Ifn α

In vitro deletion of the Hoxa locus was by IFNα (R&D Systems, Abingdon, UK) treatment at the indicated dose and time. In vivo deletion in MAFF-MA9 leukemic mice was achieved by intraperitoneal (IP) injection of Polyinosinic:polycytidylic acid (Poly I:C; GE Healthcare Life Sciences, Buckinghamshire, UK) as previously described [46 (link)]. Briefly, mice were given 10 µg/g Poly I:C or vehicle (PBS) up to a maximum of 250 µg/mouse. Ex vivo deletion was by direct exposure to MSCV-Cre-GFP or MSCV-GFP control retroviral supernatants followed by cell sorting and gene expression profiling. Mice transplanted with traceable (pSLIEW) leukemias were injected IP with 150 mg D-luciferin/kg (Gold Biotechnology, St. Louis, MO, USA) and imaged using the Xenogen IVIS 200 (PerkinElmer, Buckinghamshire, UK). Nested PCR was used to identify individual colony bands.

Per well 1 × 10⁶ isolated monocytes where plated in 12-well plates (TPP) in 1 ml IMDM with 10% human AB serum (Karolinska University Hospital). Differentiation of monocytes to immature DC (iDC) was done using a fast protocol, in which iDC formation was established by 48 h of culture in the presence of 100 ng/ml GM-CSF (Peprotech) and 20 ng/ml IL-4 (Peprotech). Mature DC (maDC) were created by incubating iDC for an additional 18 h with one of the three following cocktails. The first was the gold standard [26 (link)]: 20 ng/ml tumor necrosis factor-α (TNF-α; Peprotech), 10 ng/ml interleukin-1β (IL-1β; CellGenix), 1000 U/ml interleukin-6 (IL-6; CellGenix), and 10 ng/ml PGE2 (SIGMA). Second, the alpha-type 1 polarizing cocktail [27 (link)]: 50 ng/ml TNF-α, 25 ng/ml IL1b, 3000 U/ml IFN-α (R&D Systems), 100 U/ml IFN-γ (Imukin^®, Boehringer Ingelheim), and 250 ng/ml polyinosinic:polycytidylic acid (poly I:C, Sigma-Aldrich). Finally, the COMBIG CCK Cocktail [28 (link)] was used: 10 ng/ml LPS (Sigma-Aldrich), 20 μg/ml Hiltonol (OncoVir), 2.5 ug/ml R848 (VacciGrade™, InvivoGen), and 1000 U/ml IFN-γ. Readout was performed by flow cytometry.

1.0 × 10⁶ THP18 (WT), THP18-ΔA3A, and THP18-ΔA3A/B cells were seeded in 6-well plates, allowed to grow for 24 h, and were then induced with 1 µg/mL lipopolysaccharide (LPS) (MilliporeSigma, #L2654) and 300 U/mL of Interferon-alpha (IFN-α) (R&D Systems Inc., Minneapolis, MN, USA, #11200-1) for 24 h. The 293T and inducible T-REx-293-A3B-eGFP cells were also included in the CMA, with the latter induced for 24 h with 1µg/µL doxycycline. Cell buttons were prepared as follows: Trypsinization, centrifugation, and supernatant removals were followed by 3 washes with PBS. Following the last wash, cells were resuspended in 10% formalin for 15 min. After fixation, cells were rinsed 3× with PBS, mixed with Histogel (Thermo Fisher Scientific, #HG-4000-012) at 65 °C, and allowed to solidify at RT for approximately 10 min. The resulting cell blocks were placed in Histosette II cassettes, dehydrated, and embedded in paraffin.
Immunohistochemical staining of the CMA was performed as above for tissues. Nuclear A3B immunoreactivity was visualized with the Aperio ScanScope XT (Leica Biosystems) and quantified using the Aperio Nuclear Algorithm. Histoscore (H-Score) was calculated using the formula (3+) × 3 + (2+) × 2 + (1+) × 1, as described earlier [56 (link),57 (link)].

To investigate for phosphorylation defects in STAT1 and STAT3, PBMCs from SLT patients (n = 13) were isolated and cells were stimulated for 15 min with IL-6 (100 ng/ml), IL-21 (100 ng/ml), and IFNα (11,500 U/ml; R&D Systems, cat#11101-1) according to previously described diagnostic protocols^{71 (link),72 (link)}. Cells were fixed immediately (Cytofix Fixation Buffer; BD Biosciences, cat#554655), permeabilised (Phosflow Perm Buffer III; BD Biosciences, cat#558050), and stained for CD4 (CD4-FITC), pSTAT1 (STAT1 pY701—Alexa647; BD Biosciences, cat#612597) and pSTAT3 (STAT3 pY705 – PE; BD Biosciences, cat#612569). For each subject, the increase in phosphorylation of STAT1 and STAT3 in stimulated compared to unstimulated CD4⁺ and CD4⁻ cells were determined, and this increase was expressed as a ratio with the increase seen in a healthy control subject in each experiment.

Monocytes were enriched from PBMCs using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic-activated cell sorting (MACS), and then cultured in 96-well flat-bottomed plates in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 55 µM 2-mercaptoethanol (all from Thermo Fisher Scientific) at 37°C in a 5% CO₂ incubator. Monocytes were stimulated with recombinant human IL-3 (200 ng/ml; PeproTech, Rocky Hill, NJ, USA) and 2’3’-cGAMP (50 µg/ml; In vivoGen, San Diego, CA, USA) for 5 h, except for the immunofluorescence experiment where monocytes were stimulated for 3 h. HC monocytes used for RNA-seq experiments were pretreated with IFNα (100 U/ml) (R&D Systems, Minneapolis, MN, USA) for 18 h prior to stimulation with 2’3’-cGAMP.

The 3 × 10⁶ PBMCs from healthy donors or individuals carrying heterozygous IKZF1 mutation were cultured in the presence poly(I:C) (10 μg/ml, Invivogen), LPS (5ng/ml, Sigma), CL075 (1 μg/ml, Invivogen) and CpG (ODN 2216, 7.5 μΜ, Invivogen) with or without IFN-α (3000 IU/ml, R&D), with or without anti-CD303 and anti-CD304 (Biolegend), with or without 0.1, 1 or 10 μM lenalidomide (Sigma). Cells were cultured for 14 h at 37 ^oC, 5% CO₂, with addition of Brefaldin A (10 μg/ml, eBioscience) after 3 h. For dead-cell exclusion (usually <30%) cells were stained with Zombie amine dye (Biolegend), surface markers and then intracellular cytokines antibodies after fixation and permeabilization (eBioscience), as above.