Adrenergic receptor signaling regulates the CD40-receptor mediated anti-tumor immunity

Abstract

Inroduction: Anti-CD40 agonistic antibody (αCD40), an activator of dendritic cells (DC) can enhance antigen presentation and activate cytotoxic T-cells against poorly immunogenic tumors. However, cancer immunotherapy trials also suggest that αCD40 is only moderately effective in patients, falling short of achieving clinical success. Identifying factors that decrease αCD40 immune-stimulating effects can aid the translation of this agent to clinical reality.

Method/results: Here, we reveal that β-adrenergic signaling on DCs directly interferes with αCD40 efficacy in immunologically cold head and neck tumor model. We discovered that β-2 adrenergic receptor (β2AR) activation rewires CD40 signaling in DCs by directly inhibiting the phosphorylation of IκBα and indirectly by upregulating levels of phosphorylated-cAMP response element-binding protein (pCREB). Importantly, the addition of propranolol, a pan β-Blocker reprograms the CD40 pathways, inducing superior tumor regressions, increased infiltration of cytotoxic T-cells, and a reduced burden of regulatory T-cells in tumors compared to monotherapy.

Discussion/conclusion: Thus, our study highlights an important mechanistic link between stress-induced β2AR signaling and reduced αCD40 efficacy in cold tumors, providing a new combinatorial approach to improve clinical outcomes in patients.

Keywords: Anti-CD40 agonist antibody; adrenergic signaling; anti-tumor immunity; immunotherapy; propranalol.

Figure 1

A schematic representation of the in-vitro BMDC isolation and treatment plan is shown. Bone marrow cells derived from C57BL/6 female mice were cultured in DC conditioning media and the expression of CD11c was confirmed after 5 days of culture. BMDCs with ≥80% CD11c expression were categorized as naïve (nDCs) or induced DCs (iDCs) based on exposure to MOC2 tumor cell lysate. The cells were then treated with 1 μM ISO (a β2AR agonist) and 10 μg/ml αCD40 (a CD40 agonist) for 48 hours before analysis. Schematic created with BioRender.com.

Figure 2

The impact of ISO (1µM) treatment on the viability and surface expression of co-stimulatory molecules on CD11c+ expressing naïve DC (nDCs) and tumor lysate induced DCs (iDCs) was evaluated after 48 hours. (A) ISO reduced nDCs viability, while having no effect on iDCs. (B, C) Both nDCs and iDCs treated with ISO showed significant decreases in surface expression of co-stimulatory molecules, MHC-II+, CD86+ and CD40+. (D) iDCs exposed to ISO showed a higher decrease in MHC-II, CD86 and CD40 surface expression compared to nDCs. The fold change was calculated by comparing the ISO treated population to the respective control (untreated nDCs or lysate-only treated iDCs) using the formula [(ISO treated population/Control population)-1]. (E) The results are demonstrated by representative contour plots of MHC-II+, CD86+ and CD40+ cell populations, showing the intensity of ISO treated nDCs and iDCs overlaid with their respective controls. Statistical analysis was carried out using unpaired t-test, One-way ANOVA & Two-way ANOVA tests where applicable. P values less than 0.05 were considered significant. * P <0.05, ** P <0.005, *** P <0.0005, **** P <0.0001.

Figure 3

Viability & frequencies of MHC-II+, CD86+ & CD40+ nDCs and iDCs exposed to 10 µg/ml αCD40 and 1µM ISO for 48h. (A) A significant decrease in the viability of αCD40 treated nDCs with ISO treatment was observed in absence of tumor lysate stimulation. (B, C). The population of MHC-II+ and CD86+ nDC and iDC was reduced with ISO and αCD40 co-treatment, however, CD40+ population remain unchanged in nDCs but reduced in iDCs. (D) Decrease in the surface expression of co-stimulatory molecules was significantly higher in αCD40-treated iDCs compared to nDCs. Fold change in a cell population with ISO treatment was calculated using αCD40 only treated nDCs and iDCs as control and using the formula: [(ISO treated population/Control population)-1]. (E) The results are demonstrated by representative contour plots of MHC-II+, CD86+ and CD40+ cell populations, showing the intensity of ISO treated nDCs and iDCs overlaid with their respective αCD40 treatment controls. Statistical analysis was carried out using unpaired T-test, One-way ANOVA & Two-way ANOVA tests where applicable. P values less than 0.05 were considered significant. *** P <0.0005, **** P <0.0001.

