Fig 2: PmTriTNE@CDA transformed cold tumors into hot tumors and achieved long-term immune memory against TNBC. A) BALB/c mice were inoculated with 4T1 tumor cells, and the tumor-bearing mice were intravenously administered a-Isotype+a-Isotype-PAMAM NPs (control) (i), ?-PD-L1+?-CD3 +mPEG-HA-PAMAM (ii), PmTiTNE (iii), ?-PD-L1+?-CD3+mPEG-HA-PAMAM@CDA (iv) or PmTiTNE@CDA (v) at the indicated time points. At day 27, TILs were measured by flow cytometry. B) CD8+ TIL absolute number. C) CD8+ TIL/Treg ratio. D) Expression of Ki67 in CD8+ TILs. E) Statistical analysis of the data in (D). F) Expression of granzyme B in CD8+ TILs. G) Statistical analysis of the data in (F). H,I) mRNA expression of IFN-ß (H) and CXCL9 (I) in tumor tissue. J) Maturation of DCs. K) Statistical analysis of the data in (J). L) Percentage of TEMs and TCMs among CD8+ T cells in TDLNs. M) Statistical analysis of the data in (L). N) ALDH expression in 4T1 tumors was measured by flow cytometry. O) Tumor masses were excised and processed for ex vivo primary mammosphere-forming assays. P) BALB/c mice were treated with PmTriTNE@CDA on days 15, 18, and 21 after tumor implantation. Fifty days after treatment, the cured mice were rechallenged with 4T1 tumor cells, and naïve mice were used as a control. Then, tumor growth was monitored. Q) A total of 2 × 107 splenocytes from cured mice or naïve mice were intravenously administered to 4T1 cell-inoculated NSG mice. After 3 days, 4T1 cells were inoculated, and tumor growth was monitored. The data are presented as the means ± SDs (n = 5). p-values are calculated using one-way ANOVA with Tukey's multiple comparison. ***p < 0.001.

Fig 3: Changes in cell numbers for cell populations in unwounded skin and WIHN wounds. Cells were isolated from digested unwounded skin (pw0) or at different pw along wound healing, stained with antibodies against cell surface markers or intracellular FOXP3 and analyzed with flow cytometry. (A) Composition of skin (pw0) or wounds (other pw) in terms of dermal compartment, immune cells and epidermis. (B) Changes in epithelial cell number expressed as cell/1 × 103 CHD1+ cells. (C) Changes in suprabasal and basal Bul. (D) Changes in numbers for fib expressed as cell/1 × 103 CHD1-/PTPRC- cells. (E) Changes in DP and endothelial cells expressed as cell/1 × 103 CHD1-/PTPRC- cells. (F) Changes in numbers for DC, mono and neutrophil expressed as cell/1 × 103 PTPRC+ cells. (G) Changes in CD206(MRC1) + and - cells expressed as cell/1 × 103 PTPRC+ cells. (H) Changes in numbers for T cells expressed as cell/1 × 103 PTPRC+/CD3+ cells. (I) Exemplary dot plot of CD4 and CD8 expression of CD3+/PTPRC+ cells at pw9. (J) Changes in numbers for d?T cells expressed as cell/1 × 103 PTPRC+/CD3+ cells and Treg expressed as cell/1 × 103 PTPRC+/CD3+/CD4+ cells. Results are expressed as mean ± SEM, n = 3–8. Comparison between pw0 and subsequent pw values was carried out individually with a t-Test, p value = * = 0.05, ** = 0.01.

Fig 4: Nociceptor-released CGRP increases cytotoxic CD8+ T cell exhaustion.(a–b) Splenocytes-isolated CD8+ T cells were cultured under Tc1-stimulating condition (ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 µM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1+LAG3+TIM3+ (a) cytotoxic CD8+ T cells, while it decreased the one of IFN?+ (b). (c–d) Splenocytes-isolated CD8+ T cells were cultured under Tc1-stimulating conditions (ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 µL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8+ T cells were exposed or not to a RAMP1 blocker (CGRP8–37; 2 µg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1+LAG3+TIM3+ (c) cytotoxic CD8+ T cells, while it decreased the one of IFN?+ (d). Such effect was absent when cytotoxic CD8+ T cells were co-exposed to the RAMP1 blocker CGRP8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons (c–d). (e–f) Splenocytes-isolated CD8+ T cells from wild-type and Ramp1-/- mice were cultured under Tc1-stimulating conditions (ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8+ T cells were exposed to CGRP (0.1 µM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots (f) show that CGRP decrease RampWT cytotoxic CD8+ T cells expression of IFN?+ (e,f), TNF+ (f), and IL-2+ (f) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1+LAG3+TIM3+ in Ramp1WT cytotoxic CD8+ T cells (f). Ramp1-/- cytotoxic CD8+ T cells were protected from the effect of CGRP (e–f). (g–i) Splenocytes-isolated CD8+ T cells from naive OT-I mice were cultured under Tc1-stimulating conditions (ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×105 cells) were then cultured with or without OT-I cytotoxic CD8+ T cells (4×105 cells). Tc1-stimulated OT-I-CD8+ T cells lead to B16F10-OVA cell apoptosis (AnnexinV+7AAD+; g, measured after 48h; h–i, measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8+ T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 µM)-stimulated naive DRG neurons (g; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis (h; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP8-37 (h; 2 µg/mL; measured after 24h). CGRP (0.1 µM) challenges also reduced OT-I cytotoxic CD8+ T cells elimination of B16F10-OVA cell (i; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25th to 75th percentile and the middle line indicates the median), for which individual data points are given (a–e, g–h), or representative FACS plot (f, i). N are as follows: a: CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b: n = 5/groups, c: CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d: n = 5/groups, e: Ramp1WT CD8 + vehicle (n = 7), Ramp1WT CD8 + CGRP (n = 8), Ramp1-/- CD8 + vehicle (n = 6), Ramp1-/- CD8 + CGRP (n = 6), g: B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h: B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni (a–e, g–h). Source Data

