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

Introduction 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. Conclusion 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.


Introduction
The unique ability of dendritic cells (DCs) to cross-present antigens to CD8+ T-cells makes them the most potent antigen-presenting cells (APCs) in anti-tumor immunity cascade. This is highly promising, but DCs with dysregulated CD receptor signaling fail to respond significantly to tumor antigens, resulting in poor antigen presentation and T-cell mediated tumor clearance. To overcome this challenge, the use of anti-CD40 agonistic antibody (aCD40), an activator of APCs has grown with the goal of enhancing the proportions of functional/activated DCs and subsequent activation of cytotoxic T-cells (1,2). However, immunologically cold tumors can generate redundant immune evasive mechanisms to inhibit aCD40 immune activation by released tumor antigens, and clinical trials have shown that this approach is moderately effective as a monotherapy (3). Also, aCD40's short circulatory half-life and toxicity can further limit its clinical utility (4)(5)(6). Thus, therapeutic approaches that increase sensitivity to aCD40 immunotherapy and thereby reduce the required treatment doses are needed to improve outcomes in patients with cold tumors. Herein, we aimed to dissect the role of b2-adrenergic signaling in aCD40 treatment, thereby providing a mechanistic and pharmacological basis to improve outcomes in clinical settings.
Adrenergic signaling mediated stress and anti-tumor immunity are intricately linked and demonstrate an inverse relationship. The sympathetic nervous system is closely associated with the body's immune system since both primary and secondary lymphoid organs are permeated by post-ganglionic sympathetic neurons (7). The neurotransmitters (norepinephrine or NE) released from adrenergic neurons during stress can bind to the b2-adrenergic receptor (b2AR) present on tumor cell membranes. This binding activates anti-apoptotic pathways via adenylyl cyclase to induce rapid tumor growth rates, metastasis, chemo-and radio-resistance (8)(9)(10)(11). The released NE also engages with macrophages, DCs, or T-cells to enhance macrophage polarization from M1 to M2 type, increases the production of anti-inflammatory cytokines, and reduces the proliferative capacities of cytotoxic T-cells (8,12). Several studies have already demonstrated that b-ARs expressed on DCs decline pro-inflammatory cytokine secretion (13)(14)(15)(16), antigen uptake (17), antigen presentation (18,19), and migration capabilities (15,20). What is not known is how the complementary activation mechanisms of aCD40 and b2AR influence DC effector functions, and whether targeting these signaling pathways concurrently would translate into superior tumor control relative to monotherapies.
Among the b2AR inhibitors, Propranolol, an FDA approved Pan-Beta blocker has been shown to improve outcomes of radiation (21), Immune checkpoint inhibitors (ICI) (22-24) & c h e m o t h e r a p i e s (2 5) in p r e -c li n i c al t u m o r m o d e l s . Mechanistically, propranolol remodels tumor microenvironment by increasing the infiltration of effector CD8+ T-cells and declining suppressor cell populations. Propranolol hydrochloride is also being investigated in clinical trials as supportive therapy for prostate cancer (26) and stage IIIC-IV melanoma, and as part of combinatorial regimens of recurrent or metastatic urothelial cancer with anti-PD1 ICI (27), and radiation therapy for esophageal cancer (28). Based on these promising features, the aims of this study were two-fold. First, we assessed the implications of b-adrenergic signaling on DCs activation and maturation mediated by aCD40. Next, we evaluated the ability of propranolol to improve local aCD40 in-situ immunotherapy of poorly immunogenic head and neck tumors (MOC2). In-situ therapies utilize direct injection of immunostimulatory reagents into tumors to disrupt local immunosuppression, thereby reducing the dosage and associated toxicities. Our data shows that blocking b2AR can enhance the in-situ aCD40 efficacy against the MOC2 tumor model at suboptimal doses, thereby providing a translation basis of this approach for clinical use.

Generation of tumor cell lysate, and bone marrow-derived Dendritic cells (BMDCs) for cytotoxicity and signaling studies
The lysate was generated by sonicating 10,000 MOC2 cells using Branson Sonifier 450 and centrifuging the lysate to harvest whole or partially lysed cells. The protein content of the whole lysate was determined using a BCA assay kit.
Female mice aged 8-12 weeks were utilized to generate bone marrow-derived DCs (BMDCs) through culturing of the harvested marrow cells with RPMI media that was enriched with FBS, Penicillin-Streptomycin, and GM-CSF. On the 5th day, the BMDCs were harvested, and their CD11c expression was confirmed using flow cytometry (≥80% CD11c+) and these cells were used for subsequent experiments as described in Figure 1. Briefly, the BMDCs were divided into two groups: naïve BMDCs (nDCs, untreated with tumor cell lysate) and induced DCs (iDCs, treated with tumor cell lysate). 25,000 BMDCs were treated with 1mM Isoproterenol (non-selective b2AR agonist, ISO), 10mg/ml aCD40 or 100 mg/ml tumor cells lysate and incubated for 48h in RPMI with 100ng/ml GM-CSF to assess cytotoxicity using the MTT assay (following manufacturer's protocol), and for other mechanistic assays as described in following sections. For cotreatments, BMDCs were pretreated with 1mM ISO for 1h before being subjected to aCD40 or tumor cell lysate and incubated for 48h.

3.2
In-vitro and in-vivo immune cell analysis using flow cytometry  Figure S1) and compensations were done using unstained cells, single-stained cells, and appropriate FMOs.

RT-qPCR analysis for cytokine gene expression in nDC and iDCs
RNA was extracted from treated and untreated nDCs and iDCs using TRIzol reagent followed by DNase treatment. cDNA was prepared from 1mg of total RNA using cDNA synthesis kit following manufacturer protocol. SYBR green based real-time analysis was done to detect the expression of IL-1b, IL-6 & IL-10 genes using GAPDH as a housekeeping gene. PCR mixture contained 1ml cDNA, 10ml SYBR green master mix (2X), and 100nM of each reverse and forward primer in a total volume of 20ml. PCR was run using settings of 95°C for 15s and 60°C for 60s for 35 cycles, on 7500 Fast Real-Time PCR system, Applied Biosystem. DC T for the gene target was calculated by subtracting GAPDH C T values for each replicate and represented as 2^DC T in the bar graph. Primer sequences are given in Table 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 mM ISO (a b2AR agonist) and 10 mg/ml aCD40 (a CD40 agonist) for 48 hours before analysis. Schematic created with BioRender.com.

Western blot analysis of NFkB pathway targets and quantification of released cytokine
nDC and iDC were lysed and the total protein amount was estimated using BCA assay. An equal amount of cell lysate (30mg) was loaded on SDS-PAGE (BioRad, MiniPROTEAN Tetra System), and transferred to NC membrane (BioRad Trans-Blot Turbo) for 30min run at a constant voltage of 25V. Blots were blocked using 3% BSA solution for 1h at RT and stained with anti-pIkBa, anti-IkBa, anti-pCREB, and anti-CREB antibodies overnight at 4°C. Blots were washed in TBST buffer before incubating with appropriate secondary antibodies for 1h at RT, and imaged using BioRad, ChemiDoc ™ MP Imaging System. Blots were stripped using mild stripping buffer (Glycine, pH2) and re-stained with anti-GAPDH antibody for 1h at RT following the above-mentioned procedure. To create intensity graphs, the intensities of pIkBa, IkBa, pCREB1, CREB, and GAPDH bands were measured using ImageJ software. The background intensities were subtracted from each blot. The intensities of pIkBa, IkBa, pCREB, or CREB were then normalized by dividing them with the GAPDH band intensities and represented as ratios of the phosphorylated to unphosphorylated IkBa or CREB on a bar graph. Data were analyzed using 2 independent BMDC experiments. ELISA was also performed to quantify the released cytokines, IL-6, IL-1b, IL-10 & IL-12, in 50ml of iDC culture supernatants following the manufacturer's protocol.

MOC2 in-vivo study design
All animal associated procedures were approved by Oklahoma State University Animal Care and Use Committee. For tumor inoculation, MOC2 cells (purchased from Kerafast, Boston, MA, USA) cultured in DMEM media supplemented with 10% v/v FBS & 100U/ml PenStrep, harvested at 70-80% confluency were washed with sterile cold PBS before inoculations. 8-weeks old C57BL/6 female mice were injected subcutaneously with 1.5 X 10 5 MOC2 cells in the flank region. Propranolol-HCl (10 mg/kg B.W.) resuspended in sterile PBS was injected subcutaneously from day 5 onwards daily until mice euthanasia. Tumor volumes were measured daily using a caliper (3-in Digital caliper, UltraTECH) and calculated using the formula (L*W*W)/2. Once tumors reached an average volume of~50 mm 3 , 30 mg of aCD40 antibody was injected intratumorally. Two doses were given 8 days apart, and mice were euthanized 4-wk post-inoculation. Tumors were harvested for immune cell analysis using flow cytometry.

Statistical analysis
All analyses were performed using Prism v.9.4.0 software (GraphPad Software Inc. La Jolla, CA, USA). Treatment groups were compared using Two-tailed Unpaired T-tests. For group analysis with multiple variables, One-way ANOVA with either Tukey or Bonferroni multiple comparison test, and Two-way ANOVA were used as applicable. P values less than 0.05 was considered significant and represented as * P <0.05, ** P <0.005, *** P <0.0005, **** P <0.0001.

Results
4.1 b2AR signaling reduced the surface expression of co-stimulatory molecules on nDCs and iDCs.

b2AR
signaling reduced aCD40 priming abilities of nDCs and iDCs.
Unlike iDCs, nDCs that were co-treated with aCD40 and ISO showed significantly reduced cell viability compared to aCD40 alone ( Figure 3A). Additionally, although the MHC-II and CD86 expression on CD11c+ cells and percentage of CD40+ cell modified similarly for nDCs and iDCs ( Figure 3B, C & Figure S2), however, the MFI of CD40 expression on iDC was significantly reduced relative to nDC ( Figure  S2), indicating an overall decline in the aCD40 mediated priming of iDCs following b2AR activation ( Figures 3D, E).

b2AR signaling reengineered CD40
signaling by inhibiting phosphorylation of IkBa subunit of the Ikk complex to alter cytokine production.
The activator of NFkB1 & NFkB2 complexes, i.e. IKK complex, was analyzed by targeting phosphorylated IkBa (pIkBa, Ser32) using western blot and represented as the ratio of phosphorylated and unphosphorylated IkBa band intensities. In both nDCs and iDCs, a significant increase in intracellular levels of pIkBa vs IkBa was 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. observed with aCD40 treatment, but pIkBa levels declined significantly with aCD40+ISO cotreatment (~1.5-fold in nDCs and 4-fold in iDCs). Also, the levels of phosphorylated CREB (pCREB, Ser133) relative to unphosphorylated CREB (CREB) decreased slightly in nDCs with aCD40 treatment, but this phenomenon was more evident in iDCs. The addition of ISO (± aCD40) significantly increased the levels of pCREB in both nDCs and iDCs ( Figure 4A). To understand the association of IKK complex activation with cytokines production, the gene expression of IL-1b, IL-6 & IL-10 in iDCs was quantified. Expression of IL-10 decreased, and IL-1b & IL-6 increased in presence of aCD40 in iDCs. In contrast, ISO significantly decreased the expression of IL-1b and IL-6 and increased the expression of IL-10 in aCD40 treated iDCs. (Figure 4B). These were similarly observed in culture supernatant with significant decreases in pro-inflammatory cytokine levels of IL-1b, IL-6, and IL-12 and an increase in antiinflammatory IL-10 levels in aCD40+ISO treated iDCs relative to aCD40 treated cells ( Figure 4C).

