Single-Cell Transcriptome Sequencing Uncovers New Immune Precursor Cell

NEW YORK (GenomeWeb) – Through single-cell transcriptome sequencing, researchers have uncovered CD4⁺ cytotoxic T lymphocyte precursor cells.

CD4⁺ helper T cells generally help turn on other immune cells, but in certain situations, such as in people with chronic viral infections, a subset of CD4⁺ helper T cells turn into cytotoxic T cells (CD4­-CTLs), though not much is know about these cells.

A La Jolla Institute for Allergy and Immunology-led team of researchers performed single-cell RNA sequencing on more than 9,000 CD4⁺ T cells from donors living in locations around the world, including Sri Lanka and Nicaragua. As they reported in Science Immunology today, the researchers found that CD4-CTLs originate from a subset of T cells known as CD4-T_EMRA.

"Continually evolving genomic tools and single-cell analysis technologies are revolutionizing our understanding of the human immune system in health and disease," La Jolla's Pandurangan Vijayanand said in a statement. "By applying these tools in relevant diseases and cell types we are changing our understanding of the biology of human immune cells."

He and his colleagues collected blood samples from healthy adult donors and isolated peripheral blood mononuclear cells, which include not only T cells, but also B cells, NK cells, and monocytes. The researchers then sorted the single cells and isolated T cells for RNA sequencing.

Some of the blood donors had been exposed previously to the dengue virus or the human cytomegalovirus, both of which are known to influence CD4-CTLs. For three dengue virus-exposed donors, the researchers compared the full-length transcriptomes of CD4⁺ T cells found in their CD45RA (T_EMRA), effector memory (T_EM), and central memory (T_CM) cell fractions.

The researchers uncovered 111 transcripts that were enriched among the T_EMRA subgroup in dengue-exposed donors. A number of these transcripts — like GZMB, SPON2, and CX3CR1 — are linked to the cytotoxic abilities of CD8⁺ T lymphocytes and natural killer cells, the researchers noted, while other transcripts encode transcription factors associated with CTL function such as ZNF683 and TBX21. This confirmed to the researchers that T_EMRA cells are enriched for CD4-CTLs.

By comparing the T cell antigen receptor repertoires of single cells to cells in bulk, the researchers found that the T_EMRA subgroup exhibited greater clonal expansion, as compared to the T_EM and T_CM cells.

When they compared the clonality and transcriptomes of CD4-CTLs from people who'd been exposed to dengue to samples from unexposed people and between people from different geographical location, the researchers noted high heterogeneity among the CD4-T_EMRA cells.

Still, using clustering software, they found that CD4-T_EMRA cells could be grouped into four subsets. By analyzing and comparing their transcriptomes, the researchers found that two of the clusters likely included CD4-CTL effectors with high cytotoxic potential, while the other two clusters appeared to be memory precursor cells to those in clusters 1 and 2. Clusters 1 and 2 were marked by high levels of KLRG1 and low levels of IL-7R, while clusters 3 and 4 had low KLRG1 levels and high IL-7R levels.

Additional studies of longitudinal samples led further credence to CD4-CTL effectors arising from those precursors, the researchers said.

Vijayanand and his colleagues added that a better understanding of CD4-CTL precursors could help find ways to improve immune response to vaccines. "Understanding the origins and biology of potentially long-lived CD4­-CTL precursors may pave the way for developing strategies to boost durable CD4­-CTL immune responses after vaccination against viral infections and cancer," they wrote in their paper.