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Authors: Bortolotti, D. et al.

Link to paper: https://www.researchsquare.com/article/rs-31860/v1

Journal/ Pre-Print: Research square

Tags: Immunology/Immunity, Inflammation, Molecular biology, Cell Biology

Research Highlights 

1. SARS-Cov-2 spike proteins 1 (SP1) and 2 (SP2), as well as the full spike trimer of previous SARS-CoV promote NK migration in vitro

2. SP1 promotes downregulation of classical HLA and upregulation of non-classical HLA-E in lung epithelial cell line via GATA3

3. SP1 loaded HLA-E (IEDB prediction) promotes NK cell exhaustion via NKG2A upregulation and HLA-E/NKG2A interaction

Summary 

It has been published that SARS-Cov-2 patients have fewer NK cells that also display an exhausted phenotype. Here, Bortolotti and al. transfect bronchial epithelial cell line Beas-2B with SP1, SP2 from SARS-CoV-2 or full spike protein form SARS-CoV. SP1 expression alone downregulated classical HLAs in Beas-2B, while GATA3 driven expression of the non-classical HLA-E increased. One of the peptides in SP1 domain is predicted to bind HLA-E specifically. NK cells co-cultured with SP1 expressing Beas-2B upregulate NKG2A, an inhibitory ligand for HLA-E, and exhibit decreased CD107a expression and cytotoxic activity. Additionally, authors show that all spike proteins tested exert chemotactic activity on NK cells.

Impact for SARS-CoV2/COVID19 research efforts

Understand the immune response to SARS-CoV2/COVID19

The study shows how presentation of SP1 by non-classical HLA-E on lung epithelium could cause exhaustion of NK cells

Study Type

· In vitro study

Strengths and limitations of the paper

Novelty: The study shows for the first time how SARS-Cov-2 SP1 could inhibit NK cell activation thus causing immune evasion

Standing in the field: Supports the literature in that immune evasion and impaired lymphocytes play a role in the pathogenesis of COVID-19. Also, it has previously been shown that NKG2A on the surface of NK cells was increased during COVID-19.

Appropriate statistics: No. Kruskal- Wallis inappropriate as N is too small. It compares mean sum of the ranks to measure whether any one of the test groups are different. It doesn't tell which groups are different, so it requires a post hoc. T test used as post hoc inappropriate as we don't know about the distribution. Welch post hoc would be suitable. Another option would be One-way ANOVA.

Viral model used: purified Spike proteins (SP1 and SP2 from SARS-CoV2) and full Spike from previous SARS-CoV

Translatability: remote- suggests that preventing NK cell exhaustion trough the inhibition of NKG2A with e.g. monoclonal antibody monalizumab would be a potential therapy

Main limitations:

§ The author does not suggest a possible mechanism by which SP proteins could cause NK cell migration, there is lack of a negative control with a non-stimulating protein in high concentration in the transwell.

§ A high number of NK cells in Figure 1B are producing IFN-g even without stimulation and without BrefeldinA treatment, it is unclear what is activating this NK cells and if an isotype control has been used in the experiment.

§ In Figure 5D not only anti HLA-E and anti-NKG2A antibodies restore CD107a expression but also the isotype control, making the experiment impossible to interpret, also cytotoxicity is only analysed by %CD107a+ cells but not directly with cell killing assays

§ Authors test subunit 1 and 2 of SARS-CoV2 and the whole trimer in the case of SARS-CoV. Unclear why not the whole SARS-CoV2 trimer instead.

§ It would be nice to validate these observations regarding NK cell function with Beas 2B cells infected with SARS-CoV2