Fig 2: 4MU blocks activation and polarization of naive CD4 T cells into Th1 cells.CFSE-stained naive CD4+ T cells from C57BL/6 mice (n = 3) were stimulated for 5 days with anti-CD3E, anti-CD28, or rhIL-2 ± 4MU (100 μg/mL) and then flow analyzed to quantify expression of CD25 (A and E), CD69 (B and F), CD279 (C and G), and CFSE dilution (D and H). CFSE-stained naive CD4+ T cells from C57BL/6 mice (n = 3) were stimulated and polarized toward Th1 cells for 5 days with anti-CD3E, anti-CD28, rhIL-2, rmIL-12, or anti–IL-4 ± 100 μg/mL 4MU and then flow analyzed for expression and quantification of IFNG (I and K) and CFSE dilution (J and L) (flow gating strategy shown in Supplemental Figures 6 and 7). Data are represented as mean ± SEM and analyzed using 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Inhibition of hyaluronic acid synthesis reduces posttransplant cellular rejection.Representative images of donor left lungs stained with anti–mouse CD4 (A) and CD8 (C) followed by DAB chromogen IHC secondary stain and hematoxylin counterstain. Scale bar: 100 μm. Quantitative analysis of CD4 (B) and CD8 (D) DAB+ cells from 5 random high-powered fields per mouse lung from control (n = 6), vehicle (n = 6), 4MU (n = 8) treated mice. Single-cell suspensions of lung tissues from vehicle-treated (n = 4) and 4MU-treated (n = 4) (450 mg/kg 5% Arabic gum in PBS) mice were stained against CD3E, CD4, and IFNG (E), TNFA (F), IL-17A (G), and IL-4 (H) (flow gating strategy shown in Supplemental Figure 3). Data are represented as mean ± SEM and analyzed using 1-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (*P < 0.05) or 2-tailed Student’s t test (*P < 0.05, **P < 0.01).

Fig 4: IL-27 Deficiency Suppresses Islet Infiltration of DCs and T Cells(A) The percentages of CD45+ cells among single viable cells in the islets of 4- or 10-week-old NOD and NOD.Il27−/− females.(B) The percentages of CD3+ T cells among CD45+ cells in the islets of 4- or 10-week-old NOD and NOD.Il27−/− females. Cells were gated on viable, single, and CD45+ cells.(C) The percentages of F4/80+ single-positive (SP), F4/80+ CD11c+, and CD11c+ SP among myeloid APCs in the islets of 4- or 10-week-old NOD and NOD.Il27−/− females. Cells were first gated on viable, single, CD45+, and CD3− cells, followed by excluding F4/80− CD11c− cells. For (A)–(C), representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels. Summarized data are representative of two independent experiments. Each symbol represents a mouse. The horizontal line indicates the mean. *p < 0.05 by Mann-Whitney test.(D) Representative flow cytometry plots on the left depict I-Ag7 and CD40 expression among myeloid APC subsets in the islets of 10-week-old NOD or NOD.Il27−/− females. Summarized data on the right show geometric mean fluorescence intensity of I-Ag7 or CD40 among F4/80+ SP, F4/80+ CD11c+, or CD11c+ SP APC subsets in the islet of 10-week-old NOD or NOD.Il27−/− females. Summarized data are representative of two independent experiments. Each symbol represents a mouse. The horizontal bar depicts the mean. *p < 0.05 by Mann-Whitney test.See also Figure S2.

Fig 5: 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.