Figure 4

Analysis of Phosphorylated IkBα and CREB levels in nDCs and iDCs treated with ISO (1µM) and αCD40 (10 µg/ml) for 48h. (A) Western blots showed reduced pIkBα levels compared to unphosphorylated IkBα in the presence of ISO in both αCD40 treated nDCs and iDCs. ISO treatment increased pCREB levels in both nDCs and iDCs, with or without αCD40 treatment. The ratio of phosphorylated to unphosphorylated forms is shown as the intensity graphs. Respective GAPDH blots used to normalize the band intensities are shown in Figures S4A, B and the normalized band intensities are summarized in Figure S5C . (B) Gene expression analysis revealed that αCD40+ISO treatment significantly decreased IL-1β & IL-6 levels in iDCs compared to αCD40 treatment alone, and increased IL-10 expression in these cells. The results are expressed as 2^ΔCT with respect to GAPDH levels. (C) The release of IL-1β, IL-6 & IL-10 in the culture supernatant of co-treated iDCs showed a similar trend, with αCD40-mediated increase in released IL-12 significantly declining with ISO treatment. Statistical analysis was carried out using One-way ANOVA. P values less than 0.05 were considered significant. * P <0.05, ** P <0.005, *** P <0.0005, **** P <0.0001.

Figure 5

Treatment design of the murine efficacy and immune-evaluation study. (A) Propranolol (10mg/kg of BW) was administered subcutaneously daily 5 days post-inoculation. Two 30 µg αCD40 intratumoral injections were administered at 8 days intervals in the tumor (~50 mm³ volume). Mean tumor volume and anti-tumor immune cells were compared on day 28 post-inoculation (Timeline created with BioRender.com). (B) The combination of Prop+αCD40 demonstrated a significant reduction in tumor volume compared to the control on day 28 post-inoculation, while monotherapies did not show any significant differences. These results suggest that the combination therapy of Prop+αCD40 is more effective in reducing tumor growth compared to either Prop or αCD40 alone. Immune cells infiltrating MOC2 tumors (n=5 mice/group) analyzed by flow cytometry showed superior immunomodulation with Prop+αCD40. (C) Frequencies of CD3+ T-cells, especially CD8+ T-cells infiltrating tumors were enhanced at the highest level by combination treatment vs untreated control and monotherapies. The ratio of cytotoxic T-cells (CD8+ GZMB+) to T regulatory cells (CD4+ Foxp3+) was increased significantly in αCD40 treated groups relative to the control. (D) CD11c+, MHC-II+ CD86+ double positive (gated at CD45+ CD11c+) & MHC-II+ CD40+ double positive (gated at CD45+ CD11c+) dendritic cell frequencies showed significant enhancements in the presence of Prop and αCD40. Statistical analysis was carried out using One-way ANOVA & Two-way ANOVA multiple comparison tests. P values less than 0.05 were considered significant. * P <0.05, ** P <0.005, *** P <0.0005, **** P <0.0001. ns, nonsignificant.

Figure 6

Proposed mechanism of β2AR signaling mediated re-engineering of CD40-CD40L signaling in DCs. An increase in intracellular pIkBα (pIkBα) level with αCD40 treatment is reversed by β2AR signaling, thereby resulting in an altered cytokine production and immuno-retardation of anti-tumor response. Adapted from “NF-KB Signaling Pathway”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates.