key: cord-0787617-nqodhc2b authors: Yang, J.; Zhang, E.; Zhong, M.; Yang, Q.; Hong, K.; Shu, T.; Zhou, D.; Xiang, J.; Xia, J.; Zhou, X.; Zhang, D.; Huang, C.; Shang, Y.; Yan, H. title: Impaired T cell functions along with elevated activated Tregs at the early stage of asymptomatic SARS-CoV-2 infection date: 2020-05-26 journal: nan DOI: 10.1101/2020.05.25.20108852 sha: eb5031e8e79494e0e62d537df9e334fb0985415f doc_id: 787617 cord_uid: nqodhc2b Background Limited data are available on the T cell responses for the asymptomatic SARS-CoV-2 infection case. Methods The first imported SARS-CoV-2 infection case in Wuhan was admitted in hospital for quarantine and observation. The T cell responses were followed up by flow cytometry analysis of the peripheral blood nonnuclear cells (PBMCs) at days 7, 13, 22, and 28 after admission. Findings We found the first imported SARS-CoV-2 infection in Wuhan is an asymptomatic case. His T cell differentiation, proliferation and activation matched the classical kinetics of T cell responses induced by viral infection, but the activation maintained at a relatively low level. Function analysis indicated frequencies of IFN-{gamma} producing CD4+ and CD8+ T cells were notably lower than that of the healthy controls (HC) at day 7, and then rebound gradually. But IFN-{gamma}+CD8+ T cells were detained at a significant lower level even at day 28, when the SARS-CoV-2 virus had already become undetectable for 3 weeks. Moreover, percentage of IL-17 producing CD4+ T cells was also detained constantly at a much lower level compared to HC. At day 7, although percentage of Tregs was in normal range, the frequency of activated Treg (aTreg) was remarkably as high as 4.4-fold of that in HC. Interpretation The T cell activation in the asymptomatic SARS-CoV-2 infection experienced a significant suppression and presented impairment of Th1/Th17 and CD8+ T cell functions. Early elevation of the aTregs might play role in the activation and function of T cells in the asymptomatic SARS-CoV-2 infection. In December 2019, an epidemic of severe acute respiratory syndrome coronavirus-2 2 (SARS-CoV-2) causing human disease named coronavirus disease was firstly 3 reported in Wuhan China. Up to May 9, 2020, a total of 3,855,788 cases have been reported with 4 265,862 deaths according to WHO report. SARS-CoV-2 infection is characterized by a broad 5 range of symptoms including fever, dry cough, and general malaise in the majority of cases while 6 severe pneumonia, acute respiratory distress syndrome (ARDS), septic shock and/or multiple 7 organ failure in a minor population 1, 2 . Based on the clinical presentation, COVID-19 patients can 8 be classified into mild, moderate, severe and critical. However, a group of laboratory-confirmed 9 SARS-CoV-2 infected cases, found by assay with quantitative real-time reverse transcription PCR 10 (qRT-PCR), presented neither clinical symptom nor radiographic abnormality. This group of 11 people was designated as asymptomatic infected individuals. The suspected rate of asymptomatic 12 infections is substantially high, which might surpass 17.9% 3, 4 . Furthermore, the viral load 13 detected in the asymptomatic case was similar to that in the symptomatic patients, which suggests 14 high transmission potential of the asymptomatic individuals 5, 6 . Actually, a growing number of 15 reports have evidenced substantial asymptomatic transmissions 4, 7, 8 . 16 A study of a patient with mild-to-moderate COVID-19 disease showed that the CD4 + T and 17 CD8 + T cells were activated after SARS-CoV-2 infection and before the resolution of symptoms 9 , 18 suggesting possible potent protective T cell functions against SARS-CoV-2 infection. On the other 19 hand, several studies showed that viral infection may cause significant decrease of T lymphocytes 20 and impairment of T cell function in COVID-19 patients, especially in severe COVID-19 cases 2, 10 . 21 Exhausted function of CD8 + T cells with the increased expression of NKG2A was also reported in 22 severe cases 11 . But so far，how T cells behave and function following acute infection of 23 SARS-CoV-2 in asymptomatic infected individuals remains largely unknown. The suspected high 24 rate of asymptomatic infections and substantial asymptomatic transmissions assure high necessity 25 of answering the above questions 3, 4, 7, 8 . In this article, we reported the kinetics of CD4 + T and 26 CD8 + T cell responses in an asymptomatic SARS-CoV-2 infected case, accompanied with the 27 clinical and virological features. 