Notch4 regulatory T cells and SARS-CoV-2 viremia shape COVID19 survival outcome

Abstract

Background: Immune dysregulation and SARS-CoV-2 plasma viremia have been implicated in fatal COVID-19 disease. However, how these two factors interact to shape disease outcomes is unclear.

Methods: We carried out viral and immunological phenotyping on a prospective cohort of 280 patients with COVID-19 presenting to acute care hospitals in Boston, Massachusetts and Genoa, Italy between June 1, 2020 and February 8, 2022. Disease severity, mortality, plasma viremia, and immune dysregulation were assessed. A mouse model of lethal H1N1 influenza infection was used to analyze the therapeutic potential of Notch4 and pyroptosis inhibition in disease outcome.

Results: Stratifying patients based on %Notch4⁺ Treg cells and/or the presence of plasma viremia identified four subgroups with different clinical trajectories and immune phenotypes. Patients with both high %Notch4⁺ Treg cells and viremia suffered the most disease severity and 90-day mortality compared to the other groups even after adjusting for baseline comorbidities. Increased Notch4 and plasma viremia impacted different arms of the immune response in SARS-CoV-2 infection. Increased Notch4 was associated with decreased Treg cell amphiregulin expression and suppressive function whereas plasma viremia was associated with increased monocyte cell pyroptosis. Combinatorial therapies using Notch4 blockade and pyroptosis inhibition induced stepwise protection against mortality in a mouse model of lethal H1N1 influenza infection.

Conclusions: The clinical trajectory and survival outcome in hospitalized patients with COVID-19 is predicated on two cardinal factors in disease pathogenesis: viremia and Notch4⁺ Treg cells. Intervention strategies aimed at resetting the immune dysregulation in COVID-19 by antagonizing Notch4 and pyroptosis may be effective in severe cases of viral lung infection.

Keywords: COVID19; Notch4; pyroptosis; regulatory T cells; survival; viremia.

Figure 1.. Notch4⁺ Treg cells and plasma viremia are independently and combinatorially associated with COVID-19 disease severity and mortality.

A and B, %Notch4⁺ Treg cells among CD4⁺ T cells and plasma viremia in healthy controls, COVID-19 patients with mild, moderate, severe, and critical illness using the WHO definition for COVID-19 severity. The overall comparison of the Kruskal-Wallis’s tests for both A and B were statistically significant (P < 0.001). Only significant pairwise comparisons are included in the figure for ease of interpretation. C, Pie chart stratified by %Notch4 Treg (using a cutoff of 24.5%) and presence of plasma viremia groups. Colors indicate proportion of patients with mild, moderate, severe, and critical disease. The overall Fisher’s Exact Test P <0.001, indicating significant differences between four groups of %Notch4 Treg and plasma viremia and COVID-19 severity. Pairwise comparison of Fisher’s exact test conducted with a multiple adjustment using Bonferroni correction. Only significant pairwise comparisons are included in the figure for ease of interpretation. D-F, Kaplan-Meier curves for 90 day survival stratified by discretized %Notch4 Treg (E), by presence or absence of SARS-CoV-2 detected in plasma (F) or by both %Notch4 Treg and plasma viremia. G, predicted 90-day survival curves based on Cox proportional hazards model adjusting for age, gender, Charlson comorbidity index, IL-6 and time to blood collection. Significance value indicators. NS P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 2.. Notch4⁺ Treg cells and plasma viremia define patient subgroups with different Clinical trajectories and immunological features.

A, Clinical variables and plasma cytokine levels in healthy controls and COVID-19 patients stratified by Treg %Notch4 and plasma viremia. Colors in heatmap indicate average Z-score values for continuous variables while height of barplot indicates proportion of that group with that outcome or receiving treatment for binary variables. B. Flow-tSNE plot of Teff and Treg TCM (CCR7⁺CD45RA^–), naïve (CCR7⁺CD45RA⁺), effector memory (CCR7^–CD45RA^–) and TEMRA (CCR7^–CD45RA⁺) cells in representative samples. C, Flow-tSNE plot of Teff and Treg cells, from anti-CD3, CD4, CD25, CD127, Notch1, Notch2, Notch3, Notch4, and FOXP3 staining in representative samples. D. Heatmap of CD4+ T cell populations in healthy controls and COVID-19 patients stratified by Treg %Notch4 and plasma viremia. Colors indicate average Z-score values for that group. E. Non-metric Multi-dimensional Scaling (NMDS) plot of Gower’s distance using clinical variables, plasma cytokine levels, and flow cytometry of CD4+ T cells. Categorizing patients by Treg %Notch4 and plasma viremia explains differences in clinical and immunological features between patients (PERMANOVA R² 0.113, p < 0.001). Each symbol represents one patient.

Figure 3.. Notch4⁺ Treg cells and plasma viremia associate with distinct immunological responses.

A.B. Flow cytometry analysis (A) and cell Frequencies of Areg+ Treg cells (B) in circulating Treg cells from Healthy control (n=5) and COVID-19 patients with Notch4^Lowno viremia (n=14), Notch4^highno viremia (n=15), Notch4^Low(+)viremia (n=11) and Notch4^high(+) viremia (n=11) C, In vitro suppression assay of CD4⁺ T cells (T_eff) by T_reg cells from COVID-19 patients stratified by Treg %Notch4 and plasma viremia. D,E. Flow cytometry analysis (D) and cell Frequencies of Cleaved Gasdermin D (E) in circulating monocytes from Healthy control (n=5) and COVID-19 patients with Notch4^Lowno viremia (n=14), Notch4^highno viremia (n=16), Notch4^Low(+)viremia (n=17) and Notch4^high(+) viremia (n=16). Error bars indicate SEM. Statistical tests: One-way ANOVA with Dunnett’s post hoc analysis (B, C,E) *P<0.05, **P<0.01.

Figure 4.. Protective effect of Treg cell Notch4 and pyroptosis inhibition in severe lung virus infection.

A, Survival of Foxp3^YFPCre or Foxp3^YFPCreNotch4^Δ/Δ mice infected with a lethal dose of H1N1 virus that were either sham-treated (n=17) or treated (n=20) with LDC7559, as indicated. B, Weight indices of Foxp3^YFPCre or Foxp3^YFPCreNotch4^Δ/Δ mice infected with a sublethal dose of H1N1 virus that was either sham-treated (n=4) or treated (n=4) with LDC7559, as indicated. C, Cell frequencies of cleaved Gasdermin D expression in lung tissue CD11b⁺ of the respective mouse groups (n=4). D, Flow cytometric analysis and cell frequencies of NLRP3⁺ expression in lung tissue monocytes of respective groups infected with a sublethal dose of H1N1 virus (n=4). E, Flow cytometric analysis and cell frequencies of IFNγ and IL-17 expression in lung tissue CD4⁺ Teff cells of respective groups infected with a sublethal dose of H1N1 virus (n=4). F, Cell frequencies and numbers of lung tissue CD4⁺ Teff cells of respective groups infected with a sublethal dose of H1N1 virus (n=4). G, Flow cytometric analysis and cell frequencies of IFNγ and IL-17 expression in lung tissue CD8⁺ Teff cells of respective groups infected with a sublethal dose of H1N1 virus (n=4). H, Cell frequencies and numbers of lung tissue CD4⁺ Teff cells of respective groups infected with a sublethal dose of H1N1 virus (n=4). Each symbol represents one mouse. Numbers in flow plots indicate percentages. The results represent a pool of 2–4 experiments. Error bars indicate SEM. Statistical tests: log-rank-test (A), One-way ANOVA with Dunnett’s post hoc analysis (B, C, D, E) *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.