key: cord-0953010-gbfkn2te authors: Yang, Chen Xi; Tomchaney, Michael; Landecho, Manuel F.; Zamacona, Borja R.; Oto, Marta Marin; Zulueta, Javier; Malo, Joshua; Knoper, Steve; Contoli, Marco; Papi, Alberto; Vasilescu, Dragoş M.; Sauler, Maor; Straub, Christof; Tan, Cheryl; Martinez, Fernando D.; Bhattacharya, Deepta; Rosas, Ivan O.; Kheradmand, Farrah; Hackett, Tillie-Louise; Polverino, Francesca title: Lung spatial profiling reveals a T cell signature in COPD patients with fatal SARS-CoV-2 infection date: 2022-04-21 journal: bioRxiv DOI: 10.1101/2022.04.20.488968 sha: 20ca3017aee3dc0b2cff7631083773e53f652052 doc_id: 953010 cord_uid: gbfkn2te Rationale People with pre-existing lung diseases like chronic obstructive pulmonary disease (COPD) are more likely to get very sick from SARS-CoV-2 disease 2019 (COVID-19), but an interrogation of the immune response to COVID-19 infection, spatial throughout the lung structure is lacking in patients with COPD. Objectives To profile the immune microenvironment of lung parenchyma, airways, and vessels of never- and ever-smokers with or without COPD, whom all died of COVID-19, using spatial transcriptomic and proteomic profiling. Findings The parenchyma, airways, and vessels of COPD patients, compared to control lungs had: 1) significant enrichment for lung resident CD45RO+ memory T cells; 2) downregulation of genes associated with T cell antigen-priming and memory T cell differentiation; 3) higher expression of proteins associated with SARS-CoV-2 entry and major receptor ubiquitously across the ROIs and in particular the lung parenchyma, despite similar SARS-CoV-2 structural gene expression levels. Conclusions The lung parenchyma, airways, and vessels of COPD patients have increased T-lymphocytes with a blunted memory T cell response and a more invasive SARS-CoV-2 infection pattern, and may underlie the higher death toll observed with COVID-19. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 pandemic, which to date has infected over 175 million people and caused over six million deaths 1 . The fatality rate for Coronavirus Disease 2019 (COVID-19) is estimated at 0.5 -1%, with most people dying from respiratory failure related to diffuse acute lung injury and acute respiratory distress syndrome (ARDS) associated with ineffective viral clearance, especially in the elderly individuals 2 . There is much debate whether smoking or other comorbidities such as chronic obstructive pulmonary disease (COPD) increase susceptibility to ARDS from COVID-19. Several studies have shown a wide range in the prevalence (1.1 -38%) of COPD patients becoming infected with COVID19 3, 4 . COPD patients are more likely to be hospitalized, admitted to the intensive care unit (ICU) and receive mechanical ventilation, versus non-COPD COVID-19 patients 4 . Additionally, COPD is associated with increased susceptibility to respiratory virus infections, which are one of the most common causes of acute exacerbations of COPD 5 . In smokers with COPD, there is well-documented bystander activation of the T lymphocytes [6] [7] [8] . Interestingly, severe COVID-19 disease has been associated with low avidity T lymphocyte responses to SARS-CoV-2 9 . However, there is great uncertainty about whether the timing, composition, or magnitude of the innate and adaptive immune responses to SARS-CoV-2 are protective or pathogenic 10, 11 , and how cigarette smoke and COPD shape the immune responses during SARS-CoV-2 infection. An understanding of the adaptive immune responses to SARS-CoV2 in smokers with and without COPD is therefore essential to establish treatment strategies. The goal of this study was to spatially profile the transcriptome and proteome of innate and adaptive immune cell responses within the lung parenchyma, airways and vessels of COPD patients compared to never-and ever-smokers who died of COVID-19, to develop a biological landscape of the lung immune response from a structural, immunological and clinical standpoint. Additional information is in the Online Supplement. The study included seven never-smokers and eleven ever-smokers with and without COPD who died from COVID-19 pneumonia following SARS-CoV2 infection at the University of Navarra Hospital, Pamplona, Spain ( Table 1) . All patients ceased smoking ≥1 year prior to hospitalization. The study conformed to the Declaration of Helsinki and was approved by the University of Arizona ethics committee (IRB #1811124026). COPD patients had a prior physician diagnosis. Computed tomography (CT) scans were analyzed for tissue volume and emphysema (% lower attenuation areas [LAA] <-950 Hounsfield units [HU]), using Vida Vision software (Version 2.20, Iowa, USA). For each