key: cord-0962001-2okcuviq
authors: Huang, Lulin; Shi, Yi; Gong, Bo; Jiang, Li; Liu, Xiaoqi; Yang, Jialiang; Tang, Juan; You, Chunfang; Jiang, Qi; Long, Bo; Zeng, Tao; Luo, Mei; Zeng, Fanwei; Zeng, Fanxin; Wang, Shuqiang; Yang, Xingxiang; Yang, Zhenglin
title: Blood single cell immune profiling reveals the interferon-MAPK pathway mediated adaptive immune response for COVID-19
date: 2020-03-17
journal: nan
DOI: 10.1101/2020.03.15.20033472
sha: 20a1d878639ad14f6ecffab84a56c223946cb081
doc_id: 962001
cord_uid: 2okcuviq

The coronavirus disease 2019 (COVID-19) outbreak is an ongoing global health emergence, but the pathogenesis remains unclear. We revealed blood cell immune response profiles using 5' mRNA, TCR and BCR V(D)J transcriptome analysis with single-cell resolution. Data from 134,620 PBMCs and 83,387 TCR and 12,601 BCR clones was obtained, and 56 blood cell subtypes and 23 new cell marker genes were identified from 16 participants. The number of specific subtypes of immune cells changed significantly when compared patients with controls. Activation of the interferon-MAPK pathway is the major defense mechanism, but MAPK transcription signaling is inhibited in cured patients. TCR and BCR V(D)J recombination is highly diverse in generating different antibodies against SARS-CoV-2. Therefore, the interferon-MAPK pathway and TCR- and BCR-produced antibodies play important roles in the COVID-19 immune response. Immune deficiency or immune over-response may result in the condition of patients with COVID-19 becoming critical or severe.

The battles between humans and the pathogenic viruses are never ending. Epidemic diseases, including Zika (1), severe acute respitatory sundrome ( SARS) (2) , and Ebola (3), strike quickly, often killing hundreds of people during a single outbreak.

On December 31, 2019, a novel coronavirus (SARS-CoV-2), belonging to the Orthocoronavirinae subfamily and distinct from middle east respiratory syndrome (MERS)-CoV and SARS-CoV, emerged in Wuhan, China (4) (5) (6) . The rapid person-toperson spread of SARS-CoV-2, which causes the coronavirus disease 2019 (COVID- 19) , presents an imminent threat to public health and has caused a global health emergency (7) (8) (9) . The World Health Organization emergency committee declared this outbreak a "public health emergency of international concern" because of its 2.1% mortality rate and person-to-person transmission (10) . As of March 7, 2020, over 102,136 people have fallen ill with SARS-CoV-2, leading to 3,492 deaths in 93 countries, and the coronavirus continues to spread quickly all over the world.

Symptoms of COVID-19 included fever, myalgia, and fatigue, as well as dry cough, shortness of breath, sputum production, headache, hemoptysis, sore throat, and diarrhea. Lymphopenia, prolonged prothrombin time, and elevated lactate dehydrogenase levels have also been observed in the patients with COVID-19 (6) . A computed tomography (CT) can identify bilateral patchy shadows or ground glass opacity in the lungs (6, 8, (11) (12) (13) . Despite the high infection and mortality rates, there is no specific cure for the disease because its pathogenesis remains unclear.

The human adaptive immune system plays a critical role in defending against viral infections. Many immune cells (such as leukocytes) and immune molecules (such as specific plasma proteins) are intrinsic components of blood. Therefore, the functions of blood and the immune system are inseparable. T cells and B cells are important in the pathogenesis of many-perhaps all-immune-mediated diseases (14) (15) (16) (17) . A T cell receptor (TCR) mediates recognition of pathogen-associated epitopes through interactions with peptide and major histocompatibility complexes (pMHCs). TCRs and B cell receptors (BCRs) are generated by genomic rearrangement at the germline level, a process termed V(D)J recombination, which has the ability to generate marked diversity among TCRs and BCRs. Parameterizing the elements of antigenspecific immune repertoires across a diverse set of epitopes may generate powerful applications in a variety of research fields for the diagnosis and treatment of infectious diseases. Here, we focus on insights into blood immune responses to 4 4 single-cell resolution.

