key: cord-0916302-120allsr authors: Sharif-Askari, Narjes Saheb; Sharif-Askari, Fatemeh Saheb; Mdkhana, Bushra; Al Heialy, Saba; Alsafar, Habiba S.; Hamoudi, Rifat; Hamid, Qutayba; Halwani, Rabih title: Enhanced expression of immune checkpoint receptors during SARS-CoV-2 viral infection date: 2020-11-12 journal: Mol Ther Methods Clin Dev DOI: 10.1016/j.omtm.2020.11.002 sha: 5f923a259b32f606a7c24b658fca8691548364fa doc_id: 916302 cord_uid: 120allsr The immune system is tightly regulated by the activity of stimulatory and inhibitory immune receptors. This immune homeostasis is usually disturbed during chronic viral infection. Using publicly available transcriptomic datasets, we conducted an in-silico analyses to evaluate the expression pattern of 38 selected immunoinhibitory receptors (IRs) associated with different myeloid and lymphoid immune cells during COVID-19 infection. Our analyses revealed a pattern of overall upregulation of IRs mRNA during SARS-CoV-2 infection. A large number of IRs expressed on both lymphoid and myeloid cells were upregulated in nasopharyngeal swabs (NPs), while lymphoid associated IRs were specifically upregulated in autopsies, reflecting severe, terminal stage, COVID-19 disease. Eight genes (BTLA, LAG3, FCGR2B, PDCD1, CEACAM1, CTLA4, CD72, and SIGLEC7), shared by NPs and autopsies, were more expressed in autopsies and were directly correlated with viral levels. Single-cell data from blood and bronchioalveolar samples also reflected the observed association between IRs upregulation and disease severity. Moreover, compared to SARS-CoV-1, influenza and respiratory syncytial virus infections, the number and intensities of upregulated IRs were higher in SARS-CoV-2 infections. In conclusion, the immunopathology and severity of COVID-19 could be attributed to dysregulation of different immune inhibitors. Targeting one or more of these immune inhibitors could represent an effective therapeutic approach for the treatment of COVID-19 early and late immune dysregulations. The expression of IRs is known to be enhanced upon persistent exposure to viral antigens. To 163 investigate that in the context of SARS-CoV-2, we associated the expression of these 164 receptors with the expression of SARS-CoV-2 N viral protein. COVID-19 infection was 165 confirmed by quantitative PCR for the SARS-CoV-2 N protein. 30 We have associated the 166 expression of the 8 common IRs between autopsies and NPs with SARS-CoV-2 N reported 167 viral protein levels. As expected, higher viral level was associated with higher expression of 168 immune inhibitory receptors (Figure 2) . 169 170 After establishing an overall upregulation of IRs in autopsies of COVID-19 patients, we next 172 determined whether the observed increase in IRs is reflected on the main inflammatory cells 173 regulating COVID-19 severity. A single cell dataset of Monocyte/macrophage and CD8 + 174 cells isolated from bronchioalveolar fluid (BALF) (GSE145926) and peripheral blood 175 mononuclear cells (PBMCs) (GSE149689) of COVID-19 severe patients were used. 11, 12 176 Overall, the number of upregulated IRs and their level of expression were higher in BALF 177 isolated inflammatory cells compared to PBMCs. In the first study using BALF samples, 178 macrophages were clustered into M1-like and M2-like macrophages. Both M1 and M2 179 macrophages were higher in severe compared to moderate COVID-19 patients, and healthy 180 donors. 12 Immune inhibitory receptors were expressed by both macrophage subsets as shown 181 in figure 3A . Further, specific upregulation of KLRC1, CD244 (2B4), and PECAM was 182 observed in BALF CD8+ T cells isolated from moderate COVID-19 patients, while CTLA4, 183 HAVCR2 (TIM3), and TIGIT were upregulated in CD8+ T cells of severe COVID-19 184 patients ( Figure 3B ). 185 186 J o u r n a l P r e -p r o o f Lung autopsies presented in figure 1B included upregulation of LAG3 and CD72 that were 187 also upregulated in BALF macrophages. Additionally, CTLA4 was upregulated in both of 188 lung autopsies and BALF CD8+ T cells. We then determined the differential expression of 189 IRs on PBMCs of COVID19 patients during different stages of the disease using single cell 190 data (GSE149689). 11 On Monocytes, the expression of LILRB1 was increased during the 191 mild stage of the disease, while two immune inhibitors, CLECLT12A and PILRA, were 192 upregulated on during the severe stage ( Figure 3C ). Interestingly, LILRB1 was upregulated 193 on macrophages of BALFs and monocytes of PBMCs, but its expression was three-fold 194 higher in lung compared to blood ( Figure 3A and 3C). Furthermore, CTLA4 and HAVCR2 195 (TIM3) expression level was higher in CD8+ cells of BALFs compared to CD8+ cells of 196 PBMCs ( Figure 3B and 3D). In this same study (GSE149689), they have also compared the 197 level of expression of IRs on PBMCs from COVID19 to those of IAV infected patients. 