Daclizumab-induced normalization of Ag-specific T cell responses.

<p>Intracellular cytokine secretion of each research subject was analyzed for IFN-γ⁺, TNF-α⁺ and double positive T cell events. The sums of all cytokine positive events were normalized to beads. Ratios of intrathecal to peripheral T cell reactivities of un-treated (RR) and DAC HYP-treated RRMS patients (Dac) were calculated to foreign Ag's for CD4⁺ (<b>A</b>) and CD8⁺ T cells (<b>B</b>). Ratios greater than one (dotted line) indicate enrichment of Ag-specific T cell events in the intrathecal compartment; ratios lower than one represent less pronounced intrathecal T cell responses. Horizontal bars represent median values; vertical lines represent interquartile ranges. <i>*0.01.</i></p

Patients' demographics and clinical characteristics.

a<p> Diagnoses: Cryptococcal meningitis (7), recurrent meningitis (1), systemic lupus erythematosus (1), cyclic fever (2), neuromyelitis optica (1), autoimmune lymphoproliferative syndrome-like disorder with CELs (1), persistently enhancing C-spine lesions (1), leukodystrophy-like disorder (1), CLIPPERS (Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids) (1), unclear diagnosis (3).</p>b<p> Long-term treatment with DAC HYP.</p>c<p> EDSS (Expanded Disability Status Scale) not available for each patient.</p><p>A: Asian, AA: African American, Cau: Caucasian, MR: Mixed race, Unkn: Unknown.</p

CSF T cell recognition and confirmation assay.

<p>PBMCs were obtained from apheresis samples or blood, and monocytes were isolated via positive selection with CD14⁺ magnetic beads. For the primary proliferation assay, monocytes were differentiated into immature dendritic cells (iDC). On the sixth day of culture, iDCs were loaded with selected Ag's and stimulation cocktail was added to induce DC maturation. 48 hours later, mature DCs (mDC) were co-cultured with peripheral and intrathecal T cells. After seven days of proliferation, cultured T cells were re-stimulated overnight with newly differentiated, identically loaded mDCs. Ag-specificity was detected by flow cytometric analysis of cytokine-producing CD4⁺ and CD8⁺ T cells.</p

Classification of candidate Ag's.

<p>Optimal concentrations of candidate Ag's were selected based on pilot experiments.</p

Differential phenotypes of peripheral and intrathecal CD4⁺ T cells in response to foreign Ag's.

<p>(<b>A</b>) FACS plots illustrate intracellular TNF-α and IFN-γ secretion by peripheral (Blood) and intrathecal (CSF) CD4⁺ T cells in response to all candidate Ag's. Gating is based on the isotype controls (far left plots). Upper panels correspond to one representative OIND patient; lower panels correspond to one representative un-treated RRMS patient. MFI ratios of intrathecal to peripheral CD4⁺ (<b>B</b>) and CD8⁺ T cells (<b>C</b>) were calculated for TNF-α (upper panel) and IFN-γ (lower panel), and are shown for OIND, progressive and RRMS patients. MFI ratios are depicted as log-transformed data. <i>*0.01.</i></p

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator