Background: RUNX3 is a key transcription factor essential for immune cell development and function. Genetic studies have identified multiple RUNX3 single-nucleotide polymorphisms (SNPs) associated with spondyloarthritis (SpA) and related immune-mediated diseases, implicating RUNX3-dependent pathways in disease pathogenesis. RUNX3 exerts highly cell-type–specific functions, particularly in T-cells where it regulates lineage commitment, differentiation, and effector function. In SpA, γδ T cells are emerging as important drivers of early inflammation and tissue damage, yet the transcriptional mechanisms governing their differentiation and function remain poorly understood. The ontogeny and function of γδ T cells differ significantly between rodents and humans, precluding direct translation from mouse studies.

Objectives: This study aims to delineate the role of RUNX3 in human γδ T cells development and effector function. By comparing γδ T cells with conventional αβ CD8 T cells, we sought to identify shared and cell-specific RUNX3-dependent pathways relevant to immune dysregulation in rheumatic diseases.

Methods: CD3 ⁺ T cells were isolated from peripheral blood of healthy donors and cultured with IL-2 (30 IU/mL). After 5 days, αβ CD8 ⁺ T and γδ T cells were sorted by flow cytometry and subjected to RUNX3 CUT&RUN profiling. In another experiment, RUNX3 knockout (KO) CD3 ⁺ T cells were generated using CRISPR-Cas9. Wild-type (WT) and RUNX3-KO CD3 ⁺ T cells were cultured with IL-2 (30 UI/mL) for 14 days, followed by sorting of αβ CD8 ⁺ and γδ T cells for bulk RNA-seq and ATAC-seq. To assess RUNX3 function during ontogeny, humanized immune system (HIS) mice were generated by engrafting WT or RUNX3-KO human hematopoietic stem cells (HSCs) into NBSGW mice at birth. 16 weeks post-engraftment, thymocytes and splenocytes were analyse by flow cytometry.

Results: CUT&RUN analysis identified 41,871 RUNX3 binding sites, with 75% shared between γδ and αβ CD8 ⁺ T cells, indicating substantial overlap in genomic targeting. Transcriptomic analysis with rank–rank hypergeometric overlap revealed convergent RUNX3-dependent regulatory programs in both cell types. RUNX3 promote cytotoxic genes and type 1 effector gene expression (e.g., GZMs , GNLY , HOPX ), while repressing immune checkpoint genes ( PDCD1 ), transcriptional regulators ( TOX2 , RUNX1 , BATF3 ), and cytokine receptors ( IL2RA , IL6R , IL17R ). γδ T-cell–specific RUNX3 targets include KLRB1 (CD161) and T-cell receptor signalling components ( MAPK3 , RIPK3AP1 ), alongside repression of genes involved in homeostasis and migration ( IL7R , IL21R , CCR7 ) and TGF-β signalling ( SMAD3 ). ATAC-seq revealed extensive RUNX3-dependent chromatin remodelling in γδ T cells, with 8,881 differentially accessible regions upon RUNX3 deletion, of which only 24% overlapped with αβ CD8 T cells. In HIS mice, RUNX3 expression was detected early during γδ T-cell development and increased with maturation, mirroring human biology. As expected, RUNX3 deficiency resulted in a marked reduction in conventional human CD8 T cells in the spleen. In sharp contrast, γδ T-cell frequencies were maintained. However, both αβ CD8 and γδ T cells exhibited impaired cytotoxic function in the absence of RUNX3.

Conclusions: RUNX3 is a central regulator of human γδ T cell effector programming, with substantial overlap with αβ CD8 T-cell transcriptional network. In contrast, RUNX3 is dispensable for γδ T-cell development. Together, these findings identify RUNX3 as a key determinant of γδ T-cell functional identity and support a mechanistic link between RUNX3 genetic variants and immune dysregulation in rheumatological diseases. Further studies are warranted to determine how RUNX3 influences γδ T-cell subset diversity and pathogenic potential.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.