A humanized mouse model of chronic COVID-19

Abstract

Coronavirus disease 2019 (COVID-19) is an infectious disease that can present as an uncontrolled, hyperactive immune response, causing severe immunological injury. Existing rodent models do not recapitulate the sustained immunopathology of patients with severe disease. Here we describe a humanized mouse model of COVID-19 that uses adeno-associated virus to deliver human ACE2 to the lungs of humanized MISTRG6 mice. This model recapitulates innate and adaptive human immune responses to severe acute respiratory syndrome coronavirus 2 infection up to 28 days after infection, with key features of chronic COVID-19, including weight loss, persistent viral RNA, lung pathology with fibrosis, a human inflammatory macrophage response, a persistent interferon-stimulated gene signature and T cell lymphopenia. We used this model to study two therapeutics on immunopathology, patient-derived antibodies and steroids and found that the same inflammatory macrophages crucial to containing early infection later drove immunopathology. This model will enable evaluation of COVID-19 disease mechanisms and treatments.

Extended Data Figure 1:. Comparison of COVID-19 parameters and response to therapeutics in human patients, humanized mice, and other animal models.

Although substantial anti-viral immunity mediates viral clearance in non-severe COVID-19, robust inflammatory cytokine production, decreased circulating lymphocytes and failure to generate germinal centers characterizes the immunopathology in severe COVID-19 ^–.Dysregulated myeloid^– and lymphocyte^–, compartments, including monocytes, macrophages, neutrophils, NK cells and antibody secreting B cells, have been described as players in the observed lung immunopathology of COVID-19. Accurate model systems are essential to rapidly evaluate promising discoveries but most models currently available in mice, ferrets and hamsters do not recapitulate sustained immunopathology described in COVID-19 patients. The following table summarizes various aspects of the human disease and how well these are recapitulated in existing animal models of COVID-19 and in our humanized mouse model of COVID-19. References: 1 Imai, M. et al. Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development. Proceedings of the National Academy of Sciences 117, 16587–16595 (2020). 2 Dinnon, K. H. et al. A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures. Nature 586, 560–566 (2020). 3 Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet 395, 497–506, doi:10.1016/S0140–6736(20)30183–5 (2020). 4 Song, E. et al. Neuroinvasion of SARS-CoV-2 in human and mouse brain. Journal of Experimental Medicine 218 (2021). 5 Tian, S. et al. Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies. Modern Pathology, 1–8 (2020). 6 Menter, T. et al. Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction. Histopathology 77, 198–209 (2020). 7 Barton, L. M., Duval, E. J., Stroberg, E., Ghosh, S. & Mukhopadhyay, S. Covid-19 autopsies, oklahoma, usa. American Journal of Clinical Pathology 153, 725–733 (2020). 8 Xu, Z. et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. The Lancet. Respiratory medicine 8, 420–422, doi:10.1016/S2213–2600(20)30076-X (2020). 9 Israelow, B. et al. Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling. J Exp Med 217, doi:10.1084/jem.20201241 (2020). 10 Sun, S.-H. et al. A mouse model of SARS-CoV-2 infection and pathogenesis. Cell Host & Microbe (2020). 11 Hassan, A. O. et al. A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies. Cell 182, 744–753. e744 (2020). 12 Ter Meulen, J. et al. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets. The Lancet 363, 2139–2141 (2004). 13 Mathew, D. et al. Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science 369 (2020). 14 Tan, L. et al. Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study. Signal transduction and targeted therapy 5, 1–3 (2020). 15 Zhang, J.-Y. et al. Single-cell landscape of immunological responses in patients with COVID-19. Nature immunology 21, 1107–1118 (2020). 16 Liao, M. et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nature medicine, 1–3 (2020). 17 Lucas, C. et al. Longitudinal analyses reveal immunological misfiring in severe COVID-19. Nature 584, 463–469 (2020). 18 Chen, Z. & Wherry, E. J. T cell responses in patients with COVID-19. Nature Reviews Immunology, 1–8 (2020). 19 Kaneko, N. et al. Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19. Cell 183, 143–157. e113 (2020). 20 Woodruff, M. C. et al. Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19. Nature immunology 21, 1506–1516 (2020). 21 Group, R. C. Dexamethasone in hospitalized patients with Covid-19—preliminary report. New England Journal of Medicine (2020). 22 Cruz-Teran, C. et al. Challenges and opportunities for antiviral monoclonal antibodies as COVID-19 therapy. Advanced Drug Delivery Reviews (2020). 23 Casadevall, A. & Pirofski, L.-a. The convalescent sera option for containing COVID-19. The Journal of clinical investigation 130 (2020). 24 Chen, P. et al. SARS-CoV-2 neutralizing antibody LY-CoV555 in outpatients with Covid-19. New England Journal of Medicine (2020).

Extended data Figure 2 (matched to Figure 3):

