Enzyme-Activatable CXCL13 Chemokine Probes Enable Direct Fluorescence Detection of Hypoxic Subpopulations of Human B Cells

Oxygen availability is a key regulator of immune cell function, particularly in B lymphocytes, because they are highly sensitive to hypoxia signaling. However, current hypoxia-sensing probes lack the specificity to distinguish B cells from other leukocytes and lymphocytes in biosamples. Here, we present hCXCL13-6 as an AND-gate activatable probe for the fluorescence detection and live imaging of hypoxic human B cells. The probe hCXCL13-6 combines a site-specifically labeled analog of the human chemokine CXCL13─for selective internalization in B cells─and a bioconjugable azo-containing rhodamine fluorophore─for hypoxia sensing. Notably, hCXCL13-6 displays both CXCR5 receptor-mediated endocytosis in B cells and hypoxia-mediated enzymatic activation, which results in bright fluorescence signals being exclusively found in hypoxic B cells but not in normoxic B cells or in other immune cells. Notably, we demonstrated that hCXCL13-6 enables direct identification of hypoxic B cells in cell mixtures derived from human blood biosamples. The combination of 'clickable' fluorogenic reporters with nonperturbative ligation to chemokine proteins will create new avenues for the rational design of targeted B cell probes to study inflammatory diseases and hematological malignancies.