198 Interestingly, the expression of IRs was not increased in IAV specific groups, while two of 199 the receptors were significantly upregulated in COVID-19 specific population (CTLA4 and 200 HAVCR2). Next we used the BALF study (GSE145926) to determine the expression of two 201 T cell activation markers (CD25 and CD69) and two macrophage activation markers (HLA-202 DRA and CD86). Interestingly, T cell activation markers were not upregulated in CD8+ T 203 cells isolated from BALF. Further, HLA-DRA was not upregulated, while CD86 was slightly 204 upregulated in the M2 like macrophage cluster enriched more in BALF from severe COVID-205 19 patients ( Figure S2 Here we show an increase in myeloid related signatures particularly in nasopharyngeal 293 swabs, PBMC and BALF cells. The overexpression of IRs could, at least partially, explain 294 the delayed innate immune response and muted type I and III interferon signaling during 295 SARS-CoV-2 infection. 37-40 A large number of IRs were upregulated on BALF macrophages 296 compared to blood monocytes ( Figure 3 ). This includes LILRBs, SIGLECs, and SIRPA. 297 LILRB1 was shared between both cell types but was three-fold higher in BALF compared to 298 blood. This could reflect the expected higher viral levels in BALF compared to blood. In fact, 299 LILRBs were also broadly upregulated in NPs and whole blood cells. Interestingly, Dengue II is its main ligand to which it binds with high affinity. 52 Similar to CTLA4 and PD1, LAG3 333 negatively regulates cellular proliferation, activation, and homeostasis of T cells. 53-55 334 However, LAG3, is also considered as an activation marker. 56 It is upregulated transiently 335 during both CD4 and CD8 T cell activation, reaching the peak expression level at 48 hours 336 post infection. 56 Therefore, the observed upregulation of LAG3 in nasopharyngeal swabs 337 could be a negative feedback mechanism in response to immune activation; while its 338 enhanced expression in lung autopsies and macrophages could most likely be due to terminal 339 differentiation of these cells. Understanding the mechanism regulating these receptors, 340 however, requires further investigation. 341 342 Interestingly, the number and intensities of lymphoid IRs expressed on viral specific CD8 T 343 cells were higher during COVID-19 disease than other respiratory infections. TIM3 and 344 CTLA4 were significantly upregulated in COVID-19 specific, but not influenza specific, 345 CD8 + T cell ( Figure 3D ). This may explain the inefficient control of viral replication during 346 late stages of the disease. It also suggests that the enhanced expression of lymphoid IRs 347 observed in lung autopsies could in part be attributed to the infiltrated T cells. For the purpose of this study we first established a list of 38 immune inhibitory receptors that 392 are known to be expressed in different innate and adaptive immune cells (Table 1) A new coronavirus associated with human respiratory 497 disease in China A Novel Coronavirus from Patients with Pneumonia in China Coronavirus disease ( COVID-19) : situation report macrophages are epigenetically altered after inflammation, leading to long-term lung 641 immunoparalysis Inhibitory 646 Receptors Beyond T Cell Exhaustion Clinical and immunological features of severe and moderate 649 coronavirus disease 2019 Harnessing the 651 immunotherapeutic potential of T-lymphocyte co-signaling molecules in 652 transplantation CD4/major 654 histocompatibility complex class II interaction analyzed with CD4-and lymphocyte 655 activation gene-3 (LAG-3)-Ig fusion proteins Role of LAG-3 in Regulatory T 662 Cells Role of Lymphocyte Activation Gene-3 (Lag-3) in Conventional and 665 Regulatory T Cell Function in Allogeneic Transplantation LAG3 667 (CD223) as a cancer immunotherapy target MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the 671 epigenetic landscape On the use of immune 673 checkpoint inhibitors in patients with viral infections including COVID-19 An open-label, multiple ascending dose study of the anti-CTLA-4 677 antibody ipilimumab in viremic HIV patients Single-cell RNA-seq identifies a PD-1hi ILC 684 progenitor and defines its development pathway Disease severity-specific neutrophil signatures in blood transcriptomes stratify 688 COVID-19 patients. medRxiv: 2020 Gene expression signatures 691 diagnose influenza and other symptomatic respiratory viral infections in humans Robust meta-analysis of gene expression using the 697 elastic net limma powers differential expression analyses for RNA-sequencing and microarray 700 studies Statistical methods for 702 identifying differentially expressed genes in replicated cDNA microarray 703 experiments A LAG3-interfering oligonucleotide acts as an adjuvant to enhance the 709 antibody responses induced by recombinant protein vaccines and inactivated 710 influenza virus vaccines LAG-3 inhibits the activation of CD4+ T cells that recognize 713 stable pMHCII through its conformation-dependent recognition of pMHCII The interaction of TIGIT with PVR 717 and PVRL2 inhibits human NK cell cytotoxicity The surface protein TIGIT suppresses T cell 721 activation by promoting the generation of mature immunoregulatory dendritic cells Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus 726 infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells CD160-Associated CD8 T-Cell Functional Impairment Is 730 Independent of PD-1 Expression