A. Numbers of human T cells in the blood pre- and post-infection (2,4,7,14,28dpi). Unpaired, two-tailed t-test. P-values<0.05 are plotted. Uninfected n=5, 2dpi n=4, 4dpi n=6, 7dpi n=5, 14dpi n=5, 28dpi=4, biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are plotted. B. Numbers of human T cells in the spleens of uninfected or infected mice (2,4,7,14,28dpi). Unpaired, two-tailed t-test. P-values<0.05 are plotted. Unpaired, two-tailed t-test. Only p-values<0.05 are plotted. Uninfected n=7, 2dpi n=4, 4dpi n=6, 7dpi n=4, 14dpi n=8, 28dpi=4, biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are plotted. C. Frequencies of human CD4+ or CD8+ T cells within hCD3+ population in the blood and spleens of uninfected or infected mice at 2,4,7,14,28 dpi. Blood: N=4 biologically independent mice examined over at least 2 independent experiments. Means with SD are plotted. Spleen: Uninfected n=5, 2dpi n=4, 4dpi n=6, 7dpi n=4, 14dpi n=4 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are plotted. D. Frequencies of human CD4+ or CD8+ T cells within hCD3+ population in the lungs of uninfected or infected mice at 2,4,7,14,28 dpi. Uninfected n=4, 2dpi n=4, 4dpi n=6, 7dpi n=4, 14dpi n=4 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are plotted. E. Representative flow cytometry plots of CD8+ T cells stained with viral antigen bearing HLA-A2 tetramers. HLA-A2 tetramers bound to peptides of Nucleoprotein(N), Membrane protein(M) and Spike protein were used to characterize T cells from uninfected and infected (2,4,7,14,28dpi) mice. Representative of uninfected n=3, 2dpi n=2, 4dpi n=3, 7dpi n=3, 14dpi n=3, 28dpi n=3 biologically independent mice examined over 2 independent experiments. F. T cell numbers and viral titers following prophylactic T cell transfer in infected mice at 4dpi. Splenocytes and sorted T cells from lungs of 14dpi mice where the contribution of the antigen specific B cell response was minimal were transferred to unengrafted MISTRG-hACE2 (no human immune cells prior to transfer). Control MISTRG-hACE2 mice received splenocytes but not sorted lung T cells (n=3 biologically independent mice examined over 2 independent experiments). These mice were subsequently infected and analyzed at 4dpi for viral titers and human immune cells in lungs. In mice that received sorted lung T cells, human immune compartment in lungs comprised of T cells and few CD16+ monocytes and macrophages but no B cells (n=4 biologically independent mice examined over 2 independent experiments). G. Numbers of human B cells in the BAL of uninfected or infected mice at 2,4,7,14,28 dpi. N=3–6. Unpaired, two-tailed t-test. P-values<0.05 are plotted. Uninfected n=3, 2dpi n=3, 4dpi n=4, 7dpi n=3, 14dpi n=7, 28dpi n=4 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are plotted. H. Representative flow cytometry plots of IgG+ B cells in the BAL of uninfected and infected mice 14 and 28dpi. Representative of n=4 biologically independent mice examined over 3 independent experiments. I. CD11C and CD19 expression on human immune cells from infected lungs at 14 and 28 dpi. N=4–5. Representative of n=5 (14dpi) and n=4 (28dpi) biologically independent mice examined over at least 3 independent experiments. J. Frequencies of human B cells marked by CD19 and CD20 expression within hCD45+ population in spleen and blood of uninfected or infected mice at 2,4,7,14,28 dpi. Blood: Uninfected n=6, 2dpi n=5, 4dpi n=8, 7dpi n=5, 14dpi n=6, 28dpi n=6 biologically independent mice examined over at least 3 independent experiments. Spleen: Uninfected n=6, 2dpi n=4, 4dpi n=8, 7dpi n=5, 14dpi n=6, 28dpi n=7 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are plotted. K. IgG levels measured by ELISA in serum of uninfected or infected mice at 7,14, 28 dpi. Uninfected n=5, 7dpi n=2, 14dpi n=2, 28dpi n=5 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are plotted. Unpaired, two tailed t-test.

Extended Data Figure 3 (matched to Figure 4):

A. t-distributed stochastic neighbor embedding (t-SNE) plot with clustering results of single cell RNA sequencing of mouse cells from lungs at 4dpi. Single cell suspensions from whole infected lung at 4dpi were processed and sequenced. There were 731 cells identified as mouse cells. B. Dot plot of cluster identifying genes for mouse cell clusters described in S4A. C. Cluster distribution and expression profile of mouse inflammatory cytokines for clusters described in 4B. D. Normalized expression of human IFNB measured by qPCR in homogenized lung tissue of uninfected and infected (2,4,7,14,28dpi) mice. Expression was normalized to human HPRT1. Unpaired t-test, one-tailed since the distribution is expected to be one-sided. Uninfected n=3, 2dpi n=3, 4dpi n=6, 7dpi n=7, 14dpi n=10, 28dpi n=2 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are presented. P-values<0.05 are plotted. E. IFNA levels measured by ELISA in serum and BAL of uninfected or infected mice. Serum: Uninfected n=6, 4dpi n=3, 7dpi n=2, 14dpi n=5, 28dpi n=3; BAL: Uninfected n=3, 14dpi n=4 biologically independent mice examined over at least 2 independent experiments for all time points. Individual values (average of duplicates) for each mouse and means are plotted. F. Type I interferon levels (IFNA6 and IFNB) per cell measured by qPCR in sorted human immune cells (human CD45+): plasmacytoid dendritic cells (PDCs; CD123^hi CD11B-), macrophages and monocytes (CD11B+) and rest of human immune cells (CD123-CD11B-CD45+). Expression was normalized to human ACTB. Individual values and means are plotted. n=3 biologically independent mice examined over 2 independent experiments. Individual values with means are plotted. G. Normalized expression of human IFNG measured by qPCR in homogenized lung tissue of uninfected and infected (2,4,7,14,28dpi) mice. Expression was normalized to human HPRT1. Unpaired t-test, one tailed since distribution is expected to be one-sided. Uninfected n=10, 2dpi n=3, 4dpi n=8, 7dpi n=3, 14dpi n=15, 28dpi n=5 biologically independent mice examined over at least 3 independent experiments for all time points. H. Heatmap of normalized counts for bystander activated memory T cell signature genes (based on Low et al., JEM 2020) in lungs of MISTRG6-hACE2 mice infected with SARS-CoV-2. . Row min and max of transformed values, calculated by subtracting row mean and diving by STD for each gene across all samples, are visualized. Uninfected n=5, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=2, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points. I. Volcano plot showing foldchange and adjusted p-values (by Wald test using DESeq2) of differentially regulated genes at 28dpi compared to uninfected lungs. Genes with FC(Log2)>1 and p value<0.05 are highlighted in red. Replicates of at least 2 mice. Uninfected n=5, 28dpi n=2 biologically independent mice examined over 2 independent experiments. J. Heatmap of normalized counts for genes that are induced in B cells of patients with moderate or severe COVID-19 in comparison with healthy controls. Normalized counts in lungs of uninfected or infected MISTRG6-hACE2 were plotted over the course of infection. Row min and max of transformed values, calculated by subtracting row mean and diving by STD for each gene across all samples, are visualized. Uninfected n=5, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=2, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points. K. Heatmap of normalized counts for bystander activated memory B cell signature genes(based on Horns et al., 2020) in lungs of MISTRG6-hACE2 mice infected with SARS-CoV-2. Row min and max of transformed values, calculated by subtracting row mean and diving by STD for each gene across all samples, are visualized. Uninfected n=5, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=2, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points.

Extended Date Figure 4 (matched to Figure 5):

A. Schematic of experimental design of prophylactic antibody treatment MISTRG6-hACE2 mice received prophylactic treatment of convalescent plasma (5ml/kg) or monoclonal antibodies at 10mg/kg (clone 135 -m135 or clone 144-m144) 8 hours prior to infection or left untreated (untd). Mice were euthanized 4dpi. Convalescent plasma samples from the top 30 neutralizers in a cohort of 148 individuals were pooled to create a mixture with an NT50 titer of 1597 against HIV-1 pseudotyped with SARS-CoV-2 S protein. Sequencing the antibody genes from infected humans has revealed the expansion of closely related Receptor Binding Domain of the Spike protein (RBD)-specific B cell antibody clones in different SARS-CoV-2 infected individuals. Although most convalescent plasma samples obtained from patients who recovered from COVID-19 did not contain high levels of neutralizing activity, RBD-specific antibodies with potent antiviral activity were found in all individuals tested. Monoclonal recombinant antibodies (mAbs) used in this study were cloned from these convalescent patients and had high neutralizing activity against SARS-CoV-2 in vitro and in vivo in mouse adapted SARS-CoV-2 infection ^,. B. Weight change in convalescent plasma treated mice at 2 and 4dpi plotted as percent change compared with original weight measured just before inoculation with SARS-CoV-2. Unpaired t-test, two-tailed comparing 4dpi values. N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values per each mouse plotted. C. Representative images (10x magnification) of H&E staining of lungs (4dpi) from mice treated prophylactically with convalescent plasma or left untreated. The histopathological findings mark transition into the organizing phase, highlighted by infiltration of the inter- and intra-alveolar space by lymphocytes macrophages, and fibroblasts. Representative of N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values for each mouse are plotted. D. Frequencies of human monocytes (CD14+ classical; CD14+CD16+ intermediate, CD16+ non classical) within human CD45+ cells in the lungs of MISTRG6-hACE2 mice which received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Unpaired, two-tailed t-test. P-values<0.05 are plotted. Untreated N=5, treated n=6 N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values for each mouse and means are plotted. E. Frequencies of HLA-DR+CXCR3+ T cells within human CD3+CD45+ cells in lungs of MISTRG6-hACE2 mice which received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Unpaired, two-tailed t-test. N=5–6. P-values<0.05 are plotted. Untreated N=5, treated n=6 N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values for each mouse and means are plotted. F. Frequencies of Tγδ cells within human CD3+CD45+ cells in lungs of MISTRG6-hACE2 mice which received prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Unpaired, two-tailed t-test. N=5–6. P-values<0.05 are plotted. Untreated N=5, treated n=6 N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values for each mouse and means are plotted. G. Frequencies of human T cells within human CD45+ cells in spleens of MISTRG6-hACE2 mice received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Unpaired, two-tailed t-test. N=5–6. P-values<0.05 are plotted. Untreated N=5, treated n=6 N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values for each mouse and means are plotted. H. Frequencies of human CD3+ T cells within human CD45+ population in the blood pre- and post-infection (2,4,7,14,28dpi). Lines connect pre- and post-infection values for the same mouse. MISTRG6-hACE2 mice received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Paired, two-tailed t-test. P-values<0.05 are plotted. Untreated N=5, treated n=6 N=4 biologically independent mice examined over 2 independent experiments for all time points. Individual values for each mouse are plotted. I. Schematic of experimental design of post-infection mAb treatment. MISTRG6-hACE2 mice received a mixed cocktail of monoclonal antibodies clone 135 (m135) and clone 144(m144) at 20mg/kg or left untreated (untd). Early treatment groups were treated 11 hours post-infection and late treatment 35 hours post-infection. Monoclonal recombinant antibodies (mAbs) used in this study were cloned from the convalescent patients (whose plasma was used prophylactically before) and had high neutralizing activity against SARS-CoV-2 in vitro and in vivo in mouse adapted SARS-CoV-2 infection ^,. J. Viral titers measured by PFU in homogenized lung tissue following early or late therapeutic mAb at 4dpi using Vero E6 (standard assay) Vero-ACE2+TMPRSS2+ (sensitive assay) cells. N=4 biologically independent mice examined over 2 independent experiments measurement with Vero-ACE2+TMPRSS2+ (sensitive assay) cells. Individual values for each mouse are plotted. K. Frequencies of human T cells within human CD45+ cells in spleens of MISTRG6-hACE2 mice that received early, late or no treatment of monoclonal antibody mix. Unpaired, two-tailed t-test. Untreated n=7, treated n=5 biologically independent mice examined over 3 independent experiments for all time points. Pooled, infection-matched mice are presented. P-values<0.05 are plotted. Individual values representing each mouse are plotted. Individual values for each mouse are plotted. L. Number of CD16+ human monocytes in lungs of treated and untreated mice at 4dpi. Mice were either MISTRG6-hACE2 mice that received a mixed cocktail of monoclonal antibodies clone 135 (m135) and clone 144(m144) or left untreated (untd). Early treatment group was treated 11 hours post-infection (hpi) and late treatment 35 hpi. Unpaired, two-tailed t-test. Untreated n=7, treated n=5 biologically independent mice examined over 3 independent experiments for all time points. Pooled, infection-matched mice are presented. P-values<0.05 are plotted. Individual values representing each mouse are plotted. M. Number of human macrophages in lungs of treated and untreated mice at 4dpi. MISTRG6-hACE2 mice that received a mixed cocktail of monoclonal antibodies clone 135 (m135) and clone 144(m144) or left untreated (untd). Early treatment group was treated 11 hours post-infection and late treatment 35 hours post-infection. Unpaired, two-tailed t-test. Untreated n=7, treated n=5 biologically independent mice examined over 3 independent experiments for all time points. Pooled, infection-matched mice are presented. P-values<0.05 are plotted. Individual values representing each mouse are plotted.

