Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Institut Pasteur

The transcription factor RORγt is required for the development of several innate lymphoid populations, such as lymphoid tissue-inducer cells (LTi cells) and cells that secrete interleukin 17 (IL-17) or IL-22. The progenitor cells as well as the developmental stages that lead to the emergence of RORγt(+) innate lymphoid cells (ILCs) remain undefined. Here we identify the chemokine receptor CXCR6 as an additional marker of the development of ILCs and show that common lymphoid progenitors lost B cell and T cell potential as they successively acquired expression of the integrin α(4)β(7) and CXCR6. Whereas fetal RORγt(+) cells matured in the fetal liver environment, adult bone marrow-derived RORγt(+) ILCs matured outside the bone marrow, in a Notch2-dependent manner. Therefore, fetal and adult environments influence the differentiation of RORγt(+) cells differently.

Notch signaling is necessary for adult, but not fetal, development of RORγt(+) innate lymphoid cells.

RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size, inclusive area, and numbers of B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These findings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system.

RANKL induces organized lymph node growth by stromal cell proliferation.

Innate lymphoid cells are present at mucosal sites and represent the first immune barrier against infections, but what contributes to their circulation and homing is still unclear. Using Rag2(-/-) Cxcr6(Gfp/+) reporter mice, we assessed the expression and role of CXCR6 in the circulation of ILC precursors and their progeny. We identify CXCR6 expressing ILC precursors in the bone marrow and characterize their significant increase in CXCR6-deficient mice at steady state, indicating their partial retention in the bone marrow after CXCR6 ablation. Circulation was also impaired during embryonic life as fetal liver from CXCR6-deficient embryos displayed decreased numbers of ILC3 precursors. When injected, fetal CXCR6-deficient ILC3 precursors also fail to home and reconstitute ILC compartments in vivo. We show that adult intestinal ILC subsets have heterogeneous expression pattern of CXCR6, integrin α 4 β 7, CD62L, CD69, and CD44, with ILC1 and ILC3 being more likely tissue resident lymphocytes. Intestinal ILC subsets were unchanged in percentages and numbers in both mice. We demonstrate that the ILC frequency is maintained due to a significant increase of ILC peripheral proliferation, as well as an increased proliferation of the in situ ILC precursors to compensate their retention in the bone marrow.

CXCR6 Expression Is Important for Retention and Circulation of ILC Precursors.

The precise lineage relationship between innate lymphoid cells (ILCs) and lymphoid tissue-inducer (LTi) cells is poorly understood. Using single-cell multiplex transcriptional analysis of 100 lymphoid genes and single-cell cultures of fetal liver precursor cells, we identified the common proximal precursor to these lineages and found that its bifurcation was marked by differential induction of the transcription factors PLZF and TCF1. Acquisition of individual effector programs specific to the ILC subsets ILC1, ILC2 and ILC3 was initiated later, at the common ILC precursor stage, by transient expression of mixed ILC1, ILC2 and ILC3 transcriptional patterns, whereas, in contrast, the development of LTi cells did not go through multilineage priming. Our findings provide insight into the divergent mechanisms of the differentiation of the ILC lineage and LTi cell lineage and establish a high-resolution 'blueprint' of their development.

Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage.

T and innate lymphoid cells (ILCs) share some aspects of their developmental programs. However, although Notch signaling is strictly required for T cell development, it is dispensable for fetal ILC development. Constitutive activation of Notch signaling, at the common lymphoid progenitor stage, drives T cell development and abrogates ILC development by preventing Id2 expression. By combining single-cell transcriptomics and clonal culture strategies, we characterize two heterogeneous α4β7-expressing lymphoid progenitor compartments. αLP1 (Flt3(+)) still retains T cell potential and comprises the global ILC progenitor, while αLP2 (Flt3(-)) consists of ILC precursors that are primed toward the different ILC lineages. Only a subset of αLP2 precursors is sensitive to Notch signaling required for their proliferation. Our study identifies, in a refined manner, the diversity of transitional stages of ILC development, their transcriptional signatures, and their differential dependence on Notch signaling.

Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling.

The Notch signaling pathway is conserved throughout evolution, and it controls various processes, including cell fate determination, differentiation, and proliferation. Innate lymphoid cells (ILCs) are lymphoid cells lacking antigen receptors that fulfill effector and regulatory functions in innate immunity and tissue remodeling. Type 3 ILCs (ILC3s) reinforce the epithelial barrier and maintain homeostasis with intestinal microbiota. We demonstrated that the population of natural cytotoxicity receptor-positive (NCR(+)) ILC3s in mice is composed of two subsets that have distinct developmental requirements. A major subset depended on the activation of Notch2 in NCR(-) ILC3 precursors in the lamina propria of the small intestine to stimulate expression of the genes encoding the transcription factors T-bet, RORγt, and aryl hydrocarbon receptor (AhR). Notch signaling contributed to the transition of NCR(-) cells into NCR(+) cells, the more proinflammatory subset, in a cell-autonomous manner. In the absence of Notch signaling, this subset of NCR(-) ILC3s did not acquire the gene expression profile of NCR(+) ILC3s. A second subset of NCR(+) ILC3s did not depend on Notch for their development or for increased transcription factor abundance; however, their production of cytokines and cell surface abundance of NCRs were decreased in the absence of Notch signaling. Together, our data suggest that Notch is a regulator of the plasticity of ILC3s by controlling NCR(+) cell fate.

Sylvestre Chea

Unit for Lymphopoieseis,
Immunology Department,
INSERM U1223,
University Paris Diderot,
Sorbonne Paris Cité,
Cellule Pasteur,
Unit for Lymphopoieseis, Immunology Department, INSERM U1223, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur

Institut Pasteur

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations