Chemokines, soluble PD-L1, and immune cell hyporesponsiveness are distinct features of SARS-CoV-2 critical illness

Abstract

Critically ill patients manifest many of the same immune features seen in coronavirus disease 2019 (COVID-19), including both "cytokine storm" and "immune suppression." However, direct comparisons of molecular and cellular profiles between contemporaneously enrolled critically ill patients with and without severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited. We sought to identify immune signatures specifically enriched in critically ill patients with COVID-19 compared with patients without COVID-19. We enrolled a multisite prospective cohort of patients admitted under suspicion for COVID-19, who were then determined to be SARS-CoV-2-positive (n = 204) or -negative (n = 122). SARS-CoV-2-positive patients had higher plasma levels of CXCL10, sPD-L1, IFN-γ, CCL26, C-reactive protein (CRP), and TNF-α relative to SARS-CoV-2-negative patients adjusting for demographics and severity of illness (Bonferroni P value < 0.05). In contrast, the levels of IL-6, IL-8, IL-10, and IL-17A were not significantly different between the two groups. In SARS-CoV-2-positive patients, higher plasma levels of sPD-L1 and TNF-α were associated with fewer ventilator-free days (VFDs) and higher mortality rates (Bonferroni P value < 0.05). Lymphocyte chemoattractants such as CCL17 were associated with more severe respiratory failure in SARS-CoV-2-positive patients, but less severe respiratory failure in SARS-CoV-2-negative patients (P value for interaction < 0.01). Circulating T cells and monocytes from SARS-CoV-2-positive subjects were hyporesponsive to in vitro stimulation compared with SARS-CoV-2-negative subjects. Critically ill SARS-CoV-2-positive patients exhibit an immune signature of high interferon-induced lymphocyte chemoattractants (e.g., CXCL10 and CCL17) and immune cell hyporesponsiveness when directly compared with SARS-CoV-2-negative patients. This suggests a specific role for T-cell migration coupled with an immune-checkpoint regulatory response in COVID-19-related critical illness.

Keywords: ARDS; COVID-19; PD-L1; checkpoint pathway; pneumonia; sepsis.

Figure 1.

Interferon-induced mediators distinguish subjects with SARS-CoV-2. Volcano plots display differentially expressed proteins between SARS-CoV-2-positive and -negative subjects adjusted for age, sex, ethnicity, APACHE III score, tocilizumab, and steroid administration. Dashed line indicates Bonferroni-corrected P value = 0.05. Dotted line indicates a nominal P value = 0.05. A: proteins measured from plasma in all subjects (SARS-CoV-2-positive: n = 204; SARS-CoV-2-negative: n = 122). B: proteins measured from plasma in SARS-CoV-2-positive subjects (n = 204) vs. SARS-CoV-2-negative subjects with a primary diagnosis of pneumonia, sepsis, or ARDS (n = 62). APACHE III, acute physiology, age, chronic health evaluation; ARDS, acute respiratory distress syndrome; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

Figure 2.

Distinct associations between plasma immune mediators and ventilator-free days (VFDs) in SARS-CoV-2-positive and -negative subjects. Forrest plots display the β (95% CI) for tertiles of VFDs according to each plasma mediator adjusted for age, sex, corticosteroid administration, tocilizumab administration, and interval of days between SARS-CoV-2 test and enrollment. Mediators are ranked by P values for associations with VFDs in the SARS-CoV-2-positive subjects (point estimates for each mediator are provided in Supplemental Table S6). Dashed line (and red color) indicates Bonferroni-corrected P values < 0.05. Dotted line (and gold color) indicates nominal P values < 0.05. P values for interaction were calculated by incorporating an interaction term for the product of SARS-CoV-2 infection and log₂ plasma mediator level in the statistical model (regress “mean”: vfds ∼ log₂(mediator) X sars-cov-2-status + age + sex + covid-test-interval + corticosteroids + tocilizumab). CI, confidence interval; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

Figure 3.

SARS-CoV-2 is characterized by circulating immune cell hyporesponsiveness. The panels show individual percentages (dots); medians (thick line); and interquartile ranges (error bars). Statistical analyses were performed with Mann–Whitney tests. Biaxial gating of cell populations is shown in Supplemental Fig. S2. A: percentage of monocytes positively staining for IL-8, IL-6, or TNF-α as a proportion of total CD33⁺ monocyte events in response to LPS stimulation. B: percentage of cells positively staining for either IFN-γ or TNF-α as a proportion of all CD3⁺CD4⁺ or CD3⁺CD8⁺ events in response to PMA/ionomycin or anti-CD3/CD28 stimulation. The combined data set includes values from either stimulation. C: percentage of CXCR3⁺ (ligand: CXCL10) cells as a proportion of total CD4⁺ or CD8⁺ T cells, respectively. Percentage of PD-L1⁺ or PD-1⁺ as a proportion of total CD33⁺ monocytes or CD8⁺ T cells, respectively. LPS, Lipopolysaccharide; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.

Figure 4.

Correlations between immune cell subsets and soluble mediators in SARS-CoV-2-negative and -positive subjects. Matrices displaying the correlation coefficients (r) between immune cell subsets (Y-axis) and plasma mediator levels (X-axes). Colors represent the correlation with scale indicating value of Pearson’s r correlation.