28 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Plasma and blood cells were separated from fresh peripheral blood from the SARS-CoV-2 24 infected case and healthy volunteers. Plasma was used for SARS-CoV-2 binding IgM or IgG 25 detections. PBMCs were separated from the blood cells by Ficoll-plaque density gradient 26 centrifugation and resuspended in complete RPMI1640 medium containing 10% FBS (Gibco), 1% 27 penicillin and 1% streptomycin. PBMCs were then divided into three panels for analysis: panel 1 28 for T cell differentiation, proliferation and activation detection, panel 2 for T cell cytokine 29 production detection, and panel 3 for Treg detection. PBMCs of panel 1 and panel 3 were directly 30 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and surface-staining antibodies in PBS at 4℃ for 30 min. After washing with PBS, cells were 5 fixed with fixation/permeabilization buffer (eBioscience) at 4℃ overnight, and then stained with 6 the respective panel of intracellular markers in a permeabilization buffer at 4℃ for 30 min. 7 Antibodies used for each panel were listed in supplementary table 1. A BD LSR Fortessa flow 8 cytometer (Becton Dickinson) was used to assess the stained cells and data were analyzed using 9 FlowJo V7·0. 10 Data analysis was carried out with InStat, version 5·0 (GraphPad Software, La Jolla, CA, 12 USA) and the data of healthy controls were presented as mean ± SD with dot plots. 13 14 After admission on March, 2020, the man received regular physical and clinical examinations 16 and treatments. He was a healthy smoker taking no medications. Clinical examination revealed a 17 temperature of 36·2 °C, a pulse rate of 85 beats per minute, a blood pressure of 156/106 mm Hg, a 18 respiratory rate of 21 breaths per minute, and oxygen saturation 98% while breathing ambient air. 19 SARS-CoV-2 was again detected at days 1-2 in sputum and/or nasopharyngeal swab but was 20 undetectable since day 5 (figure 1A and Supplementary Table 2 ). No other respiratory pathogens 21 and common pathogens were detected. On day 14, the man was discharged to a designated hotel 22 for another 14-day-isolation. Within the 28-day follow-up, the man experienced no clinical 23 symptom, no fever, no lethargy, no sore throat, no chest pain, no dyspnea, and no dry cough. 24 Moreover, chest CT images taken during hospitalization did not show any significant abnormality. 25 Taken together, this man was an asymptomatic SARS-CoV-2 infected individual characterized as 26 no clinical symptom or CT abnormality but SARS-CoV-2 positive by repeated qRT-PCR tests. 27 Among the tested biochemical markers, only serum amyloid A (SAA) and high-sensitivity 28 C-reactive protein (hsCRP) increased above the upper limit of normal (ULN) at day 8 and day 14. 29 On day 1, both SAA and hsCRP were at normal level (range of SAA, 0-10mg/ml & range of 30 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint hsCRP, 0-5mg/ml). SAA was elevated to 11·7 mg/ml at day 8 and 15·07 mg/ml at day 14 while 1 hsCRP was increased to 7·7 mg/ml at day 8 and 13·5 mg/ml at day 14. In contrast to the 2 significant increase in moderate and severe COVID-19 patients 2, 10, 13 , IL-6 in the subject 3 constantly kept in the normal range. SARS-CoV-2-binding IgM was presented in plasma since day 4 7 but IgG remained undetectable at all detected time points ( figure 1A) . 5 During the follow-up period, the cell count of total lymphocytes, CD4 + T cells, CD8 + T cells 6 as well as the ratio of CD4 + /CD8 + T cells maintained at a normal level (figure S1B). By analyzing 7 the differentiation of CD4 + and CD8 + T cells, a significant and gradual increase of effector T cells 8 (CD45RO -CD27 -, Teff) in both CD4 + and CD8 + T cells were observed in the follow up (figure 1B). 9 The frequencies of naï ve T cells (CD45RO -CD27 + , Tna), central memory T cells 10 (CD45RO + CD27 + , Tcm) in either CD4 + or CD8 + T cells were not significantly changed, while the 11 effector memory subset (CD45RO + CD27 -, Tem) were slightly increased (figure 1B, S2A, S3A). The activation of CD4 + and CD8 + T cells, determined as the percentage of PD-1 + , HLA-DR + 20 or CD38 + HLA-DR + , were remarkably lower than the normal low limit at day 7, peaked at day 13, 21 and gradually decreased to a low level that was comparable as the beginning (figure 1D, 1E, the 22 middle and right panel of S2 & S3, S4). These results indicate that the activation of T cells in the 23 peripheral blood might be transiently suppressed after the initial infection, and then activated from 24 the reduced baseline. This might be due to the virus induced T cell suppression, or the 25 re-distribution of activated T cells towards the infected tissues, or unknown else. 26 To analyze the function of T cells, the PBMCs were stimulated by the polyclonal stimulator 27 PMA and Ionomycin for 4·5 hours ex vivo, and proceed by intracellular cytokine staining of IFN-γ, 28 IL-4 and IL-17. Notable low frequencies of IFN-γ producing CD4 + and CD8 + T cells were 29 detected at day 7, compared to the corresponding cell subsets of the healthy controls (figure 2, 30 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint IFN-γ panel). Although the percentages of IFN-γ producing CD4 + and CD8 + T cells gradually 1 increased from day 7 to day 28, that of CD4 + T cells (figure 2A) reached the low limit of healthy 2 controls at day 13, while that of CD8 + T cells (figure 2B) was even persistently lower than the 3 normal low limit throughout the whole following up period. These results imply a functional 4 suppression of type 1 immune responses at the beginning of infection and only a partial recovery 5 afterwards. IL-4 producing