SUMMARY PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

Chromatin structure is epigenetically altered via covalent modifications of histones to allow for heritable gene regulation without altering the nucleotide sequence. Multiple lines of evidence from rodents have established a role for epigenetic remodeling in regulating gene transcription in response to an altered gestational milieu. However, to date, it is unknown whether variations in the intrauterine environment in primates similarly induce changes in key determinants of hepatic chromatin structure. We hypothesized that a maternal high-fat diet would alter the epigenomic profile of the developing offspring, which would result in alterations in fetal gene expression. Age-and weight-matched adult female Japanese macaques were placed on control (13% fat) or high-fat (35% fat) breeder diets and mated annually over a 4-year interval. Fetuses in successive years were delivered near term (e130 of 167 days) and underwent necropsy with tissue harvest. Fetal histones were acid extracted for characterization of H3 modification and chromatin immunoprecipitation (ChIP) with differential display PCR; fetal RNA, DNA, and cytoplasmic and nuclear protein extracts were similarly extracted for comparison. Chronic consumption of a maternal high-fat diet results in a threefold increase in fetal liver triglycerides and histologic correlates of non-alcoholic fatty liver disease. These gross changes in the fetal liver are accompanied by a statistically significant hyperacetylation of fetal hepatic tissue at H3K14 (199 . 85G9 . 64 . 096). However, epigenetic modifications on fetal hepatic H3 associated with gene repression were absent or subtle (PO0 . 05). Subsequent characterization of key epigenetic determinants associated with H3 acetylation marks revealed similar significant alterations in association with a high-fat maternal diet (e.g., relative fetal histone deacetylase 1 (HDAC1) gene expression 0 . 61G0 . 25; PZ0 . 011). Consistent with our mRNA expression profile, fetal nuclear extracts from offspring of highfat diet animals were observed to be significantly relatively deplete of HDAC1 protein (36 . 07G6 . 73 vs 83 . 18G7 . 51; PZ0 . 006) and in vitro HDAC functional activity (0 . 252G0 . 03 vs 0 . 698G0 . 02; P!0 . 001). We employ these observations in ChIP differential display PCR to attempt to identify potential fetal genes whose expression is reprogramed under conditions of a high-fat maternal diet. We quantitatively confirm a minimum of a 40% alteration in the expression of several genes of interest: glutamic pyruvate transaminase (alanine aminotransferase) 2 (GPT2) (1 . 59G0 . 23-fold; PZ0 . 08), DNAJA2 (1 . 36G0 . 21; PZ0 . 09), and Rdh12 (1 . 88G0 . 15; PZ0 . 01) are appreciably increased in fetal hepatic tissue from maternal caloric-dense diet animals when compared with control while Npas2, a peripheral circadian regulator, was significantly downmodulated in the offspring of high-fat diet animals (0 . 66G0 . 08; PZ0 . 03). In this study, we show that a current significant in utero exposure (caloric-dense hi...

Intrauterine growth restriction (IUGR) decreases serum insulin growth factor-1 (IGF-1) levels. IGF-1 is an epigenetically regulated gene that has two promoters, alternative exon 5 splicing, and multiple termination sites. The regulation of gene expression involves the whole gene, as evidenced by the aforementioned IGF-1 paradigm. We hypothesized that IUGR in the rat would affect hepatic IGF-1 expression and alter the epigenetic characteristics of the IGF-1 gene along its length. IUGR was induced through a bilateral uterine artery ligation of the pregnant rat, a well-characterized model of IUGR. Pups from anesthesia and sham-operated dams were used as controls. Real-time RT-PCR and ELISA was used to measure expression at day of life (DOL) 0 and 21. Bisulfite sequencing and chromatin immunoprecipitation (ChIP) quantified IGF-1 epigenetic characteristics. A nontranscribed intergenic control was used for ChIP studies. IUGR decreased hepatic and serum IGF-1. Concurrently, IUGR modified epigenetic characteristics, particularly the histone code, along the length of the hepatic IGF-1 gene. Many changes persisted postnatally, and the postnatal effect of IUGR on the histone code was gender-specific. We conclude that IUGR modifies epigenetic characteristics of the rat hepatic IGF-1 gene along the length of the whole gene.

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To examine effects of static exercise on the arterial baroreflex control of vascular sympathetic nerve activity, 22 healthy male volunteers performed 2 min of static handgrip exercise at 30% of maximal voluntary force, followed by postexercise circulatory arrest (PE-CA). Microneurographic recording of muscle sympathetic nerve activity (MSNA) was made with simultaneous recording of arterial pressure (Portapres). The relationship between MSNA and diastolic arterial pressure was calculated for each condition and was defined as the arterial baroreflex function. There was a close relationship between MSNA and diastolic arterial pressure in each subject at rest and during static exercise and PE-CA. The slope of the relationship significantly increased by >300% during static exercise (P < 0.001), and the x-axis intercept (diastolic arterial pressure level) increased by 13 mmHg during exercise (P < 0.001). These alterations in the baroreflex relationship were completely maintained during PE-CA. It is concluded that static handgrip exercise is associated with a resetting of the operating range and an increase in the reflex gain of the arterial barorelex control of MSNA.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of adult onset insulin resistance and dyslipidemia in humans and rats. IUGR rats are further characterized by postnatal alterations in hepatic PPAR-gamma coactivator (PGC-1) and carnitine-palmitoyl-transferase I (CPTI) expression, as well as overall hyperacetylation of histone H3. However, it is unknown whether the histone H3 hyperacetylation is site specific or relates to the changes in gene expression previously described in IUGR rats. We therefore hypothesized that uteroplacental insufficiency causes site-specific modifications in hepatic H3 acetylation and affects the association of acetylated histone H3 with PGC-1 and CPTI promoter sequences. Uteroplacental insufficiency was used to produce asymmetrical IUGR rats. IUGR significantly increased acetylation of H3 lysine-9 (H3/K9), lysine-14 (H3/K14), and lysine-18 (H3/K18) at day 0 of life, and these changes occurred in association with decreased nuclear protein levels of histone deacetylase 1 (HDAC1) and HDAC activity. Chromatin immunoprecipitation using acetyl-H3/K9 antibody and day 0 chromatin revealed that uteroplacental insufficiency affected the association between acetylated H3/K9 and the promoters of PGC-1 and CPTI, respectively, in IUGR liver. At day 21 of life, the neonatal pattern of H3 hyperacetylation persisted only in the IUGR males. We conclude that uteroplacental insufficiency increases H3 acetylation in a site-specific manner in IUGR liver and that these changes persist in male IUGR animals. The altered association of the PGC-1 and CPTI promoters with acetylated H3/K9 correlates with previous reports of IUGR altering the expression of these genes. We speculate that in utero alterations of chromatin structure contribute to fetal programming.

In adult myocardium, excitation-contraction coupling is critically regulated by sarcoplasmic reticulum (SR) Ca2+ release via type 2 ryanodine receptor (RyR2), but generally, it is believed that SR-function is rudimentary in the fetal heart and in embryonic stem (ES) cell-derived cardiomyocytes (ESCMs), a possible source for cell replacement therapies. This study used wild-type (RyR2+/+) and RyR2 null (RyR2-/-) ESCMs as an in vitro model of cardiomyogenesis, together with pharmacological approaches and expression profiles of genes relevant for SR function, to elucidate the functional importance of RyR2 and SR on the regulation of Ca2+ transients and contraction during early cardiomyocyte development. During differentiation of RyR2+/+ ESCMs, SR function developed progressively with increased basal cytosolic free Ca2+ concentration ([Ca2+]i), enhanced frequency and amplitude, and decreased duration of Ca2+ transients that were inhibited by ryanodine and thapsigargin. These functional traits correlated with SR Ca2+ load and the expression of RyR2, SERCA2a, and phospholamban. RyR2-/- ESCMs, comparatively, demonstrated a significantly prolonged time-to-peak and reduced frequency of Ca2+ transients and contractions. Beta-adrenergic stimulation of RyR2+/+ ESCMs increased the frequency and amplitude of Ca2+ transients with differentiation but was much weaker in RyR2-/- ESCMs. We conclude that functional SR and control of RyR2-mediated SR Ca2+ release directly contribute to the spontaneous and beta-adrenergic receptor-stimulated contraction of ESCMs, even at very immature stages of development.

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