We present a tool to measure gene and protein expression levels in single cells with DNA-labeled antibodies and droplet microfluidics. Using the RNA expression and protein sequencing assay (REAP-seq), we quantified proteins with 82 barcoded antibodies and >20,000 genes in a single workflow. We used REAP-seq to assess the costimulatory effects of a CD27 agonist on human CD8 lymphocytes and to identify and characterize an unknown cell type.
GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of T reg -mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both na€ ve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating T regs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of T regs in vitro. In human TIL cultures, MK-4166 induced phosphorylation of NFkB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFkB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating T regs , suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the T reg -mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. Ó2017 AACR.
Objectives
The objective of this study is to analyse retrospective, observational, longitudinal growth (weight, height and BMI) data in ambulatory boys aged 5–12 years with Duchenne muscular dystrophy (DMD).
Background
We considered glucocorticoids (GC) use, dystrophin isoforms and amenability to exon 8, 44, 45, 51 and 53 skipping drug subgroups, and the impact of growth on loss of ambulation. We analysed 598 boys, with 2604 observations. This analysis considered patients from the UK NorthStar database (2003–2020) on one of five regimes: “GC naïve”, “deflazacort daily” (DD), “deflazacort intermittent” (DI), “prednisolone daily” (PD) and “prednisolone intermittent” (PI). A random slope model was used to model the weight, height and BMI SD scores (using the UK90).
Results
The daily regime subgroups had significant yearly height stunting compared to the GC naïve subgroup. Notably, the average height change for the DD subgroup was 0.25 SD (95% CI − 0.30, − 0.21) less than reference values. Those with affected expression of Dp427, Dp140 and Dp71 isoforms were 0.77 (95% CI 0.3, 1.24) and 0.82 (95% CI 1.28, 0.36) SD shorter than those with Dp427 and/or Dp140 expression affected respectively. Increased weight was not associated with earlier loss of ambulation, but taller boys still ambulant between the age of 10 and 11 years were more at risk of losing ambulation.
Conclusion
These findings may provide further guidance to clinicians when counselling and discussing GCs commencement with patients and their carers and may represent a benchmark set of data to evaluate the effects of new generations of GC.
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