Fig 2: CD28 signaling regulates ATG5 expression(A) Western blot of MM.1S after 24 h of 1 μM CC treatment ± αCD28 mAb in SF/FM.(B) Western blot of MM.1S after 24 h of SF ± αCD28 mAb. MM was pre-treated with control, 50 μM CHX, or 10 nM Btz for 2 h.(C) Western blot of siCtrl- or siHuR-treated MM.1S after 16 h in SF/FM ± αCD28 mAb.(D) Western blot of fractionation of MM.1S after 6 h of SF ± αCD28 mAb ± CC.(E and F) Percentage of ATG5 mRNA by αeIF4E (E) or αHuR (F) from MM.1S after 6 h in SF ± αCD28 mAb ± CC or 5 μM CMLD2.(G and H) Survival of CMLD2- (G) or siCtrl or siHuR (H)-treated MM.1S with moDC ± melphalan ± CTLA4-Ig. RNA immunoprecipitation/western blot: Representative results from 2 replicates; flow cytometry: mean ± SD of ≥3 replicates.(I and J) CytoID staining (I) or MM viability (J) of CD138+ cells from Vκ*MYC mice BM (n = 5 mice) ± BMDC ± 5 μM CMLD2 ± CTLA4-Ig.(K–O) Flow analysis of ATG5 (K), CytoID (L), LipidTOX (M), viability (N), and viable cell numbers (O) of primary human MM cells ± moDC ± CTLA4-Ig ± CMLD2.Paired multiple t test.(E–J) Two-way ANOVA with Bonferroni adjustment.n.s., not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Fig 3: CD28 signaling directly regulates autophagy and lipophagy(A) LD number per MM by ImageStream after 24 h in FM/SF ± αCD28 mAb.(B and C) Flow cytometry of LipidTOX staining of Ctrl/Lal2/MRT (B) or siCtrl/siATG5/siLIPA (C)-treated MM ± moDC ± CLTA4-Ig for 24 h.(D and E) MM were cultured in FM/SF ± αCD28 mAb for 24 h. Western blots of LIPA (D) and LC3 (E).(F) Flow cytometry of CytoID staining of MM.1S ± moDC ± melphalan ± CTLA4-Ig for 24 h.(G and H) Flow cytometry of mCh-GFP-LC3 expressed MM ± moDC ± CTLA4-Ig for 24 h. The representative histogram (G) and quantitation (H) are shown.(I and J) ELISA for total TG (I) or FAs (J) from MM.1S in FM/SF ± αCD28 mAb ± MRT for 16 h.(K and L) CytoID (K) or LipidTOX (L) of CD138+ cells from Vκ*MYC mice BM (n = 5 mice) ± BMDC ± CTLA4-Ig ± MRT ± Lal2 for 24 h.(M and N) Flow analysis of CytoID (M) or LipidTOX (N) of primary human MM cells ± moDC ± CTLA4-Ig ± MRT ± Lal2 for 24 h.ImageStream/western blot/ELISA: representative data from 2 replicates; flow cytometry: mean ± SD of ≥3 replicates.(A–C, F, H, K, and L) Two-way ANOVA with Bonferroni adjustment.(I and J) Student’s t test.(M and N) Paired multiple t test.n.s., not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Fig 4: SOAT2 induces Teffs cellular senescence via competitive cholesterol metabolism of Treg cells.A–C Representative images of Teffs stained for β-galactosidase (arrows) after co-cultured with Treg cells (n = 3) (A), scale bars: 50 μm; quantitation of β-galactosidase-positive Teffs were shown (B, C). D–F The protein expression of P16, P21, P53 in Teff cells in the young and old individuals were monitored by western blotting analysis in indicated groups. The experiment was repeated 3 times independently with similar results. G–J, Treg cells were co-cultured with CD8+ T lymphocytes at a 0.2:1 ratio in indicated groups, and the expression of CD27 or CD28 in human Teffs (G, H) and murine Teffs (I, J) were detected by flow cytometric analysis (n = 3). K Schematic representation of Treg cells exerts immunosuppressive path on Teffs (Created in BioRender. Major, Z. (2024) https://BioRender.com/s68z662). L, M The content of cholesterol components were measured by the Cholesterol Assay Kit in indicated groups (n = 5). N–P Representative images of Teffs stained for β-galactosidase (arrows) after co-cultured with Treg cells (N), scale bars: 50 μm; quantitation of β-galactosidase-positive Teffs were shown (n = 3) (O, P). Data represented means ± SD. Statistical difference was evaluated by one-way ANOVA test (B, C, L, M, O, P). Source data are provided as a Source Data file. (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 5: ZEA augments CD8+ T cell surface TCR stimulation(A) KEGG pathway enrichment for upregulated genes generated from KAS-seq analysis of αCD3/CD28-stimulated murine CD8+ T cells treated with 5 μM ZEA compared to cells treated with DMSO control (n = 3 biological replicates).(B) Schematic depicting experimental setup for flow cytometry and confocal microscopy detection of αTCRβ, αCD3ε, and αCD28 antibody binding in murine CD8+ T cells with or without 5 μM ZEA or LUT treatment with different stimulation strategies. This schematic was generated using BioRender.com.(C) Flow cytometry analysis showing the effects of 5 μM ZEA or LUT on αTCRβ, αCD3ε, and αCD28 antibody binding in murine CD8+ T cells at 5, 15, and 30 min after treatment and stimulation with αCD3ε and αCD28 antibodies (n = 3 technical replicates).(D) Representative confocal microscopy images showing the effects of 5 μM ZEA on αTCRβ, αCD3ε, and αCD28 antibody binding in murine CD8+ T cells at 5, 15, and 30 min after treatment and stimulation with αCD3ε and αCD28 antibodies (n = 3 technical replicates). Scale bars represent 5 μm.(E) Quantification of fluorescence intensities from confocal microscopy images shown in (D). TCRβ, CD3, and CD28 antibody signals were measured at 5, 15, and 30 min after treatment and stimulation with CD3 and CD28 antibodies (n = 3 technical replicates).(F) The chemical structure of the ZEA-based photo-affinity labeling (PAL) probe and schematic workflow for validating its binding to the TCR complex.(G) Pull-down of the TCR complex using the biotinylated ZEA probe. Mouse primary CD8+ T cells were incubated with DMSO and 50 μM ZEA probe or pre-treated with 250 μM ZEA for 1 h followed by 50 μM ZEA probe. After UV-induced crosslinking, samples were subjected to click chemistry with a biotin tag, followed by streptavidin pull-down and immunoblotting.Data are mean ± SD. p values are calculated using two-way ANOVA (ns, not significant; ∗∗∗p < 0.001). Also see Figure S3 and Table S2.