Fig 5: Total and cytoskeleton binding protein-specific tumor mutational burden in human biopsies and their association with patient survival, related to Figure 7(A) Prognostic value of presence or absence of mutational burden in F-actin binding proteins for cancer patient overall survival in the indicated TCGA datasets.(B) Prognostic value of cGSN transcript levels for overall survival comparing samples with lowest (cGSNLow) and highest (cGSNHigh) expression in the presence (Pos) or absence (Neg) of tumor mutational burden in F-actin binding proteins (FABP) in the indicated TCGA datasets.(C) Prognostic value of sGSN transcript levels for overall survival comparing samples with lowest (sGSNLow) and highest (sGSNHigh) expression in the high tumor mutational burden (top quartile) patient subcohort of the indicated TCGA datasets. In (B and C) for cGSN and sGSN segregation between the highest and lowest expressors the same cut-off was used as in (Figure 6A) for the indicated TCGA datasets.(D) Prognostic value of sGSN transcript levels for overall survival comparing samples with lowest (sGSNLow) and highest (sGSNHigh) expression in the indicated TCGA dataset. Low grade glioma (LGG), bottom (n = 103) and top (n = 103) 20% of patient cohort.(E) Prognostic value of sGSN transcript levels for overall survival comparing samples with lowest (sGSNLow) and highest (sGSNHigh) expression in the high tumor mutational burden (top quartile) patient subcohort of the indicated TCGA dataset.(F) Prognostic value of cGSN transcript levels for overall survival comparing samples with lowest (sGSNLow) and highest (sGSNHigh) expression in the presence (Pos) of tumor mutational burden in F-actin binding proteins (FABP) in the indicated TCGA dataset.(G) Prognostic value of CLEC9A expression for cancer patient overall survival comparing top and bottom quartiles in the indicated TCGA dataset.(H) Prognostic value of CLEC9A expression for cancer patient overall survival comparing top and bottom quartiles of sGSNLow and sGSNHigh subgroups in the indicated TCGA dataset.(I) Prognostic value of CLEC9A transcript levels expression for cancer patient overall survival comparing top and bottom quartiles of cGSNLow and cGSNHigh subgroups in the indicated TCGA dataset.(J) Prognostic value of cDC1 gene signature expression for cancer patient overall survival comparing top and bottom quartiles in the indicated TCGA dataset.(K) Prognostic value of cDC1 gene signature expression for cancer patient overall survival comparing top and bottom quartiles of sGSNLow and sGSNHigh subgroups in the indicated TCGA dataset. In (E, F, H, I, K) for cGSN and sGSN segregation between the highest and lowest expressors the same cut-off was used as in (D) for the indicated TCGA dataset.(L) Comparison between Pearson r correlation values, obtained from correlation of CLEC9A – ER phagosome pathway signature with individual CD8 - MHC class I (cross)-presentation related signature between sGSNLow and sGSNHigh subgroups in the indicated TCGA dataset.In (B-F, H, I, K) all data are presented as mean of log2 normalized expression ± SEM Hazard ratios (HR) with 95% confidence interval showed in brackets have been calculated in (A-G, J) as a ratio of low expressed transcript or absent mutational burden / high expressed transcript or present mutational group, in (H) as a ratio of each group / sGSNHighCLEC9ALow, in (I) as a ratio of each group / cGSNHighCLEC9ALow and in (K) as a ratio of each group / sGSNHighcDC1Low. Survival (Kaplan-Meier) curves in (A-K) were compared using Log-rank (Mantel-Cox) test. In (L) the dotted line indicates a p value of 0.05 obtained by Pearson’s r correlation. *p = 0.05, ***p < 0.001. ns, not significant.