γδ T Cells Support Antigen-Specific αß T cell-Mediated Antitumor Responses during BCG Treatment for Bladder Cancer.

Bacillus Calmette-Guérin (BCG) is the most effective intravesical agent at reducing recurrence for patients with high-grade, non-muscle-invasive bladder cancer. Nevertheless, response to BCG is variable and strategies to boost BCG efficacy have not materialized. Prior work demonstrated a requirement for either conventional αß or nonconventional γδ T cells in mediating BCG treatment efficacy, yet the importance of T-cell antigen specificity for BCG's treatment effect is unclear. Here, we provide direct evidence to show that BCG increases the number of tumor antigen-specific αß T cells in patients with bladder cancer and protects mice from subsequent same-tumor challenge, supporting BCG induction of tumor-specific memory and protection. Adoptive T-cell transfers of antigen-specific αß T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen-specific αß T cells contributed to BCG efficacy. BCG-specific antitumor immunity, however, also required nonconventional γδ T cells. Prior work shows that the mTOR inhibitor rapamycin induces the proliferation and effector function of γδ T cells. Here, rapamycin increased BCG efficacy against both mouse and human bladder cancer in vivo in a γδ T cell-dependent manner. Thus, γδ T cells augment antitumor adaptive immune effects of BCG and support rapamycin as a promising approach to boost BCG efficacy in the treatment of non-muscle-invasive bladder cancer.

Rapamycin enhances BCG-specific γδ T cells during intravesical BCG therapy for non-muscle invasive bladder cancer: a randomized, double-blind study.

BACKGROUND: Although intravesical BCG is the standard treatment of high-grade non-muscle invasive bladder cancer (NMIBC), response rates remain unsatisfactory. In preclinical models, rapamycin enhances BCG vaccine efficacy against tuberculosis and the killing capacity of γδ T cells, which are critical for BCG's antitumor effects. Here, we monitored immunity, safety, and tolerability of rapamycin combined with BCG in patients with NMIBC. METHODS: A randomized double-blind trial of oral rapamycin (0.5 or 2.0 mg daily) versus placebo for 1 month was conducted in patients with NMIBC concurrently receiving 3 weekly BCG instillations (NCT02753309). The primary outcome was induction of BCG-specific γδ T cells, measured as a percentage change from baseline. Post-BCG urinary cytokines and immune cells were examined as surrogates for local immune response in the bladder. Secondary outcomes measured were adverse events (AEs) and tolerability using validated patient-reported questionnaires. RESULTS: Thirty-one patients were randomized (11 placebo, 8 rapamycin 2.0 mg, and 12 rapamycin 0.5 mg). AEs were similar across groups and most were grade 1-2. One (12.5%) patient randomized to 2.0 mg rapamycin was taken off treatment due to stomatitis. No significant differences in urinary symptoms, bowel function, or bother were observed between groups. The median (IQR) percentage change in BCG-specific γδ T cells from baseline per group was as follows: -26% (-51% to 24%) for placebo, 9.6% (-59% to 117%) for rapamycin 0.5 mg (versus placebo, p=0.18), and 78.8% (-31% to 115%) for rapamycin 2.0 mg (versus placebo, p=0.03). BCG-induced cytokines showed a progressive increase in IL-8 (p=0.02) and TNF-α (p=0.04) over time for patients on rapamycin 2.0 mg, whereas patients receiving placebo had no significant change in urinary cytokines. Compared with placebo, patients receiving 2.0 mg rapamycin had increased urinary γδ T cells at the first week of BCG (p=0.02). CONCLUSIONS: Four weeks of 0.5 and 2.0 mg oral rapamycin daily is safe and tolerable in combination with BCG for patients with NMIBC. Rapamycin enhances BCG-specific γδ T cell immunity and boosts urinary cytokines during BCG treatment. Further study is needed to determine long-term rapamycin safety, tolerability and effects on BCG efficacy.

Rapamycin Prevents Surgery-Induced Immune Dysfunction in Patients with Bladder Cancer.

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients (P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction.

Altered expression of hepatic ß-adrenergic receptors in aging rats: implications for age-related metabolic dysfunction in liver.

Increased ß-adrenergic receptor (ß-AR)-mediated activation of adenylyl cyclase (AC) in rat liver during aging has been linked to age-related increases in hepatic glucose output and hepatosteatosis. In this study, we investigated the expression of ß-ARs, individual receptor subtypes, and G protein-coupled receptor (GPCR) regulatory proteins in livers from aging rats. Radioligand-binding studies demonstrated that ß-AR density increased by greater than threefold in hepatocyte membranes from senescent (24-mo-old) compared with young adult (7-mo-old) rats and that this phenomenon was blocked by food restriction, which is known to retard aging processes in rodents. Competition-binding studies revealed a mixed population of ß1- and ß2-AR subtypes in liver membranes over the adult life span, with a trend for greater ß2-AR density with age. Expression of both ß-AR subtype mRNAs in rat liver increased with age, whereas ß2- but not ß1-AR protein levels declined in livers of old animals. Immunoreactive ß2- but not ß1-ARs were preferentially distributed in pericentral hepatic regions. Levels of GRK2/3 and ß-arrestin 2 proteins, which are involved in downregulation of agonist-activated GPCRs, including ß-ARs, increased during aging. Insofar as sympathetic tone increases with age, our findings suggest that, despite enhanced agonist-mediated downregulation of hepatic ß-ARs preferentially affecting the ß2-AR subtype, increased generation of both receptor subtypes during aging augments the pool of plasma membrane-bound ß-ARs coupled to AC in hepatocytes. This study thus identifies one or both ß-AR subtypes as possible therapeutic targets involved in aberrant hepatic processes of glucose and lipid metabolism during aging.

ß2-Adrenergic receptor ablation modulates hepatic lipid accumulation and glucose tolerance in aging mice.

Catecholamines acting through ß-adrenergic receptors (ß(1)-, ß(2)-, ß(3)-AR subtypes) modulate important biological responses in various tissues. Our previous studies suggest a role for increased hepatic ß-AR-mediated signaling during aging as a mediator of hepatic steatosis, liver glucose output, and insulin resistance in rodents. In the current study, we have utilized ß(2)-AR knockout (KO) and wildtype (WT) control mice to define further the role of ß(2)-AR signaling during aging on lipid and glucose metabolism. Our results demonstrate for the first time that age-related increases in hepatic triglyceride accumulation and body weight are attenuated upon ß(2)-AR ablation. Although no differences in plasma triglyceride, non-esterified fatty acids or insulin levels were detected between old WT and KO animals, an age-associated increase in hepatic expression of lipid homeostasis regulator Cidea was significantly reduced in old KO mice. Interestingly, we also observed a shift from reduced glucose tolerance in young adult KO animals to significantly improved glucose tolerance in old KO when compared to age-matched WT mice. These results provide evidence for an important role played by ß(2)-ARs in the regulation of lipid and glucose metabolism during aging. The effect of ß(2)-AR ablation on caloric intake during aging is currently not known and requires investigation. Future studies are also warranted to delineate the ß(2)-AR-mediated mechanisms involved in the control of lipid and glucose homeostasis, especially in the context of a growing aging population.

Effects of maternal nutrient restriction, intrauterine growth restriction, and glucocorticoid exposure on phosphoenolpyruvate carboxykinase-1 expression in fetal baboon hepatocytes in vitro.

BACKGROUND: The objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (i) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase-1 (PEPCK-1) is upregulated in hepatocytes isolated from fetuses of nutrient-restricted mothers (MNR) compared with ad libitum-fed controls (CTR), and (ii) glucocorticoids stimulate PEPCK-1 expression. METHODS: Hepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK-1 protein and mRNA levels in hepatocytes were determined by Western blot and quantitative PCR, respectively. RESULTS: Fetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK-1 protein levels were increased in hepatocytes isolated from IUGR fetuses, and PEPCK-1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes. CONCLUSIONS: Cultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function.

Role of ß-adrenergic receptors in regulation of hepatic fat accumulation during aging.

Excessive fat accumulation in liver (hepatic steatosis) predisposes to hepatic functional and structural impairment and overall metabolic risk. Previous studies noted an association between hepatic steatosis and age in humans and rodents. However, the mechanisms leading to age-associated hepatic fat accumulation remain unknown. Earlier work from our group showed that ß-adrenergic receptor (ß-AR) levels and ß-AR-stimulated adenylyl cyclase activity increase in rat liver during aging. Here we investigated whether age-associated increases in ß-AR signaling play a role in augmenting hepatic lipid accumulation. We demonstrate an increase in hepatic lipid content during senescence and a significant correlation between hepatic fat content and stimulation of adenylyl cyclase activity by the ß-AR agonist isoproterenol in rat liver. Isoproterenol administration to young and old rodents in vivo increased hepatic lipid accumulation. Furthermore, in vitro overexpression of ß1- and ß2-AR subtypes in hepatocytes from young rodents increased cellular lipid content, whereas inhibition of ß-ARs by receptor subtype-specific inhibitors reduced lipid levels in hepatocytes from senescent animals. Isoproterenol-induced hepatic lipid accumulation in vivo was prevented by the ß-AR nonselective blocker propranolol, suggesting a novel therapeutic effect of this class of drugs in hepatic steatosis. Acipimox, which inhibits adipose tissue lipolysis, did not alter isoproterenol-mediated hepatic fat accumulation; thus ß-AR responsive hepatic lipid accumulation does not appear to be related primarily to altered lipolysis. These findings suggest that augmented hepatic ß-AR signaling during aging may increase lipid accumulation in liver and advocate a possible role for ß-adrenergic blockers in preventing or retarding the development of hepatic steatosis.