subject, the nucleocapsid (N) and envelope (E) SARS-CoV-2 gene expression levels were quantified in nasal swabs and blood using rt-PCR. In order to assess whether the up-down-regulation of proteins in COPD patients with COVID-19 was associated with SARS-CoV-2 infection or with the presence of underlying COPD, a second cohort of uninfected never-smokers (n= 7) and ever-smokers with or without COPD (n =11) was studied, who underwent thoracic surgery at the University of Arizona 6 before the SARS-CoV-2 pandemic started (Supplemental Table E1 ). In patients who died of COVID-19, lung biopsies were obtained through a 5 cm incision between the 3 rd and 5 th intercostal space between the midclavicular and midaxillary lines at the time of death, and samples formalin-fixed and paraffin-embedded (FFPE). Lung sections were prepared for use with the GeoMX digital spatial profiler (DSP), and the GeoMX Transcriptome and COVID-19 Immune Response panels, according to the manufacturer's instructions (Nanostring, WA, USA). For each lung section, we randomly selected 16 total regions of interest (ROI) from the parenchyma, airways and vessels, uniformly spread throughout the section to ensure sample randomization (Figure 2A) . Each ROI was subdivided into compartments based on fluorescent cell-specific markers, and serial UV illumination of each compartment was used to sequentially collect the probe barcodes from the different cell types as described elsewhere 3 . Once all of the indexing oligonucleotides were collected into a 96 well plate they were counted using nextgeneration sequencing. For each ROI, the indexing oligonucleotides were counted using Illumina's i5xi7 dual-indexing system, and cDNA libraries pair-end sequenced. Consecutive lung sections were incubated with a GeoMX DSP 41 oligo-labelled antibody panel (See Supplemental Table E2 ), to spatially profile proteins in the same ROIs. In order to validate the main spatial proteomic findings, double immunofluorescence staining for ACE2 or CD45RO, with epithelial cell adhesion molecule (EPCAM) or endothelial cell marker (von Willebrand Factor), was performed on consecutive lung sections from the COVID-19 cohort. The number of positive cells were counted by two blinded observers and normalized using Metamorph software. The demographic patient data were assessed using a Kruskal-Wallis H test for continuous variables and a Fisher's exact test for categorical variables. The days of survival from day one of COVID-10 symptoms and day of death in hospital was compared pair-wisely using a Mann-Whitney U test. Differential protein and RNA expression analysis were performed to compare disease conditions (never-, ever-smokers, or COPD) tissue structures (parenchyma, airways, or vessels), using generalized estimating equations (GEE) to account for multiple ROIs per subject, and adjusted for age using R (version 3.5.0). Multiple hypothesis testing in all the analyses was controlled using the Benjamini-Hochberg procedure. A false discovery rate (FDR) of <0.05 was used as the significance threshold. The cellular proportions of the immune cell types between patient groups were inferred using the R package "SpatialDecon". "SpatialDecon" is a referencebased deconvolution method that is designed specifically for the NanoString GeoMx platform. By using log-normal regression and modelling background noise, "SpatialDecon" outperformed classical least-squares methods. "SpatialDecon" provides pre-defined cell profile matrices generated using published single-cell RNA-seq for 75 tissue types. In our study, we performed the deconvolution analysis using the "Lung plus neutrophil" panel. Immunohistochemical staining was assessed using Kruskal-Wallis and Mann Whitney tests, a P<0.05 was considered significant. The COVID-19 cohort demographics are reported in Table 1 . There were no differences in age or sex between the never-and ever-smoker controls and COPD patient groups, however, due to the large variation in ages of the COPD group, RNA and protein analyses were adjusted for age. COPD patients (purple) with SARS-CoV-2 infection died within a mean of 14.0 days following initial symptoms, compared to ever-smoker controls (green) within 17.3 days (p=0.78), and neversmokers (yellow) within 25.6 days (p=0.03) ( Figure 1A) . The gene expression analysis for SARS-CoV-2 Gene N (nucleocapsid) and Gene E (envelope) showed no significant differences in the viral load on hospital admission between the never-and ever-smoker controls and patients with COPD ( Figures 1B-C) . The high-resolution CT coronal and sagittal views of a representative never-smoker, ever-smoker, and patient with COPD, four days before death demonstrate the extensive