Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: Munc13-4 deficiency in tumor cells enhances T cell infiltration and activation (A–C) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells among total cells in the tumors (A) , spleens (B) , and draining lymph nodes (C) of BALB/c mice (n = 5), 21 days after mammary gland injection with 3 × 10 5 control or Munc13-4 KO 4T1 cells per mouse. (D–F) Quantification of the percentage of granzyme B + (GzmB + ) (D) , Ki67 + (E) and IFNγ + (F) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within tumors from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (G–I) Quantification of the percentage of granzyme B + (G) , Ki67 + (H) and IFNγ + (I) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within spleens from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). (J–L) Quantification of the percentage of granzyme B + (J) , Ki67 + (K) and IFNγ + (L) cells among CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + T cells within the draining lymph nodes from orthotopic mouse models of breast cancer generated by control or Munc13-4 KO 4T1 cells (n = 5). Box plots show all data points, all p -values were calculated by Multiple t tests. See also Figure S3.

Article Snippet: Mouse CD8 + T cells were purified from splenocytes using the Mouse CD8 T Cell Isolation Kit (Vazyme, CS103-01) and stimulated for 24 hours with anti-CD3 (Biolegend, 300301, 1 μg/ml) and anti-CD28 (Biolegend, 117003, 1 μg/ml) antibodies.

Techniques: Activation Assay, Injection, Control, Generated

Journal: bioRxiv

Article Title: Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes

doi: 10.1101/2025.03.22.644518

Figure Lengend Snippet: A peptide disrupting PD-L1 – Munc13-4 interaction inhibits tumor growth (A) Prediction of the interaction between PD-L1 (residues 253–290) and Munc13-4 (residues 1049–1080). Top insets show five ensembles generated by AlphaFold multimer, where N- and C-terminus of the proteins are indicated. PD-L1 and Munc13-4 are colored in blue and red, respectively. Residues that show potential contacts with each other (within 4 Å) are shown in sticks. Bottom inset displays the statistics of the residues in PD-L1 that show contacts with Munc13-4. The absolute and averaged contact numbers for each ensemble are illustrated by stacked histograms and line-scatter plot. The potential Munc13-4–interacting sequence of PD-L1 is shaded in magenta. (B) Diagram of the sequences for P-pep and S-pep. P-pep comprises a cell-penetrating peptide (CPP) fused to the human PD-L1 256–273 motif, whereas S-pep consists of a CPP linked to a scrambled sequence containing the same amino acid composition as the human PD-L1 256–273 motif. (C) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to examine the effect of P-pep on Munc13-4–PD-L1 interaction (n = 3). (D) Co-IP and IB analysis in HEK293T cells transfected with indicated constructs and incubated with P-pep or S-pep to assess the effect of P-pep on the interactions of HRS with PD-L1 and Munc13-4 (n = 3). (E) Western blot analysis of PD-L1, Alix, CD63 and CD81 abundance on the same number of exosomes secreted from equal number of SUM159 cells treated with S-pep or P-pep. (F and G) Assessment of in vivo anti-tumor efficacy of P-pep. (F) Schematic of experimental design. (G) Tumor growth curves of orthotopic mouse models of breast cancer treated with P-pep or S-pep (n = 9). (H and I) Flow cytometric quantification of the percentage of CD45 + CD3 + CD4 + (H) and CD45 + CD3 + CD8 + (I) T cells among total cells in tumors (n = 5). (J and L) Representative contour plots depicting CD45 + CD3 + CD4 + (J) and CD45 + CD3 + CD8 + (L) T cell populations within tumors, showing the expression of granzyme B. (K and M) Quantification of the percentage of granzyme B + cells among CD45 + CD3 + CD4 + (K) and CD45 + CD3 + CD8 + (M) T cells within tumors (n = 5). Data are represented as means ± SEM (G), box plots show all data points (H, I, K and M), p -values were all calculated by unpaired t test. See also Figure S12.

Article Snippet: Mouse CD8 + T cells were purified from splenocytes using the Mouse CD8 T Cell Isolation Kit (Vazyme, CS103-01) and stimulated for 24 hours with anti-CD3 (Biolegend, 300301, 1 μg/ml) and anti-CD28 (Biolegend, 117003, 1 μg/ml) antibodies.

Techniques: Generated, Sequencing, Co-Immunoprecipitation Assay, Transfection, Construct, Incubation, Western Blot, In Vivo, Expressing