The objective of this study was to identify functional drug targets in SCZ using patient primary blood cells and high-content phospho-specific flow cytometry and subsequently use the identified targets as a basis for DR and clinical treatment response prediction. This work was performed in four stages (Fig. 1). First, samples from eight healthy donors were analyzed in a time-course exploration of T cell responses to 70 ligands across 78 cell signaling epitopes (5460 responses) to select the most active nodes and time point (KP; Fig. 1B). Next, responses to a selection of 56 ligands across 66 cell signaling epitopes (3696 in total) were compared across three clinical groups [healthy controls (n = 12), antipsychotic drug-naïve patients with SCZ (n = 12), and the same patients following 6 weeks of clinical treatment with the atypical antipsychotic olanzapine (n = 10)] to identify functional SCZ drug targets (TI; Fig. 1C). The identified target, attenuated response to thapsigargin at PLC-γ1, was then modeled in blood cells from healthy control donors (n = 6 to 12; limited clinical sample availability did not allow the use of patient PBMCs at this stage) and used for screening of 946 FDA-approved drugs (repurposing) and experimental neuropsychiatric compounds (extended FDA-approved DR; Fig. 1D), in addition to hypothesis-driven genotype association analysis between selected SCZ risk genes and target response. Last, the candidate compounds were validated in the SH-SY5Y human neuronal cell line (n = 3 replicates) and blood cells from an independent cohort of drug-naïve patients with SCZ (n = 30) treated with two of the study compounds, aripiprazole and risperidone, to investigate the relationship between the ex vivo and in vivo drug efficacy (CV; Fig. 1E). Methods relevant to specific substudies (KP, TI, DR, and CV) are indicated by abbreviations. All samples were randomized across different plate positions and experimental days to minimize measurement-related batch effects. To allow randomization, investigators were not blinded to sample characteristics. Samples in which the lymphocyte gate contained less than 30% of events (e.g., because of low viability; one sample in the TI study) were excluded from statistical analysis. Data permutation procedures (n = 10,000 permutations) were applied to control for small sample sizes, non-normal distribution of data, presence of outliers, and simultaneous testing of multiple hypotheses. Sample sizes were based on the availability of viable PBMCs from strictly defined drug-naïve first-onset patients with SCZ, controlled for treatment status and with longitudinal in vivo treatment follow-up information available, as well as previous studies (12, 31). At 12 versus 12 and with a 20% coefficient of variation, the TI study was sufficiently powered (0.8) to detect a 23% difference between the groups at α = 0.05.

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