patchy consolidation, extensive ground-glass opacities, interlobular septal thickening and interlobular pleural thickening in the lungs, consistent with changes found in ARDS ( Figure 1D ) 12 . The CT scans confirmed that the patients with physician-diagnosed COPD had a greater percentage of emphysema (%LAA950), compared to the never-and ever-smoker controls (Supplemental Figure E1A ). In the COVID-19 cohort, the lung tissue sections were stained with pan-cytokeratin (epithelial marker), CD45 (leukocyte marker), and SYTO13 (DNA marker) to enable sampling of parenchymal, airways, and vessels (Figure 2A) . We confirmed across the multiple ROIs sampled, that the parenchyma, airways, and vessels expressed specific gene signatures for each structure (FDR<0.05, Supplemental Figure E2A ). Of the total of 1839 genes, 1803 were reliably measured across the dynamic range of gene expression panels (Supplemental Figure E2B ). The parenchyma of COVID-19 COPD patients had greater expression of 246 genes compared to never-smokers, and 186 genes compared to ever-smoker controls (FDR<0.05, Notably, these included ACE2, TMPRSS2 which cleaves SARS-CoV-2 Spike protein to allow entry into host cells 13 , and ORF1ab encoding for the SARS-CoV-2 replicase 14 . Further, in the same comparison, we identified 71 and 92 down-regulated genes respectively (FDR<0.05), including IL2RA and HLA-DR, markers of antigen-specific activated T cells 15, 16 , and IRF-7 and IRF-9, main regulators of type-I interferon-dependent immune responses to virus 17, 18 . The airways of COVID-19 COPD patients had greater expression of 129 genes compared to never-smokers, and 58 genes compared to ever-smoker controls (FDR<0.05, Figures 2D and 2E ). These included ACE2 and ORF1ab. In the same comparison, we also identified downregulated expression of 40 and 49 genes, respectively (FDR<0.05). These included ID2, which allows the antigen-driven differentiation of memory T cells 19 , and SOD2, which clears mitochondrial reactive oxygen species 20 . The pulmonary blood vessels of COVID-19 COPD patients had greater expression of 78 genes compared to never-smokers, and 47 genes compared to ever-smoker controls (FDR<0.05, Notably, the same ACE2 and ORF1ab genes increased in the airways, were also upregulated in the pulmonary blood vessels. Additionally, in the same comparison, we identified down-regulated expression of 44 and 37 genes, respectively (FDR<0.05). These included DUSP-1, which is thought to lead to steroid resistance in SARS-CoV-2 infection 21 , and LAIR-2, an inhibitory immune receptor expressed by T cells 22 . There were less than 10 differentially expressed genes between never-and ever-smoker controls in the lung parenchyma, airways, and vessels (Supplemental Figure E2C ). We next identified the overlap of up-and down-regulated genes found in the parenchyma, airways, and vessels of COPD patients compared to ever-and never-smoker patients (Figure 2H -I). The alteration in gene expression in COPD lung tissue compared to never-and ever-smoker controls was not due to increased viral load, as the SARS-CoV-2 gene N and E expression (Supplemental Figures E3A and B) , was not different among the groups. In the COVID-19 cohort, we next performed GeoMX spatial proteomic profiling for the same ROI regions on a consecutive FFPE section to validate the transcriptomic findings. We confirmed that ACE2, one of the most upregulated genes, was also elevated at the protein level in the airways, and vessels of COPD patients compared to never-and ever-smoker controls ( Figure 3A ). Immunofluorescence staining of alveolar, and airway epithelial cells and endothelial cells further confirmed elevated ACE2 protein expression in the parenchyma ( Figure 3B ) and airways ( Figure 3C ) of COPD patients compared to never-and ever-smoker controls. Protein expression of ACE2 was also elevated in the vessels of ever-smoker compared to never-smoker controls ( Figure 3D ). In the COVID-19 cohort, gene ontology pathways identified immune pathways involved in antigen processing and presentation of endogenous peptide antigen (FDR<0.05) enriched in the differentially expressed genes between controls and COPD patients. Thus, in order to assess the immune cell composition within the parenchyma, airways, and vessels of COVID-19 never-and ever-smoker controls and COPD patients, a lung cell deconvolution analysis was used ( Figure 4A ). The cell deconvolution analysis identified an increased number of T lymphocytes in the lung parenchyma, airways, and vessels of COPD patients compared to never-and ever-smoker controls ( Figure 4B) . The T lymphocyte proportion within the lung was negatively correlated with the absolute T lymphocyte count in the blood of patients admitted for COVID-19 (Figure 4C) , suggesting increased T lymphocyte recruitment from the blood into the lung in response to SARS-CoV-2 infection with the highest recruitment being in COPD patients. To validate the cell deconvolution data, the GeoMX spatial proteomic profiling of T lymphocyte markers was performed in the COVID-19 cohort. Interestingly, of all the T lymphocyte protein markers measured, CD45RO was the only one upregulated in the COPD lung tissue structures compared to never-and ever-smoker controls (Supplemental Figure E4A ). Most naïve human T lymphocytes express CD45RA, whereas CD45RO is a CD45 isoform specifically expressed on memory T lymphocytes. Spatial profiling of CD45RO protein revealed that COPD patients had increased expression of CD45RO in the parenchyma, airways, and vessels compared to both ever-and never-smoker controls ( Figure 5A) . Similarly, CD45RO protein expression was also elevated in the parenchyma, airways, and vessels of ever-smokers compared to neversmokers, indicating a possible effect of smoking on the lung tissue that persists in former smokers ( Figure 5A) . Further, we found a positive correlation between CD45RO and ACE2 protein in the parenchyma (Rho= 0.359, p<0.01), airways (Rho= 0.549, p<0.001), and vessels, (Rho= 0.473, p<0.001) (Supplemental Figure E4B) . We next validated our findings using immunofluorescence staining of alveolar and airway epithelial cells and endothelial cells, which confirmed elevated CD45RO protein expression in the parenchyma (Figure 5B) , airways ( Figure 5C ) and vessels (Figure 5D ) of COVID-19 COPD patients and ever-smokers, compared to never-smokers. To determine if the changes observed in COPD patients who died of COVID-19 were caused by SARS-CoV-2 infection or to the underlying inflammation in COPD, the same protein panels were assessed in a second cohort of lung tissue collected prior to the pandemic from neverand ever-smokers with and without COPD (see Supplemental Table 1 for demographics) . Surprisingly, CD45RO expression was down-regulated in the parenchyma, airways and vessels of non-COVID-19 COPD patients compared to never-smokers and the airways of ever-smoker controls (supplemental Figure E5) . These data suggest that the presence of both SARS-CoV-2 infection and pre-existing COPD underlie the increase in memory T cells observed in the lungs of COPD patients who died of COVID-19. We report to our knowledge, the first spatial transcriptomic and proteomic profiling in the lung parenchyma, airways, and vessels of fatal COVID-19 patients with COPD versus never-smoker and ever-smoker controls. These data indicate that COPD patients have lower survival despite similar levels of lung and blood SARS-CoV-2 viral load, which is associated with an upregulated inflammatory response involving increased numbers of memory T lymphocytes, but downregulation of genes associated with antigen-dependent T lymphocytes differentiation. These Increased numbers of CD4+ and CD8+ T lymphocytes have been detected in patients who have recovered from SARS-CoV-2 infection who had diffuse lung disease 10 . In particular, lung resident memory T lymphocytes positioned within the respiratory tract are required to limit SARS-CoV-2 spread and COVID-19 severity 26 . Tissue-resident memory T lymphocytes responses affect the susceptibility to and the pathogenesis of SARS-CoV-2 infection by secreting pro-inflammatory interferon (IFN) γ and anti-inflammatory interleukin (IL)-10 molecules 27 . We observed an increased memory T lymphocytes-immune infiltration within the lungs of COPD patients, compared with ever-and never-smoker controls who all died of COVID-19, which was associated with an upregulation of crucial genes associated with SARS-CoV2 entry and infection (e.g., ACE2, TMPRSS, and ORF1ab). Interestingly, the increase in the memory T lymphocyte population, confirmed both by spatial proteomics data and immunohistochemical staining, was accompanied by the downregulated expression of genes associated with memory function and antigen-driven maturation. To confirm that our findings were associated with SARS-CoV-2 infection and not the pre-existing immune milieu associated with COPD itself, CD45RO staining was performed on a separate cohort of never-smokers, ex-smokers, and patients with COPD that underwent lung resection surgery prior to the COVID-19 pandemic. In this tissue cohort, in the absence of SARS-CoV-2 infection, the number of memory T lymphocytes in COPD parenchymal, airways and vessels were reduced, compared to ever-and never-smoker controls. These data together indicate that the increase in memory T lymphocytes observed in the COVID-19 COPD patients is specific to SARS-CoV-2 infection. Lung resident memory T lymphocytes in host immunity are crucial for mucosal responses against pathogens including SARS-CoV-2 (e.g., in airways and interstitial tissue), and limit reinfections locally 26 . In stable COPD, the T lymphocyte responses may be impaired 7,28,29 , thus explaining the lower levels of memory T lymphocytes in uninfected COPD patients. However, during a viral challenge such as SARS-CoV-2 infection, the memory T lymphocytes, although upregulated as suggested by elevated CD45RO, may be off-targeted in COPD, thus explaining the downregulation of key genes involved in memory T lymphocyte functioning and antigen priming observed in COVID-19 COPD patients. Our findings are in line with recent reports indicating that SARS-CoV-2 and other beta coronaviruses induce multi-specific and long-lasting T lymphocyte immunity responses with strong upregulation of memory T lymphocytes against the viral structural proteins 27 . However, it is important to note that the T lymphocyte immune profile in COPD lungs was different from the one in never-and ever-smoker controls. Specifically, we found increased numbers of memory T lymphocytes, but reduced expression of key genes involved in antigenspecific T lymphocyte priming and regulation of type-I interferon-dependent immune responses to the virus, suggesting off-targeted lung resident memory T lymphocytes responses to SARS-CoV-2 in COPD. Upregulation of the memory T lymphocytes was associated with upregulation of ACE2 protein levels measured by spatial protein profiling and immunostaining. Although the upregulation of ACE2 has been reported in COPD lungs by several studies, to the best of our knowledge, this is the first study that investigated the levels of ACE2 in the lung parenchyma, airways and vessels of ever-and ex-smokers and patients with COPD who died from COVID-19. Interestingly, we found a positive association between ACE2 and CD45RO protein levels across all the three lung structures studied. These results suggest that the ACE2-dense lung regions, that likely represent the SARS-CoV-2 entryways into the lung, may represent immune "hot spots" This study has limitations to note: first, the sample size of the COVID-19 patients is small, due to ongoing challenges that limit the access to tissue from these patients. However, spatial profiling generates a high amount of multiplexed information on different regions of interest within the lung structure. Second, recent spirometry data are lacking for 15 out of 18 subjects as most of the COVID-19 patients were admitted in critical condition. However, the patients had a prior clinical diagnosis of COPD, and emphysema was confirmed on the CT scans of the COPD patients. Third, previous cigarette smoke exposure can affect the gene expression profile within the lung 31 . However, all our study subjects ceased smoking at least one year prior to the study, and there was no difference in pack/years between COPD and ever-smoker controls, which minimizes the effect of cigarette smoke on the transcriptomic data. Fourth, it is difficult to distinguish how much of the immune profile found in COPD patients versus controls is driven by the presence of COPD, and how much it is driven by the presence of SARS-CoV2 infection. To address this, we profiled CD45RO expression in a non-infected cohort of never-and ever-smokers with and without COPD, these data confirmed that the upregulation of CD45RO expressing memory T lymphocytes in COPD patients who died of COVID-19, is likely driven by SARS-CoV-2 infection. Last, our main study cohort is skewed towards the most severe cases of SARS-CoV-2 infection, who are the ones who died of COVID-19. This was due to the fact that lung autopsy is the only source of COVID-19 lungs tissue for research, and it is not possible to study lung biopsies from patients with milder COVID-19 symptoms who survived. We report for the first time that within the lung parenchyma, airways and vessels of COPD patients with SARS-CoV-2 infection that there is a significant enrichment for memory T lymphocytes with a blunted memory phenotype, associated with an increase in genes crucial for SARS-CoV-2 entry and infection. Using spatial transcriptomic and proteomic characterization of parenchyma, airways, and vessels provides a biologically interpretable landscape of COVID-19 lung pathology and serves as an important resource for future treatment of patients with COPD and SARS-CoV-2 infection. Tables Table 1 Data are expressed as the median [interquartile range] for continuous variables and n (%) for categorical variables. 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