Effect of Thalidomide analogues on immune cells and cells derived from HIV-1+ infected donors

Flow cytometry data was obtained with BD LSR Fortessa and analyzed by FlowJo v.10. Description of experiments: Primary CD4+ T cells isolated from buffy coats of healthy volunteers were treated with 10uM of iberdomide, 500nM of JQ1, or both compounds, with PMA/ionomycin used as a positive control. The cells were examined by flow-cytometry at 24 and 48 hours, live cells were gated using forward scatter area versus side scatter area profiles (FSC-A, SSC-A). Cells were then stained for expression of Annexin V to examine the percentage of cells undergoing apoptosis and with the surface receptor CD69 to measure T cells activation. For Annexin V and CD69 staining, 106 cells were washed with PBS supplemented with 3% FBS and 2.5 mM CaCl2 followed by staining with Annexin V-PE (Becton and Dickinson), CD69-FITC (eBIOSCIENCE) for 20 minutes at 4C in the presence of 2.5 mM CaCl2. Cells were then washed with PBS/FBS/CaCl2 and analyzed by flow cytometry. Between 2 - 4x105 events were collected per sample within 3 hours after staining on an LSRFortessa (BD Biosciences, 4 lasers, 18 parameters) and analyzed using FlowJo software (version 9.7.4, Tree Star). To analyze T cell proliferation capacity, 1 million CD8+ or CD4+ T cells were stained with 0,07 uM CellTrace Far Red Cell Proliferation dye (ThermoFisher Scientific) following manufacturer?s instructions. Cells were then cultivated for 72 hours with either unstimulated or stimulated conditions in the presence of the LRA, and analyzed by flow cytometry. Stimulation of T cells was performed using Anti-CD3/CD28 coated Dynabeads (ThermoFisher Scientific) following manufacturer?s protocol. Proliferation capacity was determined by a decrease in proliferation dye intensity in daughter cells upon cell division. To analyze T cell functionality by means of cytokine expression 1 million CD8+ or CD4+ T cells were left untreated or treated with the LRA for 18 hours. Cells were then left unstimulated or stimulated with 50 ng/mL PMA and 1uM Ionomycin for 7 hours in the presence of a protein transport inhibitor (BD GolgiPlugTM, BD Biosciences). To stain for intracellular cytokines cells were washed with PBS supplemented with 3% FBS followed by a fixation and permeabilization step with FIX & PERM Kit (Invitrogen) following manufacturer?s protocol and incubated with 1:25 BV510 anti-IL2 (563265, BD Biosciences) and PE-Cy7 anti-IFNg (27-7319-41, eBioscience) in permeabilization buffer for 45 minutes at 4C. Stained cells were washed with PBS supplemented with 3% FBS and analyzed by flow cytometry. To analyze production of viral RNA by FISH-Flow, cells were collected, fixed, permeabilised and subjected to the PrimeFlow RNA assay (Thermo Fisher Scientific) following the manufacturer?s instructions and as described in (Rao, 2021, Nat Comm)(21). Primary CD4+ T cells were first stained in Fixable Viability dye 780 (Thermo Fisher Scientific) for 20 minutes at room temperature (1:1000 in dPBS). HIV unspliced mRNA was thenlabelled with a set of 40 probe pairs against the GagPol region of the vRNA (catalogue number GagPol HIV-1 VF10-10884, Thermo Fisher Scientific) diluted 1:5 in diluent provided in the kit and hybridized to the target mRNA for 2 hours at 40°C. Samples were washed to remove excess probes and stored overnight in the presence of RNAsin. Signal amplification was then performed by sequential 1.5 hours, 40°C incubations with the pre-amplification and amplification mix. Amplified mRNA was labelled with fluorescently-tagged probes for 1 hour at 40°C. Samples were acquired on a BD LSR Fortessa Analyzer and gates were set using the uninfected CD4+ T cells. The analysis was performed using the FlowJo V10 software (Treestar).
Conclusion:
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Notes:
Labeling of FCS files: FCS files are labelled as follows: Experiment ID number_Donor number_Treatment_Timepoint(if applicable)_Number of replicate(if applicable) ID number: - E1: Experiments in Figure 4C-E and Supplementary Figure S10A-C - E2: Experiments in Figure Supplementary Figure S11A-B, E-F - E3: Experiments in Figure 4H-I and Supplementary Figure S11C-D - E4: Experiments in Figure 4F-G - E5: Experiments in Supplementary Figure S10D-E - E6: Experiments in Supplementary Figure S12A-B - E7: Experiments in Supplementary Figure S12C-E - E8: Experiments in Figure 5E-F - E9: Experiments in Supplementary Figure S12F-H - E10: Experiments in Figure 5G-I - E11: Experiments in Figure 5A-B and Supplementary Figure S11A Legends: - Len 1: Lenalidomide 1 micromolar - Len 10: Lenalidomide 10 micromolar - Pom 1: Pomalidomide 1 micromolar - Pom 10:Pomalidomide 10 micromolar - PMA: Phorbol 12-myristate 13-acetate - PMA + Iono: PMA and Ionomycin - GTX 200: Gliotoxin 200 nanomolar - Ibe 10: Iberdomide 10 micromolar - JQ1 500: JQ1 500 nanomolar - Ibe+JQ1: Combination of Iberdomide and JQ1 - Stim: Stimulated - Unstim: Unstimulatede -