Integrative scATAC-seq and scRNA-seq analyses map thymic iNKT cell development and identify Cbfβ for its commitment.

Area of Science:

• Immunology
• Developmental Biology
• Transcriptomics

Background:

• Invariant natural killer T (iNKT) cells are crucial immune cells that undergo terminal differentiation in the thymus.
• The molecular mechanisms governing the differentiation of iNKT cell subsets (iNKT1, iNKT2, iNKT17) remain largely undefined.

Purpose of the Study:

• To elucidate the developmental trajectories and molecular programs guiding invariant natural killer T (iNKT) cell subset differentiation in the thymus.
• To identify novel regulators of early iNKT cell commitment and effector function.

Main Methods:

• Single-cell RNA sequencing (scRNA-seq) of over 17,000 iNKT cells.
• Single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) of over 39,000 iNKT cells across four thymic developmental stages.
• Conditional gene deletion to assess the function of identified transcription factors.

Main Results:

• iNKT2 and iNKT17 lineage commitment may initiate as early as Stage 0 (ST0) via distinct molecular programs.
• iNKT1 commitment appears to occur after ST0, with significant heterogeneity observed in iNKT1 and iNKT2 cells, unlike more homogenous iNKT17 cells.
• A novel transcription factor, Cbfβ, was identified as highly expressed during iNKT progenitor commitment and is essential for iNKT cell development and differentiation.

Conclusions:

• Distinct molecular programs govern iNKT cell subset differentiation, with early commitment for iNKT2/iNKT17 lineages.
• Cellular heterogeneity exists within iNKT1 and iNKT2 subsets, while iNKT17 cells are more uniform.
• The transcription factor Cbfβ is a key regulator of early iNKT cell commitment and effector phenotype development.