β-adrenergic signaling in mice housed at standard temperatures suppresses an effector phenotype in CD8⁺ T cells and undermines checkpoint inhibitor therapy

Mice

Female BALB/cAnNcr (BALB/c) and C57BL/6NCr (C57BL/6) were purchased from Charles River and C.B. Igh-1b Icr Tac Prkdc scid (SCID) mice from the Laboratory Animal Resource at RPCI. BALB/c mice globally deficient in β₂-ARs (Adrb2^−/−) were provided by David Farrar (University of Texas Southwestern Medical Center) (24). OT-1 (B6.129S7-Rag1tm1Mom Tg[TcraTcrb]1,100Mjb) mice were provided by Dr. Minhyung Kim (RPCI). Mice were 8–12 weeks old and were maintained in specific pathogen-free facilities. All studies followed approved protocols and were performed under the guidelines established by the Institutional Animal Care and Use Committee at RPCI.

Cell culture and tumor models

4T1 tumor cells were purchased from and authenticated by ATCC in 2014. B16-OVA tumor cells were provided by Dr. Protul Shrikant in 2012 and have not been genetically authenticated by our laboratory. Cell lines were confirmed to be Mycoplasma negative using the Mycoplasma Plus PCR Primer Set (Aligent Technologies, 302008). Both cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS, 1% L-glutamine, and 1% Penicillin/Streptomycin. Once thawed, cells were passed twice prior to use. 1×10⁴ 4T1 cells in 100μl PBS were injected into 4^th mammary fat-pad. 2×10⁵ B16-OVA cells in 100μl PBS were injected subcutaneously into the lower left abdomen. Tumor growth was monitored throughout experiments; perpendicular diameters (width/length) were measured 2–3x/week and tumor volume calculated = W² × L/2 mm³.

Ambient temperature manipulation

Mice were housed five/cage in Precision Refrigerated Plant-Growth Incubators (Thermo Fisher Scientific) maintained at either standard temperature (~22°C) or thermoneutral temperature (~30°C) as previously described (14–16). Humidity was controlled using a Top Fin Air Pump AIR 1000 with Top Fin tubing. Mice were acclimated to the assigned temperature for at least 2 weeks.

Propranolol (β-blocker) studies

For studies in which propranolol was used to assess the impact of adrenergic signaling on the endogenous anti-tumor immune response, mice were acclimated to 22°C or 30°C housing temperature for 2 weeks; propranolol treatment in mice at 22°C and 30°C was initiated 4 days prior to tumor cell implantation and given daily until the experimental endpoint. Mice received 200μg propranolol (P0884, Sigma-Aldrich) in 100μl of PBS by i.p. injection; control mice received 100μl PBS for 4 days. Tumor cells were implanted (see above) and treatment continued for the duration of the experiment.

Immunotherapy and propranolol combination studies

Anti-mouse PD-1 antibody (RMP1-14) and rat IgG2a isotype control antibody (2A3) were purchased from BioXCell. The experimental design detailed above was modified for these studies in that treatment was not initiated until tumors were detectable to ensure that tumors in all groups were the same size when treatment was started. This experimental strategy was also designed to mimic clinical implementation of combining β-blockers with checkpoint inhibitors in patients who present to clinic with a tumor. For combination immunotherapy +/− propranolol studies, tumor cells were implanted into mice housed at 22°C prior to starting treatment and mice monitored daily for tumor formation. On a rolling basis beginning the day after tumors became detectable, mice were randomized to receive either PBS (100μl) + isotype antibody (200μg), propranolol (200μg) + isotype antibody (200μg), PBS (100μl) + anti-PD-1 (200μg), or propranolol (200μg) + anti-PD-1 (200μg). Mice received 5–6 injections of antibody spaced 2–3 days apart while propranolol and PBS were given daily.

CD8⁺ and CD4⁺ T cell depletion

Mice received weekly i.p. injections (400μg in 100 μl PBS) of CD8⁺ T cell depleting antibody (53-6.72, BioXCell) or CD4⁺ T cell depleting antibody (GK1.5, BioXCell) beginning 4 days prior to tumor implantation. Control animals received either rat IgG2a (2A3, BioXCell) or rat IgG2b (LTF-2, BioXCell) isotype controls for CD8⁺ and CD4⁺ T cell depletion studies respectively. Depletion was confirmed using flow cytometry.

