NEW YORK (GenomeWeb) – Using single-cell RNA sequencing to uncover transcriptional clusters, a Swedish team reporting in Nature Immunology has identified new sub-populations within at least one group of innate lymphoid cells (ILC), the diverse group of immune cells tasked with maintaining mucosal barriers, contributing to homeostasis, and helping to mediate inflammation.
In an effort to characterize ILC heterogeneity, the researchers did RNA sequencing on hundreds of individual immune cells from the human tonsil region, focusing on non-cytotoxic CD127+ ILCs, and natural killer (NK) cells from another branch of the ILC family with similar but distinct functions and developmental pathways. The resulting transcript clusters coincided with four ILC populations: the ILC1, ILC2, ILC3, and NK cells, they explained.
But within the ILC3 group, the team saw variable gene expression profiles pointing to the presence of three sub-populations with slightly different transcriptional signatures and potential functional capabilities. These sub-populations were marked by enhanced expression of the NKp44 protein, the CD62L protein-coding gene SELL, and the genes coding for HLA-DR and HLA-DP human leukocyte antigens.
"We've identified three new subgroups of ILC3s that evince different gene expression patterns and that differ in how they react to signaling molecules and in their ability to secrete proteins," co-corresponding author Jenny Mjösberg, an infectious medicine researcher at the Karolinska Institute, said in a statement. She also noted that "our study has taught us a lot about this relatively uncharacterized family of cells."
Along with their apparent role in lymph node organization in developing mice, the team noted, human CD127+ ILCs have been implicated in homeostasis and inflammation processes in tissues with and without mucosal features. ILC1, ILC2, and ILC3 differ in their cytokine features and transcription factor profiles, though past studies point to plasticity within each of these immune cell populations.
To delve into this heterogeneity and get a better look at functional differences between these populations, the researchers used single-cell RNA sequencing to characterize CD127+ ILCs and NK cells from tonsil tissue obtained from three adults who'd undergone tonsillectomy for sleep apnea and did not show signs of tonsil inflammation or infection.
With the help of flow cytometry, the researchers isolated and sorted immune cells in these samples, using a modified Smart-seq2 protocol to prepare hundreds of individual ILC cells for indexed and pooled single-cell RNA sequencing on the Illumina HiSeq 2000 instrument.
Based on transcriptional patterns for an average of around 3,000 genes in 648 single cells, the team put together clusters containing ILC1, ILC2, ILC3, and NK cells. These clusters broadly corresponded with flow cytometry-based analyses of protein abundance in individual immune cells.
The researchers then took a closer look at types of genes with distinct expression signatures in each ILC population, which offered a glimpse at potential differences in population functions. Their transcriptional clustering also uncovered the three sub-populations within ILC3, each with slightly different expression profiles that were explored in more detail through in vitro immune cell stimulation experiments.
The study's authors emphasized the need for additional studies to delve into the functional contributions that these sub-populations may make during various inflammatory immune responses.
"Our systematic comparison of single-cell transcriptional variation within and between ILC populations provide new insight into ILC biology during homeostasis," they wrote, "with additional implications for dysregulation of the immune system."