Journal: Scientific Reports

Article Title: Chemotherapy-induced high expression of IL23A enhances efficacy of anti-PD-1 therapy in TNBC by co-activating the PI3K-AKT signaling pathway of CTLs

doi: 10.1038/s41598-024-65129-7

Figure Lengend Snippet: IL23A was significantly upregulated by chemotherapy and associated with a favorable prognosis in TNBC patients. ( A ) The whole transcriptomic sequencing of TNBC cells treated with chemotherapeutic drugs. Upper panel, the experimental design. Lower panel, overlapping immune- and secretome-related genes, and significantly affected genes in TNBC cells treated with chemotherapeutic drugs (log2|Fc|≥ 2, P < 0.05). ( B ) Heapmap of 18 genes identified in Fig. 1A, ranked by normalized log2Fc. IL23A was identified as the most upregulated gene in response to chemotherapeutic drugs. ( C ) mRNA levels of two subunits of IL-23 (IL23A and IL12B) in TNBC cells treated with chemotherapeutic drugs, detected by qRT-PCR. ( D ) Protein expressions and quantifications of IL-23 in TNBC cells treated with chemotherapeutic drugs, detected by western blot. The PVDF membranes were cropped at 35 kDa before antibody incubation. Original blots are presented in Supplementary Fig. . ( E ) Left panel, representative immunohistochemistry (IHC) staining of IL-23A and CD8 on resected tumor tissues from TNBC patients who did (n = 27) or did not (n = 37) receive neoadjuvant chemotherapy. Scale bars 50 μm. Middle and right panel, quantitative analysis. ( F ) Spearman correlation analysis revealing a correlation between IL-23A staining score and CD8 + T cell counts in resected tissues from TNBC patients (n = 64, r = 0.465, p = 0.0001). ( G ) Upper panel, representative CT scans before and after neoadjuvant chemotherapy from one of the patients whose resected tumor tissues exhibited high scores of IL-23A staining, as well as the representative IHC staining. Scale bars, 50 μm. Lower panel, the slope chart shows the overall response rate (ORR) of TNBC patients who received neoadjuvant chemotherapy (n = 22). Blue bars represent patients from the IL-23A low group with an IL-23A IHC score of 0–6 (n = 8); Red bars represent patients from the IL-23A high group with an IL-23A IHC score of 7–12 (n = 14). (H) The correlation between IL23A mRNA levels and overall survival of patients with breast cancer (HR = 0.73, P = 0.0048, left panel) or Triple-negative breast cancer (HR = 0.22, P < 0.0001, right panel). Data from the Kaplan–Meier plotter database ( https://www.kmplot.com/ ). Error bars represent standard deviation (SD), *P < 0.05; ** P < 0.01; ****P < 0.0001.

Article Snippet: After blocking with 1% BSA (Beyotime, China), the slides were incubated with primary antibodies [IL-23A (1:500; Proteintech, China), CD8α (1:200; Abcam, UK)] overnight at 4 °C and then the corresponding secondary antibody (ZSGB-BIO, China) for 30 min at room temperature.

Techniques: Sequencing, Quantitative RT-PCR, Western Blot, Incubation, Immunohistochemistry, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: Chemotherapy-induced high expression of IL23A enhances efficacy of anti-PD-1 therapy in TNBC by co-activating the PI3K-AKT signaling pathway of CTLs

doi: 10.1038/s41598-024-65129-7

Figure Lengend Snippet: Clinical parameters of patients for response evaluation in this study.

Article Snippet: After blocking with 1% BSA (Beyotime, China), the slides were incubated with primary antibodies [IL-23A (1:500; Proteintech, China), CD8α (1:200; Abcam, UK)] overnight at 4 °C and then the corresponding secondary antibody (ZSGB-BIO, China) for 30 min at room temperature.