Flow Cytometry

Single-cell suspensions were created by excising and cutting mouse tumors into 2–3mm pieces. 4T1 tumors were dissociated with collagenase/hyaluronidase (STEMCELL Technologies, 07912) and B16-OVA tumors with a murine tumor dissociation kit (Miltenyi, 130-096-730) following the manufacturers’ protocols prior to passing samples through a 70μm nylon cell strainer (Corning). Spleens were mechanically disrupted and directly passed through a 70μm nylon cell strainer (Corning). Red blood cells were lysed using ACK buffer (Gibco). Cells were then washed with flow running buffer (0.1% BSA in PBS) and incubated with anti-CD16/32 (Fc receptors blocker, 1:200) at 4°C for 15 minutes.

Cells were then stained with the following extracellular antibodies: anti-CD8α BUV395 (53-6.7), anti-CD8β PE (H35-17.2), anti-CD4 BV786 or BUV395 (GK1.5), anti-CD3 BV786, or APC-Cy7 (145-2C11), anti-CD45 FITC or BUV395 (30-F11), anti-PD-1 BV605 (J43), anti-CD11b BUV395 (M1/70), and anti-Gr-1 BV605 (RB6-8C5) all purchased from BD biosciences. CD8⁺ T cells specific for B16-OVA tumors were identified using the H-2K^b OVA (SIINFEKL) tetramer (APC or BV421) from MBL International Corporation; OT-1 splenocytes were used as a positive control for tetramer staining. Live/dead violet, aqua, or yellow dyes (Thermo Fisher) were used to gate out dead cells.

For intracellular staining, cells were surface-stained as above, then fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (eBiosciences) as per the manufacturer’s protocol. Cells were then stained with anti-FoxP3 Ax647 (MF23), anti-T-bet Ax647 (4B10), or anti-IFN-γ BV421 (XMG1.2) from BD biosciences, anti-IFN-γ APC (XMG1.2) from eBiosciences, or anti-granzyme B Ax647 (GB11) from Biolegend.

All data was collected on a LSR Fortessa flow cytometer (BD biosciences) and analyzed with FlowJo v10 software (Tree Star, Inc).

ELISA

Noradrenaline Research ELISA kits were purchased from Rocky Mountain Diagnostics (BA E-5200). Blood was collected at the experimental endpoint by retro-orbital bleeding and NE was determined in serum.

Statistical analysis

Students t-test was used to compare data between 2 groups and tumor growth statistics were calculated using two-way ANOVA with Tukey analysis using GraphPad Prism. One-way ANOVA with Tukey posthoc tests was used to compare data between 4 groups using SAS v9.4 (Cary, NC). All data are depicted as mean ± SEM.

Tumor growth in mice housed at ST is promoted by β-adrenergic signaling in non-tumor host cells and can be inhibited by propranolol treatment

We previously showed that several syngeneic tumors and a carcinogen-induced tumor grow more slowly when mice were housed at thermoneutrality (~30°C) than at standard temperatures (~22°C) and this is dependent on the adaptive immune system (14). However, the mechanism(s) mediating immune suppression at 22°C was not identified. We later showed that pancreatic tumor-bearing mice housed at 22°C have significantly higher norepinephrine (NE) levels than those housed at 30°C (16).

In this study, NE levels were also found to be significantly elevated in 4T1 tumor-bearing mice at 22°C compared to 30°C, indicating that this is not a tumor-type dependent phenomenon (Fig. 1A). To test the hypothesis that increased β-AR signaling at 22°C caused accelerated tumor growth when compared to 30°C, we acclimated mice to 22°C or 30°C, began treatment with propranolol (a pan-β-AR antagonist) or PBS 4 days prior to tumor implantation, then implanted tumor cells and monitored tumor formation and growth. A detailed experimental design is depicted in Fig. 1B. Pharmacologic blockade of adrenergic signaling by propranolol significantly slowed both B16-OVA (Fig. 1C) and 4T1 (Fig. 1D) tumor growth in mice at 22°C to a degree comparable to that achieved by housing mice at 30°C. Furthermore, addition of propranolol to mice at 30°C did not improve tumor growth control which is likely because NE levels are already significantly lower at baseline.

We next utilized a β₂-AR global receptor knockout BALB/c mouse (Adrb2^−/−) to assess the role of β₂-AR in host cells by comparing tumor growth between Adrb2^−/− and wildtype (WT) BALB/c mice (8). First, we physiologically manipulated adrenergic stress by housing mice at 22°C or 30°C. Then we implanted 4T1 tumor cells, which we and others have shown to lack functional β-ARs (25) (data not shown), and monitored tumor growth. Housing temperature had no impact on tumor growth in Adrb2^−/− mice and grew at a rate similar to that observed in WT mice housed at 30°C. However, tumors in WT mice at 22°C grew significantly faster than the other three groups (Fig. 1E) indicating that accelerated tumor growth at 22°C is dependent on functional β₂-ARs on host cells.

