Intravital lung microscopy unveils T cell dynamics in mouse tuberculosis lesions

Protective immunity to Mycobacterium tuberculosis ( Mtb ) depends on the ability of T cells to access and engage infected cells within lung lesions, yet these spatiotemporal interactions remain poorly defined. Direct intravital imaging of Mtb -infected lungs has historically been limited by biosafety and technical constraints, preventing real-time visualization of immune dynamics in situ . Here, we present LiveLung-TB, a biosafety level 3–compatible lung intravital imaging platform that enables high-resolution imaging of immune cell behavior in Mtb -infected lungs. Using this approach, we demonstrate that although most CD4⁺ T cells infiltrate the infected parenchyma, only a small fraction forms transient (~11%) or stable (~5%) contacts with infected macrophages. Strikingly, the majority remain immobilized or display intermittent migration within uninfected, collagen-rich regions or at the periphery of macrophage clusters, independently of antigen recognition. These findings uncover previously unrecognized physical and microenvironmental barriers that restrict T cell motility and limit productive effector engagement. LiveLung-TB thus provides a powerful framework to elucidate and ultimately overcome tissue-imposed constraints on immune efficacy in tuberculosis and other pulmonary infections.