Techniques: Significance Assay

Journal: Scientific reports

Article Title: Chemotherapy-induced high expression of IL23A enhances efficacy of anti-PD-1 therapy in TNBC by co-activating the PI3K-AKT signaling pathway of CTLs.

doi: 10.1038/s41598-024-65129-7

Figure Lengend Snippet: Figure 1. IL23A was significantly upregulated by chemotherapy and associated with a favorable prognosis in TNBC patients. (A) The whole transcriptomic sequencing of TNBC cells treated with chemotherapeutic drugs. Upper panel, the experimental design. Lower panel, overlapping immune- and secretome-related genes, and significantly affected genes in TNBC cells treated with chemotherapeutic drugs (log2|Fc|≥ 2, P < 0.05). (B) Heapmap of 18 genes identified in Fig. 1A, ranked by normalized log2Fc. IL23A was identified as the most upregulated gene in response to chemotherapeutic drugs. (C) mRNA levels of two subunits of IL-23 (IL23A and IL12B) in TNBC cells treated with chemotherapeutic drugs, detected by qRT-PCR. (D) Protein expressions and quantifications of IL-23 in TNBC cells treated with chemotherapeutic drugs, detected by western blot. The PVDF membranes were cropped at 35 kDa before antibody incubation. Original blots are presented in Supplementary Fig. S6. (E) Left panel, representative immunohistochemistry (IHC) staining of IL-23A and CD8 on resected tumor tissues from TNBC patients who did (n = 27) or did not (n = 37) receive neoadjuvant chemotherapy. Scale bars 50 μm. Middle and right panel, quantitative analysis. (F) Spearman correlation analysis revealing a correlation between IL-23A staining score and CD8 + T cell counts in resected tissues from TNBC patients (n = 64, r = 0.465, p = 0.0001). (G) Upper panel, representative CT scans before and after neoadjuvant chemotherapy from one of the patients whose resected tumor tissues exhibited high scores of IL-23A staining, as well as the representative IHC staining. Scale bars, 50 μm. Lower panel, the slope chart shows the overall response rate (ORR) of TNBC patients who received neoadjuvant chemotherapy (n = 22). Blue bars represent patients from the IL-23Alow group with an IL-23A IHC score of 0–6 (n = 8); Red bars represent patients from the IL-23Ahigh group with an IL-23A IHC score of 7–12 (n = 14). (H) The correlation between IL23A mRNA levels and overall survival of patients with breast cancer (HR = 0.73, P = 0.0048, left panel) or Triple-negative breast cancer (HR = 0.22, P < 0.0001, right panel). Data from the Kaplan–Meier plotter database (https://www.kmplot. com/). Error bars represent standard deviation (SD), *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: After blocking with 1% BSA (Beyotime, China), the slides were incubated with primary antibodies [IL-23A (1:500; Proteintech, China), CD8α (1:200; Abcam, UK)] overnight at 4 °C and then the corresponding secondary antibody (ZSGB-BIO, China) for 30 min at room temperature.

Techniques: Sequencing, Quantitative RT-PCR, Western Blot, Incubation, Immunohistochemistry, Staining, Standard Deviation

Journal: Journal of molecular biology

Article Title: Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

doi: 10.1016/j.jmb.2023.168300

Figure Lengend Snippet: Figure 4. Effects of IL-23a structural optimization on chaperone recruitment and IL-23 heterodimer assembly. (a) IL-23a proteins were equipped with a KDEL retention signal and FLAG-tag, the latter allowing immunoprecipitation (IP) from cell lysates. Interaction with Hsp70 family chaperone immunoglobulin heavy-chain binding protein (BiP) and protein disulfide isomerase (PDI) family members ERp72, ERp5, and ERp44 was analyzed via co-IP and immunoblotting. Mock, empty vector transfection. Iso., isotype control. MW, molecular weight. (b) Quantification of immunoblot IP signals, with unspecific signals subtracted and signal normalization to the wildtype (n = 8–12, ±SEM, * p < 0.05, multiple t-test) shows that chaperones interact differently with IL-23aKDEL variants. (c) Isothermal titration calorimetry (ITC) measurement of purified subunits IL-23aDfree cys and IL-12bC177S reveals thermodynamic parameters of wildtype-like IL-23 formation. Schematic depicts titration of IL-12b (orange) with its two fibronectin type III domains (ellipses) and the N-terminal immunoglobulin-like domain (hexamer) to the four-helix bundle IL-23a (green circle). (d) ITC measurement like in (c), with IL-23astabilized and IL-12bC177S, assessing respective thermodynamic parameters of subunit interaction during heterodimer formation. A fourfold lower KD-value for the engineered, stabilized IL-23 depicts higher binding affinity of IL-23astabilized in assembly with IL-12b compared to IL-23 formation with a subunit IL-23aDfree cys in vitro.