Lastly, we housed mice at 22°C and reduced adrenergic signaling pharmacologically with propranolol. As shown in Fig. 1F, propranolol treatment significantly slowed tumor growth in WT mice but had minimal effect in Adrb2^−/− mice, further supporting the conclusion that signaling through β₂-ARs in host cells drives immune suppression at 22°C. Unlike housing at 30°C however, there was a the small, but significant, reduction in tumor growth in Adrb2^−/− mice treated with a pan-β-AR antagonist suggesting a minor role for other β-AR isoforms not found on immune cells (i.e., β₁-AR and β₃-AR). These data suggest that increased signaling through β₂-ARs on host cells causes tumors to grow faster in mice housed at 22°C compared to those housed at 30°C.

Reduced tumor growth following β-AR blockade results from enhanced CD8⁺ T cell-mediated anti-tumor immune response

NE signaling through β₂-ARs on lymphocytes has recently been shown to suppress immune cell function in non-oncological settings (17,23,24), therefore we asked whether this signaling pathway mediates immune suppression in mice housed at 22°C. We repeated the experiments shown above (Fig. 1C, 1D) in SCID mice and found that neither physiologic (30°C housing) nor pharmacologic (propranolol) reductions in adrenergic signaling had any effect on tumor growth (Fig. 2A, 2B), indicating that the inhibition of tumor growth observed in Fig. 1C, 1D is dependent on the adaptive immune system.

In order to determine which immune cell(s) were responsible for the improved tumor growth control following β-blockade, we selectively depleted either CD8⁺ or CD4⁺ T cells from WT mice. Depletion of CD8⁺ T cells abrogated the effect of propranolol on both B16-OVA and 4T1 tumor growth (Fig. 2C, 2D), indicating dependence on CD8⁺ T cells. Furthermore, depletion of CD8⁺ T cells in Adrb2^−/− mice also accelerated tumor growth rates similar to that seen in WT mice following CD8⁺ T cell depletion (Fig. 2E). In contrast to CD8⁺ T depletion, the benefit of β-blockade was not lost following CD4⁺ T cell depletion (Supplementary Fig. S1) indicating that propranolol was slowing tumor growth in mice housed at 22°C in a CD8⁺ T cell-dependent manner.

Since T cell killing is dependent upon contact with tumor cells in the tumor microenvironment (TME), we examined the functional orientation of tumor infiltrating lymphocytes (TILs) (5,10,11). We evaluated the frequency and relative number (# cells/mg tumor) of CD8⁺ T cells in tumors using flow cytometry. Gating on all CD8⁺ TILs (independent of effector markers) revealed no difference in the frequency of CD8⁺ TILs from propranolol treated mice bearing 4T1 or B16-OVA tumors compared with controls (Supplementary Fig. S2A, S2B). Moreover, using an OVA-tetramer to evaluate antigen-specific CD8⁺ TILs in the B16-OVA tumor model further showed no difference in the frequency or relative number of antigen-specific CD8⁺ TILs (Supplementary Fig. S2C–S2E).

We next assessed the frequency and relative number of CD8⁺ TILs expressing the transcription factor T-bet which drives expression of the effector molecules IFN-γ and granzyme B (GzmB) in CD8⁺ T cells (26). As shown in Fig. 3A–3C for B16-OVA (4T1 data shown in Supplementary Fig. S3A–S3C), the frequency and relative number of CD8⁺ TILs expressing T-bet was significantly elevated in propranolol treated mice compared with controls. Similar observations were also made regarding the frequency and relative number of CD8⁺ TILs expressing IFN-γ (Fig. 3D–3F; 4T1 data in Supplementary Fig. S3D–S3F). We did not observe differences in the frequency or relative number of CD8⁺ TILs that expressed GzmB (Fig. 3G–3I). However, further analysis of the GzmB⁺ fraction of CD8⁺ TILs revealed that the GzmB MFI was significantly increased in the CD8⁺ T cell population of propranolol treated mice (Fig. 3J, 3K), indicating that CD8⁺ TILs with detectable GzmB were producing more GzmB in propranolol treated mice than PBS controls.

