Anti il 12

To study the effect LRA effect of UMB-136 we slightly modified a primary CD4+ T cell model of HIV-1 latency, which we had used previously (Bosque and Planelles, 2011 (link); Huang et al., 2015 (link)). In brief, primary T cells were activated using CD3/CD28 antibodies and then infected with full-length HIV-1 NL4-3 (X4) viruses. Cells were treated with TGF-β, anti-IL12, and anti-IL4 antibodies (R & D Systems) for non-polarization to allow for the establishment of latent HIV-1. These cells were treated with compounds 2.5 UMB-136 or 1 μM JQ1 day 16 post infection. Twenty-four hours later the compounds were washed away and cells were re-suspended in fresh complete RPMI media. On day 21, 5 days post treatment; the supernatant was collected for quantification of newly produced viruses using the Lent-X™ qRT-PCR Titration Kit (Clontech) following the manufacturer's instruction.

In vitro knock-downs were generated from human naïve CD4+ T-cells isolated from healthy donor buffy coats using magnetic bead separation (Miltenyi Biotec, Sweden). Typically, 1x10⁶ cells were transfected with 600nM on-target plus SMART pool against IRF4 (Dharmacon, USA), MAF, GATA3, USF2, COPEB (Thermo Fisher Scientific Inc, USA), or non-targeting siRNA (Dharmacon, USA) using Amaxa transfection (U-014). After six hours of incubation, the cells were washed and subsequently activated with plate-bound anti-CD3 (500 ng/ml), soluble anti-CD28 (500 ng/ml) and polarized towards Th2 using IL-4 (10 ng/ml), IL-2 (17 ng/ml) and anti-IL-12 (5 μg/ml, R&D Systems, USA) for 24 hours for each of the TFs siRNA (12h for IRF4). The cells were then harvested in Qiazol and total RNA was extracted using the RNeasy mini kit (QIAGEN, Germany). Expression was analyzed using the Human GE 4x44K v2 Microarray Kit from Agilent Technologies. All siRNA-induced knock-downs were followed by a short Th2 polarization (24h for all except IRF4, which had 12h) and microarray analysis, and control experiments corresponding Th2 polarization using scrambled non-targeting siRNA.

Initially, on day 0, NSG (NOD.Cg-Prkdc^scidIl2rg^em1Sm°^c, Shanghai Model Organisms) female mice (n = 5) were injected with 5 × 10⁶ PBMCs from human sources for 4-6 weeks through the tail vein to complete the humanized reconstruction. On the seventh day, human small cell lung cancer cells (5 × 10⁶ cells/mouse) were subcutaneously injected into the neck and back of humanized NSG mice. From around the 10^th day, cisplatin (8mg/kg) was administered every seven days; durvalumab (AstraZeneca, 10mg/kg), Recombinant Human IL-12 (huIL12, R&D, 100ng/kg), and human IL-12 affinity-purified polyclonal Ab (antiIL12, R&D, 500ng/kg) were administered every three days, respectively. Tumor volume was calculated using the following equation: tumor volume (mm³) = 0.5 × length (mm) × width mm². Around the 40^th day, the mice were euthanized and the tumor and spleen were collected for further analysis. All mice were housed in a specific pathogen-free (SPF) Animal Center of Shanghai Chest Hospital. Animal experiments were approved by Shanghai Chest Hospital and performed according to the Laboratory regulations.

Total CD4+ cells were isolated from PBMCs using isolation kits from Miltenyi (Bergisch Gladbach, Germany) according to the manufacturer’s instruction. The isolated cells were washed, activated, and the Th2 cells were polarized as previously described [35 (link)]. Briefly, the isolated total CD4+ cells were activated with plate-bound anti-CD3 (500ng/ml), 500ng/ml soluble anti-CD28, 5μg/ml anti-IL-12, 10ng/ml IL-4 and 17ng/ml IL-2 (R&D Systems). For microarray experiments, 200 ng of total RNA was labeled and transcribed to cRNA using the Quick Amp Labeling Kit (Agilent, USA), then purified using the RNeasy mini kit and hybridized to Agilent Human GE 4x44 K v2 slides, according to the manufacturer’s instructions. Slides were scanned using the Agilent Microarray scanner 2505C, and raw data were obtained using the Feature Extraction Software (Agilent). Moreover, we only considered data for genes that were deemed to respond to the stimulation, such that we could reject the changes of expression over time from being white noise measurements, i.e. reject if the sum of residuals from the null-model < χ²_0.95(df = 4).