Extended data Figure 5 (matched to Figure 6):

A. Schematic of experimental design of dexamethasone treatment: SARS-CoV-2 infected MISTRG6-hACE2 mice were either treated with dexamethasone on days 7,8,9 post-infection or left untreated. Mice were analyzed either at 14dpi or 28dpi. Mice were treated intraperitoneally (i.p.) with dexamethasone at 10mg/kg dose. B. Representative flow cytometry plots of Ly6G expressing SSChi cells (mouse neutrophils) within the mouse immune cell population (mouse CD45+) in the BAL of dexamethasone treated or control untreated mice. Representative of 4 biologically independent mice examined over at least 2 independent experiments. C. HLA-DR expression on lung T cells 28dpi in dexamethasone treated or control mice. Representative of 3 biologically independent mice examined over at least 2 independent experiments. D. Representative flow cytometry plots of Surface IgG and CD19 expression on human immune cells gated on hCD45+ cells in lungs of untreated or dexamethasone treated mice at 28dpi. N=3 of biologically independent mice examined over at least 2 independent experiments. E. Frequencies of IgM+ and IgG+ B cells in lungs of untreated or dexamethasone treated mice at 28dpi. Mice were treated with dexamethasone on days 7,8,9 post-infection. N=3 biologically independent mice examined over at least 2 independent experiments. Unpaired t-test, two-tailed. F. Viral RNA in the lung homogenates of dexamethasone treated or control untreated mice at 28dpi. Mann-Whitney, two-tailed test. N=5 biologically independent mice examined over 3 independent experiments. G. Schematic of experimental design of SARS-CoV-2 infected MISTRG6-hACE2 mice either treated with dexamethasone on days 3,4,5 dpi or left untreated. H. CD206 and CD68 expression in lung human immune cells in mice treated with dexamethasone or left untreated at 7dpi. CD206hi+ CD68+ cells are alveolar macrophages. Treated N=4, untreated N=6 biologically independent mice examined over 2 independent experiments. Alveolar macrophages are marked by high CD206 expression. Inflammatory macrophages are enriched in the CD206 negative population which also express CD86 (not shown).

Figure 1.. MISTRG6 humanized mice that transiently express hACE2 can be infected with SARS-CoV-2.

A. Schematic of experimental design. MISTRG6 mice were neonatally reconstituted with human CD34+ cells. After confirmation of human immune cell humanization in circulation, reconstituted MISTRG6 were injected AAV-hACE2 (10¹¹ genomic copies/ml) intratracheally (MISTRG6-hACE2). Following a two-week acclimation and recovery period, MISTRG6-hACE2 mice were infected intranasally with SARS-CoV-2 (10⁶ PFU). B. Viral RNA (quantification of N gene) C. Viral titers measured by PFU in homogenized lung tissue at 2, 4, 7, 14, 28 days post-infection (dpi) in B6 control or reconstituted or unengrafted MISTRG6 mice expressing or lacking human ACE2 (using standard Vero E6 cells). Group 1: n=4(2dpi), 3(4dpi), 3(7dpi), 3(14dpi), 3(28dpi) biologically independent mice examined over 2 independent experiments. Group 2: n=4(2–28dpi) biologically independent mice examined over 2 independent experiments. Group 3: n=4(2dpi), 4(4dpi), 7(7dpi), 4(14dpi), 2(28dpi) biologically independent mice examined over 3 independent experiments. Group 4: n=(2dpi), 26(4dpi), 6(7dpi), 8(14dpi), 8(28dpi) biologically independent mice examined over at least 4 independent experiments. Group 5: n=3(2dpi), 11(4dpi), 3(7dpi), 3(14dpi), 2(28dpi) biologically independent mice examined over at least 2 independent experiments; Significance in viral RNA was determined by Mann-Whitney, two-tailed test. One-sample t and Wilcoxon test was utilized for comparison of viral titers. Individual values for each mouse and means are presented. D. Weight change during the course of infection plotted as percent change compared with original weight measured just before inoculation with SARS-CoV-2. Group A-D: N=7, Group E: N= 8 biologically independent mice examined over at least 3 independent experiments for 28 days. Means with SD experiments are presented. Ordinary one-way ANOVA compared to the mean of uninfected reconstituted MISTRG6 mice (Group A) with Dunnett’s multiple comparison test was utilized. Group E p-value <0.0001; Group D p-value= 0.2889; Group C p-value= 0.8926, Group B p-value=0.9974. Means with SD are plotted. Individual values for Group E are presented in Fig S1C. E. Representative images of H&E staining (2x and 10x magnification) and box and whisker plot (min to max) of the histopathological scores and percent area affected of infected (2, 4, 7, 14, 28 and 35dpi) or uninfected lungs. The whiskers go down to the smallest value (min) and up to the largest (max). The box extends from the 25th to 75th percentiles. The median is shown as a line in the center of the box. Uninfected n=9, 2dpi n=5, 4dpi n=14, 7dpi n=6, 14dpi n=11, 28dpi n=10, 35dpi n=6 biologically independent mice examined over at least 3 independent experiments. Ordinary one-way ANOVA compared to uninfected lungs was utilized. P value was adjusted by Šídák’s multiple comparisons test. F. Trichrome staining of infected (2, 4, 7, 14, 28 and 35dpi) or uninfected lungs. Representative images (40X) and box and whisker plot (min to max) of the histopathological scores are presented. Arrows indicate areas with Collagen deposition. The extend of fibrosis was determined by the thickness of collagen bundles. The whiskers go down to the smallest value (min) and up to the largest (max). The box extends from the 25th to 75th percentiles. The median is shown as a line in the center of the box. Uninfected n=7, 2dpi n=3, 4dpi n=4, 14dpi n=12, 28dpi n=8, 35dpi n=4 biologically independent mice examined over at least 3 independent experiments. Ordinary one-way ANOVA compared to uninfected lungs was utilized. P value was adjusted by Šídák’s multiple comparisons test. Representative images of all time points are presented as part of Fig S1F.