Thus far, these data indicate that β-AR blockade reduces tumor growth in a CD8⁺ T cell-dependent manner and increases the frequency of effector CD8⁺ TILs in both the 4T1 and B16-OVA tumor models.

β-AR blockade significantly increases the ratio of effector CD8⁺ T cells to Tregs in B16-OVA tumors and decreases the accumulation of MDSCs in the spleens of 4T1 tumor-bearing mice

Tumor infiltration by effector CD8⁺ T cells is a favorable prognostic indicator in many tumor types (5,10,11). However, the presence of CD4⁺ regulatory T cells (Tregs; CD4⁺ FoxP3⁺), as well as a low CD8⁺ T cell to Treg ratio, is often a negative prognostic indicator (5,27,28). We therefore asked whether propranolol treatment decreased numbers of Tregs and, if so, did this favorably alter the CD8:Treg ratio. At endpoint, we found that the frequency CD4⁺ T cells in B16-OVA tumors with a Treg phenotype was significantly reduced in propranolol treated mice (Fig. 4A, 4B). We next evaluated the ratio of total CD8⁺ T cells to Tregs, but did not observe a difference between groups (Fig. 4C). However, we did observe a significant increase in the ratio of effector (IFN-γ⁺) CD8⁺ T cells to Tregs in propranolol treated mice compared to controls (Fig. 4D).

In 4T1, we assessed the frequency of another potent suppressor cell type, myeloid derived suppressor cells (MDSC; CD11b⁺ Gr-1⁺), since they accumulate in the spleens of mice bearing 4T1 and other mammary tumors (29). Using flow cytometry, we observed that the mass of the spleens in propranolol treated mice was significantly less than that of untreated mice (Supplementary Fig. S4A). We also observed a significant reduction in the frequency and absolute number of MDSCs in the spleens of 4T1 tumor-bearing mice housed at 22°C treated with propranolol compared with PBS controls (Supplementary Fig. S4B, S4C).

Since the MDSC frequency correlates with tumor burden (29), we next asked whether adrenergic signaling played a role in the accumulation of MDSCs in addition to tumor burden. Here, we evaluated spleens of both non-tumor-bearing and 4T1 tumor-bearing mice treated with propranolol or PBS following the experimental outline detailed in Supplementary Fig. S5A. In the absence of a tumor, propranolol treatment did not alter MDSC frequency. However, analysis of spleens at three different time-points post 4T1 tumor implantation (days 7, 12, and 20) revealed that the frequency and absolute number of MDSCs were decreased in the propranolol treated cohorts despite the fact that tumor volumes were starting to separate but were not yet significantly different (Supplementary Fig. S5B–S5F).

Overall, these data indicate that β-blockade reduces the frequency of Tregs and increases the ratio of IFN-γ⁺ CD8 TILs to Tregs while also decreasing the accumulation of MDSCs in the spleens of 4T1 tumor-bearing mice.

Fewer effector CD8⁺ TILs express PD-1 in β-blocker treated mice compared to controls

Because we found that β-blockade reversed immunosuppression at 22°C, we next examined whether this would also change the expression of immune checkpoint molecules which naturally suppress the anti-tumor immune response, specifically PD-1 (programmed death receptor-1). Recent work indicates that tumors, as well as suppressive cells in the TME, can express the ligand PD-L1 which can bind to PD-1 and suppress anti-tumor immune responses. Moreover, one recent study concluded that the percentage of PD-1 expressing CD8⁺ T cells in murine tumors is one factor that dictates the responsiveness of tumors to anti-PD-1 (30). Specifically, responsive tumors had a low frequency of PD-1⁺ CD8⁺ TILs while non-responding tumors had a high frequency of PD-1⁺ CD8⁺ TILs.

Therefore, prior to testing anti-PD-1 checkpoint blockade in our stress reducing murine tumor models, we first investigated whether β-AR signaling had any impact on PD-1 expression. Following treatment of mice housed at 22°C with propranolol, we observed that the percentage of CD8⁺ TILs expressing PD-1 was decreased in both 4T1 (Fig. 5A, 5B) and B16-OVA tumors (Fig. 5C, 5D). Further analysis of the PD-1 MFI on the PD-1⁺ CD8⁺ TIL population from 4T1 tumors revealed that the expression of PD-1 was significantly decreased when mice were treated with propranolol compared with controls (Fig. 5E, 5F). In contrast, the PD-1 MFI was increased on PD-1⁺ CD8⁺ TILs isolated B16-OVA tumors in mice treated with propranolol compared with control animals (Fig. 5G, 5H).