Article Snippet: After blocking the membrane with Tris-buffered saline (25 mM Tris/HCl, pH 7.5, 150 mM NaCl; TBS) containing 5% (w/v) skim milk powder and 0.05% (v/v) Tween-20 (M-TBST), binding of primary antibody was carried out o/n at 4 C with anti-Hsc70 (Santa Cruz Biotechnology, sc-7298, 1:1,000), anti-IL-12b (abcam, ab133752, 1:500), anti-IL-23a (Santa Cruz Biotechnology, sc-271279, 1:250–1:500), anti-myc-tag (Sigma-Aldrich, clone 4A6, 05- 724MG, 1:1,000), anti-FLAG-tag (Sigma-Aldrich, F7425, 1:1,000), anti-HA-tag (Biolegend, 902,302 1:500), anti-ERp70 (Proteintech, 14712-1-AP, 1:1,000), anti-PDIA6/ERp5 (Proteintech, 18233–1- AP, 1:1,000), anti-ERp44 (B68,19 1:1,000), antiBiP (Cell Signaling Technology, C50B12, 1:500) in M-TBST containing 0.002% NaN3.

Techniques: FLAG-tag, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Transfection, Control, Molecular Weight, Isothermal Titration Calorimetry, Titration, In Vitro

Journal: Journal of molecular biology

Article Title: Assembly-dependent Structure Formation Shapes Human Interleukin-23 versus Interleukin-12 Secretion.

doi: 10.1016/j.jmb.2023.168300

Figure Lengend Snippet: Figure 6. Assembly-dependent structure formation influences the human interleukin secretion ratio. Left: IL-23a (bright green) is highly flexible (red regions) with free cysteines (-SH), forming erroneous disulfide-linked species of high molecular weight (HMW) in the endoplasmic reticulum (ER) in isolation. It strongly interacts with a diverse chaperone repertoire (yellow) involved in ER quality control (ERQC) processes. Assembly-induced folding by IL-12b rescues this a subunit from ER-associated degradation (ERAD), resulting in IL-23 heterodimer formation and secretion (Extracellular). For IL-12a, chaperone interaction, misfolding to HMW species in isolation, and degradation by ERAD are depicted in gray and adopted from4. Chaperone interactions not analyzed in this study, but reported in other studies, are marked with one7 or two2,4 asterisks. a subunits compete for IL-12b interaction, with arrowhead sizes illustrating the IL-23:IL-12 ratios based on IL-23 and IL-12 ELISA of HEK 293 T supernatants. Right: For the engineered IL-23astabilized (dark green), an extra intramolecular disulfide bond structurally stabilizes its fold, leading to reduced chaperone binding, less HMW redox species formation, and slower protein turnover rates by ERAD. It can fold and secrete assembly-independent as well as interact with IL-12b with a higher affinity than the wild-type IL-23a to form IL-23stabilized. Thus, presence of the folding-competent IL-23astabilized subunit in the engineered setting shifts the assembled cytokine ratio (Extracellular, arrows) compared to the wild-type setting. Secreted heterodimeric cytokines induce different signaling in the target cell by receptor binding, with sizes of receptor chains demonstrating the relative cytokine secretion. IL-12 and IL-23 stimulate distinct immune responses by T helper (Th) subset differentiation (Th1 versus Th17) and other cytokine secretion (IFNc versus IL-17), resulting in strong pro- inflammatory (IL-23) or pro-inflammatory (IL-12) immunity.

Article Snippet: After blocking the membrane with Tris-buffered saline (25 mM Tris/HCl, pH 7.5, 150 mM NaCl; TBS) containing 5% (w/v) skim milk powder and 0.05% (v/v) Tween-20 (M-TBST), binding of primary antibody was carried out o/n at 4 C with anti-Hsc70 (Santa Cruz Biotechnology, sc-7298, 1:1,000), anti-IL-12b (abcam, ab133752, 1:500), anti-IL-23a (Santa Cruz Biotechnology, sc-271279, 1:250–1:500), anti-myc-tag (Sigma-Aldrich, clone 4A6, 05- 724MG, 1:1,000), anti-FLAG-tag (Sigma-Aldrich, F7425, 1:1,000), anti-HA-tag (Biolegend, 902,302 1:500), anti-ERp70 (Proteintech, 14712-1-AP, 1:1,000), anti-PDIA6/ERp5 (Proteintech, 18233–1- AP, 1:1,000), anti-ERp44 (B68,19 1:1,000), antiBiP (Cell Signaling Technology, C50B12, 1:500) in M-TBST containing 0.002% NaN3.

Techniques: High Molecular Weight, Isolation, Control, Enzyme-linked Immunosorbent Assay, Binding Assay