Fig 2: Subcutaneous injection of AAT in healthy volunteers.a H&E staining of AAT implants excised from healthy volunteers at early, mid, and late timepoints during a first-in-human Phase 1 study. High magnification insets show cell migration from the adjacent host tissue into the AAT matrix. b Multispectral immunohistochemistry (IHC) reveals formation of blood vessels (CD31+) and migration of adipose stem cells (CD34+). c CD4+ and CD8+ T cell infiltration by multispectral IHC. d Flow cytometry analysis was performed on excised AAT implants from six subjects to quantify immune cells (CD45+) recruited within AAT implant(s) relative to multiple samples of subject-matched adipose tissue collected distally from the injection site. Results for each recovered AAT implant were normalized to the average of multiple samples of subject-matched adipose tissue. Data are shown pooled for all subjects. e Relative abundance of different immune cell subsets including granulocytes (CD45+CD11b+CD15+), macrophages (CD45+CD15−CD11c+MHCII+CD11b+CD14+CD68+), and T cells (CD45+CD3+). Data are shown pooled for all subjects. f Relative expression of intracellular FoxP3 as well as TH1, TH2, and TH17 cytokines in T cells isolated from AAT and matched adipose tissue, shown pooled for all subjects. g Expression of macrophage polarization markers (CD163 and CD80) in AAT and matched adipose tissue. (h) Average distribution of M1 (CD80+CD163−), M2 (CD163+CD80−), double-positive and nonpolarized macrophages in AAT and matched adipose tissue as a frequency of total macrophages. i Expression of macrophage polarization (CD163, CD80) and lineage markers (CD11b, CD14) by MFI, pooled for all subjects. Significance (p-values): * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Median and quartiles are shown in violin plots. Scale bars represent 5 mm in whole sections and 200 µm in enlarged regions.

Fig 3: Differences in lymphocytes counts of peripheral blood mononuclear cells (PBMCs) in peripheral blood in lung cancer patients and healthy controls. (a) Flow cytometry was performed to detect lymphocyte subsets in PBMCs and to analyze the proportions of T cells (CD3+CD56−), B cells (CD19+), natural killer T (NKT) cells (CD3+CD56+), and natural killer (NK) cells (CD3−CD56−) among CD45+ cells and the proportions of CD4+ T cells and CD8+ T cells among T cells. The results are presented using scatter plots. (b) Differences in T cells (CD3+CD56−), B cells (CD19+), NKT cells (CD3+CD56+), and NK cells (CD3−CD56−) among CD45+ cells and the proportions of CD4+ T cells and CD8+ T cells among T cells between lung cancer patients and healthy controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 4: AAT scaffolds skew macrophages towards an M2-phenotype in muscle wounds.a CD206 and CD86 expression in macrophages (CD45+SiglecF-CD11b+CD3-Ly6g−Ly6c−F4/80+) isolated from healthy quadriceps muscle and scaffold- or saline-treated muscle wounds after 1-week post-op. b Populations of alternately activated “M2” (CD206+CD86−), classically activated “M1” (CD206−CD86+), double-polarized (CD206+CD86+) and nonpolarized (CD206−CD86−) macrophages quantified by flow cytometry. c Expression of canonical M2 (Il4, Arg1) and M1 (Ifng, Nos2) genes at the wound site by qRT-PCR. Significance relative to vehicle control is depicted (p-values), see Supplementary Table 2 for other significance values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Error bars represent ±1 standard deviation.

Fig 5: High IRF8 Expression Defines LMPP-Associated DC Progenitors(A) Flow gating strategy used to define and FACS-purify components of the CD34+ lin(CD3,14,16,19,20,7)- compartment of human BM. HSC, hematopoietic stem cell; MPP, multipotent progenitor; MEP, megakaryocyte-erythroid progenitor; MLP, multilymphoid progenitor; LMPP, lymphoid-primed multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor.(B) Heatmap of intracellular IRF8 protein expression across CMP and GMP as defined in (A) (gate 1).(C) Monocyte and DC subset output from purified BM CD34+ populations at day 14 of culture gated as in Figure 2A. Populations were quantified as percentage of the total cells captured by all DC and monocyte gates. Absolute output is shown in Figure S3C. Bulk CD34+ (22 experiments from 13 donors: 22;13); CMP (7;5); GMP33+ (7;6); LMPP (7;6); GMP33- (6;6); GMP123lo (3;3); GMP123int (8;7) (Table S4). Bars represent mean + SEM, and circles represent individual experiments. Significant differences in the proportional output of DC2s versus DC3s are indicated in red; *p < 0.05; **p < 0.01; ****p = 0.0001 (paired two-tailed t tests).(D) Unsupervised hierarchical clustering of transcriptomes of single cells within the GMP index-sorting gate, using all protein-coding, non-cell-cycle genes, independent of surface antigen expression. Marker genes for four clusters identified within the single-cell consensus clustering 3 (SC3) tool (p < 0.1, AUROC > 0.75) and IRF8 are displayed. The top rows show fluorescence intensity of surface antigens from index-sorted cells. Flow annotation (“Flow annot”) denotes the classification of cells by their surface phenotype (Figures 3A and S3F).(E–G) tSNE visualization of the first 10 principal components (25% of total variance) of the transcriptomes of 262 CD34+ progenitor cells, independently of their surface phenotype. tSNE plots are shown annotated by (E), gate of origin from index-linked flow (Figure S3F), or (F), 10 clusters from hierarchical clustering (Figure S3J), Heatmaps (G) show flow surface antigen expression (“SA”) and log2 expression of key DC TFs, IRF8, TCF4, SPIB, and SPI1(PU.1), displayed across the tSNE plot (E and F). Black circles represent regions of high (“A”) or low (“B”) IRF8 expression.(H and I) Diffusion map using all protein-coding, non-cell-cycle genes. (H) The key specifies the designated cluster color, identity, and cluster number from Figure S3J. (I) IRF8 expression. Diff Comp, diffusion component.(J) Violin plot of differential IRF8 expression (log2) in progenitor clusters 5 (HSCs and MPPs), 1 (monocyte enriched), and 8 (DC related). **p = 0.001 by Mann-Whitney U.(K) Median fluorescence intensity (MFI) of intracellular IRF8 by flow analysis across gates identifying HC BM CD34+ HSCs and CD123neg-lo CD33+ and CD123int GMPs (n = 4) as defined in (A). *p = 0.028 by Mann-Whitney U.See also Figure S3.