Fig 2: Characterization of the Phenotype of Human Hemogenic Endothelial Cells on a Single-Cell Level(A) Schematic representation of single-cell blood-forming assay.(B) Representative photographs showing single human hemogenic endothelial cell on D0 (i), hemogenic endothelial cell colony on D3 (ii), DiI AcLDL uptake of hemogenic endothelial cells on D3 (iii), and adherent hemogenic endothelial cells among blood cells on D14 (iv and v). Scale bars, 35 µm for (i), (ii), and (iii). Scale bars, 170 µm for (iv) and (v).(C) Representative photographs showing BFU-E (i and ii) and CFU-E (iii and iv) on D8, marked by CD235ab. Scale bars, 35 µm.(D) Representative photographs showing CFU-GM on D14, marked by CD66b and CD14. Scale bars, 35 µm.(E) Representative photographs showing CFU-GEMM on D14, marked by CD66b, CD235ab, CD14, and CD41. Scale bars, 70 µm.

Fig 3: CD3+CD14+ cells are tightly bound T cell:monocyte complexes that represent in vivo association.(A) Gating strategy and (B) random gallery of events for monocytes (CD14+CD3), T cells (CD3+CD14-), CD3+CD14hi cells and CD3+CD14mid cells determined by imaging flow cytometry (ImageStreamX, MkII Amnis Amnis). CD14+ cell debris were identified within CD3+CD14mid cells (C) by imaging flow cytometry and (D) confocal microscopy after bulk population cell sorting. (E) Plots and (F) Ratio of Aspect ratio vs Area of the brightfield parameter for monocytes (CD14+CD3-), T cells (CD3+CD14-), CD3+CD14hi cells and CD3+ CD14 mid cells, determined by imaging flow cytometry. (G) Non-parametric Spearman correlation of the frequency of T cell:monocyte complexes in paired fresh PBMC vs cryopreserved PBMC derived from 45 blood draws of healthy subjects. T cell:monocyte complexes were defined as the CD3+CD14+ cell population gated from live singlets as represented in Figure 1—figure supplement 2. (H) Single z-plan (0 µm) images (left) and z-plane stacks (right) of the region marked (dashed rectangle) from one sorted CD3+ CD14+ T cell:monocyte complex displaying accumulation of LFA1 and ICAM1 at the interface. Images show expression of CD14 (blue), CD3 (green), ICAM1(Cyan), and LFA1 (Magenta). Relative z-positions are indicated on the right, and scale bars represent 2 µm. Imaging flow cytometry data was derived from 10 subjects across three independent experiments and microscopy data was representative of the analysis of n = 105 CD3+CD14+ complexes isolated from three subjects across three independent experiments.

Fig 4: Enhanced DC antigen uptake and processing by immunogenic mAbs. CD14+ monocyte-derived dendritic cells from 13 healthy individual donors were incubated with the therapeutic antibodies and the matched isotype control antibody (hIgG1-EN) in the presence of an activatable fluorescence-quencher probe consisting of goat-anti-human IgG Fab-fragments conjugated with the TAMRA (fluorophore)–QSY7 (quencher) pair. Endocytosis of the internalized antibody was measured by an increase in % TAMRA by flow cytometry. (a) Representative flow plots are gated on live single cells. (b) Scatter plot summarizing the %TAMRA+ from 13 donors incubated with the test antigens, hIgG1-EN, Control mAb, mAb1, mAb2, and mAb3

Fig 5: 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.