Fig 2: Neuronal infection is required for lethal SARS-CoV-2 infection in hACE2fl mice.(A, B) Weight loss and survival of hACE2fl/y and Baf53b-Cre; hACE2fl/y mice after infection with 104 PFU of SARS-CoV-2. N = 10 (hACE2fl/y) and 12 (Baf53b-Cre; hACE2fl/y), three independent experiments. (C) Pulse oximetry measured in WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 days after exposure to 104 PFU of SARS-CoV-2 virus. (D) Immunoblotting of whole brain lysates from hACE2fl/y and Baf53b-Cre; hACE2fl/y and WT mice was performed using anti-panACE2, anti-hACE2, and anti-ß-actin antibodies. Each lane represents a single animal. n = 4, two independent experiments. (E) Immunohistochemistry of SARS-CoV-2 nucleocapsid, epithelial cell Krt8, and OSN OMP in the OE of WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 DPI. Representative of n = 4 animals per genotype. (F) Immunohistochemistry of SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP in the lungs of WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 DPI. (G) Immunohistochemistry of SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP in the cerebral cortex at low and high magnification of the same tissue section of WT, hACE2fl/y, and Baf53b-Cre; hACE2fl/y mice 6 DPI. Representative of N = 5–6 animals per genotype and time point. Scale bars in all images 50 µm. ****p < 0.0001 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding S1 Metadata tab. AT1, alveolar type 1; AT2, alveolar type 2; DPI, days postinfection; OE, olfactory epithelium; OMP, olfactory marker protein; OSN, olfactory sensory neuron; PDPN, Podoplanin; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; WT, wild-type.

Fig 3: Selective hACE2 expression in OE and neurons is sufficient to confer hypoxemia and death following SARS-CoV-2 infection.(A-C) Immunohistochemistry of WT and Foxg1Cre/+;LSL-hACE2+/0 mouse OE (A), OB, and cerebral cortex (B) and lung (C) 6 days after infection with 105 PFU of SARS-CoV-2 using antibodies that recognize viral nucleocapsid, sustentacular cell E-cadherin, olfactory sensory neuron ßIII-tubulin, neuronal NeuN, glial GFAP, AT1 PDPN, or AT2 DC-LAMP. Asterisk indicates OB with nucleocapsid staining. Arrows indicate nucleocapsid staining colocalized with ßIII-tubulin+ OSNs (OE in A) or NeuN+ neurons (OB and cerebral cortex in B). White dotted lines trace the border between the OE and OB. Representative of n = 5 per genotype. (D) HE staining of WT and Foxg1Cre/+; hACE2fl/y lung tissue 6 days after exposure to 105 PFU of SARS-CoV-2 virus. Arrows, sites of focal consolidation. Asterisks, intravascular thrombi. Representative of n = 5 animals per genotype. (E, F) Immunohistochemistry of WT and Foxg1Cre/+; hACE2fl/y lung tissue 6 days after exposure to 105 PFU of SARS-CoV-2 virus using antibodies against ICAM-1 and PDPN, or vWF and PECAM. Arrows in I identify vWF-positive microvasculature of the lung in Foxg1Cre/+; hACE2fl/y animals. Representative of n = 5 animals per genotype. (G, H) Weight loss and survival of LSL-hACE2+/0 and Foxg1Cre/+; LSL-hACE2+/0 mice after infection with 105 or 104 PFU of SARS-CoV-2. N = 4 (LSL-hACE2+/0), 8 (Foxg1Cre/+; LSL-hACE2+/0 105 PFU), 5 (Foxg1Cre/+; LSL-hACE2+/0 104 PFU) and two independent experiments. Asterisks indicate time points at which significant differences in weight (C) or survival (D) were observed between infected Foxg1Cre/+; LSL-hACE2+/0 and LSL-hACE2+/0 mice animals. (I) Pulse oximetry of WT (LSL-hACE2+/0 or Foxg1Cre/+) and Foxg1Cre/+; LSL-hACE2+/0 mice infected with 104 viral titer at the time of harvest (Day 6–8). ***p < 0.001; ****p < 0.0001 determined by unpaired, two-tailed t test or log-rank Mantel Cox test. Scale bars in all images 50 µm. Numerical data in corresponding S1 Metadata tab. hACE2, human ACE2; ICAM-1, intracellular adhesion marker 1; OB, olfactory bulb; OE, olfactory epithelium; OSN, olfactory sensory neuron; PDPN, Podoplanin; PECAM, platelet endothelial cell adhesion molecule; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; vWF, von Willebrand’s Factor; WT, wild-type.

