In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13 perivascular and CXCL12LTB and PD-L1 epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Furthermore, TLS density correlated with GC formation and expression of adaptive immune response-related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar, but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are coadministered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naïve LSCC patients treated with corticosteroids before surgery, compared with untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment. Corticosteroid treatment during chemotherapy negatively affects the development of tertiary lymphoid structures and abrogates their prognostic value in patients with lung cancer. .

SARS‐CoV‐2 is a newly emerged coronavirus that caused the global COVID‐19 outbreak in early 2020. COVID‐19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS‐CoV‐2–host cell interactions and entry mechanisms remain poorly understood. Investigating SARS‐CoV‐2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS‐CoV‐2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH‐independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS‐CoV‐2 entered the cytosol via acid‐activated cathepsin L protease 40–60 min post‐infection. Overexpression of TMPRSS2 in non‐TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS‐CoV‐2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS‐CoV‐2 sorting into either pathway.

SARS-CoV-2 is a newly emerged coronavirus (CoV) that spread through human populations worldwide in early 2020. CoVs rely on host cell proteases for activation and infection. The trypsin-like protease TMPRSS2 at the cell surface, cathepsin L in endolysosomes, and furin in the Golgi have all been implicated in the SARS-CoV-2 proteolytic processing. Whether SARS-CoV-2 depends on endocytosis internalization and vacuolar acidification for infectious entry remains unclear. Here, we examined the dynamics of SARS-CoV-2 activation during the cell entry process in tissue culture. Using four cell lines representative of lung, colon, and kidney epithelial tissues, we found that TMPRSS2 determines the SARS-CoV-2 entry pathways. In TMPRSS2-positive cells, infection was sensitive to aprotinin, a TMPRSS2 inhibitor, but not to SB412515, a drug that impairs cathepsin L. Infectious penetration was marginally dependent on endosomal acidification, and the virus passed the protease-sensitive step within 10 min. In a marked contrast, in TMPRSS2-negative cells cathepsin L and low pH were required for SARS-CoV-2 entry. The cathepsin L-activated penetration occurred within 40-60 min after internalization and required intact endolysosomal functions. Importantly, pre-activation of the virus allowed it to bypass the need for endosomal acidification for viral fusion and productive entry. Overall, our results indicate that SARS-CoV-2 shares with other CoVs a strategy of differential use of host cell proteases for activation and infectious penetration. This study also highlights the importance of TMPRSS2 in dictating the entry pathway used by SARS-CoV-2.SignificancePreventing SARS-CoV-2 spread requires approaches affecting early virus-host cell interactions before the virus enters and infects target cells. Host cell proteases are critical for coronavirus activation and infectious entry. Here, we reconcile apparent contradictory observations from recent reports on endosomal acidification and the role of furin, TMPRSS2, and cathepsin L in the productive entry and fusion process of SARS-CoV-2. Investigating authentic virus in various cell types, we demonstrated that SARS-CoV-2 developed the ability to use different entry pathways, depending on the proteases expressed by the target cell. Our results have strong implications for future research on the apparent broad tropism of the virus in vivo. This study also provides a handle to develop novel antiviral strategies aiming to block virus entry, as illustrated with the several drugs that we identified to prevent SARS-CoV-2 infection, some with low IC50.