Fig 2: Activation of DC and T lymphocytes by Nano-DOX-treated 4T1 cells. a–d Immunostaining of CD40, CD80, CD83 and MHC-II (markers of DC activation) in bone marrow-derived DC (BMDC) assayed by FACS. Representative FACS histograms are presented in Additional file 1: Fig. S6. e Spleen-derived CD4+ and CD8+ T lymphocytes characterized by immunofluorescent staining and FACS.f, g Proliferation of CD4+ and CD8+ T lymphocytes, assayed by CFSE staining and FACS. Representative FACS histograms are presented in Additional file 1: Fig. S7. h, i Immunostaining of CD69 (a marker of T lymphocyte activation) in CD4+ and CD8+ T lymphocytes, assayed by FACS. Representative FACS dot plots are presented in Additional file 1: Fig. S8. j Immunohistochemical staining of CD4, CD8, CD69 and foxp3 (a marker of Treg) in 4T1 tumor xenografts at the end of 3-week treatment of Nano-DOX or DOX. In the ex vivo cell experiments, BMDC were first treated with Nano-DOX (2 μg/mL) or culture medium conditioned by Nano-DOX (2 μg/mL)-treated 4T1 cells (ND-CM) for 24 h. Spleen-derived lymphocytes were then co-cultured with the BMDC in the same system for another 24 h. In FACS analysis, geometric means were used to quantify fluorescence intensity. Values were mean ± SD (n = 3, *p < 0.05, **p < 0.01)

Fig 3: CD83 influences tumor growth in the pB-IUE system. Kaplan–Meier survival curve of pB-IUE tumors control (n = 17, Med OS = 102 days), CD83KO (n = 20, Med OS = 94 days), and CD83OE (n = 16, Med OS = 116 days; log-rank, all three curves, P value = 0.018; A). Age-matched P60 brains demonstrating decreased growth of CD83OE tumors compared with control and CD83KO tumors (B). Hematoxylin and eosin stain of pB-IUE control, CD83OE, and CD83KO tumors (C and D). IHC for Ki67 of pB-IUE control, CD83OE, and CD83KO tumors (one-way ANOVA P value = 0.0133. Tukey MCT: CD83KO vs. CD83OEP value = 0.0158, CD83KO vs. control P value = 0.8075, and CD83OE vs. control P value = 0.0454; E and F). EdU staining of in vitro cultured control, CD83OE, and CD83KO tumors (one-way ANOVA P value < 0.0001. Tukey MCT: CD83KO vs. CD83OEP value = 0.0193, CD83KO vs. control P value < 0.0001, and CD83OE vs. control P value < 0.0001; G and H). *, P value < 0.05; ****, P value < 0.0001. (Created with Biorender.com.)

Fig 4: Antigen presenting–like tumor cells express Cd83 in human and mouse glioma. Expression of GO:0002291: T-cell activation via T-cell receptor contact with antigen bound to MHC molecule on APC and GO:0002468: dendritic cell antigen processing and presentation in human IDHWT and IDHmut glioma tumor cells that were differentially expressed (A). scRNA-seq from five IDHmut and seven IDHWT gliomas demonstrate a rare population of CD83+ tumor cells (B and C). Percentage of IDHmut and IDHWT tumor cells from scRNA-seq that expressed different combinations of CD45 and CD83 (D). Immunofluorescence imaging for IDH1R132H tumor cells that express CD83 (E). scRNA-seq from pB-IUE glioma isolates similar murine tumor cells (F). Percentage of murine tumor cells from scRNA-seq that expressed different combinations of CD45 and CD83 (G). Multispectral flow cytometry isolates a distinct population of GFP+CD83+ tumor cells (H). (Created with Biorender.com.)

Fig 5: Tumoral CD83 influences expression of T-cell exhaustion markers. Immunofluorescence for CD8 and CD3 in pB-IUE control, CD83OE, and Cd83KO tumors (A). Increased number of CD3 and Cd8 T cells in CD83KO compared with CD83OE (two-way RM ANOVA P value < 0.0001, tumor type P value = 0.0183. Tukey MCT CD3: CD83KO vs. CD83OEP value = 0.0094, CD83KO vs. control P value = 0.0897, and OE CD83OE vs. control P value = 0.6658. Tukey MCT CD8: CD83KO vs. CD83OEP value = 0.0270, CD83KO vs. control P value = 0.1466, and CD83OE vs. control P value = 0.7514; B). Expression of T-cell exhaustion markers in subset T-cells from scRNA-seq in pB-IUE IUE control, CD83OE, and CD83KO tumors (C). Immunofluorescence for PD-1 and TIM3 in pB-IUE control, CD83OE, and CD83KO tumors (D and F). Increased number of PD-1–positive cells in CD83KO compared with control and CD83OE tumors and in control compared with CD83OE OE (one-way ANOVA P value < 0.0001. Tukey MCT: CD83KO vs. CD83OEP value < 0.0001, CD83KO vs. control P value = 0.0120, and CD83OE vs. control P value = 0.0003; E). Increased number of TIM3-positive cells in CD83KO compared with control and control compared with CD83OE tumors and in control compared with CD83OE (one-way ANOVA P value < 0.0001. Tukey MCT: CD83KO vs. CD83OEP value < 0.0001, CD83KO vs. control P value = 0.9737, and CD83OE vs. control P value < 0.0001; G). *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001. CTLA4, cytotoxic T lymphocyte–associated antigen 4. (Created with Biorender.com.)