aCD40 and propranolol combination achieved superior MOC2 tumor suppression and induction of anti-tumor immunity
Propranolol mediated efficacy of aCD40 in-situ vaccination (ISV) in the MOC2 model was evaluated by comparing tumor growth up to 4 weeks post-inoculation (see Figure 5A schematic).
aCD40 and propranolol induced partial to moderate reduction of tumor growth compared to the untreated control, but the combined treatment achieved a significant suppression of tumor growth rates compared to the control (p<0.05; Figure 5B). We also evaluated the percentage of T-cells and their functional counterparts in the tumor. Data showed a significant increase in the frequency of tumorinfiltrating CD45+ CD3+ T-cells in the combination regimen compared to control which was not observed with monotherapies ( Figure 5C). Notably, a significantly higher infiltration of CD8+ Tcells in Prop+aCD40 treated tumors (~24%) relative to untreated tumors (9%) and aCD40 (~15%) was noted. A significantly higher numbers of CD8+ T cells vs CD4+ T cells was observed with combination treatment compared to control and Prop alone treated tumors. A non-statistically significant decrease in regulatory T-cells (CD4+ Foxp3+) populations and an increase in cytotoxic T-cells (CD8+ GZMB+) was observed with all therapies compared to control but their ratio demonstrated a significantly higher number of cytotoxic T-cells vs Tregs in aCD40 and Prop+aCD40 groups compared to the untreated control. Further, the addition of Prop enhanced CD45+ CD11c+ cells by 2.5-fold in treated tumors compared to untreated and aCD40 treated tumors ( Figure 5D Analysis of Phosphorylated IkBa and CREB levels in nDCs and iDCs treated with ISO (1µM) and aCD40 (10 µg/ml) for 48h. (A) Western blots showed reduced pIkBa levels compared to unphosphorylated IkBa in the presence of ISO in both aCD40 treated nDCs and iDCs. ISO treatment increased pCREB levels in both nDCs and iDCs, with or without aCD40 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.

