Fig 2: Impaired antibody class switching in B cells treated by Iµ exon dss ASO. (A) Antisense oligonucleotide targeting the donor splice site of Iµ exon (Iµ dss ASO) was designed and synthetized as “vivo-morpholino ASO” permitting passive administration of ASO in the cells (Gene Tools, LLC). Targeted µ GLT (uppercase: exon sequence; lowercase: intron sequence) and Iµ dss ASO sequences are indicated. Iµ, µ I exon; Sµ, µ switch region; CH1µ, µ constant exon 1; dss, donor splice site. (B–K) Splenic B cells were isolated from C57BL/6 mice, stimulated with LPS + IL4 (B–E) or LPS (F–K) and treated with 2 µM (B–E), 3 µM (F,G,J) or 4 µM (H,I,K) Iµ dss ASO or an irrelevant control ASO for 2 days (B,E) or 3 days (C,D,F–K). (B) Constitutively and alternatively spliced µ transcripts analyzed as described in Figure 2 using Iµ-for and Cµ-rev primers, described in schema A. One experiment out of three is shown. Relative quantification of amplification products was done using ImageJ software and expressed after normalization to actin band intensity. (C) Quantification of IgG1 in culture supernatants by ELISA. (D) Percentage of IgG1-positive cells determined by flow cytometry as described in Figure 2. (E) Unspliced ?1 GLT expression monitored as described in Figure 2. (F,I) Flow cytometry analysis of purified B-cell populations using the indicated cell surface markers. Plots are gated on live cells. The percentage of B220+IgG3+ (F) or B220+IgG2b+ (I) cells is indicated. One experiment out of three is shown. (G,H,J,K) Percentage of IgG3-positive (G,H) or IgG2b-positive (J,K) cells determined by flow cytometry. (B–K) Data are means ± SEM, n = 3 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, *P <0.05, **P <0.01, ****P <0.0001.

Fig 3: SLP76 Is Differentially Expressed in BMPCs versus SPPCs(A) Lcp2 mRNA expression in B cell and PC subsets (probe 205269_at) in the Amazonia database.(B) Lcp2 expression in B220+ B cells from naive mice, SP B220+CD138+ plasmablasts, and B220-CD138+ PCs from LPS-treated mice (D3) by Illumina microarray. **p < 0.01, ***p < 0.001.(C) Analysis from purified BMPC and SPPC samples using Affymetrix probe 1418641_at, normalized and annotated with the Affy and Limma Bioconductor packages in R. Statistical analysis was done between non-transformed expression values of BMPCs and SPPCs using Student’s t test. *p < .05.(D) Total WT SP and BM mononuclear cells (MNCs) stained for CD138, B220, and CD3, then for intracellular SLP76, and analyzed using flow cytometry of the indicated populations versus FMO (full minus one control). Quantification of SLP76 expression in BMPCs and SPPCs as normalized mean fluorescence intensities (MFIs) (MFI of SLP76 minus FMO) pooled from two independent experiments is shown on the right. *p < .05.Statistical analysis by two-way ANOVA; annotation key in STAR Methods. Error bars represent ± SD.

Fig 4: CD28 Regulates Glucose Uptake in LLPCs(A) BM and SP MNCs from two WT mice were stained for CD138 and B220 and cultured in NBDG to assess glucose uptake using flow cytometry. Data are representative of three individual experiments. **p < 0.01.(B) SP T cells were purified from WT or age-matched CD28-AYAA mice, activated with bead-bound anti-CD3 + anti-CD28 mAb + IL-2 ×24 h, and analyzed using Seahorse XF Glycostress test. Data are representative of three independent experiments. **p < 0.01.(C) Level of NBDG uptake between WT and CD28KI-AYAA PCs evaluated as in (A). *p < .05.(D) Purified BMPCs and SPPCs were treated with isotype control or anti-CD28 mAb × 16 h and evaluated for GLUT1 expression using flow cytometry. Results reflective of two independent experiments with values normalized to isotype control-treated MFI. Statistical analysis performed by analyzing raw MFI between control and anti-CD28 mAb-treated cells of SPPCs and BMPCs separately. **p < 0.01.(E) Glucose uptake assessed by isolation and activation of SPPCs and BMPCs as in (C), evaluating NBDG uptake using flow cytometry. Analysis performed on pooled data from two independent experiments, normalizing to control-treated MFI between SPPCs and BMPCs. *p < .05.Statistical analysis by Student’s t test. Error bars represent ± SD.

Fig 5: SDT plus aPD-L1 therapy activating systematic antitumor immunity. A Representative flow-cytometry plots showing the tumor-infiltrating leucocyte cells, including CD45 + cells (CD45 +) (A), CD8 + T cells (CD45 + CD3 + CD8 +) (B) and NK cells (CD45 + CD3-CD49b +) (C) in orthotopic tumors and the quantitative data (D–F). The tumor sections were stained using H&E, TUNNEL, Ki-67, and CRT (G) and the quantitative data (H–J)