Programmed death-ligand 1 is a glycoprotein expressed on antigen presenting cells, hepatocytes, and tumors which upon interaction with programmed death-1, results in inhibition of antigen-specific T cell responses. Here, we report a mechanism of inhibiting programmed death-ligand 1 through small molecule-induced dimerization and internalization. This represents a mechanism of checkpoint inhibition, which differentiates from anti-programmed death-ligand 1 antibodies which function through molecular disruption of the programmed death 1 interaction. Testing of programmed death ligand 1 small molecule inhibition in a humanized mouse model of colorectal cancer results in a significant reduction in tumor size and promotes T cell proliferation. In addition, antigen-specific T and B cell responses from patients with chronic hepatitis B infection are significantly elevated upon programmed death ligand 1 small molecule inhibitor treatment. Taken together, these data identify a mechanism of small molecule-induced programmed death ligand 1 internalization with potential therapeutic implications in oncology and chronic viral infections.

Knowledge of the nitrogen-vacancy center formation kinetics in diamond is critical to engineering sensors and quantum information devices based on this defect. Here we utilize the longitudinal tracking of single NV centers to elucidate NV defect kinetics during high-temperature annealing from 800-1100 $^\circ$C in high-purity chemical-vapor-deposition diamond. We observe three phenomena which can coexist: NV formation, NV quenching, and NV orientation changes. Of relevance to NV-based applications, a 6 to 24-fold enhancement in the NV density, in the absence of sample irradiation, is observed by annealing at 980 $^\circ$C, and NV orientation changes are observed at 1050 $^\circ$C. With respect to the fundamental understanding of defect kinetics in ultra-pure diamond, our results indicate a significant vacancy source can be activated for NV creation between 950-980 $^\circ$C and suggests that native hydrogen from NVH$_y$ complexes plays a dominant role in NV quenching, in agreement with recent {\it ab initio} calculations. Finally, the direct observation of orientation changes allows us to estimate an NV diffusion barrier of 5.1~eV.

Chronic hepatitis B (CHB) infection functional cure is defined as sustained loss of HBsAg and several therapeutic strategies are in clinical development designed to pharmacologically reduce serum HBsAg, break immune tolerance, and increase functional cure rates. However, little is known about pre-treatment HBsAg levels as an indicator of HBV immune potential. Here, we compared the phenotypes and HBV-specific response of lymphocytes in CHB patients stratified by serum HBsAg levels <500 (HBs lo ) or >50,000 IU/ml (HBs hi ) using immunological assays (flow cytometry, ICS, ELISPOT). HBs hi patients had significantly higher expression of inhibitory PD-1 on CD4 + T cells, particularly among TEMRA subset, and higher FcRL5 expression on B cells. Upon HBcAg(core) or HBsAg(env)-stimulation, 85% and 60% of HBs lo patients had IFNγ + TNFα + and IFNγ + IL2 + CD4 + T cell responses respectively, in comparison to 33% and 13% of HBs hi patients. Checkpoint blockade with αPD-1 improved HBV-specific CD4 + T cell function only in HBs lo patients. HBsAg-specific antibody-secreting cells (ASCs) response was not different between these groups, yet αPD-1 treatment resulted in significantly higher fold change in ASCs among patients with HBsAg <100 IU/ml compared to patients with HBsAg >5,000 IU/ml. Thus, serum HBsAg correlates with inhibitory receptor expression, HBV-specific CD4 + T cell responses, and augmentation by checkpoint blockade.

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.

We report on the observation of an increase in the concentration of negatively charged nitrogen-vacancy (NV−) defects in a sample of electronic grade single crystal, chemical vapor deposition (CVD) grown diamond after repeated annealing. The diamond sample is annealed at high temperature and under vacuum. Confocal microscopy is utilized to image the NV− photoluminescence in the same region of the diamond after each anneal cycle. Rotation of linearly polarized 532nm laser excitation is used to record NV− orientation. Comparison of these images allows us to track behavior of each individual NV− center through nine annealing cycles at different temperatures and durations. Analysis reveals total number of NV− in the region, individual appearances, disappearances and orientation changes. We observe a four fold increase in overall density of NV− with no preferential orientation. Accepted theory points to vacancy migration as the source of NV formation. In the absence of extrinsic vacancy incorporation via irradiation prior to annealing, the origin of excess mobile vacancies that could result in increased NV density in our sample is unknown. Such behavior could provide a useful mechanism for increasing NV− density without introducing additional lattice damage.