PD-L1 is expressed in tumor cells and its interaction with PD-1 plays an important role in evading immune surveillance; this can be overcome using PD-L1 or PD-1 immunotherapy antibodies. This study reports a novel approach for targeting PD-L1. In human breast cancer cell lines and 4T1 mouse mammary tumor cells, PD-L1 expression was regulated by the nuclear receptor NR4A1/Sp1 complex bound to the proximal germinal center (GC)-rich region of the PD-L1 gene promoter. Treatment of breast cancer cells with bis-indole-derived NR4A1 antagonists including 1,1-bis(3 0indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (Cl-OCH3) decreased expression of PD-L1 mRNA, promoterdependent luciferase activity, and protein. In in vivo studies using a syngeneic mouse model bearing orthotopically injected 4T1 cells, Cl-OCH3 decreased tumor growth and weight and inhibited lung metastasis. Cl-OCH3 also decreased expression of CD3 þ /CD4 þ / CD25 þ /FoxP3 þ regulatory T cells and increased the Teff/Treg ratio. Therefore, the potent anticancer activities of NR4A1 antagonists are also accompanied by enhanced antitumor immunity in PD-L1-expressing triple-negative breast cancer and thus represent a novel class of drugs that mimic immunotherapy.Significance: These findings show that the orphan nuclear receptor NR4A1 controls PD-L1 expression and identify a chemical probe capable of disrupting this regulatory axis.

We present detailed simulations of the kilonova and gamma-ray burst (GRB) afterglow and kilonova luminosity function from black hole–neutron star (BH–NS) mergers, and discuss the detectability of an electromagnetic (EM) counterpart in connection with gravitational wave (GW) detections, GW-triggered target-of-opportunity observations, and time-domain blind searches. The predicted absolute magnitude of BH–NS kilonovae at 0.5 days after the merger falls in the range [−10, −15.5]. The simulated luminosity function contains potential information on the viewing-angle distribution of the anisotropic kilonova emission. We simulate the GW detection rates, detectable distances, and signal duration for future networks of 2nd/2.5th/3rd generation GW detectors. BH–NSs tend to produce brighter kilonovae and afterglows if the BH has a higher aligned spin, and a less massive NS with a stiffer equation of state. The detectability of kilonovae is especially sensitive to the BH spin. If BHs typically have low spins, the BH–NS EM counterparts are hard to discover. For 2nd generation GW detector networks, a limiting magnitude of m limit ∼ 23–24 mag is required to detect kilonovae even if high BH spin is assumed. Thus, a plausible explanation for the lack of BH–NS-associated kilonova detection during LIGO/Virgo O3 is that either there is no EM counterpart (plunging events) or the current follow-ups are too shallow. These observations still have the chance to detect the on-axis jet afterglow associated with a short GRB or an orphan afterglow. Follow-up observations can detect possible associated short GRB afterglows, from which kilonova signatures may be studied. For time-domain observations, a high-cadence search in redder filters is recommended to detect more BH–NS-associated kilonovae and afterglows.

Over the past decade, time-domain astronomy in optical bands has developed rapidly with the operations of some wide-field survey facilities. However, most of these surveys are conducted with only a single band, and simultaneous color information is usually unavailable for the objects monitored during the survey. Here we present introductions to the system of Tsinghua University-Ma Huateng Telescopes for Survey (TMTS), which consists of an array of four optical telescopes installed on a single equatorial mount. Such a system is designed to get multiband photometry simultaneously for stars and transients discovered during the survey. The optics of each telescope is a modified Hamilton-Newtonian system, covering the wavelengths from 400 nm to 900 nm, with a field of view (FoV) of about 4.5 deg 2 and a plate scale of 1.86 ′′ /pixel when combining with a 4K×4K QHY4040 CMOS detector. The TMTS system can have a FoV of about 9 deg 2 when monitoring the sky with two bands (i.e., SDSS g and r filters) at the same time, and a maximum FoV of ∼18 deg 2 when four telescopes monitor different sky areas in monochromatic filter mode. For an exposure time of 60s, the average 3-σ detection limit of the TMTS system can reach at ∼19.4 mag in Luminous filter and at ∼18.7 mag in SDSS r filter. The preliminary discovery obtained during the first few months' survey is briefly discussed. As this telescope array is located at the Xinglong Observatory of NAOC, it can have an excellent synergy with the spectroscopic survey by the LAMOST (with a FoV of about 20 deg 2) at the same site, which will benefit the studies of stellar and binary physics besides the transient sciences.

<p>Supplemental Figure S2. Quantitation of results in Figure 1F and 1G. and mRNA results A. Breast cancer cells were treated with Cl-OCH3 (Fig. 1F) and effects on PD-L1, NR4A1 and Sp1 were determined by western blots and quantitation of the results are summarized. B. MDA-MB-231 and 4T1 cells were treated with NR4A1 antagonists, MG132 and their combinations, whole cell lysates were analyzed by western blots (Fig. 1G) and quantitation of the results are summarized. Results of both studies (A, B) are means {plus minus} SD for at least 3 replicate determinations for each treatment groups and changes in expression were determined relative to the control group (set ratio) significantly (p<0.05) decreased effects are indicated (*) and inhibition (reversal of degradation) by MG132 is also indicated (**). C. Basal PD-L1 and NR4A1 mRNA levels in cancer cell lines were determined as described in the Methods.</p>

<p>Supplemental Figure S5. A-F. Gating Strategy for Teff (CD3+/CD8+) and Treg (CD3+/CD4+/CD25+/FoxP3+) cells. In panel A, cells are selected within the "cells" gate from the Forward Scatter versus Side Scatter dot plot. In panel B, cells from within the "cells" gate are plotted on a dot plot showing Side Scatter Area versus Side Scatter Height in which the single cells are selected within the "singlets" gate. In panel C, single cells from within the "cells" and "singlets" gates are plotted on a dot plot depicting Side Scatter versus Live/Dead Near IR dye. Viable, single cells are selected within the "viable" gate. In panel D, viable, single cells are shown on a dot plot depicting Side Scatter versus CD3 PE-Cy5.5 in which the CD3+ cells are selected within the "CD3+" gate. In panel E, CD3+, live, single cells are visualized on a dot plot with CD8 PE-eFluor 610 on the x-axis and CD4 Alexa Fluor 488 on the y-axis. The CD4+ cells are selected within "CD4+" gate and the CD8+ cells are selected within the "CD8+" gate. In panel E, the CD4+, CD3+, viable, single cells are plotted on a dot plot showing FoxP3 BV 421 versus CD25 Alexa Fluor 647. The regulatory T cells are selected within the "T reg cells" gate. G/H. GraphPad PRISM software was used to calculate Effective concentration (EC50) of PD-L1 mRNA (G) and PD-L1 protein (H) after treatment with different concentrations of CDIM-8 and Cl-OCH3 and the values were calculated by regression analysis using the dose-response curves generated from the experimental data.</p>