In this study, 16 participants were recruited from the Sichuan Province of China.

The basic information and clinical features of the patients are listed in Table S1 . The lung CT images of these subjects are shown in Fig. S1 . There was one critical case (patient 1, male, 34 years old), one severe case (patient 2, male, 43 years old), and six moderate cases (patients 3-8) comprised of two males and four females aged 25-62 years old (Table S1 ). The two cured patients (patient 9, male, 40 years old and patient 10, female, 20 years old) were on the discharged day from hospital after they tested negative for SARS-CoV-2 and the disease signs disappeared. Three healthy people were included as normal controls (NC 1-3, Table S1 ). In order to differentiate the COVID-19 immune response from other infectious diseases, thereby identifying the unique COVID-19 immune response, we also recruited one case of influenza A (patient 11), one case of acute pharyngitis (patient 12), and one case of cerebral infarction (patient 13) as controls (Table S1 ).

The peripheral blood mononuclear cells (PBMCs) of each individual were isolated from the whole blood, and the single-cell mRNA sequencing (ScRNA-Seq) analysis of the PBMCs was performed on the 10 X genomics platform with Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (Fig. 1A ). After quality control, we obtained 134,620 PBMC cells, 83,387 TCR clones, and 12,601 BCR clones from the 16 samples. In these samples, we identified 17 completely different PBMC cell types, which could be further divided into 56 cell subtypes according to marker genes of the 134,620 cells (Fig. 1B, 1C, 1D and Table S2 ). The 17 cell types included CD4+ T cells identified by LTB and IL7R markers, CD8+ T cells identified by LEF1 and CD8A, CD1C+_B dendritic cells identified by BSET1 and CD1D, CD4+ cytotoxic T cells identified by CST7 and CCL4, CD8 cytotoxic T cells identified by CD3D and CD8A, and other cell types (Fig. 1C, Table S2 ). This suggests that human PBMCs have evolved highly differentiated cell subtypes to resist the invasion of pathogens (18) . There were three main states in the analysis process for reconstructing the developmental trajectories of the 56 cell subtypes, which showed a continuous trajectory of differentiating PBMCs (Fig. 1E, Fig. S2 ). In addition, 23 totally new candidate marker genes linked to 22 cell subtypes were identified (Fig. S3) , suggesting potential roles for these newly discovered cell markers in the identification and functional study of these subtypes.

To investigate the immune cell behaviors of the 56 cell subtypes in different All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in In the severe condition (patient 2), 18 signaling pathways were significantly changed in more than half of the cell subtypes compared to the normal controls.

Among these pathways, the most important was over-activation of the interferonrelated signaling pathways, including 2'-5'-oligoadenylate synthase and type I interferon signaling pathway(naive CD8+ T cells, CD4+ T cells, and other cells) and interferon alpha/beta/gamma signaling (naive CD8+ T cells, CD4+ T cells, and other cells) (Fig. 2C , Table S3 ). In other COVID-19 conditions, the interferon signal was also over-activated to varying degrees. However, in the cured patients, the interferon signal was just mildly activated compared to the patients with severe and moderate conditions. In the cured condition, the signal pathway related to mRNA translation of the virus was suppressed, while the signal pathway related to MAPK was significantly enriched (Fig. 2C , Table S3-6). To detect whether these signaling pathway changes were specific to COVID-19, we compared the COVID-19 patients with the patients with influenza A (patient 11), acute pharyngitis (patient 12), and cerebral infarction (patient 13) and found that the signaling pathways were still enriched after filtering with these related disease controls (Fig. S14 ). Among these pathways, the interferon-MAPK signaling pathway was the major blood immune response for COVID-19

infection.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The activation of the MAPK signaling pathway by interferon involves a set of defense mechanisms evolved by human beings to fight against viral infections (19) (20) (21) .

