Journal: iScience

Article Title: VPS33B regulates MHC class II antigen presentation in dendritic cells to drive CD4 T cell immunity

doi: 10.1016/j.isci.2026.116052

Figure Lengend Snippet: Mice lacking VPS33B in the myeloid compartment have defective CD4 and CD8 T cell priming in response to protein immunization (A) Schematic of experimental set-up for in vivo immunizations. Mice were immunized with 50 μg of OVA and 5 μg of LPS in IFA (100 μL/mouse; hock injection). Draining Ig LNs were collected, processed, and cultured with OVA (100 μg/mL) for 72 h. (B) Representative flow plots of CD44 expression on CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-II T cells to the right. (C) Representative flow plots of Tbet expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-II T cells to the right. (D) Representative flow plots of IFNγ expression in CD4 T cells (pre-gated on live, CD90.2 + , CD4 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-II T cells to the right. (E) Representative flow plots of CD44 expression on CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % CD44 + OT-I T cells below. (F) Representative flow plots of Tbet expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R Ig LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Graphical quantification of % Tbet + OT-I T cells below. (G) Representative flow plots of IFNγ expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from immunized VPS33B WT and VPS33B ΔCSF1R IgLNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. Cells were rested with IL-2 for 48 h and re-stimulated with Phorbol 12-myristate 13-acetate (PMA) + Ionomycin for 6 h. Graphical quantification of % IFNγ + OT-I T cells below. (H) Graphical quantification of % granzyme B (GrzB) + expression in CD8 T cells (pre-gated on live, CD90.2 + , CD8 + ) from VPS33B WT and VPS33B ΔCSF1R IgLNs in the presence or absence of OVA (100 μg/mL) for 72 h. (I) IFNγ was quantified by ELISA from supernatants taken from total immunized LNs cultured in the presence or absence of OVA (100 μg/mL) for 72 h. NP = no protein (OVA); error bars shown as mean ± SEM. n = 3 biological replicates for one independent experiment. Statistical analysis was performed by two-way ANOVA. ∗p < 0.05, ∗∗ p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗ p < 0.0001, n.s. = not significant.

Article Snippet: Half of the cells were subject to CD44, Tbet, and GrzB flow cytometry staining while the other half was resuspended in low dose (10 U/mL) IL-2 (BioLegend; 575402) for an additional 48-h. Rested cells were then stimulated with PMA (Invivogen; tlrl-pma) and Ionomycin (Invivogen; inh-ion) with Brefeldin A (BioLegend; 420601) for 6 h and intracellular IFNγ was measured via flow cytometry.

Techniques: In Vivo, Injection, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay