Objective To investigate the dynamic changes of the immune microenvironment during critical-sized bone

defect repair and elucidate the regulatory role of T cells in bone regeneration. Methods A critical-sized bone defect model was established in pigs and mice. Regenerating tissues from steady state and multiple postoperative time points were collected for single-cell RNA sequencing （scRNA-seq）. After data preprocessing， dimension reduction， clustering， and cell-type annotation， T cells and myeloid populations were further extracted for subcluster identification， differential gene expression analysis， pathway enrichment， and intercellular communication analysis. Functional validation was performed in T celldeficient mice， and bone regeneration outcomes were assessed using Micro-CT. Results scRNA-seq analysis identified 10 major cell types， with T cells and myeloid cells rapidly recruited during the early regenerative phase and exhibiting timedependent functional reprogramming. T cells were classified into 7 subclusters， among which CD8 ⁺ T cell subclusters expanded markedly during bone repair and showed elevated expression of the chemokine CCL5. Myeloid cells were subdivided into 5 subpopulations， and osteoclasts were prominently increased and activated during the bone reconstruction phase. Intercellular interaction analysis indicated that CD8⁺ T cells promoted osteoclast migration and functional activation through the CCL5-CCR5 signaling axis. In T cell-deficient mice， excessive bone formation and ectopic ossification were observed， accompanied by a reduced osteoclast population and hyperactivation of osteogenic pathways. Conclusion During  critical-sized bone defect repair， T cells and myeloid cells undergo temporally coordinated recruitment and functional transitions. CD8⁺ T cells regulate osteoclast recruitment and activation via the CCL5-CCR5 signaling axis， maintaining the balance between bone formation and resorption， thereby ensuring orderly bone regeneration and structural reconstruction.

Critical-sized bone defect, Osteoimmunology, Bone regeneration, Single-cell sequencing, T cells,