PMC8635722_01
Evolution of clinical, laboratory, and radiological parameters. (A) Timeline of the duration of patient stays in the intensive care unit (ICU), shown as illness days. OTI, orotracheal intubation; MV, mechanical ventilation; UC-MSC, umbilical cord-derived mesenchymal stromal cells. Arrows represent the time of MSC infusions; respective samples were collected prior to MSC infusion. *Day of orotracheal intubation. (B) Data are represented as single values or mean +- SD of the values of different measurements performed each day. (C) The representative images of chest CT scans were performed at different time points.
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Therapy, Sao Rafael Hospital, Salvador, Brazil and cryopreserved at passage 3 in 50 ml of a cryopreservation solution containing Plasmalyte, 3% human albumin, and 5% dimethyl sulfoxide (DMSO) and stored in cryobags at <-135 C. The identity of MSC was assessed by flow cytometry (Stemflow Human MSC Analysis kit, BD Biosciences, NJ, USA) and in vitro trilineage differentiation assays (StemPro Osteogenesis and Adipogenesis kits, Thermo Fisher Scientific, MA, USA) (Supplementary Figures 1A-E). Genetic stability was evaluated by G-band karyotype, as previously described (Supplementary Figure 1F). The variations in PaO2/FiO2, Sequential Organ Failure Assessment (SOFA) score, lymphocyte counts, CRP, ferritin, and D-dimer, along with the dynamic changes seen in chest CT are shown (Figure 1).
Expression profile of cytokines, chemokines, and growth factors by Luminex assay. (A) A heatmap was designed to depict the overall profile of biomarkers among times, according to log10 of Mean Fluorescent Intensity (MFI). A one-way hierarchical cluster analysis (Ward's method) was performed. The dendrograms represent Euclidean distance. (B) Enrichment analysis for each cluster at NCI Nature database. The top four pathways are shown, ranked by the p-value. (C) Interlukin (IL)-2Ra, IL-6, IL-18, and macrophage colony-stimulating factor (M-CSF) showed a higher fold-change (+-0.5) of MFI over time. The samples were collected on the day of the first MSC infusion (D1, pre-infusion), the second MSC infusion (D3, pre-infusion), and the 7th day following cell therapy (D7).
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Figure 2A), as described previously. Finally, the product hemocompatibility was tested by evaluating tissue factor (CD142) expression by flow cytometry and by performing thromboelastography (TEG) studies in the citrate blood samples obtained from three different donors, as previously described (Supplementary Figures 2B-D). Finally, the cell viability was checked before cryopreservation, 48 h after, and at the time of infusion, by flow cytometry with 7AAD (BD Biosciences) (Supplementary Figure 2E). On day 7, the levels of most cytokines approached the first measured value (Figures 2A,B). On the third cluster, the increase was more accentuated and was maintained at day 7 (Figure 2A). While the third was enriched to IL-12 signaling events (Figure 2B). The biomarkers that showed the greatest discrepancies (+-0.4-fold change) at day 3 were IL-2RA, IL-18, IL-6, and M-CSF (Figure 2C).
The classical monocytes (CD14++CD16-) are more prevalent up to 7 days after treatment initiation. (A) The frequency of classical (CD14++CD16-), inflammatory (CD14++CD16+), and patrolling (CD14+CD16+) monocytes (pink, green, and purple, respectively), exhibits the same overall profile until day 7. On day 14, there is an increase in the patrolling monocytes frequency, that exceeds 50% of all the monocyte populations. (B) Observing the frequencies, we notice that CCR5 and CCR7 are the most prevalent markers over time. (C) Median fluorescence intensity of HLD-DR among the monocytes subpopulation over period post-treatment. The assessment of monocyte polyfunctionality following overnight lipopolysaccharide (LPS) stimulation. (D) The frequency of IL-6, TF, TNF-a, and IL-1B expression is shown. (E) The frequencies of cytokine producer subpopulations are shown. (F) A heatmap was designed to depict the overall profile of cytokine producer subpopulation of monocytes overtimes. A one-way hierarchical cluster analysis (Ward's method) was performed. The dendrograms represent Euclidean distance. The samples were collected on the day of the first MSC infusion (D1, pre-infusion), second MSC infusion (D3, pre-infusion), and the 7th and 14th days following cell therapy (D7 and D14).