To further explore the expression differences of genes involved in different cell types in the various COVID-19 conditions, we analyzed the gene expressions in patients with COVID-19 and compared them to those of the controls (Fig. 2G-J, Fig. S7 ). First, we observed up-regulation expression of the interferon signal genes in the COVID-19

patients. Among them, interferon α-inducible protein 27 (IFI27), originally known to involve in innate immunity and to intervene in cell proliferation (22) , was increasingly expressed in many cell types, including CD1C+_B dendritic cells, Table S8 ). These genes were previously shown to regulate cell invasion, migration, proliferation and innate immunity by inhibiting pro-inflammatory cytokine production (27, 28) . In three groups of inpatients with different conditions, the up-regulated expression of these genes was observed in CD1C+_B dendritic cell, CD1C-CD141dendritic cell, CD141+CLEC9A+ dendritic cell, CD8 cytotoxic T cells, and plasmacytoid dendritic cells. Interestingly, in contrast to the three groups of inpatients, the expression pattern of these genes in the two cured patients was the opposite. In the cured patients, down-regulated expression was detected, indicating that the MAPK signaling pathway was inhibited in these cells. These results indicate that downregulation of the MAPK signal pathway may be one sign of patient recovery.

To further explore the antibody clonal proliferation of TCR and BCR in COVID-19, we conducted TCR and BCR V(D)J single cell transcriptome analysis. Using integration analysis, we detected 83,387 TCR cell clones and 12,601 BCR clones in the 16 subjects (Fig. 3A) . This result revealed the antibody repertoire in patients with All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

In the critical condition (patient 1), the fewest TCR clones (1,232) were detected, while, in the severe condition (patient 2), more TCR clones (7, 161) were detected. In one moderate patient (patient 8), the most TCR clones (20,370) were detected.

According to these findings, the fewer the TCR clones, the more severe the COVID-19 condition. The distribution of all TCR clones and the number of TCR clones of each patient are shown in Fig. 3B and Table S9 . Using evolutionary analysis of the top TCR antibody sequences revealed by TCR sequencing, we found that TCRα sequences of CASSEGVGTPFDEQFF (patient 2), CASSLGLAGDLDEQFF and CASNQGLAGGRLYNEQFF (patient 4), CASSQERGVYNEQFF (patient 6) and CASSEVWASDHEQYF (patient 10) were closely related (Fig. S15) , suggesting that these antibody sequences may have a special activity for SARS-CoV-2 (and influenza A). Compared to TCR, the number of BCR clones detected was much lower (Fig. 3C , Table S10 ). However, very strong BCR antibodies were not induced in most of the patients except in two moderate patients (patients 6 and 8).

In one moderate patient (patient 9) (Fig. 3D-G) , we detected 361 clones of CDR3 IGLorK/IGH sequences of CQQYGSSPWTF/CARVAESYYDFWSAKWEGAFDIW and 119 clones of CQQYNSYSF/CARSGPITIFGVVIKGRGGDAFDIW (Fig. 3C , Table S10 ). In another moderate patient (patient 7), four high frequency clones were identified, including CQQYYSIPSLTF/CATHRTYFDWLLPFDYW (179) (Fig. 3C , Table S10 ). These results suggest that the antibodies produced by patients 7 and 9 may have a SARS-CoV-2 specific affinity.

