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. 2022 Mar 11;7(1):83.
doi: 10.1038/s41392-022-00919-x.

ACE2-independent infection of T lymphocytes by SARS-CoV-2

Xu-Rui Shen #  1   2 , Rong Geng #  1   2 , Qian Li #  1   2 , Ying Chen #  1   2 , Shu-Fen Li #  1 , Qi Wang  1   2 , Juan Min  1 , Yong Yang  1   2 , Bei Li  1 , Ren-Di Jiang  1 , Xi Wang  1   2 , Xiao-Shuang Zheng  1   2 , Yan Zhu  1 , Jing-Kun Jia  1   2 , Xing-Lou Yang  1 , Mei-Qin Liu  1   2 , Qian-Chun Gong  3   4 , Yu-Lan Zhang  1 , Zhen-Qiong Guan  1 , Hui-Ling Li  1   2 , Zhen-Hua Zheng  1 , Zheng-Li Shi  1 , Hui-Lan Zhang  5 , Ke Peng  1   2 , Peng Zhou  6   7
Affiliations

ACE2-independent infection of T lymphocytes by SARS-CoV-2

Xu-Rui Shen et al. Signal Transduct Target Ther. .

Abstract

SARS-CoV-2 induced marked lymphopenia in severe patients with COVID-19. However, whether lymphocytes are targets of viral infection is yet to be determined, although SARS-CoV-2 RNA or antigen has been identified in T cells from patients. Here, we confirmed that SARS-CoV-2 viral antigen could be detected in patient peripheral blood cells (PBCs) or postmortem lung T cells, and the infectious virus could also be detected from viral antigen-positive PBCs. We next prove that SARS-CoV-2 infects T lymphocytes, preferably activated CD4 + T cells in vitro. Upon infection, viral RNA, subgenomic RNA, viral protein or viral particle can be detected in the T cells. Furthermore, we show that the infection is spike-ACE2/TMPRSS2-independent through using ACE2 knockdown or receptor blocking experiments. Next, we demonstrate that viral antigen-positive T cells from patient undergone pronounced apoptosis. In vitro infection of T cells induced cell death that is likely in mitochondria ROS-HIF-1a-dependent pathways. Finally, we demonstrated that LFA-1, the protein exclusively expresses in multiple leukocytes, is more likely the entry molecule that mediated SARS-CoV-2 infection in T cells, compared to a list of other known receptors. Collectively, this work confirmed a SARS-CoV-2 infection of T cells, in a spike-ACE2-independent manner, which shed novel insights into the underlying mechanisms of SARS-CoV-2-induced lymphopenia in COVID-19 patients.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Peripheral blood lymphocytes are infected by SARS-CoV-2 in COVID-19 patients. a Percentage of different types of lymphocytes in the healthy donors (n = 15) or in COVID-19 patients (n = 22). (b) Percentage of CD4 + and CD8 + T lymphocytes in healthy donor (n = 8) or in COVID-19 patients (n = 9). c, d Immunofluorescent test of the presence of SARS-CoV-2 viral antigen in T cells. PBCs (c) or postmortem lung section (d) from COVID-19 patients were stained with T lymphocytes (CD3, green), SARS-CoV-2 (NP, red) and nuclei (DAPI, blue). In-house-made pAb against SARS-CoV-2 NP was used. White arrows indicate areas T lymphocytes that were infected by SARS-CoV-2. Pictures were taken under confocal microscopy with a bar = 50 μm (c) or 200 μm (d). Comparison of mean values (a, b) between two groups was analyzed by Student’s t test. *P < 0.05; **P < 0.01; ****P < 0.0001; NS no significance
Fig. 2
Fig. 2
SARS-CoV-2 in vitro infection of T cell lines or primary T cells. a Schedule of experiments. b Unactivated or activated Jurkat cells were infected with SARS-CoV-2 (MOI = 0.1) and samples were collected at 0, 24, 48, and 72 h post infection. Viral load in cells or cell supernatant was then quantified by qPCR detection of total viral RNA (RBD of spike gene as target) or subgenomic RNA (sgRNA, M gene as target). c Depth and coverage comparison for SARS-CoV-2 0 h or 24 h-infected activated Jurkat cells. For each sample, virus quantity was normalized with its total reads number of sequencing. Two replicates are shown for each time point. d Viral NP in the infected activated Jurkat cells and cell supernatant was analyzed by western blot at 0, 24, 48, and 72 h post infection. e Viral NP 72 h-infected cells from (d) were analyzed by flow cytometry and the number of replicates that are represented in the bar graph is three. f Viral particles in infected activated Jurkat or MT4 cells were observed by transmission electron microscope. bar = 1 μm or 500 nm, magnification: 3500-folds and 9600-folds for Jurkat cell, 5000-folds or 11500-folds for MT4 cell. g Unactivated or activated primary T cells were infected with SARS-CoV-2 (MOI = 0.01) and samples were collected at 0, 4, 8, and 12 h post infection. Viral load in cells was then quantified by qPCR. h Colon organoids were infected with SARS-CoV-2 (MOI = 0.01). Zero hour and 24 h samples were harvested and quantified by qPCR. The data were analyzed by Student’s t test and statistical significance is indicated by the asterisks (*P < 0.05; **P < 0.01; ****P < 0.0001; NS no significance)
Fig. 3
Fig. 3
SARS-CoV-2 infection of T cell is spike-ACE2/TMPRSS2-independent. a The ACE2 expression level of Scramble or ACE2-knockdown Caco2 or Jurkat cells was analyzed by qPCR or WB. b ACE2 stably knockdown Caco2 or activated Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01). Viral RNA or viral NP in cells was analyzed by qPCR or WB at 24 h post infection. c The ACE2 expression level of control or ACE-knockout Caco2 or Jurkat cells were quantified by qPCR or detected by WB. d ACE2 stably knock-out Caco2 or activated Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01). Viral RNA or viral NP was detected using qPCR or WB. e For ACE2 blocking, Caco2 or activated Jurkat cells were pre-incubated with anti-ACE2 Ab (3.33 ng/μl final) before infected with SARS-CoV-2. For virus blocking, ACE2-Fc protein (10 μg/μl final) or RD#4-anti-Spike Ab (160 ng/μl final) were incubated with SARS-CoV-2 at a volume of 1:1 at 37 °C for 30 min. Cells were infected at 0.01 MOI for 24 h before they were quantified for SARS-CoV-2 viral RNA or NP protein by qPCR or WB. f The TMPRSS2 expression level of Caco2, Jurkat and activated Jurkat cells was analyzed using qPCR. g Caco2 or activated Jurkat cells were pre-incubated with Camostat (2 μM or 20 μM) for 1 h and then infected with SARS-CoV-2 (MOI = 0.01). Viral RNA or NP proteins were quantified. The results were derived from three independent experiments. Statistical analyses were carried out using Student’s t test (*P < 0.05; **P < 0.01; ****P < 0.0001; NS no significance)
Fig. 4
Fig. 4
SARS-CoV-2 infection-induced apoptosis in T cells. a Detection of apoptotic T lymphocytes in human PBCs. PBCs were prepared from COVID-19 patients or from healthy donors. Apoptosis in virally infected T lymphocytes were determined using CD3, SARS-CoV-2 NP antibodies, and TUNEL assay. Detection results for two patients and one healthy donor, or the statistics of apoptotic cells or TUNEL/NP double-positive cells were shown for healthy donors (H, n = 3) or patients (P, n = 5). Comparison of mean values between two groups was analyzed by Student’s t test. *P < 0.05. b Unactivated or activated primary T cells were infected with SARS-CoV-2 (MOI = 0.01) for 8 h and cell apoptosis was analyzed with TUNEL assay. **P < 0.01. c Activated Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01) for 0, 24, 48, and 72 h. The ratio of apoptotic cells in 72 h mock or SARS-CoV-2-infected cells were shown (flow chart). The ratio of apoptotic cells in different time points were also compared (plot). d Samples from C were subjected for RNA-seq analysis. The top ten upregulated GO pathways in 48 h compared to 24 h group are shown. Heatmap shows the normalized expression of genes that were enriched from PID HIF1 pathway. The data were analyzed by Student’s t test (*P < 0.05; **P < 0.01; NS no significance). e Compared to healthy donors, differentially expressed genes of severe COVID-19 patients were shown in the volcano plot, and the top ten differential expressed pathways were shown in the right panel
Fig. 5
Fig. 5
Exploration of potential receptors in T cells. a The expression of ACE2, TMPRSS2, and ITGB2 (LFA-1) in blood T cells from healthy donors and COVID-19 patients. The analysis is dependent on public single-cell NGS data. The expression of the three genes is indicated in SARS-CoV-2 viral RNA-positive T cells for patient penal. b BEAS-2B and activated Jurkat cells were incubated with different concentrations of AXL proteins (25, 50, or 100 μg/ml) at 37 °C for 30 min before infected by SARS-CoV-2 (MOI of 0.01). Intracellular viral RNA (RBD) at 24 h post infection was quantified using qPCR. c Knockdown or overexpression of AXL were performed on Jurkat cells and the expression level of AXL was detected using qPCR. Cell lines were infected by SARS-CoV-2 at an MOI of 0.01 for 24 h and viral RNA was quantified. d LFA-1 was stably overexpressed on ACE2 knockdown Caco2 (Caco2-ACE2-shRNA) or Jurkat cells, and the RNA level was quantified using qPCR. e Caco2, activated Jurkat and their respective LFA-1-overexpression cells were infected by SARS-CoV-2 at an MOI of 0.01 and harvested at 24 h post infection. Intracellular viral RNA was detected using qPCR. f, g Caco2, Caco2-ACE2-shRNA, and its LFA-1 overexpression cell line were infected by SARS-CoV-2 at an MOI = 5 for 8 h. The high content microscope was used to observe (f) and quantify (g) the viral NP-positive cells. h LFA-1 knockdown Jurkat cells were infected with SARS-CoV-2 (MOI = 0.01) for 24 h, and the expression of LFA-1 and intracellular viral RNA was analyzed using qPCR. i Activated Jurkat cells were pretreated with Lifitegrast, an inhibitor of LFA-1 at different concentrations (50, 100, or 200 nM) at 37 °C for 30 min before infection (MOI = 0.01). Intracellular viral RNA was quantified at 24 h post infection. The statistics was performed using Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS no significance)
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