Fig 2: Targeting SERPINB3 sensitizes tumors to RT and enhances T cell response.(A) Growth curves of LL2/B3 tumors treated with siNC (red lines) and siB3 (purple lines) with or without RT (sham: solid lines; RT: dotted lines). ***P < 0.001, by 2-way ANOVA. (B) The chemokines CXCL1 and S100A8/A9 in tumor homogenates were assessed by ELISA. Data were normalized to the protein concentration for each tumor homogenate. (C and D) Cumulative data from FACS analysis show the (C) frequencies of immune cell populations including CD11b+Ly6G-Ly6Chi M-MDSCs, CD11b+Ly6G+ PMN-MDSCs, CD11b+Ly6G-F4/80+ TAMs, and CD11b+Ly6G-F4/80+CD163+ M2 macrophages, as well as (D) CD3+CD8+ T cells in total TILs and the ratio of CD3+CD8+ T cells to CD4+CD25+Foxp3+ Tregs. (E) Intracellular cytokine staining for granzyme B and perforin in CD8+ T cells was analyzed by flow cytometry. (F) CellTrace-labeled intratumoral T cells were stimulated with anti-CD3/anti-CD28 antibody for 4 days, and cell proliferation was determined by the dilution of CellTrace. (G) The expression of PD-1 and CTLA-4 was examined by flow cytometry and is shown as MFI. Data in B–G are shown as the mean ± SEM, and each dot represents a biologically independent sample. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test.

Fig 3: Depleting CD11b+ myeloid cells in SERPINB3 tumors improves T cell activity.(A) Representative plots show the depletion of CD11b+ cells, gated on CD45+CD11b+ cells, in tumors and spleens on day 15 and day 21 after tumor inoculation. (B) Tumor growth of LL2/Ctrl tumors (blue line) and LL2/B3a tumors treated with anti-CD11b antibody (red dotted line) or anti-IgG2b antibody (red solid line). ***P < 0.001, by 2-way ANOVA. (C) The numbers of infiltrating CD8+ T cells in 5 × 105 total tumor cells were determined by flow cytometry. (D) CellTrace-labeled intratumoral T cells were stimulated with anti-CD3/anti-CD28 antibody for 4 days, and cell proliferation was determined by the dilution of CellTrace. (E) Representative histograms of intracellular cytokine staining of granzyme B and perforin in CD8+ T cells. (F) PD-1 and CTLA-4 expression was examined by flow cytometry and is shown as MFI. Data in C–F are shown as the mean ± SEM, and each dot represents a biologically independent sample. *P < 0.05 and ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test.

Fig 4: Glycosylation accounts for the different size bands of CD11a, CD11b, and CD18.A, NOD cell lysates were either assayed as is (NOD whole cell lysates) or subjected to immunoprecipitation by an OVA-peptide (OVA-pep IP; a control for KGYY15 peptide) or by CD3 antibody (CD3-ab IP; a control for CD40 antibody 4F11) then assayed for CD11a, CD11b, CD18, or CD40 in Western blots. CD3-ab immunoprecipitation eluates were also assayed for CD3. B, NOD PBMC lysates were subjected to deglycosylation by PNGase F (removal of N-linked deglycosylation) or by PNGase F and total O-linked deglycosylation (Total deglyc.), or not (Lysates), then Western blots for CD11a, CD11b, and CD18 were performed. Completely deglycosylated bands are indicated (compl. deglyc.). C, lysates were subjected to deglycosylation by PNGase F alone, PNGase F and total O-linked deglycosylation (Total deglyc.), deglycosylation with four different enzymes that remove O-linked glycosylations (O-linked deglyc.), or the four different enzymes that remove O-linked glycosylations separately (O-glycosidase, neuraminidase, ß-galactosidase, or glucosaminidase). Western blots were then done for CD11a. Data are representative of three experiments.

Fig 5: Heightened GAL-7 expression delineates human non-melanoma cutaneous malignancies.A, B GAL-7 expression in human NMSC and normal skin, evaluated in two different cohorts. A LGALS7 gene expression in a panel of 4 normal skin samples and 25 NMSC lesions from E-MTAB-5678 dataset. SCC, squamous cell carcinoma (n = 5 patients); IEC, intraepidermal carcinoma (n = 7 patients); AK actinic keratosis (n = 13 patients). B GAL-7 protein expression evaluated by immunohistochemistry in skin samples from a cohort of 20 NMSC patients and 7 healthy donors. Representative images (left panel; bar represents 1 mm) and quantification of staining intensity (right panel; mean ± SEM) are shown. C–E RNAseq dataset from 4 human normal skin samples and 25 NMSC lesions (E-MTAB-5678) was analyzed. C Heatmap analysis of tumor-infiltrating cells in each sample. D Heatmap analysis of human M-MDSCs (CD11b and CD14) and PMN-MDSCs (CD66b and AQP9) marker expression levels in NMSC lesions compared to normal skin samples. E Heatmap analysis of chemokine and cytokine genes expression in human NMSC samples. CXCL1, S100A8, CCL7 and CSF1 mRNA expression levels (F) and correlations with LGALS7 expression (G) in human NMSC analyzed from RNAseq data set (E-MTAB-5678). H Cxcl1 and phospho c-Met (p-c-Met) levels, determined by immunohistochemistry, in a cohort of patients diagnosed with NMSC (N = 20) and healthy donors (N = 7). Representative images (left panel; quantification across dotted line, bar represents 100 µm) and quantification of Cxcl1+ cells (right panel; mean ± SEM) are shown.