Figure 2.. Immune landscape in MISTRG6-hACE2 mice infected with SARS-CoV-2 is characterized by inflammatory macrophages and monocytes.

A. Humanization measured by ratio of human CD45+ (human immune cells) cells in total CD45+ cells (mouse and human CD45+ combined) in blood, lungs, and BAL of uninfected and infected MISTRG6-hACE2 mice. Uninfected n=8, 2dpi n=4, 4dpi n=7, 7dpi n=4, 14dpi n=9, 28dpi n=4 biologically independent mice examined over at least 3 independent experiments. Ordinary one-way ANOVA compared to uninfected lungs was utilized. P value was adjusted with Dunnett’s multiple comparison test. Distribution of individual data points is displayed in a violin plot. B. Human immune cell numbers in lungs and BAL of uninfected and infected mice at 2,4,7,14,28 dpi. Lung: Uninfected n=11, 2dpi n=6, 4dpi n=7, 7dpi n=4, 14dpi n=8, 28dpi n=5 over at least 3 independent experiments. BAL: Uninfected n=4, 2dpi n=5, 4dpi n=6, 7dpi n=4, 14dpi n=7, 28dpi n=4 over at least 3 independent experiments. Unpaired, two-tailed t-test. P values <0.05 are plotted. Individual values for each mouse and means are presented. C. Human immune lineages lungs and BAL of uninfected and infected mice at 2,4,7,14, 28 dpi within the human CD45+ population. Classical monocytes (CD14+), Intermediate monocytes (CD14+CD16+), non-classical monocytes (CD16+CD14-), macrophages (CD68+), NK cells (NKP46+), T cells (CD3+), B cells (CD19+ and/or CD20+). Statistical significance was deemed by ordinary one-way ANOVA compared to uninfected lungs. P values were adjusted with Dunnett’s multiple comparison test. P values represented by: ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P ≤ 0.0001. Lung: Uninfected n=8, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=9, 28dpi n=5 biologically independent mice examined over at least 3 independent experiments. BAL: Uninfected n=4, 2dpi n=4, 4dpi n=6, 7dpi n=3, 14dpi n=6, 28dpi n=3 biologically independent mice examined over at least 3 independent experiments. P values <0.05 are provided: Lung: CD14+CD16+ monocytes 4dpi=0.0003, 7dpi =0.030; CD16+ monocytes 4dpi<0.0001; Macrophages 14dpi= 0.0114. BAL: T cells 2dpi<0.0001, 7dpi=0.0003, 14dpi=0.003, 28dpi=0.001; Means with SD are plotted. D. Number of human macrophages in lungs of uninfected and infected mice at 2, 4, 7, 14, 28 dpi. Unpaired, two-tailed t-test. Lung: Uninfected n=8, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=9, 28dpi n=6 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are presented. E. Frequency and number of human alveolar macrophages marked by CD206hi, CD86+, CD169+ expression within the hCD45+CD68+ population in the lungs of uninfected and infected mice at 2, 4, 7, 14, 28 dpi. N=3–10. Unpaired, two-tailed t-test. Uninfected n=8, 2dpi n=4, 4dpi n=4–6, 7dpi n=3, 14dpi n=7, 28dpi n=6 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are presented. F. Frequency of inflammatory human lung macrophages marked by CD206-/lo, CD86hi macrophages within the hCD45+CD68+ population in the lungs of uninfected and infected mice at 2,4,7,14,28 dpi. Unpaired, two-tailed t-test. P values<0.05 plotted. Uninfected n=4, 2dpi n=4, 4dpi n=6, 7dpi n=4, 14dpi n=7, 28dpi n=4 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. G. Frequency of CD16+ human lung macrophages marked by CD16+ cells within the hCD45+CD68+ population in the lungs of uninfected and infected mice at 2,4,7,14,28 dpi. N=4–8. Unpaired, two-tailed t-test. P values<0.05 plotted. Uninfected n=6, 2dpi n=4, 4dpi n=8, 7dpi n=3, 14dpi n=7, 28dpi n=6 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. Unpaired t-test, two-tailed. P values <0.05 are plotted. H. Frequency and number of human plasmacytoid dendritic cells (pDCs) marked by CD123+CD11b-CD11c-/lo cells within hCD45+ population in the lungs of uninfected and infected mice at 2,4,7,14,28 dpi. Unpaired t-test, two-tailed. Uninfected n=3, 2dpi n=2, 4dpi n=2, 7dpi n=3, 14dpi n=7, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments. Individual values for each mouse and means are presented. I. Frequencies of activated, mature (CD83+ CCR7+) pDCs in infected lungs at 14dpi or uninfected lungs. Uninfected n=4, 14dpi n=5 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. Unpaired t-test, two-tailed. P values <0.05 are plotted.

