Negative costimulation on T cells is exploited by both prostate cancer and melanoma to evade antitumor immunity. Blocking such mechanisms restores antitumor immunity as was demonstrated by the improved survival of patients with metastatic melanoma after treatment with an antibody blocking the CTLA-4 inhibitory receptor (ipilimumab). Enhanced expression of another inhibitory immunoreceptor, programmed death-1 (PD-1), and its ligand, PD-L1, was found to correlate with a poor prognosis in prostate cancer and melanoma. PD-1-blocking antibodies are being developed to modulate antitumor immune responses. To support preclinical and clinical development of anti-PD-1 therapy, we sought to develop biomarker assays that can detect the effect of PD-1-blocking agents in whole blood and peripheral blood mononuclear cells. In this study, we assessed the effect of PD-1 blockade in modulating super antigen (staphylococcus enterotoxin B)-induced and recall antigen (tetanus toxoid)-induced T-cell reactivity in vitro using whole blood and peripheral blood mononuclear cells from patients with advanced melanoma, prostate cancer, and healthy controls. PD-1 blockade was found to shift antigen-induced cellular reactivity toward a proinflammatory Th1/Th17 response, as evidenced by enhanced production of interferon γ, interleukin (IL)-2, tumor necrosis factor α, IL-6, and IL-17 and reduced production of the Th2 cytokines IL-5 and IL-13. It is interesting to note that suppression of Th2 responsivity was seen with whole blood cells only from patients with cancer. Taken together, we identified novel biomarker assays that might be used to determine the functional consequences of PD-1 blockade in peripheral blood cells from patients with cancer. How these assays translate to the local antitumor response remains to be established in a clinical setting.

BackgroundT lymphocytes are orchestrators of adaptive immunity. Naïve T cells may differentiate into Th1, Th2, Th17 or iTreg phenotypes, depending on environmental co-stimulatory signals. To identify genes and pathways involved in differentiation of Jurkat T cells towards Th1 and Th2 subtypes we performed comprehensive transcriptome analyses of Jurkat T cells stimulated with various stimuli and pathway inhibitors. Results from these experiments were validated in a human experimental setting using whole blood and purified CD4+ Tcells.ResultsCalcium-dependent activation of T cells using CD3/CD28 and PMA/CD3 stimulation induced a Th1 expression profile reflected by increased expression of T-bet, RUNX3, IL-2, and IFNγ, whereas calcium-independent activation via PMA/CD28 induced a Th2 expression profile which included GATA3, RXRA, CCL1 and Itk. Knock down with siRNA and gene expression profiling in the presence of selective kinase inhibitors showed that proximal kinases Lck and PKCθ are crucial signaling hubs during T helper cell activation, revealing a clear role for Lck in Th1 development and for PKCθ in both Th1 and Th2 development. Medial signaling via MAPkinases appeared to be less important in these pathways, since specific inhibitors of these kinases displayed a minor effect on gene expression. Translation towards a primary, whole blood setting and purified human CD4+ T cells revealed that PMA/CD3 stimulation induced a more pronounced Th1 specific, Lck and PKCθ dependent IFNγ production, whereas PMA/CD28 induced Th2 specific IL-5 and IL-13 production, independent of Lck activation. PMA/CD3-mediated skewing towards a Th1 phenotype was also reflected in mRNA expression of the master transcription factor Tbet, whereas PMA/CD28-mediated stimulation enhanced GATA3 mRNA expression in primary human CD4+ Tcells.ConclusionsThis study identifies stimulatory pathways and gene expression profiles for in vitro skewing of T helper cell activation. PMA/CD3 stimulation enhances a Th1-like response in an Lck and PKCθ dependent fashion, whereas PMA/CD28 stimulation results in a Th2-like phenotype independent of the proximal TCR-tyrosine kinase Lck. This approach offers a robust and fast translational in vitro system for skewed T helper cell responses in Jurkat T cells, primary human CD4+ Tcells and in a more complex matrix such as human whole blood.

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The importance of thyroid hormone, catecholamines, and insulin in modification of the thermogenic effect of glucose (TEG) was examined in 34 healthy and 32 hypothyroid subjects. We calculated the energy expenditure at rest and during oral glucose tolerance test. Blood samples for determinations of glucose, plasma insulin, adrenaline (A), and noradrenaline (NA) were collected. It was found that TEG was lower in hypothyroid than in control group (19.68 ± 3.90 versus 55.40 ± 7.32 kJ, resp., P < 0.0004). Mean values of glucose and insulin areas under the curve were higher in women with hypothyroidism than in control group (286.79 ± 23.65 versus 188.41 ± 15.84 mmol/L·min, P < 0.003 and 7563.27 ± 863.65 versus 4987.72 ± 583.88 mU/L·min, P < 0.03 resp.). Maximal levels of catecholamines after glucose ingestion were higher in hypothyroid patients than in control subjects (Amax—0.69 ± 0.08 versus 0.30 ± 0.07 nmol/L, P < 0.0001, and NAmax—6.42 ± 0.86 versus 2.54 ± 0.30 nmol/L, P < 0.0002). It can be concluded that in hypothyroidism TEG and glucose tolerance are decreased while the adrenergic response to glucose administration is enhanced. Presumably, these changes are related to decreased insulin sensitivity and responsiveness to catecholamine action.

Purpose Angiopoietin-like protein 4 (ANGPTL4) regulates lipid metabolism by inhibiting lipoprotein lipase activity and stimulating lipolysis in adipose tissue. The aim of this study was to find out whether the mountain ultra-marathon running influences plasma ANGPTL4 and whether it is related to plasma lipid changes. Methods Ten healthy men (age 31 ± 1.1 years) completed a 100-km ultra-marathon running. Plasma ANGPTL4, free fatty acids (FFA), triacylglycerols (TG), glycerol (Gly), total cholesterol (TC), low (LDL-C) and high (HDL-C) density lipoproteincholesterol were determined before, immediately after the run and after 90 min of recovery. Results Plasma ANGPTL4 increased during exercise from 68.0 ± 16.5 to 101.2 ± 18.1 ng/ml (p < 0.001). This was accompanied by significant increases in plasma FFA, Gly, HDL-C and decreases in plasma TG concentrations (p < 0.01). After 90 min of recovery, plasma ANGPTL4 and TG did not differ significantly from the exercise values, while plasma FFA, Gly, TC and HDL-C were significantly lower than immediately after the run. TC/HDL-C and TG/HDL-C molar ratios were significantly reduced. The exercise-induced changes in plasma ANGPTL4 correlated positively with those of FFA (r = 0.73; p < 0.02), and HDL-C (r = 0.69; p < 0.05). Positive correlation was found also between plasma ANGPTL4 and FFA concentrations after 90 min of recovery (r = 0.77; p < 0.01). Conclusions The present data suggest that increase in plasma FFA during mountain ultra-marathon run may be involved in plasma ANGPTL4 release and that increase in ANGPTL4 secretion may be a compensatory mechanism against fatty acidinduced oxidative stress. Increase in plasma HDL-C observed immediately after the run may be due to the protective effect of ANGPTL4 on HDL. Keywords ANGPTL4 • Ultra-marathon • Triacylglycerols • Free fatty acids • Cholesterol Abbreviations ANGPTL4 Angiopoietin like protein 4 FFA Free fatty acids Gly Glycerol HDL High-density lipoprotein HDL-C High-density lipoprotein-cholesterol LPL Lipoprotein lipase LDL-C Low-density lipoprotein-cholesterol TC Total cholesterol TG Triacylglycerols VLDL Very low-density lipoproteins VO 2max Maximal oxygen uptake