SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway

Abstract

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.

Keywords: HLA-E; NK cell; NKG2A; SARS-CoV-2.

Figure 1

Peripheral blood NK cells from four control subjects negative for both SARS-CoV-2 and SARS-CoV viremia were cultured NK cells in the presence of SP1, SP2 from SARS-CoV-2, or spike protein (S) from SARS-CoV. (A) NK cell migration is reported as the cell number. We used CXCL12 as a positive control for NK cell migration. NK cells with no treatment were used as negative control (NK cells). (B) Secretion of IFN-gamma evaluated by ELISA. IL-12 treatment was used as the control of IFN-gamma secretion. (C) Representative intracellular expression of SP1, SP2, and S proteins in K562 cells after transfection. (D) K562 viability, assessed by the LDH assay, in basal and transfected conditions. The transfection with 0.5 µg of control fluorescent antibody was used as a positive control (null-transfected). (E) Representative histograms of NK cell degranulation towards the K562 cell target. NK cells were marked with CD56-PECy7 (see Figure S1C). The degranulation was assessed by CD107a-PE expression. (F) Mean fluorescence intensity (MFI) of CD107a-positive NK cells after the co-culture with K562 target cells. (G) CFSE+/7-AAD+ cell percentage in NK cell/K562 co-cultures. K562 alone and treated with Triton x-100 (0.8%) were used as negative and positive controls, respectively. The values are presented as mean ± standard deviation. *, ** significant p values Student t test.

Figure 2

(A) Representative intracellular expression of SP1, SP2, and S proteins and surface expression of the epithelial marker CK8-PE in Beas-2B cells after transfection. (B) Beas-2B viability, assessed by the LDH assay, in basal and transfected conditions. The transfection with 0.5 µg of control fluorescent antibody was used as a positive control (null-transfected). The values are presented as mean ± standard deviation.

Figure 3

(A) Representative histograms of NK cell degranulation towards the Beas-2B cell target. NK cells were marked with CD56 and gated as reported in Figure S1C. The degranulation was assessed by CD107a-PE expression. Beas-2B was transfected with SP1, SP2, and S proteins or control fluorescent antibody was used as the positive control (null-transfected), or treated with GATA3 inhibitor (anti-GATA). (B) MFI of CD107a-PE-positive NK cells after the co-culture with Beas-2B cells. (C) CFSE+/7-AAD+ cell percentage in NK cell/Beas-2B co-cultures. Beas-2B alone and treated with Triton-x100 (0.8%) were used as negative and positive controls, respectively. The values are presented as mean ± standard deviation. (D) Representative histograms of HLA-I expression on the surface of transfected Beas-2B. (E) The histograms showed the mean ± standard deviation MFI (mean fluorescence intensity) values of HLA-I expression in three independent experiments. * significant p values Student t test; (F) Representative histograms of HLA-E expression on the surface of transfected Beas-2B cells. (G) The histograms showed the mean ± standard deviation MFI (mean fluorescence intensity) values of HLA-E expression in three independent experiments. * significant p values t test. (H) Relative ratio of Real-Time PCR of HLA-E expression in transfected Beas-2B cells. SP1 transfected Beas-2B cells were also treated with GATA inhibitor (anti-GATA). * significant p values Student t test.

Figure 4

(A) Representative histograms of GATA3 expression on the surface of Beas-2B cells after transfection with SP1, SP2, and S proteins or control fluorescent antibody was used as a positive control (null-transfected) or treated with GATA inhibitor (anti-GATA). (B) The histograms showed the mean ± standard deviation MFI (mean fluorescence intensity) values of GATA3 expression in three independent experiments. * significant p values Student t test. (C) Relative ratio of real-time PCR of GATA3 expression in transfected Beas-2B cells. SP1-transfected Beas-2B cells were also treated with GATA inhibitor (anti-GATA). (D) Relative ratio of real-time PCR of GATA3 target gene Eotaxin3/CCL26 in transfected Beas-2B cells. SP1-transfected Beas-2B cells were also treated with GATA inhibitor (anti-GATA). * significant p values Student t test.

Figure 5

HLA-E expression in Beas-2B cells was characterized by immunofluorescence (Nikon Eclipse TE2000S, equipped with a digital camera). The evaluation was assessed after SP1 transfection (SP1) without or with GATA inhibitor (antiGATA inhibitor). (A) The cells were stained with Hoechst for nuclear detection and anti-HLA-E-PE monoclonal antibody (clone MEM-E/08, Exbio, Praha, Czech Republic). Original magnification 40×. (B) Percentage of HLA-E positive (+) cells in the Beas-2B and after SP1 transfection (SP1) without or with GATA inhibitor (antiGATA).

Figure 6

(A) Representative dot plots of NKG2A/CD94 and CD107a expression on the surface of NK cells after co-culture with Beas-2B transfected with SP1, SP2, and S proteins or control fluorescent antibody was used as a positive control (null-transfected). (B) The histograms show the mean ± standard deviation percentage of NKG2A/CD94-positive cells in three independent experiments. * significant p values Student t test. (C) Relative ratio of real-time PCR of NKG2A expression in transfected Beas-2B cells. * significant p values Student t test.

Figure 7

(A) Representative histograms of NK cell degranulation towards Beas-2B cells transfected with SP1 protein without or with anti-HLA-E, anti-NKG2A, or anti-isotype control. NK cells were marked with CD56 and gated as reported in Figure S1C. The degranulation was assessed by CD107a-PE expression. (B) MFI of CD107a in NK cells after the co-culture with Beas-2B cells transfected with SP1 protein without or with anti-HLA-E, anti-NKG2A, or anti-isotype control. The values are presented as mean ± standard deviation. * significant p values Student t test. (C) Secretion of IFN-gamma evaluated by ELISA. IL-12 treatment was used as a control of IFN-gamma secretion.