We then analyzed top TCR encoded antibody sequences for known antigens, and some of them were annotated in the VDJdb antigen categories database (Fig. 3H ). In patients with COVID-19, we detected immune responses to known antigens, including Epstein-Barr virus (EBV), HIV-1, influenza A, yellow fever, and other RNA viruses, as well as DNA viruses such as cytomegalovirus (CMV). In addition, autoantigens were detected in the severe condition (patient 2) and the moderate condition (patients 4, 8, and 9). In patient 2, the autoantigen response was obvious activation, which indicated that this severe disease condition may be related to strong autoimmunity. On the contrary, in the critical condition (patient 1), only two antibodies against the Ebola EBV antigen were detected. These results further suggest that the critical condition may be caused by insufficient immunity, while the severe condition may be aggravated by excessive autoimmunity. In the normal condition, no All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in immune response to these antigens was detected for NC 1 and NC 3; for the NC 2, two antigens were detected (HIV-1 and influenza A) (Fig. 3H) . For the patient with influenza A (patient 11), the antigen and autoimmune responses of the yellow fever virus were detected. For the patient with acute pharyngitis (patient 12), EBV, HIV-1, influenza A and yellow fever virus antigens were detected, but there was no autoimmune response. For the patient with cerebral infarction (patient 13), no known antigens were detected. These results indicate that humans might use known immune recognition (mainly known RNA viruses) to fight against SARS-CoV-2.

Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors (29) . In this study, our single cell transcriptome results showed that IRF27 was up-regulated 8.1 times more in critical patients, 51.7 times in severe patients, 39.9 times in moderate patients, 38.6 times in mild patients, and 29.5 times in cured patients compared to the normal controls (Fig. 4A) . These results suggest that IRF27 is a candidate marker gene for SARS-CoV-2 infection. BST2, which was previously shown to play a role in pre-B-cell growth (31) , also showed upexpression in COVID-19 patients. Consistent with our single cell transcription data, the expression profile of FOS, which is a transcription factor mediating MAPK pathway signaling, showed up-regulation in COVID-19 patients but down-regulation in cured patients. This result suggests that FOS is a candidate marker gene for cured COVID-19 patients. We further performed PBMC immunofluorescence testing to compare the protein expressions of IFI27 between patients with COVID-19 and controls. Immunofluorescence staining showed that IFI27 was highly expressed in the whole lymphocyte of patients with COVID-19, but only partially expressed on the lymphocyte of the controls (Fig. 4B) .

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 17, 2020. . https://doi.org/10.1101/2020.03. 15.20033472 doi: medRxiv preprint In summary, we demonstrated that SARS-CoV-2 can enter the blood through the circulatory system and cause a blood immune response after infection through the respiratory system. Therefore, in patients with COVID-19, the damage is related to multiple organs (32) . The leukocyte immune responses involve many cell subtypes, including CD8+ T cells, CD8 cytotoxic T cells, CD4+ T cells, CD4+ cytotoxic T cells, CD1C+_B dendritic cells and other cell subtypes. After the virus reaches the blood immune cells, it might replicate-mainly in AXL+SIGLEC6+ dendritic cells, CD4+ T cells, and naive CD8+ T cells and might activate multiple cell subtypes of the interferon signal pathway to produce effectors, such as IFI27, IFITM1 and IFITM3, to fight the SARS-CoV-2 virus. The similar process also happened to Influenza and West Nile Virus infection in human (23, 24) . Through downstream activation of MAPK, the expression of downstream effectors is activated by key transcription factors, such as FOS, JUN, and JUNB, which lead to a wide range of antiviral responses in the blood system (Fig. 4C) . These results suggested that the immune response may come back the normal situation when the patients recovery, and the MAPK signal is thus inhibited.

