Participant characteristics.

Median values or summary of the characteristics are reported in bold in the last line. The ID of individuals with HIV starts with “H”, the HIV-negative individuals start with “N”. 3TC = lamivudine, ART = antiretroviral therapy, AZT = zidovudine, CNS = central nervous system, DTG = dolutegravir, EFV = efavirenz, HAND = HIV-associated neurocognitive disorder, LPV/r = lopinavir/ritonavir, NA = not applicable, TB = tuberculosis, TDF = tenofovir disoproxil fumarate.

Cell-type specific of sorted data.

A) PCA plot displaying clustering of top 500 genes of bulk-sorted samples per cell-type. BC) Uniform manifold approximation and projection (UMAP) plots displaying the clustering of the single-nuclei data labeled per B) cell type and C) cluster. D) Normalized marker gene expression of different cellular subsets. Clustering distance of columns is “Euclidean”, clustering method is “complete”. In bold text the genes that encode the transcription factors used for cell-type specific nuclear staining. (RBFOX3 is the gene coding for NeuN). Excluded samples H5-O and N2-O are not shown.

The presence of HIV reservoir by proviral LTR copies and expression of inflammatory and neurodegeneration risk genes in different cell-type derived nuclear fractions.

A) Number of proviral LTR copies for the different cellular subsets. B) The expression of CD3-epsilon and C) Heatmap of the VST normalized and batch corrected counts of inflammatory genes. Columns are clustered by Euclidean distance. Individual H5 was excluded in the analyses for C and D. D) Heatmap of the VST normalized and batch corrected counts of neurodegenerative risk genes. Columns are clustered by Euclidean distance. Individual H5 was not included in this analysis.

Differential gene expression per cell type-specific nuclear fraction for each comparison.

A-D: Heatmaps for each cell-type, showing the top 50 genes per contrast with an adjusted p-value < 0.05 and LFC > 0.25 for one of the comparisons, colored by log2FC. Significant adjusted p-values are marked with asterisks (p<0.05=*, p<0.01=**, p<0.001=***).

Differential expression analysis for 4 different cell types of bulk-RNA-seq data comparing viremic with aviremic DPWH.

A: GSEA analysis with GO pathways of activated and suppressed pathways genes for all 4 cell types combined. The Normalized Enrichment Score (NES) quantifies the extent to which a specific pathway is upregulated (positive NES) or downregulated (negative NES) which is then normalized using the mean enrichment scores obtained from perputations of the same pathway, enabling to compare values between pathways. B: Overrepresentation analysis of up- and downregulated DEGs within GO pathways. All cell types with pathways with FDR≤0.05 included. DEG count identifies the number of DEGs in a pathway.

Differential expression analysis for 4 different cell-types of bulk-RNA-seq data comparing aviremic DPWH with HIV-negative individuals.

A: GSEA analysis for microglia with GO pathways of activated and suppressed pathways genes. The NES quantifies the extent to which a specific pathway is upregulated (positive NES) or downregulated (negative NES) which is then normalized using the mean enrichment scores obtained from perputations of the same pathway, enabling to compare values between pathways. B: Overrepresentation analysis of up- and downregulated DEGs within GO pathways. All cell types with pathways with FDR≤ 0.05 included. DEG count identifies the number of DEGs in a pathway.

Differential expression analysis for 4 different cell types of bulk-RNA-seq data comparing viremic DPWH with HIV-negative individuals.

A: analysis for multiple cell types with GO pathways of activated and suppressed pathways genes. The NES quantifies the extent to which a specific pathway is upregulated (positive NES) or downregulated (negative NES) which is then normalized using the mean enrichment scores obtained from perputations of the same pathway, enabling to compare values between pathways. B: Overrepresentation analysis of up- and downregulated DEGs within GO pathways. All cell-types with pathways with FDR≤ 0.05 are included. DEG count identifies the number of DEGs in a pathway.

Workflow of nuclei isolation and sorting.

Representative FANS plot for sorted nuclear fractions are shown. Created with Biorender.com

© 2026, BioRender Inc. Icons used in this image are taken from BioRender and are not made available under a CC-BY license.

Confocal microscope images of isolated brain nuclei.

The nuclear membrane (mCLING) and cell-type specific transcription factors are visualized to evaluate nuclei intactness and FANS sorting markers. Scale of the nuclei is visualized in the upper right corner. A) mCLING-647 (red) and NeuN (green), B) mCLING (green) and SOX10 (red), C) mCLING (red) and IRF5 (green).

Normalized expression of neurodegeneration markers in the cellular fractions.

A) Microglia, B) Neurons, C) Oligodendrocytes, D) Rest fraction.

Detailed clinical records and cause of death of the study participants.

The ID of individuals with HIV starts with “H”, the HIV-negative individuals start with “N”. CSF = cerebrospinal fluid, CXR= chest-x-ray, LAM=lipoarabinomannan

Overview of quality of bulk-sequencing data.

After filtering on sample level per cell type, the number of genes that is left per subset is indicated in the last column. IQR values are given between brackets.

Differently expressed genes per cell type for viremic versus aviremic individuals.

Genes differently expressed within more than 1 cellular subset are underlined. Green labeled genes are also DEGs when comparing aviremic individuals and HIV-negative individuals, orange labeled genes are also DEGs when comparing viremic individuals with HIV-negative individuals and yellow labelled genes are DEGs in all 3 comparisons.

Literature search of DEGs in viremic versus aviremic individuals.

Literature research overview of DEGs occurring in multiple cell types with search terms “HIV”, “inflammation” and “infection” or “CNS” in combination with the DEG. A summary of the findings and the associated PMID of the article are given. M = Microglia, N = Neurons, O = Oligodendrocytes, R= Rest fraction

Differently expressed genes per cell type for aviremic individuals versus HIV-negative individuals.

Genes differently expressed within more than 1 cellular subset are underlined Blue labeled genes are also DEGs when comparing viremic and aviremic, green labeled genes are also DEGs when comparing viremic individuals with HIV-negative individuals and yellow labelled genes are DEGs in all 3 comparisons.

Literature search of DEGs in viremic or aviremic individuals versus HIV-negative individuals.

Literature research overview of DEGs occurring in multiple cell types with search terms “HIV”, “inflammation” and “infection” or “CNS” in combination with the DEG. A summary of the findings and the associated PMID of the article are given. M = Microglia, N = Neurons, O = Oligodendrocytes, R= Rest fraction

Differently expressed genes per cell type for viremic individuals versus HIV-negative individuals.

Genes differently expressed within more than 1 cellular subset are underlined. Blue labeled genes are also DEGs when comparing aviremic individuals with the HIV-negative individuals, orange labeled genes are also DEGs when comparing viremic and aviremic individuals and yellow labelled genes are DEGs in all 3 comparisons.