CD4 + T cells gradually increased from day 7 to day 22 but dropped to 6 the baseline at day 28, indicating an activation of Th2 type immune response after viral infection 7 Compared to the rTreg which maintained at a sustained level as the healthy controls, the 25 composition of aTregs was at a high level at day 7，as much as 4·4-fold of that in healthy controls, 26 then gradually decreased to normal range at day 22 (figure 3B). As CTLA-4 is one of the most 27 important co-inhibitory molecules expressed by Treg, we further determined CTLA-4 expression 28 on the total Treg and the two Treg subsets. Consistent with the previous publications, the aTregs 29 possessed the highest CTLA-4 expression. Notably, the frequency of CTLA-4 + cells in aTregs was 30 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint over 4-fold of that in total Tregs or rTregs. The dynamics of CTLA-4 + in total Tregs and the two 1 subsets were similar in which the CTLA-4 + population peaked at day 13 and then dropped but 2 without significant change compared with healthy controls (figure 3C). functions might be impaired. We noted that the CD4 + T cell was suppressed in activation, but the 28 accompanied function impairment only detected in the Th1 and Th17 subsets, but not in the Th2, 29 suggesting a selective suppression on CD4 T cells ( figure 2A) . We observed that the CD8 + T cell 30 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint was also suppressed in activation especially at early infection stage, and the accompanied function 1 impairment was significantly presented in the IFN-γ + CD8 T cell subset. Furthermore, the 2 suppression of CD8 + T cell was even lasted until day 28, in which the IFN-γ expression was still 3 kept at much lower level compared to the healthy controls (figure 2B), suggesting a persistent 4 function impairment on CD8 T cells. As we tested the function of T cells ex-vivo under the 5 polyclonal (non-specific) stimulation, the observed functional impairment is not limited to the 6 viral-specific T cells, but to the total reservoirs of CD4 + and CD8 + cells. Thus, the function 7 impairment indicated by the decreased cytokine productions might also suggest a featured T cell 8 response bias in this asymptomatic individual. It should be noted that the persistent suppression of 9 Th17 cells, which usually was involved in the pulmonary inflammation, might be one key aspect 10 for the explanation of this asymptomatic case who did not progress into radiographically 11 detectable pulmonary inflammatory lesion. More intensive studies on the T cell response in the 12 asymptomatic case should be conducted to give more insights into the protective immune 13 responses to the SARA-CoV-2 infection. For understanding the mechanism of the suppressed T cell activation and function of the 24 asymptomatic case, we focused on the regulatory T (Treg) cells, which suppress immune 25 responses to a broad range of antigens, and limit immune response by employing multiple 26 mechanisms 19 . According to the expression of FoxP3 and CD45RA, Treg cells are classified into 27 two suppressive subsets: a slight inhibitory CD45RA + FoxP3 lo rTreg and a strongest inhibitory 28 CD45RA-FoxP3 hi aTreg 14 . In exerting function of Tregs, CTLA-4 on aTregs can capture its 29 ligands CD80 and CD86 from the surface of antigen presenting cells (APCs), denying their 30 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint availability for co-stimulation of CD4 + T and CD8 + T cells 20 . In this asymptomatic case, aTreg 1 which possesses the highest inhibitory molecule CTLA-4, was markedly elevated at the early 2 infection stage. The elevation of aTregs accompanying with the low activation and impaired 3 function of T cells at early infection stage of this asymptomatic case suggested that aTregs might 4 suppress T cell activation and the following function in the T cell priming stage. However, it was 5 reported that the frequency of Tregs was significantly reduced in the severe and moderate cases 2, 10 . 6 The current knowledge of the kinetics of Treg activation in either the asymptomatic or the 7 symptomatic patients is still limited. More detailed investigations on the functional regulation of 8 Tregs in the SARS-CoV-2 infection are urgently needed. 9 The present study has some limitations. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint COVID-19: immunopathology and its implications for therapy Clinical and immunological features of 4 severe and moderate coronavirus disease 2019 Estimating the asymptomatic proportion of 7 coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship Euro surveillance : bulletin Europeen sur les maladies transmissibles = European 9 communicable disease bulletin COVID-19: the case for health-care 11 worker screening to prevent hospital transmission SARS-CoV-2 Viral Load in Upper 13 Respiratory Specimens of Infected Patients. The New England journal of medicine Asymptomatic Transmission, the Achilles' Heel of Current 16 Strategies to Control Covid-19. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.25.20108852 doi: medRxiv preprint