Single-Cell RNA Sequencing Reveals T Helper Cells Synthesizing Steroids De Novo to Contribute to Immune Homeostasis

Abstract

SummaryT helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a “lymphosteroid,” a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.

Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells

“…Here, we reanalyzed data from a single-cell RNA-seq experiment that was originally designed to study the differentiation of naive T cells into T H 2 cells 25 . Briefly, a population of naive Cd4 + T helper cells were activated and polarized with interleukin (IL)-4 to induce differentiation toward a T H 2 subtype.…”

Single‐cell technologies to study the immune system

“…Both the huge cell‐to‐cell variability and the amount of data these new technologies have brought to light constitute a great resource and at the same time a challenge for scientists. The parallel development of more sophisticated algorithms, faster computational approaches and new data visualization methods has already allowed scientists to gain new insights into immune system diversity 18, 23, 55, 56. Despite this incredible progress, there are still some issues that need to be solved and most of the works involving single‐cell sequencing are methodological.…”

“…2)23, 24, but the discrimination between cell state and cell type still needs to be further validated experimentally. In other words, the distinction between physiological fluctuations of gene expressions without phenotypic changes, and different cells types cannot be made solely by analysis of gene expression pattern.…”

“…revealed the existence of a Th2 subpopulation identified by the single‐cell sequencing technologies. This specific subset of Th2 cells is distinguished from the rest of the population by the expression of a specific enzyme ( Cyp11a1 ) that is at the basis of steroid biosynthesis 23. The comprehensive data obtained by the single‐cell transcriptomic approach allowed not only identification, but also purification of these cells based on new markers.…”

“…Such an approach is expected to reveal novel genes correlated with ones already known to play an important role in the immune response. We have applied such an approach to identify cell surface receptors that were specifically enriched in the steroid‐producing Th2 cells 23. Further application of this approach is anticipated to be beneficial for immunological studies.…”