In conclusion, in this study, we revealed the immune response process of SARS-CoV-2 entering the blood circulation system using immune profiling analysis with single-cell resolution. In the critical condition, the number of different cell types, expressed genes, and TCR/BCR responses indicated a relationship between the critical nature of the patient's condition and his immunodeficiency. On the contrary, the severe condition was shown to be related to a strong autoimmune response and over-activated interferon pathway. These results suggested that immune deficiency or immune over-reacts may make patients with COVID-19 go to worse condition because of their imbalance adaptive immune response (33) (34) (35) . Our study also shows that the activation of the interferon-MAPK signaling pathway and TCR-and BCRproduced antibodies play key roles in combating SARS-CoV-2 infection. These results will provide important information for COVID-19 treatment and drug development.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in LGL E. IGK Table S1 Clinical and blood routine test information of all participants, analyzed using V(D)J transcriptome analysis Table S2 List of known marker genes, new marker genes, and highly expressed genes of 56 cell (sub) groups, identified by integrated analysis of all sequenced samples Table S7 The differential expression of interferon-related genes in each cell (sub)type of the COVID-19 patients compared with the normal controls Table S8 The differential expression of MAPK signal genes in each cell (sub)type of the COVID-19 patients compared with the normal controls Table S9 List of the most enriched clone antibodies in each patient using TCR single cell transcriptome analysis Table S10 List of the most enriched clone antibodies in each patient using BCR single cell transcriptome analysis External Database S1 Differential expressed genes in each cell subtype in a COVID-19 critical patient and the normal controls External Database S2 Differential expressed genes in each cell subtype between a COVID-19 severe patient and the normal controls External Database S3 Differential expressed genes in each cell subtype between COVID-19 moderate patients and the normal controls External Database S4 Differential expressed genes in each cell subtype between COVID-19 cured patients and the normal controls All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in FOS is up-regulated in hospitalized patients but down-regulated in cured patients. (B) Immunofluorescence staining of IFI27 in PBMCs of patients with COVID-19 and normal controls. (C) Anti-SARS-CoV-2 response of the blood system. After the virus reaches the blood immune cells, it can activate multiple cell subtypes of the interferon signal pathway to produce effectors, such as IFI27, IFITM1, and IFITM3, to fight the virus. Through downstream activation of MAPK, the expression of downstream effectors is activated by key transcription factors, FOS, JUN, and JUNB, which lead to a wide range of antiviral responses in the blood system. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 17, 2020. . https://doi.org/10.1101/2020.03. 15.20033472 doi: medRxiv preprint 

Zika virus outbreak on Yap Island, Federated States of Micronesia

Marburg and Ebola--arming ourselves against the deadly filoviruses

A Novel Coronavirus from Patients with Pneumonia in China

A new coronavirus associated with human respiratory disease in China

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

First Case of 2019 Novel Coronavirus in the United States

Clinical Characteristics of Coronavirus Disease 2019 in China

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

The Novel Coronavirus Originating in Wuhan, China: Challenges for Global Health Governance

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Analysis of the B cell receptor repertoire in six immunemediated diseases

A cell atlas of human thymic development defines T cell repertoire formation

High frequency of shared clonotypes in human B cell receptor repertoires

Identifying specificity groups in the T cell receptor repertoire

Single-cell transcriptomics to explore the immune system in health and disease

The expanding role of NLRs in antiviral immunity

The interferon response to intracellular DNA: why so many receptors?

Regulation of interferondependent mRNA translation of target genes

Interferon alpha-inducible protein 27 is an oncogene and highly expressed in cholangiocarcinoma patients with poor survival

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus

IFITM3 restricts the morbidity and mortality associated with influenza

Amphotericin B increases influenza A virus infection by preventing IFITM3-mediated restriction

Differential responses of innate immunity triggered by different subtypes of influenza a viruses in human and avian hosts

MKP-1 is necessary for T cell activation and function

West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells

West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells

A novel immune biomarker IFI27 discriminates between influenza and bacteria in patients with suspected respiratory infection

Beyond Tethering the Viral Particles: Immunomodulatory Functions of Tetherin (BST-2)

Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study

The balance between persistent virus infection and immune cells determines demyelination

ADARs and the Balance Game between Virus Infection and Innate Immune Cell Response

The role of porcine reproductive and respiratory syndrome virus infection in immune phenotype and Th1/Th2 balance of dendritic cells

We thank all participants for supporting in this study. Furthermore, we gratefully acknowledge the patients and control subjects for their donation of peripheral blood samples. We thank Genergy Bio for support with V(D)J sample processing and sequencing library preparation. We thank Y.B. Mei for helpful discussions. The authors have no competing interests to declare. Data and material availability:All sequencing data will be deposited in a public database and can be accessed online through a web portal.