To determine if the cells that expressed PD-1 had an effector phenotype, as evidenced by expression of T-bet and IFN-γ, we analyzed CD8⁺ TILs from 4T1 tumors that co-expressed effector molecules and PD-1. Here, we observed that the frequency of CD8⁺ TILs co-expressing PD-1 and either T-bet⁺ (Fig. 5I) or IFN-γ⁺ (Fig. 5J) was decreased in propranolol treated mice compared with PBS controls.

Taken together, these data indicate that the frequency of CD8⁺ TILs isolated from 4T1 and B16-OVA tumors which express PD-1 is decreased in mice housed at 22°C and treated with propranolol. In addition, fewer effector CD8⁺ TILs from 4T1 tumors express PD-1 in β-blocker treated mice, suggesting that not only does adrenergic signaling suppress the function of CD8⁺ TILs, but these effector cells are also more susceptible to suppression via PD-1/PD-L1 signaling.

β-adrenergic stress signaling impairs the efficacy of anti-PD-1 checkpoint blockade in murine tumor models

After determining that β-AR blockade facilitates development of endogenous anti-tumor immunity in murine tumor models, which was associated with a decreased percentage of PD-1 expressing CD8⁺ TILs, we next evaluated whether anti-PD-1 checkpoint blockade would have greater efficacy when adrenergic signaling was reduced. Since all previously reported murine studies assessing checkpoint inhibitor efficacy have been conducted at 22°C, we first used a physiologic approach to test whether housing temperature induced adrenergic signaling influenced anti-PD-1 efficacy in mice bearing B16-OVA and 4T1 tumors. Here, we acclimated mice to 22°C or 30°C, implanted tumor cells prior to initiation of treatment to ensure that tumors in all groups were established and of comparable size, and subsequently began treatment with anti-PD-1 or isotype antibodies as tumors became detectable. The treatment protocol is depicted in Supplementary Fig. S6A.

As shown in Supplementary Fig. S6B, although anti-PD-1 was modestly effective in slowing B16-OVA tumor growth in mice housed at 22°C, its efficacy was significantly improved in mice which had been acclimated to 30°C housing prior to tumor implantation. In the 4T1 model, anti-PD-1 had no impact on tumor growth in mice housed at 22°C (Supplementary Fig. S6C), whereas significantly improved efficacy of anti-PD-1 was observed in mice housed at 30°C.

We next tested the hypothesis that treating mice housed at 22°C with propranolol should recapitulate the beneficial effects on anti-PD-1 efficacy observed when housing mice at 30°C. To ensure that the tumors in each group were of comparable size, tumors were again implanted prior to treatment. In this clinically relevant therapeutic model, we began propranolol treatment after tumors became detectable. Mice treated with propranolol or PBS were then randomized to receive anti-PD-1 or isotype antibodies as detailed in Fig. 6A and Supplementary Fig. S7A.

In the B16-OVA tumor model, we again observed that anti-PD-1 alone had limited efficacy (Supplementary Fig. S7B) at 22°C. However, the addition of propranolol significantly improved responses to anti-PD-1 (Supplementary Fig. S7B). In 4T1 tumor-bearing mice, treatment with anti-PD-1 alone had no impact on tumor growth (Fig. 6B). In contrast, combining anti-PD-1 with propranolol significantly slowed 4T1 tumor growth (Fig 6B). Note that propranolol treatment alone had little effect on the ability of the immune response to control these established tumors in contrast to the beneficial effect seen when propranolol was given prior to tumor implantation.

Next, we sought to determine whether the improved efficacy of anti-PD-1 and propranolol in 4T1 tumor-bearing mice at 22°C was associated with changes in the immune contexture of the TME. We analyzed the effector phenotype of CD8⁺ TILs in tumor tissue taken from the experiment in Fig. 6B. We observed that propranolol, anti-PD-1, or a combination of the two had no influence on the frequency of total CD8⁺ T cells (Supplementary Fig. S8). However, the frequency and relative number of effector CD8⁺ TILs (IFN-γ⁺) in tumors from mice receiving combination therapy was significantly higher than all other groups (Fig. 7A–7C).

Altogether, these data show that combining propranolol and anti-PD-1 in mice housed at 22°C significantly changes the immune contexture of tumors to a more inflamed TME as evidenced by an overall increase in the frequency and relative number of effector CD8⁺ TILs. Importantly, these changes correlate with a significant improvement in the efficacy of anti-PD-1 therapy.