Enrichment of CD103+ CD4+ T cells in bone metastases compared to non-malignant controls

• CD103⁺ CD4⁺ T cells are markedly enriched in bone metastases (BoM) • CD103⁺ CD8⁺ T cells in BoM are depleted, raising CD4⁺/CD8⁺ ratio • In BoM CD103⁺ CD4⁺ subset shifts to central-memory phenotype • In BoM, coregulatory receptor expression is increased on CD103⁺ T cells, with distinct co-expression signatures in both CD4⁺ and CD8⁺ cells Bone metastasis (BoM) is a frequent complication of solid tumors, leading to poor prognosis and reduced survival. CD103⁺ tumor-infiltrating lymphocytes (TILs) are critical for antitumor immunity, yet studies have largely focused on CD8⁺ subsets, leaving CD103⁺CD4⁺ cells poorly characterized. Bone aspirates containing malignant cells were obtained from patients with symptomatic spinal and/or pelvic metastases of breast cancer (BC), prostate cancer (PC), or non-small cell lung cancer (NSCLC). Age-matched bone marrow samples from individuals without malignancy (NMCs) were controls. Multiparametric flow cytometry (MFC) was utilized to assess the expression of CD103 and exhaustion markers (TIGIT, PVRIG, KIR2DL5, CD39) in CD3 + cells. CD103⁺CD3 + T cells are significantly elevated in BoM compared to NMC, driven by an increase in CD103⁺CD4⁺ cells, despite a relative decrease in frequency of CD103⁺CD8⁺ cells. Furthermore, CD103 + CD4 + T cells from BoM displayed a significantly increased fraction of the central memory (CM) phenotype. Expression and co-expression of TIGIT, PVRIG, KIR2DL5 and CD39 on CD103 + cells both in the CD4 and CD8 compartment was significantly increased in BoM, compared within CD103 - within BoM and CD103 + in NMC. In conclusion, BoM exhibit a distinct T-cell composition, highlighted by an increase in CD103⁺CD4⁺ T cells displaying an increased CM phenotype. In BoM, coregulatory receptor expression is increased on CD103⁺ T cells, with distinct co-expression signatures in both CD4⁺ and CD8⁺ cells. Functional studies will determine whether targeting these checkpoint pathways can improve immunotherapy for metastatic bone disease.