Fig 2: Convergent phenotypes of microglia- and bone marrow-derived myeloid cells in glioma.a) Schematics of the MAESTER analysis pipeline for determining the origin of myeloid cells in the glioma microenvironment. b) Dot plot exhibiting the enrichment difference between PBMC-specific and Resident-specific variants. Each dot represents the enrichment level of the indicated identities (left) in each patient. X-axis denotes the scaled difference between GSVA enrichment of PBMC-specific variants and Resident variants. c) Stacked bar charts indicating the average usage of the indicated myeloid programs in the key across the four patients. The “other programs” category encompasses the other identities and activities. d) Schematic (left) and flow cytometry plots (right) of myeloid cells from indicated condition. T cells are used as gating control for P2RY12 and TMEM119. e) Immunofluorescence image showing matched patient derived PBMC cells infiltrated into a glioblastoma organoid.

Fig 3: Characterization of mature microglia and BO morphology in response to pro-inflammatory stimuli. A H&E staining of 4-month-old BO. The image shows typical BO morphology with classical rosette structures. The magnified section indicates the location of neuro stem cells (NSCs) and mature neurons (Neu) within the rosette structure. The immunofluorescence of the rosette structure confirm the presence of NSCs (Nestin) and Neu (MAP2) B Characterization of one BO by obtained by flow cytometry analysis, identifying different populations: 5.48% GFAP + reactive astrocytes (Ast), 69.3% Tuj-1 + neurons (Neu), 0% Iba-1 + /P2RY12 + microglia (Mic), and 9.7% PAX6 + /SOX2 + neuro NSCs. C p-NF-KB levels in one BO (n = 1) stimulated with TNFa 40 ng/ml + 100ng/ml IFN in presence of 100 nM calyculin versus a BO treated with vehicle (DMSO) for 1 h, obtained by flow cytometry analysis. The dot plot shows the changes of p-NF-κB in the different BOs population characterized by CNS markers: GFAP + (reactive astrocyte), TUJ-1 (neurons) and SOX2 + (neuro NSCs). D Characterization of fully differentiated microglia (HPCs -derived microglia) after 11 days shows typical microglial morphology. Representative flow cytometry dot plot showing the expression of classical microglial markers: CD11b (82.7%), CD45 (low), Iba-1 (80.8%), P2RY12 (low), and TREM2 (22%). E Analysis of Gag expression in HIV-1 infected microglia by immunofluorescence 24 h post-infection (top images). The arrows in the images indicate syncytia structures with high Gag protein expression, characteristic of HIV-1 infection (n = 1). F Quantification of levels of p24 detected in the supernatant of HIV-1 infected microglia by ELISA over 9 days of incubation (graph) (n = 1) G Quantification of pro-inflammatory cytokine levels associated with HIV-1-NCI and IRIS in the supernatant of three different microglia batches at day 3 post-infection (n = 3)

Fig 4: HIV-1-infected microglia infiltrate BOs and promote an inflammatory response associated with HIV-1-NCI. A Breakdown of the experimental design, including differentiation of microglia, infection with HIV-1, co-culture with mature BOs to obtain M-BOs, experimental treatment conditions: Uninfected, HIV-1, HIV-1 + ART, and HIV-1 + ART Interrupted. B Image showing cluster of microglia cells labeled with cell tracker orange inside the M-BO 5 days post-co-culture captured by microscopy (top panel) and a magnification of labeled cells cluster in a different M-BO (middle panel). M-BO not infiltrated with labeled microglia as a ctrl of fluorescence signal (bottom panel) C Dot plot showing the percentage of orange cells detected (orange dots) IBA-1 and P2RY12 positive (n = 1) detected by flow cytometry analysis. D The graph displays the percentage of cells in M-BOs in the four treatment conditions positive for IBA-1 detected by flow cytometry analysis. E Graph of ddPCR analysis of BOs in the four treatment conditions, showing detected copies of HIV-1 psi-gag per 106 cells. F Quantification of p24 levels detected in the supernatant of M-BOs in the four treatment conditions over 25 days of incubation. Each curve corresponds to one M-BO, with two M-BOs per condition evaluated (n = 2) G Quantification of pro-inflammatory cytokines detected in the supernatant of M-BOs for the four treatment conditions performed in the experiment. Two M-BOs per condition were evaluated (n = 2). H Quantification of the percentage of double positive cells for IBA-1 and p-NF-kB in M-BOs after 25 days of incubation. I Immunofluorescent staining for Dapi (blue), p-NF-KB (red) and CCL2 (green), on slides of M-BOs in the 4 treatment conditions. Each organoid treated is an independent experiment and is represented by a single point in the graphs for the four treatment conditions: Uninfected (n = 4), HIV-1 (n = 3), HIV-1 + ART (n = 3), and HIV-1 + ART Interrupted (n = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig 5: Cytoplasmic expression of HIV-1 icRNA in iMGs induces inflammasome activation.(A) Schematic of iMGs differentiation protocol. (B) Flow cytometry profiles of iMGs showing expression of myeloid cell markers (CD45 and CD11b) and microglia-specific markers (TREM2 and P2RY12) at day 28 of differentiation. Top panels represent isotype IgG control staining, and bottom panels represent specific antibodies against indicated cell-specific markers. (C) iMGs were infected with HIV-1 Lai/YU-2env (MOI 0.1, 0.3, or 1) ± EFV (1 μM). Alternatively, iMGs were stimulated with LPS (0.25 µg/ml, 4 h) followed by Nigericin (15 nm, 30 min). Cell culture supernatants were harvested at 3 dpi for quantifying p24Gag (left panel) and IL-1β (right panel) release by ELISA. RT-qPCR analysis of multiply-spliced (tat/rev) viral transcripts (D) and unspliced (usRNA) transcripts (E) in iMGs infected with HIV-1 WT, M10, or ΔGag-pol at 3 dpi. The CT values were normalized first to that of GAPDH, and then to uninfected (mock) control. (F) Quantitation of IL-1β secretion in the culture supernatants of HIV-infected iMGs at 3 dpi, compared to uninfected (mock) control. The means ± SD are shown, and each symbol represents an independent experiment. Statistical significance was determined by two-way ANOVA followed by the Dunnett’s post-test comparing to EFV-treated (C, left panel) or mock (C, right panel); or one-way ANOVA followed by the Dunnett’s post-test comparing to HIV-1 WT (D–F). P-values: **** < 0.0001; *** < 0.001; ** < 0.01; ns: not significant (p ≥ 0.05). The data underlying this figure can be found in S3 Data.