ICOS-ICOSL pathway enhances NKT-like cell antiviral function in pregnant women with COVID-19

Abstract

Objective: The immune response initiated by SARS-CoV-2 infection in pregnancy is poorly elucidated. We aimed to access and compare the antiviral cellular responses and lymphocytes activation between healthy pregnancies and pregnant women infected with SARS-CoV-2. Methods: We detected the immunological changes of lymphocytes in peripheral blood of healthy non-pregnant women, non-pregnant women with COVID-19, healthy pregnant women, pregnant women with COVID-19 and convalescent group by flow cytometry. In vitro blockade was used to identify NKT-like cell activation through ICOS-ICOSL pathway. Results: We found that CD3⁺CD56⁺ NKT-like cells decreased significantly in COVID-19 positive pregnant women compared to healthy pregnant women. NKT-like cells of pregnant women expressed higher level of activating receptors CD69 and NKp46 after SARS-CoV-2 infection. Particularly, they also increased the expression of the co-stimulatory molecule ICOS. NKT-like cells of pregnant women with COVID-19 up-regulated the expression of IFN-γ, CD107a and Ki67. Meanwhile, we found that ICOSL expression was significantly increased on pDCs in pregnant women with COVID-19. Blocking ICOS in vitro significantly decreased the antiviral activity of NKT-like cells in COVID-19 positive pregnant women, suggesting that ICOS-ICOSL may play an important role in the virus clearance by NKT-like cells. Conclusions: During SARS-CoV-2 infection, NKT-like cells of pregnant women activated through ICOS-ICOSL pathway and played an important role in the antiviral response.

Keywords: CD3+CD56+ NKT-like cells; COVID-19; ICOS; Pregnancy.

Figure 1

CD3⁺CD56⁺ NKT-like cells decrease dramatically in pregnant women with COVID-19 compared with healthy pregnant women. (A-C) The cellularity of lymphocytes, monocytes and neutrophils in peripheral blood of healthy control (HC group), non-pregnant women with COVID-19 (COVID-19 group), healthy pregnant women control (PHC group), pregnant women with COVID-19 (PCOVID-19 group) and convalescent patients (PConvalescent group). (E-F) Representative flow cytometry graphs of CD4⁺ T cells, CD8⁺ T cells, CD56^bri NK cells, CD56^dim NK cells, CD3⁺CD56⁺ NKT-like cells and T cells. (D, G-K) Flow cytometry detected the percentages and absolute number of (D and G) CD8⁺ T cells, (H-I) CD3⁺CD56⁺ NKT-like T cells and (J-K) CD19⁺ B cells among HC group, COVID-19 group, PHC group, PCOVID-19 group, and PConvalescent group. Data are shown as mean ± SEM. One-way ANOVA was used to determine statistical significance. *P< 0.05; **P <0.01; ***P <0.001; ns, not significant.

Figure 2

The phenotype of CD3⁺CD56⁺ NKT-like cells in COVID-19 positive pregnant women. (A) The representative flow cytometry graphs of CD69⁺, NKp46⁺, ICOS⁺, NKG2A⁺, TIGIT⁺, PD-1⁺ NKT-like cells among the HC group, COVID-19 group, PHC group, PCOVID-19 group and PConvalescent group. (B-G) The expression of (B) CD69, (C) NKp46, (D) ICOS, (E) NKG2A, (F) TIGIT and (G) PD-1 on NKT-like cells among the HC group, COVID-19 group, PHC group, PCOVID-19 group and PConvalescent group. Results are shown as mean ± SEM. One-way ANOVA was used to determine statistical significance. *P< 0.05; **P <0.01; ***P <0.001; ns, not significant.

Figure 3

NKT-like cells of pregnant women enhanced their antiviral ability and proliferation rate after SARS-CoV-2 infection. (A) The representative flow cytometry graphs of CD107a⁺ NKT-like cells, IFN-γ⁺ NKT-like cells and Ki67⁺ NKT-like cells among the HC group, COVID-19 group, PHC group, PCOVID-19 group, PConvalescent group. (B-D) The expression of (B) CD107a, (C) IFN-γ and (D) Ki67 on NKT-like cells among the HC group, COVID-19 group, PHC group, PCOVID-19 group, PConvalescent group. Results are shown as mean ± SEM. One-way ANOVA was used to determine statistical significance. *P< 0.05; **P <0.01; ***P <0.001; ****P <0.001; ns, not significant.

Figure 4

The apoptotic levels of NKT-like cells among the HC group, COVID-19 group, PHC group, PCOVID-19 group and PConvalescent group. (A) Representative flow graphs of Annexin V and 7-AAD staining of NKT-like cells. (B-C) The statistical diagram of (B) Annexin V⁺7-AAD^- NKT-like cells and (C) Annexin V⁺7-AAD⁺ NKT-like cells among the HC group, COVID-19 group, PHC group, PCOVID-19 group and PConvalescent group. Results are shown as mean ± SEM. One-way ANOVA was used to determine statistical significance **P <0.01; ns, not significant.

Figure 5

Expression of ICOSL on plasmacytoid DCs and myeloid DCs in COVID-19 positive pregnant women. (A) Gating strategy for myeloid DCs (mDCs): Lin^- (CD3, CD19, CD20, CD56, CD66b) HLA-DR⁺CD123^-CD11c⁺ and plasmacytoid DCs (pDCs): Lin^- HLA-DR⁺CD123⁺CD11c^-. (B-C) The proportion of (B) mDCs and (C) pDCs among the HC group, COVID-19 group, PHC group, PCOVID-19 group, PConvalescent group. (D-E) The expression of ICOSL on (D) mDCs and (E) pDCs among the HC group, COVID-19 group, PHC group, PCOVID-19 group, PConvalescent group. (F)The representative flow cytometry graphs of ICOSL on pDCs among the HC group, COVID-19 group, PHC group, PCOVID-19 group and PConvalescent group. Expression of CD80 on (G) mDCs and (I) pDCs among the HC group, COVID-19 group, PHC group, PCOVID-19 group and PConvalescent group. (H) Representative histogram graphs for the expression of CD80 on pDCs. Results are shown as mean ± SEM. One-way ANOVA was used to determine statistical significance. *P <0.05; **P <0.01; ns, not significant.

Figure 6

ICOS blockade reduced the cytokine secretion level of NKT-like cells in COVID-19 positive pregnant women. (A) In vitro blocking effect of anti-ICOS on NKT-like cells. (B-D) The frequency of (B) CD107a⁺ NKT-like cells, (C) Granzyme B⁺ NKT-like cells and (D) IFN-γ⁺ NKT-like cells of COVID-19 positive pregnant women in the presence of anti-ICOS mAb or isotype mAb. Data are shown as mean ± SEM. Paired Student's t-tests were performed. *P< 0.05; **P <0.01; ns, not significant.