Fig 2: Cultivation and characterization of MSCs. (A) 1 shows P0 of cells after one week of the first culture; 2 shows P1 of cells after three days; 3 shows P1 of cells after one week (40× magnification was used). (B) Flow cytometry of hAF-MSCs for surface markers. Expression of surface markers detected by flow cytometric analysis in P4 of MSCs, with CD73 and CD105 as positive markers and CD14 and CD45 as negative markers for hAF-MSCs.

Fig 3: The constant of association Ka between monocytes and T cells (and T cell subsets) varies with the presence and nature of immune perturbations.(A) Non-parametric spearman correlation between the frequency of T cell:monocyte complexes and the product of singlet T cells and monocyte frequencies in healthy subjects (n = 59). (B) Formula for the calculation of the T cell:monocyte constant of association Ka. T cell:monocyte complexes constant of association Ka in (C) active TB subjects at diagnosis and 2 months post treatment (n = 15), (D) individuals with acute dengue fever (n = 18), acute dengue hemorrhagic fever (n = 24) or previously infected (n = 47) and (E) previously vaccinated healthy adults (n = 16) before and three days post boost with Tdap vaccine, calculated as explained in B). (F) The constant of association Ka between monocytes and T cell subsets in active TB subjects at diagnosis (n = 25), individuals with acute dengue hemorrhagic fever (n = 24) and previously vaccinated healthy adults three days post boost with Tdap vaccine (n = 16), calculated as explained in B). Statistical differences over time and across cell populations within subjects were determined using the non-parametric paired Wilcoxon test; other statistical differences were determined using the non-parametric Mann-Whitney test; *, p<0.05; **, p<0.01; ***, p<0.01; ****, p<0.0001. Plots represent individual data points, median and interquartile range across all subjects within each cohort. Raw frequencies of T cell:monocyte complexes for the different disease cohorts are available on Figure 4—figure supplement 4. T cell:monocyte complexes were defined as the CD3+CD14+ cell population gated from live singlets as represented in Figure 1—figure supplement 2. CD4 and CD8 subsets within T cell:monocyte complexes were defined as presented in Figure 3E.

Fig 4: GAPDH exposed on apoptotic cells and phagocyte CD14 mediate efferocytosis.A Co-localization of apoptotic cell surface GAPDH with CD14 on phagocyte. When cells were incubated together the two signals co-localize at points of intercellular contact. Scale bar, 5 µm. B Co-immunoprecipitation (Co-IP) of GAPDH from mixed membrane fractions of apoptotic cells (J774) and phagocytes (THP-1). Right side panels are Western blots of membrane fractions as positive control for both antibodies. C Western blot to confirm the CD14 K/D in THP-1 cells. Cell lysates were prepared from THP-1 empty vector and CD14 knockdown cells and samples were run on 10% SDS-PAGE. Separated proteins were transblotted onto PVDF membrane and probed with mouse anti-CD14 antibody (Abcam) followed by secondary antibody anti-mouse peroxidase (Sigma). D–E Phagocytosis of apoptotic cells is dependent upon both GAPDH on apoptotic cell surface and CD14 on phagocytes. D Representative confocal microscopy images of live and apoptotic J774 cells that were phagocytosed by THP-1 phagocytes. Live cells, apoptotic empty vector, and GAPDH K/D J774 cells are labeled with Vybrant DiD dye (Red). Phagocyte THP-1 cells, empty vector, and CD14 K/D cells are labeled with CFSE (Green). Scale bar, 20 µm. E Bar graph represents phagocytosis as percentage of control (extent of phagocytosis by THP-1 empty vector cells incubated with J774 empty vector cells as 100%), ***P < 0.0001, n = 150 cells, also see Fig. S2C–D. F Disruption of the GAPDH–CD14 interaction utilizing a blocking antibody against CD14 results in a significant decrease in phagocytes ability to engulf apoptotic cells. This corroborates and complements the results of our knockdown studies.

Fig 5: Conventional flow cytometry parameters and expression of T cell/monocyte canonical markers cannot differentiate between T cells and monocytes in a complex vs. not in a complex.(A) 2D density plots of A, H and W from FSC and SSC parameters for CD3-CD14+ Monocytes (red), CD3+CD14- T cells (green) and CD3+CD14+ T cell:monocyte complexes (T:M, blue). Representative staining of one healthy individual. (B) Frequency of T cell:monocyte complexes cells with or without addition of CD45-SSC filtering gate (see Figure 3 – figure supplement 1 for gating strategy). Expression of canonical markers for (C) monocytes and (D) T cells in CD3-CD14+ Monocytes (red), CD3+CD14- T cells (green) and CD3+CD14+ T cell:monocyte complexes (T:M, blue). (E) Expression of CD4 and CD8 and division into T cell subsets within T cell:monocyte complexes. (F) Expression of CD45RA and CCR7 and division into naïve, central memory (Tcm), effector memory (Tem) and effector memory re-expressing CD45RA (Temra) subsets within T cell:monocyte complexes. Data derived from frozen PBMC of n=30 (A, E, F), n=8 (B) and n=4 (C, D) healthy individuals. Unless otherwise stated, T cell:monocyte complexes were defined as the CD3+CD14+ cell population gated from live singlets as represented in Figure 1—figure supplement 2.