Fig 4: Selective hACE2 expression in lung epithelial cells confers nonlethal pneumonia after SARS-CoV-2 infection.(A) Generation of LSL-hACE2 mice using gene targeting of the mouse Rosa26 locus. WPRE, SV40, PGK-NeoR. (B) Immunoblotting of whole lung lysates from WT, LSL-hACE2+/0, and ShhCre/+;LSL-hACE2+/0 mice was performed using anti-hACE2 and anti-ß-actin antibodies. Each lane represents a single animal. Representative of n = 3 per genotype and two independent experiments. (C) Immunohistochemistry of lung from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using anti-panACE2 and anti-hACE2 antibodies. Representative of N = 3 per genotype and two independent experiments. (D) Immunohistochemistry of lung from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP 2 days after infection with 104 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. (E) Immunohistochemistry of lung from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP 6 days after infection with 104 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. (F) Immunohistochemistry of cerebral cortex from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP 6 days after infection with 104 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. (G) HE staining of LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 lung tissue 2 days after exposure to 104 PFU of SARS-CoV-2 virus. Arrows, sites of inflammatory cell infiltrate. Hashtag, acute emphysematous changes. Representative of n = 4 animals per genotype. (H) HE staining of LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 lung tissue 6 days after exposure to 104 PFU of SARS-CoV-2 virus. Arrows, sites of alveolar inflammatory infiltrate and hyalinosis. Representative of n = 4 animals per genotype. (I) Pulse oximetry measured in LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice 6, 10, and 14 days after exposure to 105 PFU of SARS-CoV-2 virus. (J, K) Weight loss and survival of LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice after infection with 105 PFU of SARS-CoV-2. N = 6 (LSL-hACE2+/0) and 7 (ShhCre/+;LSL-hACE2+/0 mice), two independent experiments. Scale bars in all images 50 µm. Note: Images in each panel were taken at lower and/or higher magnification from the same tissue section respective to genotype and highlight different pathology. ns, not significant, p > 0.05 **, p < 0.01 by unpaired two-tailed t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding S1 Metadata tab. AT1, alveolar type 1; AT2, alveolar type 2; hACE2, human ACE2; HE, hematoxylin–eosin; PDPN, Podoplanin; PFU, plaque-forming unit; PGK-NeoR, Phosphoglycerate kinase promoter-driven Neomycin resistance cassette; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SV40, simian virus 40; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; WT, wild-type.

Fig 5: IHC analyses of tumor HEVs and immune-cell infiltration. A, Immunostaining of TLS-rich and TLS-free tumors for MECA-79 (magenta) and CD3 (dark brown) was performed to determine the abundance of HEVs and T cells. The dotted line shows the edge of each tumor. Arrowheads indicate the MECA-79+ HEVs. The higher magnification of each left panel (black square) is shown on the right. Scale bar, 500 µm. B, HEV and CD3+ T-cell densities were quantified in specimens of 10 patients for each tumor type (TLS-rich and TLS-free). C, Immunostaining of CD3 and MECA-79. Green arrowheads indicate several CD3+ cells on or in the endothelial wall of HEVs. Scale bar, 50 µm. D, Comparison of CD8+ T-cell, CD20+ B-cell, and DC-LAMP+ dendritic-cell densities between the two tumor groups analyzed in B. Error bar; SEM. E, Immunofluorescence detection of HEVs associated with lymphocyte clusters in TLS-rich tumors. Consecutive sections of the same tumors were stained for CD31/MECA-79 (immunofluorescence), CD8, or CD20 (dark brown) to orient the positions of immune cells and blood vessels. A representative TLS is shown here for TLS-rich tumors. CD31, EC marker (red). Scale bar, 100 µm. P value was calculated by Student t test.