to screen and characterize 133 physiologically-relevant chemicals in a high-throughput manner. The screening outcome was further validated in a second, independent in vivo assay assessing embryonic viability. In total, 13 chemicals were classified as reproductive toxicants with the two most active chemicals belonging to the large family of Quaternary Ammonium Compounds (QACs) commonly used as disinfectants but with limited available reproductive toxicity data. We compared the results from the C. elegans assay with ToxCast in vitro data compiled from 700+ cell response assays and 300+ signaling pathways-based assays. We did not observe a difference in the bioactivity or in average potency (AC50) between the top and bottom chemicals. However, the intended target categories were significantly different between the classified chemicals with, in particular, an over-representation of steroid hormone targets for the high Z-score chemicals. Taken together, these results point to the value of in vivo models that scale to high-throughput level for reproductive toxicity assessment and to the need to prioritize the assessment of QACs impacts on reproduction.

Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells.

In atherosclerosis, some regulatory T (Treg) cells become exTreg cells. We crossed inducible Treg and exTreg cell lineage-tracker mice (FoxP3eGFP-Cre-ERT2ROSA26CAG-fl-stop-fl-tdTomato) to atherosclerosis-prone Apoe-/- mice, sorted Treg cells and exTreg cells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse Treg cells and exTreg cells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exTreg cell signature genes as CST7, NKG7, GZMA, PRF1, TBX21 and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exTreg cells as CD3+CD4+CD16+CD56+, which was validated by flow cytometry. Bulk RNA-seq of sorted human exTreg cells identified them as inflammatory and cytotoxic CD4+T cells that were significantly distinct from both natural killer and Treg cells. DNA sequencing for T cell receptor-ß showed clonal expansion of Treg cell CDR3 sequences in exTreg cells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exTreg cells as cytotoxic CD4+T cells.

Titration of 124 antibodies using CITE-Seq on human PBMCs.

Single-cell RNA-sequencing (scRNA-Seq) is widely used to characterize immune cell populations. However, mRNA levels correlate poorly with expression of surface proteins, which are well established to define immune cell types. CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) utilizes oligonucleotide-tagged antibodies to simultaneously analyze surface phenotypes and transcriptomes. Considering the high costs of adding surface phenotyping to scRNA-Seq, we aimed to determine which of 188 tested CITE-Seq antibodies can detect their antigens on human peripheral blood mononuclear cells (PBMCs), a commonly interrogated cell population in immunology, and find the optimal concentration for staining. The recommended concentration was optimal for 76 antibodies, whereas staining quality of 7 antibodies improved when the concentration was doubled. 33 and 8 antibodies still worked well when the concentration was reduced to 1/5 or 1/25, respectively. 64 antigens were not detected at any antibody concentration. Optimizing the antibody panel by removing antibodies not able to detect their target antigens and adjusting concentrations of the remaining antibodies will improve the analysis and may reduce costs. In conclusion, our data are a resource for building an informative and cost-effective panel of CITE-Seq antibodies and use them at their optimal concentrations in future CITE-seq experiments on human PBMCs.

Single-cell transcriptomes and T cell receptors of vaccine-expanded apolipoprotein B-specific T cells.

Atherosclerotic cardiovascular diseases are the major cause of death worldwide. CD4 T cells responding to Apolipoprotein B (ApoB), the core protein of most lipoproteins, have been identified as critical disease modulators. In healthy individuals, ApoB-reactive (ApoB+) CD4 T cells are mostly regulatory T cells (Tregs), which exert anti-inflammatory effects. Yet, they may obtain pro-inflammatory features and thus become proatherogenic. Evidence from animal studies suggests that vaccination against certain major histocompatibility complex (MHC) II-binding ApoB peptides induces an expansion of ApoB+ Tregs and thus confers atheroprotection. To date, in-depth phenotyping of vaccine-expanded ApoB+ T cells has not yet been performed. To this end, we vaccinated C57BL/6J mice with the ApoB-peptide P6 (ApoB978-993 TGAYSNASSTESASY) and performed single-cell RNA sequencing of tetramer-sorted P6+ T cells. P6+ cells were clonally expanded (one major, two minor clones) and formed a transcriptional cluster distinct from clusters mainly containing non-expanded P6+ and P6- cells. Transcriptomic profiling revealed that most expanded P6+ cells had a strong Treg signature and highly expressed genes mediating suppressive functions. Yet, some expanded P6+ cells only had a residual Treg signature and expressed genes related to T helper 1 (TH1) cells, which are proatherogenic. Modeling the T cell receptor (TCR) and P6:MHC-II interaction showed that only three amino acid residues in the α and ß chain contact the P6 peptide in the MHC-II groove and thus determine the specificity of this TCR to P6. Our data begin to reveal the vaccination-induced response to an ApoB epitope.