Discussion
Prior research has shown that NE-mediated adrenergic receptor activation inhibits the activation, differentiation, and effector functions of T-cells (29)(30)(31)(32) and modulates the cytokines and chemokines production from macrophages, monocytes, and DCs (7,33,34). b2AR signaling can also polarize macrophages from M1 to M2 type, thereby enhancing anti-inflammatory cytokines like IL-10, IL-4 & IL-13, and decreasing pro-inflammatory cytokines like INFg, TNFa, IL1b, CCL2, CCL3 and CCL4 to cause tumor progression (35)(36)(37)(38)(39). Considering that CD40 pathways mainly impact APCs, the reported roles of b2AR signaling on macrophages and DCs piqued our interest in understanding their ability to modulate their functions and impact therapeutic outcomes. We found that b2AR signaling limits DCs function in the presence of tumor antigens by decreasing the expression of MHC-II, CD86, and CD40.
Our data disagrees with a previous study (40) that showed no effects on MHC-II & CD86 expression in NE and LPS stimulated DCs. We believe that unlike LPS that works via TLR4, DAMPs & PAMPs present in tumor cell lysate can induce DC activations through multiple signaling mechanisms (TLR3, TLR4, RAGE, etc.) on DCs (41, 42), thereby generating dramatic different immunoactivities with b2AR activation vs LPS alone. To demonstrate this, we compared the stimulation of BMDCs with two types of cancer cell lysates (B16F10 melanoma and MOC2 oral squamous carcinoma) to that of LPS treatments. Our results showed that LPS-treated BMDCs did not exhibit any changes in MHC-2 and CD86 surface expression, while BMDC stimulation with B16F10 and MOC2 tumor cell lysates resulted in differential MHC-2 expression in response to ISO treatment (as shown in Figure S3). While we did not investigate the specific mechanisms behind the differences in MHC-2 and CD86 expression on DCs with the various types of cell lysates, our data still provides strong evidence that the expression of immunomodulatory markers on DCs is dependent on the composition of the ligand/antigen pool.
The use of Pan Beta-blocker like propranolol enhances the efficacy of immunotherapy by blocking b2AR signaling on progenitor and functional immune cells to result in better therapeutic outcomes (22,27,31). We looked at the effects of b2AR activation on DCs in presence of aCD40 in BMDCs. b2AR signaling can regulate NFkB pathways by inhibiting phosphorylation of IkBa through enhanced b-Arrestin2 protein production (43,44). We found that activation of b2AR on aCD40 treated DCs significantly decreased the levels of pIkBa and enhanced the accumulation of unphosphorylated IkBa in cells, thereby suggesting the suppression of DC maturation and CD40mediated NFkB activation. b2AR signaling has also been shown to activate cAMP/PKA pathways and subsequent CREB phosphorylation. We observed higher levels of pCREB in ISO treated nDCs and iDCs ( Figure 4A). Phosphorylated (p) CREB can compete with activated NFkB for the DNA binding sites. Thus, we propose that pCREB may indirectly interfere with aCD40 mediated DC priming (see Figure 6 schematic), to decrease the production of pro-inflammatory cytokines and co-stimulatory molecules (e.g. decreased IL-6 and IL-1B expression and enhanced IL-10 production in iDCs) (45,46). We noticed similar trends in the cytokines produced in the culture supernatants. The ability of DCs to activate T cells is often evaluated by measuring IL-12 production and co-stimulatory molecules (47,48), so we examined the IL-12 released from these cells and found that ISO treatment had a significant impact on the release of IL-12 from aCD40-treated induced DCs (iDCs). Thus, we propose that activation of b2AR signaling in aCD40-treated naïve and induced DCs transforms the DC population into an immune-tolerant type, both by blocking NFkB activation and indirectly promoting CREB activation. Propranolol has been shown to improve the functions of naïve and activated immune cells (22,27,31). Since b2AR activation subverted aCD40 signaling in BMDCs, we also investigated the effects of pharmacological b2AR blocking on therapeutic outcomes of aCD40 in immunologically cold MOC2 tumors (49). Despite the improvements made in the potency of CD40 monoclonal antibodies (mAbs) through approaches such as engineering the Fc region, the use of aCD40 immunotherapy in clinics is still faced with challenges due to its associated toxicities. These toxicities include liver damage, low platelet count, cytokine release syndrome (CRS), and hyper-immune activation (2,50,51). Also, the widespread expression of the CD40 receptor on both immune and non-immune cells in tumors and other organs leads to broad activation of CD40-expressing cells, limiting the treatment dose of aCD40 mAbs and causing non-specific toxicity. Therefore, we employed a lower dose of aCD40 antibody (30µg; 2X) with Propranolol via intratumoral route to overcome the dose-limiting toxicities associated with higher quantities of aCD40 (4). b-blocker with aCD40 treatment significantly suppressed tumor growth at suboptimal doses compared to untreated control, and the outcomes were comparable to a prior report administering higher aCD40 dosage (50µg) and greater frequency (3X) of treatment in the melanoma model (52). MOC2 tumors show a high presence of Tregs and minimal populations of CD8 T-cells (53,54). Blocking b2AR in mice tumor models can increase lymphocyte infiltration, and lower M1 to M2 polarization of macrophage and Treg population (22, 30, 55, 56). Our findings demonstrate that the combination of Prop+aCD40 effectively modulates the tumor microenvironment, leading to a pronounced increase in CD8+ T cell and cytotoxic T cell infiltration, and a reduction in Tregs compared to tumors treated with monotherapies or untreated control. Surface expression of MHC and co-stimulatory molecules on DCs are known to correlate with enhanced T cell activation and its effector functions (57)(58)(59)(60)(61). Blocking b2AR signaling with Prop significantly increased intratumoral CD11c+ populations, and when combined with aCD40 treatment, the number of double-positive MHC-II+ CD40+ DCs significantly increased. This suggests that the combination treatment facilitated robust anti-tumor immune cell priming and maturation. The Proposed mechanism of b2AR signaling mediated re-engineering of CD40-CD40L signaling in DCs. An increase in intracellular pIkBa (pIkBa) level with aCD40 treatment is reversed by b2AR 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.
increased presence of dendritic cells in the tumor microenvironment, facilitated by Prop treatment, provided a stronger foundation for aCD40 immune therapy. As a result, the combination treatment showed a significant improvement in adaptive anti-tumor immunity compared to control, unlike monotherapies. For in-vivo studies, our goal was to include a true untreated control to simulate clinical conditions and evaluate the immunological synergism between aCD40 & Propranolol. PBS is not a standard treatment for head and neck tumors and other types of tumors. Therefore, previous pre-clinical studies, including ours, have used untreated controls relative to PBS to assess the effectiveness of in-situ vaccination or parenteral treatment (52,(62)(63)(64). Additionally, studies by Hu et al., Singh et al., and others have shown that PBS or non-relevant/ isotype control antibody treatments do not enhance tumor control compared to propranolol or aCD40 alone (64)(65)(66)(67)(68). Future studies may include PBS (or an isotype control antibody) to further confirm the feasibility of the proposed combination therapy in the MOC2 murine model.
Thus, our investigation provides the foundational basis for improving aCD40 immunotherapy by the use of b-blockers. This combination can be particularly relevant for cold TMEs and can reverse b2AR signaling mediated tumor cell survival seen previously (69,70). We found that the combination of Prop+aCD40 enhanced therapeutic and anti-tumor immune responses compared to control, however, some changes were not statistically different from monotherapies. Further studies are needed to optimize treatment dosages and timelines to achieve a pronounced tumor remission. Gender-specific differences in b-blocker response and immune cell characteristics should also be explored to gain a deeper understanding of the therapeutic outcomes (71).