Expression and 734 function of the B and T lymphocyte attenuator The Human 737 FcγRII (CD32) Family of Leukocyte FcR in Health and Disease. Frontiers in 738 Immunology 10 Differential expression of Fc gamma RIIA, Fc gamma RIIB 741 and Fc gamma RIIC in hematopoietic cells: analysis of transcripts CTLA4 expression is an indicator and 745 regulator of steady-state CD4+ FoxP3+ T cell homeostasis The diverse functions of the PD1 inhibitory 748 pathway Role of PD-1 during effector CD8 T cell 751 differentiation FDF03, a novel inhibitory receptor of the 754 immunoglobulin superfamily, is expressed by human dendritic and myeloid cells 758 recruits SHP-1 upon tyrosine phosphorylation and is paired with the truncated 759 counterpart PILRbeta CD5 expression promotes multiple intracellular signaling 764 pathways in B lymphocyte PECAM-1 (CD31) cloning and relation to adhesion molecules of 767 the immunoglobulin gene superfamily CD31 signals confer immune privilege to the vascular 770 endothelium The role of CD22 and Siglec-G in B-cell tolerance 772 and autoimmune disease Homomultimeric 774 complexes of CD22 in B cells revealed by protein-glycan cross-linking The past and future of CD33 as 780 therapeutic target in acute myeloid leukemia Epitope mapping, 783 expression and post-translational modifications of two isoforms of CD33 (CD33M 784 and CD33m) on lymphoid and myeloid human cells The inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1) is expressed at high 787 levels by human naive T cells and inhibits TCR mediated activation Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is differentially 791 expressed during human B cell differentiation and inhibits B cell receptor-mediated 792 signaling The inhibitory collagen receptor LAIR-1 (CD305) Tuning Natural Killer 796 Cell Anti-multiple Myeloma Reactivity by Targeting Inhibitory Signaling via KIR 797 and NKG2A. Frontiers in Immunology 9 The role of CD94/NKG2 in innate and 799 adaptive immunity Opposing effect of 801 IFNgamma and IFNalpha on expression of NKG2 receptors: negative regulation of 802 IFNgamma on NK cells The CD47-804 SIRPα signaling axis as an innate immune checkpoint in cancer Macrophage Checkpoint Blockade in Cancer 807 -Back to the Future Human signal-regulatory protein is expressed on normal, 810 but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving 811 its counterreceptor CD47 The CD200-CD200R1 inhibitory signaling 813 pathway: immune regulation and host-pathogen interactions CD200R signaling in tumor tolerance and 816 inflammation: A tricky balance The 818 Inhibitory Receptor IRp60 (CD300a) Is Expressed and Functional on Human Mast 819 Cells Molecular and functional 822 characterization of IRp60, a member of the immunoglobulin superfamily that 823 functions as an inhibitory receptor in human NK cells IREM-1 is a novel inhibitory receptor expressed by myeloid cells CD300a and CD300f differentially 828 regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation 829 of SHP-1 and/or SHP-2 in human monocytic cell lines The inhibitory receptor LILRB1 modulates the differentiation and regulatory 832 potential of human dendritic cells Impact of the NK Cell Receptor 835 LIR-1 (ILT-2/CD85j/LILRB1) on Cytotoxicity against Multiple Myeloma The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-839 mediated signaling in monocytes Leukocyte Ig-like receptor B4 (LILRB4) is a potent inhibitor of FcgammaRI-842 mediated monocyte activation via dephosphorylation of multiple kinases A novel inhibitory receptor (ILT3) expressed on monocytes, 846 macrophages, and dendritic cells involved in antigen processing Diversity of the human LILRB3/A6 locus encoding a myeloid inhibitory and 850 activating receptor pair The Expanding Spectrum of Ligands for 852 Leukocyte Ig-like Receptors Immune inhibitory receptors: Essential 854 regulators of phagocyte function Expression of the innate immune 856 receptor LILRB5 on monocytes is associated with mycobacteria exposure Signal inhibitory receptor on leukocytes-1 (SIRL-860 1) negatively regulates the oxidative burst in human phagocytes Characterization of Siglec-5, a 864 Novel Glycoprotein Expressed on Myeloid Cells Related to CD33 Siglec-mediated regulation of 867 immune cell function in disease Sialic Acid Mimetics to Target 869 the Sialic Acid–Siglec Axis Siglec-9, a novel sialic acid 871 binding member of the immunoglobulin superfamily expressed broadly on human 872 blood leukocytes Identification and characterization of a novel siglec, siglec-7, 875 expressed by human natural killer cells and monocytes Identification, characterization and leucocyte 879 expression of Siglec-10, a novel human sialic acid-binding receptor Cloning and characterization of human Siglec-11. A recently evolved signaling 883 molecule that can interact with SHP-1 and SHP-2 and is expressed by tissue 884 macrophages, including brain microglia Pivotal role of the carbohydrate 887 recognition domain in self-interaction of CLEC4A to elicit the ITIM-mediated 888 inhibitory function in murine conventional dendritic cells in vitro Human MICL (CLEC12A) is differentially 892 glycosylated and is down-regulated following cellular activation