18The ability of Mycobacterium tuberculosis to form serpentine cords is intrinsically related to 19 its virulence, but specifically how M. tuberculosis cording contributes to pathogenesis 20 remains obscure. We show that several M. tuberculosis clinical isolates form intracellular 21 cords in primary human lymphatic endothelial cells (hLEC) in vitro and also in the lymph 22 nodes of patients with tuberculosis. We identified via RNA-seq a transcriptional programme 23 in hLEC that activates cellular pro-survival and cytosolic surveillance of intracellular 24 pathogens pathways. Consistent with this, cytosolic access of hLEC is required for 25 intracellular M. tuberculosis cording; and cord formation is dependent on the M. 26 tuberculosis ESX-1 type VII secretion system and the mycobacterial lipid PDIM. Finally, we 27show that M. tuberculosis cording is a novel size-dependent mechanism used by the 28 pathogen to evade xenophagy in the cytosol of endothelial cells. These results provide a 29 mechanism that explains the long-standing association between M. tuberculosis cording and 30 virulence. 31Bacterial xenophagy is the process that regulates the removal of cytosolic bacteria after 64 damage to phagosomal membranes during selective macroautophagy (Galluzzi et al., 2017). 65This pathway constitutes one of the first cell autonomous defence pathways against 66 intracellular pathogens (Deretic and Levine, 2009; Gutierrez et al., 2004). A fraction of the 67 M. tuberculosis population damage phagosomes to access the cytosol and are subsequently 68 recognised by autophagic adaptors and the xenophagy machinery. This process targets M. 69 tuberculosis into autophagosomes and thus the lysosomal degradation pathway (Watson et 70 al., 2012). Whereas there is a large body of literature demonstrating autophagy as an anti-71 mycobacterial pathway (Deretic et al., 2009), recent evidence shows that M. tuberculosis 72 can eventually block the fusion of autophagosomes with lysosomes (Lerner et al., 2016; 73 Romagnoli et al., 2012) and in mice, M. tuberculosis can evade autophagic responses in vivo 74 (Kimmey et al., 2015). 75 76 M. tuberculosis mostly infects macrophages although there is compelling evidence that a 77 minor proportion of M. tuberculosis is found infecting various non-myeloid cells in the lungs 78and lymph nodes in vivo (Ganbat et al., 2016; Lerner et al., 2015; Nair et al., 2016; Randall et 79 al., 2015). The role that these M. tuberculosis subpopulations play in TB pathogenesis in 80 different cell types (e.g. immune vs non-immune) is unclear. We previously showed in 81 extrapulmonary tuberculosis that a subpopulation of M. tuberculosis is found in human 82 lymphatic endothelial cells (hLEC) in lymph node biopsies and these cells could represent a 83 reservoir for M. tuberculosis in infected patients (Lerner et al., 2016). 84 85 Here we discovered that M. tuberculosis forms large intracellular cords consisting of up to 86 thousands of individual bacteria arranged end-to-end in hLEC in vitro and in biopsies of 87 ...