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Table 1; gating strategy in Supplementary Figures 3, 4). To evaluate the overall profile of biomarkers, an unsupervised hierarchical cluster with the Luminex and flow cytometry assay values was performed using Ward's method. The flow cytometry analysis demonstrated that up to 7 days following treatment initiation, classical monocytes (CD14++CD16-) were enriched in the peripheral blood, whereas on day 14, the patrolling monocytes (CD14+CD16++) were the most prevailing monocyte subpopulation (Figure 3A). The substantial alterations of chemokine receptors expression over time post-treatment, with an increase on CCR5+ receptors and decrease of CCR7+ (Figure 3B). Additionally, the degree of monocyte activation was substantially altered following the treatment in the inflammatory monocyte subpopulation, where it is possible to observe a decrease in these activated monocytes on day 3 and day 7, with a subsequent increase on day 14, where the profile is similar to the baseline (Figure 3C). Regarding monocyte polyfunctionality in response to a toll-like receptor-4 (TLR-4) agonist [lipopolysaccharide (LPS), 1 mug/ml], there was a peak of multiple cytokine producer monocytes on day 3, suggesting higher polyfunctional activity in this period (Figures 3D-F).
Dynamics of CD4+ T helper subsets and dendritic cells turnover following treatment initiation. (A) Frequencies of CD4+ and CD8+ T Lymphocytes over time. (B) The differential expression of chemokine receptors CCR6 and CXCR3 in CD4 T cells are shown, further indicating the frequencies of Th1 (CXCR3+CCR6-), Th1 Star (CXCR3+CCR6+), Th2 (CXCR3-CCR6-), and Th17 (CXCR3-CCR6+) subpopulations. (C) The differential expression of chemokine receptors CCR6 and CXCR3 in activated CD4 T cells are shown, further indicating the frequencies of Th1 (CXCR3+CCR6-), Th1 Star (CXCR3+CCR6+), Th2 (CXCR3-CCR6-), and Th17 (CXCR3-CCR6+) subpopulations. (D) The frequencies of naive (CD45+CD27+), central memory (CD45-CD27+), effector (CD45-CD27-) and terminally differentiated cells (CD45+CD27-) are shown, without activation. (E) The frequencies of naive (CD45+CD27+), central memory (CD45-CD27+), effector (CD45-CD27-), and terminally differentiated cells (CD45-CD27+) are shown in activated (HLA-DR+) cells. (F) Conventional dendritic cells subtype 2 (cDC2) frequencies increased on day 3. The frequency of conventional DC2 (HLA-dr+CD11c+CD141-), plasmacytoid DCs (HLA-dr+CD11c-CD123+), and conventional DC1 (HLA-dr+CD11c+CD141+) (pink, brown and light purple, respectively) are shown. Samples were collected on the day of the 1st MSC infusion (D1, pre-infusion), 2nd MSC infusion (D3, pre-infusion) and on the 7th and 14th days following cell therapy (D7 and D14).
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Table 1; gating strategy in Supplementary Figures 3, 4). To evaluate the overall profile of biomarkers, an unsupervised hierarchical cluster with the Luminex and flow cytometry assay values was performed using Ward's method. In both cases, there was observed a slight increase at day 3, with progressive reduction at day 7 and day 14 (Figure 4A). The evaluation of differential chemokine receptor expression of CCR6 and CXCR3 on circulating CD4+ lymphocytes revealed higher frequencies of CXCR3-CCR6- (Th2) subpopulation following treatment (Figure 4B). In activated cells, the profile changes and CXCR3+CCR6- are more frequent (Figure 4C). T-cells frequencies over time along with the increased frequencies of terminally differentiated CD4+ T cells (Figure 4D). The activated TCD4+ cells were more terminally differentiated and effector on the first day, comparing with the other days (Figure 4E). Of note, the conventional dendritic cells 2 (cDC2) frequency peaked on day 3 after treatment, returning to the basal levels afterward (Figure 4F).