Fig 1: In vitro cytokine production scales with programmed chimeric antigen receptor (proCAR) oligomeric state including tetramers.(a) SDS-PAGE migration of programmed membrane protein (proMP) C4.1 is consistent with a tetramer. Design model and peptide sequence shown for reference. (b) Rosetta ab initio structure prediction calculations predict that proMP C4.1 preferentially forms a tetramer. (c) CAR surface expression on primary mouse CD8+ T cells stably expressing CD28TM and proCAR-4 analyzed by c-Myc staining on flow cytometry. HER2 proCAR-4 was designed using the proMP C4.1 sequence without the final C-terminal leucine as a transmembrane domain (TMD), inserted as shown in Figure 3a. (d) IncuCyte killing assay over 24 hr of no CAR, CD28TM, and proCAR1-4 T cells on MC57-HER2 target cells at 1:1 effector to target ratio. Comparison of maximum killing for n = 6 independent experiments shown between CD28TM vs. ProCAR-4. Data points represent individual experiments, with mean ± SEM error bars plotted. (e) Tumor growth over time using the same experimental design in Figure 5a for No CAR (empty vector), CD28TM WT, and proCAR-4 T cell groups (n = 5–6 mice/group). Data points represent mean ± error bars showing SEM. Statistical analysis performed using a two-way ANOVA at day 10. (f, g) Linear correlation of proCAR oligomeric state vs. IFN?, IL-2, TNFa, and GM-CSF cytokine production (normalized to CD28TM reference) from 24 hr co-culture with (e) MC57-HER2 and (f) antigen-negative MC57 tumor cells. Individual data points are colored, mean values in white box and error bars indicate SEM.
Fig 2: Screen for genes that modulate in vitro antigen cross-presentation by DCCAS9 expressing-inducible immortalized dendritic cells (iniDC CAS9) were transfected with specific CRISPR RNAs (crRNA) to perform gene knockout during differentiation into de-iniDC CAS9. Gene edited de-iniDC CAS9 were incubated with ovalbumin (OVA) and cocultured with B3Z hybridoma cells to quantify interleukin-2 (IL-2) secretion as an indicator for antigen cross-presentation capacity. A violin plot is used to depict the IL-2 concentrations (mean value of duplicates) (A). The nonantigen loaded- and SIINFEKL peptide (SL8) loaded-DCs were used as negative and positive controls, respectively. Candidate gene whose KO leads to defective (blue color) or increased (red color) antigen cross-presentation are highlighted. De-iniDC CAS9 cells that are KO for Clec4a2 (Clec4a2−/−) were tested for cross-presentation of OVA antigen or the SINFKEL peptide (SL8) (B), and phagocytosis of MCA205 cells that were untreated (UT) or crizotinib (CRIZO)-treated (C). Cytokine concentration and phagocytic events (CD11C+ CellTracker+ cells) are reported as bar charts (means ± SD of triplicates). Statistical differences were calculated by means of the ANOVA test (Dunnett's multiple comparisons test), with ∗∗∗p < 0.001 as compared to UT, ###p<0.001 or $$$p<0.001 as compared between Fpr1−/− and wild type (wt) de-iniDC CAS9 cells with the same stimuli. Differentiated iniDC and iniDC CAS9 cells (de-iniDC) were pulsed with tumor cell lysates and intratumorally (i.t.) injected into subcutaneous MCA205 tumors grafted on immunocompetent mice to test their immunotherapeutic potential. Tumor growth curves are reported in (D) as mean ± SEM (n = 6). Statistical significance was calculated by means of the ANOVA Type 2 (Wald test) and significant results are indicated as ∗p<0.05 and ∗∗∗p<0.001 compared to the PBS injection group.
Fig 3: Characterization of the CAS9-expressing inducible immortalized dendritic cells (iniDC CAS9)(A) Validation of CAS9 expression in iniDC CAS9 clones by immunoblot.(B) Hmgb1 expression in iniDC CAS9 cells that were transfected with crRNA targeting murine Hmgb1 gene (two gRNA sequence labeled as H1 and H2), or the non-target control gRNA (NT). Different transfection conditions (0.25 or 0.5×106 cells/well in 12-well plate, 1 or 2×106 cells/well in 6-well plate) were tested.(C–M) Maturation and activation of DCs. BMDC, de-iniDC and de-iniDC CAS9 were treated with LPS or recombinant TNFα for 16 h. Supernatants were collected for ELISA quantification and cytokine release is reported as bar charts (C–G). Cells were subjected to antibody staining of surface markers and analyzed by flow cytometry. Representative histograms indicating the increase in the expression of surface marker and normalized median fluorescence intensity (MFI) are shown in (H–M).Comparison of IL-2 production (N) as a proxy of OVA antigen cross-presentation to B3Z hybridoma by DCs was evaluated as schematically depicted in (O). Nonantigen loaded- and SIINFEKL peptide (SL8)-incubated DCs were used as negative and positive controls, respectively. Bar charts express means ± SD of triplicate assessments, ND, nondetectable. Statistical differences were calculated by means of an ANOVA test (Dunnett's multiple comparisons test), with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 as compared to the untreated condition (UT).
Fig 4: Cytokine expression in splenic culture supernatant and in serum. (A-G) Cytokine levels in splenic culture supernatant. (A) IFN-γ, (B) TNF-α, (C) TGF-β, (D) IL-2, (E) IL-4, (F) IL-10, and (G) IL-12. (H-N) Serum cytokine levels. (H) IFN-γ, (I) TNF-α, (J) TGF-β, (K) IL-2, (L) IL-4, (M) IL-10, and (N) IL-12. Data are presented for the blank group (Blank), tumor-bearing model control group (Control), and drug injection group (CAS). *P<0.05, **P<0.01, ***P<0.001.
Fig 5: Cellular immune responses in CSFV-E2 mRNA-LNP-vaccinated mice. A Schematic representation of the experiments. Mice (n = 5 per group) were immunized intramuscularly with 5 μg of E2-ECD or E2-ECD-N mRNA-LNPs via a prime-only regimen. The control group received a placebo (PBS). Spleens were harvested 10 days post-vaccination. B Frequencies of IFN-γ- and IL-2-producing splenic CD4+ and CD8+ T cells following E2-ECD protein restimulation were detected via ICS assays and analysed by flow cytometry. Splenocytes were isolated on day 10 and stimulated with the recombinant E2-ECD protein for 48 h. C Measurement of CSFV-E2-specific IFN-γ- and IL-2-producing splenocytes by ELISpot assay. Splenocytes (5 × 105) were stimulated with E2-ECD protein for 36 h, and the spots were then counted. D Measurement of CSFV-E2-specific IL-2 and IL-4 responses in splenocytes by ELISA. IL-2 and IL-4 secretion by splenocytes after stimulation with the E2-ECD protein for 72 h. The cell culture supernatant was collected for detection by ELISA. The data shown are mean ± SEM of five mice per group. The data were analysed using two-way ANOVA with GraphPad Prism 8 (*P < 0.05; **P < 0.01; ns, not significant; ND, not detected).
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