PMC8089458_01
Timelines of progression of the clinical courses of the 2 patients with confirmed SARS-CoV-2 infection. Red boxes show the events for patient 1 and green boxes show the events for patient 2. ABL, amphotericin B liposomal; ARDS, acute respiratory distress syndrome; CT, computed tomography; ED, emergency department; FiO2, fraction of inspired oxygen; HCQ, hydroxychloroquine; LMWH, low-molecular-weight heparin; Lop/R, lopinavir/ritonavir; MPS, methylprednisolone; MSSA, methicillin-sensitive Staphylococcus aureus; MV, mechanical ventilation; OS, oxygen saturation; PE, pulmonary embolism; q.i.d., 4 times a day; QTc, corrected QT interval; TC, tocilizumab.
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F1
PMC8089458_02
Gal-9/TIM-3 axis showing disparate levels and patterns for patients 1 and 2. a The plasma levels of soluble Gal-9 (sGal-9) from patients 1 and 2, collected until exitus or discharge, respectively, and the mean levels for 10 healthy volunteers (HVs) with negative SARS-CoV-2 RT-PCR results are shown. b A final reading from patient 2, obtained 70 days after disease onset, is also shown. The mean fluorescence intensities (MFI) of TIM-3 on gated CD3+ cells are shown. c Percentage of TIM-3+ cells on gated CD3+ cells. d The percentages of CD3+ cells from total leukocytes are shown. e, f The percentages of CD4+ (e) and CD8+ (f) cells on CD3+ cells are shown. g, h The MFI of TIM-3 (g) and the percentage of TIM-3+ cells (h) on gated CD4+ cells are shown. i, j The MFI of TIM-3 (i) and the percentage of TIM-3+ cells (j) on gated CD8+ cells are shown. k The t-SNE map clustering expression of TIM-3 on CD4+ and CD8+ populations is shown. a-j Data from patient 1 are shown in red, and data from patient 2 are shown in green. Dashed lines represent the calculated means from the analysis of a single sample from 10 HVs (mean +- SD). Arrows indicate important clinical events (patient 1: ICU admission, sepsis, and septic shock; patient 2: discharge).
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F2
However, a significantly different progression was observed after 8 days of illness (Fig. 2a). IC at the levels reported for 10 healthy volunteers (Fig. 2b, c). In addition, the percentages of circulating CD3+ cells as well as CD4+ and CD8+ subpopulations from both patients were different, with a patent low level in patient 1, which remained low during the whole longitudinal temporal series (Fig. 2d-f). Moreover, TIM-3 levels were markedly higher on both the CD4+ and CD8+ T cells from patient 1 compared with patient 2 (Fig. 2g-j), an effect that was more pronounced over time. t-SNE cluster map analysis of TIM-3 expression on CD4+ and CD8+ T cells also illustrated these differences (Fig. 2k).
Soluble plasma levels of immune checkpoints from patients 1 and 2. a-f The plasma levels of soluble immune checkpoints from patients 1 and 2, collected until exitus or discharge, respectively, and the calculated means from the analysis of a single sample from 10 healthy volunteers (HV) with negative SARS-CoV-2 RT-PCR results. A final reading from patient 2, obtained 70 days after disease onset, is also shown. Soluble levels of PD-L1 (a), PD-1 (b), LAG-3 (c), CTLA-4 (d), 4-1BB (e), and CD86 (f) are shown in the respective panels. g The mean fluorescence intensities (MFI) of HLA-DR on gated CD14+ monocytes are shown. a-g Data from patient 1 are shown in red, and data from patient 2 are shown in green. Dashed lines represent the calculated means from the analysis of a single sample from 10 HVs (mean +- SD). Arrows indicate important clinical events (patient 1: ICU admission, sepsis, and septic shock; patient 2: discharge).
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ICs and ligands (Fig. 3a-f), reinforcing the findings of disparate immune responses observed in the patients throughout their illness. Interestingly, sCD86 and monocyte expression of HLA (human leukocyte antigen)-DR isotype showed similar patterns, pointing to a drastic reduction in the antigen presentation ability of patient 1 from onset (Fig. 3f, g).
Effector memory T-cell analysis. a Gating strategy (TEM, effector memory T cells; TCM, central memory T cells; TEM-RA, terminally differentiated T cells; Tnaive, naive T cells). b, c The analyses of TEM on both gated CD4+ (b) and CD8+ (c) cells are shown. Left panels: the percentages of TEM are shown. Central left panels: the mean fluorescence intensities (MFI) of PD-1 on gated CD4+ and CD8+ TEM are shown. Central right panels: the percentages of CD57+ cells on gated CD4+ and CD8+ TEM are shown. Right panels: the MFI of CD95 on gated CD4+ and CD8+ TEM are shown. b, c Note that data from patient 1 are shown in red, while the data from patient 2 are shown in green. The dashed lines represent the calculated means from the analysis of a single sample from 10 healthy volunteers (HVs) (mean +- SD). Arrows indicate important clinical events (patient 1: ICU admission, sepsis, and septic shock; patient 2: discharge).
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F4
Due to the impact of effector memory T cells (TEM, defined as CD3+CD45RA-CD27-, see gating strategy in Fig. 4a) on resolving SARS-CoV-2 infection, we analyzed their percentages in circulating blood from the study patients. Both CD4+ and CD8+ TEM levels showed a downward curve for patient 1 but not for patient 2 (Fig. 4b, c, left panels). TEM was added to this effect in the latter points and on CD8+ TEM some time earlier (Fig. 4b, c, central left panels). The expression of CD57, another reported senescence marker, did not show high levels in patient 1, except at the final point on CD8+ TEM (Fig. 4b, c, central right panels). Eventually, upregulation of the proapoptotic factor CD95 was patent on CD8+ TEM from an early point in patient 1, which was also evident on CD4+ TEM after sepsis onset (Fig. 4b, c, right panels).
Gal-9/TIM-3 axis showing disparate levels in patients with different outcomes. Blood samples from 57 COVID-19 patients, recruited on admission and before any treatment, were analyzed. a Plasma levels of soluble Gal-9 (sGal-9) from COVID-19 patients classified according to their outcome (survivors or exitus) and healthy volunteers (HVs) are shown. b-d The percentages of TIM-3+ cells on CD3+ (b), CD4+ (c), and CD8+ (d) cells are shown. a-d Kruskal-Wallis statistics and multiple comparisons are shown.
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F5
As Figure 5 shows, levels of soluble Gal-9 were significantly higher in exitus patients than in survivors.