Fig 2: Cal/ICG@MPs-induced CD8+ T-cell tumor infiltration in H22 tumor-bearing mice upon 808 nm laser irradiation.a Schematic schedule for CD8+ T-cell tumor infiltration analysis in stroma-rich H22 tumor-bearing mice after intravenous injection of PBS, ICG, Cal/ICG, ICG@MPs, Cal@MPs, or Cal/ICG@MPs derived from H22 cells at the ICG dosage of 8 mg kg-1 and Cal dosage of 120 µg kg-1, followed with or without 808 nm laser irradiation (1.5 W cm-2, 10 min) at 2 h after the last injection. b–e Numbers of CD11c+CD80+CD86+ cells (b), CD8+CD69+ T cells (c), CD8+IFN-?+ T cells (d), and CD8+IL-2+ T cells (e) in tumor tissues of stroma-rich H22 tumor-bearing mice after treatment indicated in a. Data are presented as means ± s.d. (n = 6 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). f Immunofluorescence images of CD8+ T cells (labeling with Cy3-conjugated CD8 antibody, green) and CAFs (labeling with Cy5-conjugated a-SMA antibody, red) in tumor tissues of stroma-rich H22 tumor-bearing mice after intravenous injection of PBS, ICG, Cal/ICG, ICG@MPs or Cal/ICG@MPs at the ICG dosage of 8 mg kg-1 and Cal dosage of 120 µg kg-1, followed with 808 nm laser irradiation indicated in a. The tumor margin was marked with a white dotted line. Images are representative of three biologically independent mice. Scale bar: 100 µm. g CD8+ T-cell numbers in tumor tissues of stroma-rich H22 tumor-bearing mice after treatment indicated in f analyzed by StrataQuest software. Data are presented as means ± s.d. (n = 3 fields in total from two mice; one-way ANOVA followed by Tukey’s HSD post hoc test). h CD8+ T-cell numbers within 50 µm distance around CAFs in tumor tissues of stroma-rich H22 tumor-bearing mice after treatment indicated in f. Data are presented as means ± s.d. (n = 3 fields in total from two mice; one-way ANOVA followed by Tukey’s HSD post hoc test). i CD8+ T-cell numbers from the margin to center areas in tumor tissues of stroma-rich H22 tumor-bearing mice after treatment indicated in f. Data are presented as means ± s.d. (n = 6 fields in total from three mice; two-way ANOVA followed by Tukey’s multiple comparisons post test). Source data and exact P values for b, c, i are provided as a Source Data file.

Fig 3: Kinetics of Early T Cell Differentiation in Murine ATOs(A) Schematic of the M-ATO system.(B) Representative kinetic analysis of T cell differentiation from bone marrow lineage-negative, Sca1+, cKit+ (LSK) isolated from C57BL/6 wild-type (WT) mice at the indicated time points, gated on CD45+ cells to exclude non-hematopoietic cells. Middle and bottom rows: cells are gated on CD11b-, CD11c-, Gr1-, CD19-, B220-, and NK1.1- (Lin neg) and TCR?d- cells. Bottom row: cells are further gated on double-negative (DN) cells CD8- CD4- to analyze DN subsets: DN1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44- CD25+), and DN4 (CD44- CD25-) (Figure S1C).(C) Fold increase and frequencies of cell populations in M-ATOs over time. Left graph shows fold increase in total cell number from LSK cells seeded in M-ATOs at day 0. Frequencies of DN cells (CD8-CD4-), immature single-positive CD8+ (ISP8) cells (CD8+CD4-CD3-), and double-positive (DP) cells (CD8+CD4+) are shown as percentage of total CD45+ TCR?d- Lin- cells. Error bar denotes ±SD (n = 6 independent experiments).(D) Representative kinetic analysis of early T cell development within the DN population during the first 2 weeks of culture in M-ATOs.(E) Frequencies of early T cell populations over the first 2 weeks in M-ATOs. Frequencies of early thymic progenitors (ETPs) (ckitHi, CD44+CD25-), DN2a (ckitHi, CD44+CD25+), DN2b (ckitmed, CD44+CD25+), DN3 (CD44-CD25+), and DN4 (CD44-CD25-) are shown as percentage of total DN cells. Error bar denotes ±SD (n = 3 independent experiments).

Fig 4: 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 5: TRPV1 deficiency promotes the function of dermal macrophages. a Representative FACS plots of macrophages in the ear preparations isolated from wt, Trpa1 −/−, Trpv1 −/−, and Trpa1 −/−/Trpv1 −/− dKO mice. Macrophages were defined as I-Ab-lo/- F4/80+ CD11b+ CD11c-; b, c Quantification of the number (b) and the percentage (c) of macrophages based on flow cytometry analysis. n = 3. Data are presented as mean ± SEM. NS, not significant. Asterisks indicate statistical significance. **p < 0.01; ***p < 0.001, ANOVA; d–f Expression of proinflammatory cytokines TNFɑ (d), IL-1β (e) and IL-6 (f), in the SADBE-treated ear preparations of wt, Trpa1 −/−, Trpv1 −/−, and Trpa1 −/−/Trpv1 −/− dKO mice. Data are presented as mean ± SEM. NS, not significant. Asterisks indicate statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ANOVA