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by Oklahoma State University Animal Care and Use Committee.

Author contributions
AR and AS conceived the project and designed the study goals. AS performed the experiments and analyzed the data. AR and AS wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding
We acknowledge the National Cancer Institute of the National Institutes of Health under award number R01CA260974, and the Kerr Endowed Chair at Oklahoma State University for supporting this research.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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SUPPLEMENTARY FIGURE 1
Gating strategy used for the analysis of BMDCs.

SUPPLEMENTARY FIGURE 3
MHC-II and CD86 expression on CD11c+ BMDCs under different stimulation. (A) 10,000 BMDC (>80% CD11c+) were stimulated with 1 µg/ml LPS and in combination with 10 µg/ml aCD40 for 48h showed a significant increase in MHC-II and CD86 expression. Upon ISO treatment, LPS alone stimulated BMDCs demonstrated no change in MHC-II and CD86 expression whereas it decreased in LPS+aCD40 treated BMDCs. (B) BMDCs treated with 100 µg/ml B16F10 tumor cell lysate showed a significant decrease in MHC-II expression which increased with the addition of aCD40. With ISO treatment, MHC-II expression further increased in these cells whereas CD86 expression decreased. (C) BMDCs treated with 100 µg/ml MOC2 tumor cell lysate alone and with aCD40 showed a similar pattern of MHC-II expression as B16F10 lysate stimulated cells but MHC-II and CD86 expression decreased upon 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.

SUPPLEMENTARY FIGURE 4
Western blots of phosphorylated and unphosphorylated IkBa (A) & CREB (B), presented in Figure 4A, are shown with their respective GAPDH blots used for normalization. (C) Table summarizing normalized band intensities of target proteins represented as a ratio of phosphorylated and unphosphorylated forms of IkBa & CREB in Figure 4A.

SUPPLEMENTARY TABLE 1
Frequencies of immune cells analyzed in murine MOC2 tumor model treated with b2AR antagonist (Prop) and CD40 agonist (aCD40).