Figure 3.. Adaptive immune responses in MISTRG6-hACE2 mice infected with SARS-CoV-2 are characterized by virus specific, activated T cells, IgG+ B cells and systemic lymphopenia.

A. Frequencies of human CD3+ T cells within human CD45+ population in the blood pre- and post-infection (2,4,7,14,28dpi). Lines connect pre- and post-infection values for the same mouse. Paired, two-tailed t-test. 2dpi n=4, 4dpi n=7, 7dpi n=4, 14dpi n=6, 28dpi n=4 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. P-values<0.05 are plotted. B. Frequencies of human CD3+ T cells within human CD45+ population in the spleens of uninfected and infected mice at 2,4,7,14,28 dpi. Uninfected n=8, 2dpi n=4, 4dpi n=14, 7dpi n=4, 14dpi n=6, 28dpi n=4 biologically independent mice examined over at least 3 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. C. Representative flow cytometry plots of HLA-DR and CXCR3 expression on human lung T cells and frequencies of HLA-DR+CXCR3+ lung T cells in uninfected and infected mice at 2,4,7,14,28dpi. Uninfected n=7, 2dpi n=4, 4dpi n=8, 7dpi n=4, 14dpi n=9, 28dpi n=6 biologically independent mice examined over at least 3 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. D. Representative flow cytometry plots of ICOS and PD1 expression on human lung T cells and frequencies Icos+ PD1+ or PD1+ T cells in uninfected and infected mice (4,7,14,28dpi). N=4 biologically independent mice examined over at least 3 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. E. Frequencies of TCRgamma/delta T cells (Tγδ) among human lung T cells in uninfected and infected mice (4,14,28dpi). Uninfected n=3, 4dpi n=5, 14dpi n=5, 28dpi n=4 biologically independent mice examined over at least 3 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are plotted. P-values<0.05 are plotted. F. Frequencies of anti-viral CD8+ T cells in uninfected and infected mice at 2,4,7,14,28dpi. Human CD8+ T cells from mice that had been engrafted with an HLA-A2 positive source of progenitor cells were stained with viral antigen (peptides of M and S viral proteins)-bearing HLA-A2 tetramers. Uninfected n=3, 2dpi n=2, 4dpi n=3, 7dpi n=3, 14dpi n=3, 28dpi n=3 biologically independent mice examined over 2 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. G. Frequencies and numbers of human B cells within hCD45+ population in the lungs of uninfected or infected mice at 2,4,7,14,28 dpi. Uninfected n=8, 2dpi n=4, 4dpi n=8, 7dpi n=4, 14dpi n=4, 28dpi n=4 biologically independent mice examined over at least 2 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. H. Representative flow cytometry plots and frequencies of IgM+ B cells in the lungs of uninfected and infected mice 2,4,7,14,28dpi. N=3–8. Unpaired, two-tailed t-test. P-values<0.05 are plotted. Uninfected n=3, 2dpi n=2, 4dpi n=4, 7dpi n=4, 14dpi n=6, 28dpi n=8 biologically independent mice examined over at least 2 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. I. Representative flow cytometry plots and frequencies of IgG+ B cells in the lungs of uninfected and infected mice 7,14,28dpi. N=2–8. Unpaired, two-tailed t-test. P-values<0.05 are plotted. Uninfected n=2, 7dpi n=2, 14dpi n=6, 28dpi n=8 biologically independent mice examined over at least 2 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are presented. P-values<0.05 are plotted. J. Representative plot of CD11c expression on CD19+ B cells from spleen and lungs of infected mice at 28dpi. N=5. Representative of n=6 for at least 3 independent experiments. All attempts resulted with similar findings.

Figure 4.. SARS-CoV-2 lungs sustain ISGs and display SLE like features

A. Heatmap of differentially regulated human and mouse genes (combined list of genes Log2, FoldChange >1 in each infected time point vs uninfected lungs; adjusted p value<0.05; mean normalized count>5). Transformed normalized counts in lungs of uninfected or infected MISTRG6-hACE2 plotted over the course of infection were clustered using Spearman Correlation. Row min and max of transformed values, calculated by subtracting row mean and dividing by STD for each gene across all samples, are visualized. For the adjusted P values the Bonferroni correction was used. Human genes: Uninfected n=5, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=2, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points. Mouse genes: Uninfected n=5, 2dpi n=4, 4dpi n=9, 7dpi n=3, 14dpi n=4, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points. Whole tissue lung samples with less than 1% human reads were excluded from analysis (pre-established criterium). B. t-distributed stochastic neighbor embedding (t-SNE) plot with clustering results of single cell RNA sequencing of human immune cells using the 10X Genomics platform from lungs at 4dpi. Single cell suspensions from whole infected lung at 4dpi were processed and sequenced. There were 421 cells identified as human immune cells. C. Dot plot of cluster identifying genes for human immune cell clusters described in Fig. 4B. D. Cluster distribution and expression profile of human inflammatory cytokines for clusters described in Fig. 4B–C. E. Distribution of ISGs within human and mouse DEGs. F. Normalized expression of human IFNA1 and IFNA6 mRNA measured by qPCR in homogenized lung tissue of uninfected and infected (2, 4, 7, 14, 28dpi) mice. Expression was normalized to human HPRT1. Unpaired t-test, one-tailed since the distribution is expected to be one-sided. Uninfected n=6, 4dpi n=4, 7dpi n=3, 14dpi n=9, 28dpi n=3 biologically independent mice examined over at least 3 independent experiments. Individual values for each mouse and means are presented. P-values<0.05 are plotted. G. Normalized counts for inflammatory cytokines implicated in COVID-19 patients. Counts were reported separately for human(red) and mouse(blue) cytokine genes. Based on whole tissue transcriptome data presented in Fig. 4A. Human genes: Uninfected n=5, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=2, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points. Mouse genes: Uninfected n=5, 2dpi n=4, 4dpi n=9, 7dpi n=3, 14dpi n=4, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points. H. Heatmap of genes that are implicated in SLE-like B cells based on GSE10325 in infected lungs of MISTRG6 mice at 2,4,7.14.28 dpi. Row min and max of transformed values, calculated by subtracting row mean and diving by STD for each gene across all samples, are visualized. Uninfected n=5, 2dpi n=4, 4dpi n=7, 7dpi n=3, 14dpi n=2, 28dpi n=2 biologically independent mice examined over at least 2 independent experiments for all time points.

Figure 5.. Human monoclonal recombinant antibodies as prophylactic and therapeutic interventions impact disease outcome.

A. Viral titers measured by PFU in homogenized lung tissue at 4 dpi in MISTRG6-hACE2 mice that received prophylactic treatment of convalescent patient plasma or left untreated. Paired, two-tailed t-test. Untreated controls: n=6, convalescent treated group n=4 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. B. Human immune cells at 4 dpi in lungs of MISTRG6-hACE2 mice that received prophylactic treatment of convalescent patient serum or left untreated. Paired, two-tailed t-test. Untreated controls: n=6, convalescent treated group n=4 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. C. Human macrophages (hCD45+ hCD68+) at 4 dpi in lungs of MISTRG6-hACE2 mice that received prophylactic treatment of convalescent patient serum or left untreated. Paired, two-tailed t-test. Untreated controls: n=6, convalescent treated group n=4 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. D. Viral RNA and viral titers measured by PFU in homogenized lung tissue at 4 dpi in MISTRG6-hACE2 mice that received prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). N=6. Mann-Whitney, two-tailed test was used for comparison of viral RNA. One-sample Wilcoxon signed rank test was used to determine significance in the viral titer quantification (effect size=0.9, W=21). E. Human immune cells in lungs of MISTRG6-hACE2 mice received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). N=6 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. Unpaired t-test, two-tailed. F. Human immune cells in BAL of MISTRG6-hACE2 mice received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Untreated control n=4, treated group n=5 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. Unpaired t-test, two-tailed. G. Human immune lineages lungs and BAL of mAb treated or untreated mice at 4 dpi within the human CD45+ population. Classical monocytes (CD14+), Intermediate monocytes (CD14+CD16), non-classical monocytes (CD16+CD14-), macrophages (CD68+), NK cells (NKP46+), T cells (CD3+), B cells (CD19+ and/or CD20+). MISTRG6-hACE2 mice received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). In lungs, N=6 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. In BAL, Untreated control N=4, treated group n=5 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. Unpaired t-test, two-tailed. Statistical significance was deemed by comparison to uninfected group. P values represented by: ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P ≤ 0.0001. Only changes in frequencies of lung macrophages (m135 p value=0.029, m144 p value =0.037) and BAL macrophages (m144 p value =0.0042) and monocytes (CD16+ (m135 p value=0.0051, m144 p value=0.0058) were statistically significant. H. Human macrophages (hCD45+ hCD68+) at 4 dpi in lungs and BAL of MISTRG6-hACE2 mice that received prophylactic treatment of mAbs (clone 135 or 144) or left untreated. Lungs: Untreated control N=6, treated group n=5 biologically independent mice examined over 3 independent experiments. BAL: Untreated control N=5, treated group n=4 biologically independent mice examined over 2 independent experiments. Individual values for each mouse and means are presented. Unpaired t-test, two-tailed. I. Weight change in mAb treated mice (prophylaxis) at 2 and 4dpi plotted as percent change compared with original weight measured just before inoculation with SARS-CoV-2. N=6 biologically independent mice examined over 2 independent experiments. RM-one-way ANOVA with Dunnett’s multiple comparison test comparing weight change at 4dpi to untreated group was utilized. P-value for m144=0.01, p-value for m135=0.98. Individual values for each mouse and means are presented. J. Viral RNA and viral titers measured by PFU in homogenized lung tissue at 4 dpi in MISTRG6-hACE2 mice that received post-infection treatment of a mixed cocktail of monoclonal antibodies clone 135 (m135) and clone 144(m144) or left untreated (untd). Early treatment groups were treated 11 hours post-infection and late treatment 35 hours post-infection. Mann-Whitney, two-tailed test was used for comparison of viral RNA. One-sample Wilcoxon signed rank test was used to determine significance in the viral titer quantification (effect size=0.9, W=21). Untreated control N=6, early and late treated groups n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. K. Human immune cells in lungs of MISTRG6-hACE2 mice that received early, late or no treatment of monoclonal antibody mix. Untreated control N=6, early and late treated groups n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. Unpaired, two-tailed t-test. P-values<0.05 are plotted. L. Human immune cells in BAL of MISTRG6-hACE2 mice that received early, late or no treatment of monoclonal antibody mix. Untreated control N=4, early and late treated groups n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. Unpaired, two-tailed t-test. P-values<0.05 are plotted. Untreated control N=6, early and late treated groups n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. M. Weight change upon mAb therapeutic treatment at days and days post-infection plotted as percent change compared with original weight measured just before inoculation with SARS-Cov-2. N=6 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. One-way ANOVA (Šídák’s multiple comparisons test) comparing weight change at 4dpi to untreated group was utilized. Early treatment vs untreated p value=0.8 and late treatment vs untreated p value=0.49. N. Human immune lineages lungs and BAL of mAb treated or untreated mice at 4 dpi within the human CD45+ population. Classical monocytes (CD14+), Intermediate monocytes (CD14+CD16), non-classical monocytes (CD16+CD14-), macrophages (CD68+), NK cells (NKP46+), T cells (CD3+), B cells (CD19+ and/or CD20+). MISTRG6-hACE2 mice received a prophylactic treatment of monoclonal antibody clone 135 (m135) or clone 144(m144) 8 hours prior to infection or left untreated (untd). Lung: untreated control N=6, early and late treated groups n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. BAL: Untreated control N=4, early and late treated groups n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are presented. Statistical significance was deemed by unpaired t-test compared to uninfected group. P values represented by: ns P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P ≤ 0.0001. Only changes in frequencies of monocytes (CD14+CD16+) were statistically significant (p value=0.01). Means with SD are plotted. In Figure 5, MISTRG6 mice were engrafted with CD34+ cells neonatally isolated from at least 2 donors. Pooled or infection matched representative results of at least 2 independent experiments are presented. Only P-values <0.05 are shown. Mean with SD or individual values are plotted.

Figure 6.. Accurate timing of corticosteroids is necessary to balance viral clearance and prevent immunopathology.

A. Weight change in dexamethasone treated or control mice during SARS-CoV-2 infection plotted as percent change compared with original weight prior to viral inoculation. Mice were treated with dexamethasone at 7,8,9 dpi. Untreated n=8, treated n=6 biologically independent mice examined over 3 independent experiments. Unpaired, two-tailed t-test determined comparing 14dpi values (p=0.0041). Means with SD are plotted. B. Human immune cells in 14dpi lungs and BAL of MISTRG6-hACE2 mice treated with dexamethasone at 7, 8 9 dpi or left untreated. Untreated n=8, treated n=6 biologically independent mice examined over 3 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are plotted. C. Human immune lineages lungs of dexamethasone treated or untreated mice within the human CD45+ population. Classical monocytes (CD14+), Intermediate monocytes (CD14+CD16), non-classical monocytes (CD16+CD14-), macrophages (CD68+), NK cells (NKP46+), T cells (CD3+), B cells (CD19+ and/or CD20+). MISTRG6-hACE2 mice were treated with dexamethasone at 7,8,9dpi. N=4–6. Unpaired, two-tailed t-test. Untreated n=6, treated n=5 biologically independent mice examined over 3 independent experiments. Means with SD are plotted. D. Number of human macrophages in lungs of dexamethasone treated and untreated mice at 14dpi. N=6. Unpaired, two-tailed t-test. Untreated n=8, treated n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are plotted. E. Frequencies of alveolar or inflammatory macrophages in the lungs of MISTRG6-hACE2 mice that were treated with dexamethasone or left untreated. N=6. Unpaired, two-tailed t-test. Untreated n=7, treated n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are plotted. F. Numbers of alveolar or inflammatory macrophages in the lungs of MISTRG6-hACE2 mice that were treated with dexamethasone or left untreated. N=6. Unpaired, two-tailed t-test. Untreated n=6, treated n=5 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are plotted. G. Frequencies (left) and numbers (right) of pDCs at 14dpi in the lungs of dexamethasone treated or control mice. N=6. Unpaired, two-tailed t-test. Untreated n=5, treated n=4 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are plotted. H. Representative histograms for HLA-DR expression in lung T cells at 14dpi in dexamethasone treated or control mice. N=6. Unpaired, two-tailed t-test. Representative of n=4 for examined over 2 independent experiments. All attempts resulted with similar findings. I. Frequencies of HLA-DR+ or ICOS+ T cells at 14dpi in the lungs and BAL of dexamethasone treated or control mice. N=4–6. Unpaired, two-tailed t-test. Lung: untreated n=5, treated n=4 biologically independent mice examined over 3 independent experiments. BAL: untreated n=4, treated n=3 biologically independent mice examined over 3 independent experiments. Individual values for each mouse and means are plotted. J. Weight change in dexamethasone treated or control mice during SARS-CoV-2 infection plotted as percent change compared with original weight prior to viral inoculation. Mice were treated with dexamethasone at 3,4,5dpi. Untreated n=6, treated n=4 biologically independent mice examined over 2 independent experiments. Unpaired, two-tailed t-test. Means with SD are plotted. K. Human immune cells in lungs of MISTRG6-hACE2 mice treated with dexamethasone at 3, 4 5 dpi or left untreated (ctrl). N= 4 biologically independent mice examined over 2 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are plotted L. Viral RNA and viral titers measured by PFU in homogenized lung tissue at 7 dpi in dexamethasone treated or control mice. N= 4 biologically independent mice examined over 2 independent experiments. Unpaired, two-tailed t-test. Individual values for each mouse and means are plotted. Mann-Whitney test, two-tailed. MISTRG6 mice were engrafted with CD34+ cells neonatally isolated from at least 2 donors. Pooled or infection matched representative results of at least 2 independent experiments are presented. Only P-values <0.05 are plotted. Mean with SD or individual values are plotted.