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Research ArticleAIDS/HIVImmunologyMetabolism
Open Access | 
10.1172/jci.insight.198810
1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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1Department of Medicine, Division of Infectious Diseases, and
2Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
3New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, Louisiana, USA.
4Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
5Center for Pathogen Genomics and Microbial Evolution, Northwestern University Havey Institute for Global Health, Chicago, Illinois, USA.
6Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Address correspondence to: Elena Martinelli, Department ofMedicine, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-277, Chicago, Illinois 60611, USA. Phone: 312.503.7639; Email: elena.martinelli@northwestern.edu. Or to: Ramon Lorenzo-Redondo, Department of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Lurie 9-125, Chicago, Illinois 60611, USA. Phone: 847.467.2372; Email: ramon.lorenzo@northwestern.edu.
Authorship note: RA and JHG contributed equally to this work.
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Published February 23, 2026 - More info
Published in Volume 11, Issue 4 on February 23, 2026
AbstractWe previously demonstrated that blocking TGF-β with galunisertib, a safe, orally available small drug, reactivated latent SIV in vivo by shifting T cells toward a transitional effector phenotype. Here, we investigated the mechanisms underlying this effect using single-cell RNA sequencing, metabolic profiling, and high-dimensional spectral flow cytometry of samples from SIV-infected, antiretroviral therapy–treated (ART-treated) macaques before and after galunisertib. To characterize virus-transcribing, infected cells during ART, we developed a novel, sensitive SIV Transcripts Capture Assay (SCAP) that detected 127 SIV-expressing cells within lymph node single-cell transcriptome libraries. Galunisertib drove broad metabolic reprogramming in CD4+ T cells, with transcriptional upregulation of inflammatory and mitochondrial biosynthesis pathways, confirmed by Seahorse profiling. Metabolomics revealed increased energy metabolites and amino acids and enhanced metabolic flux without proliferation. SIV transcript–positive cells before galunisertib were metabolically quiescent compared with cells without detectable viral transcripts. After galunisertib, virus-expressing cells showed a dramatic metabolic activation, with upregulation of glycolysis, fatty acid metabolism, and TNF-α signaling. High-dimensional flow cytometry demonstrated effects beyond CD4+ T cells, including fewer tissue-resident memory T cells, but more inflammatory macrophages. In conclusion, SCAP represents a specific tool for characterizing rare SIV-infected cells transcribing virus during ART, and it reveals TGF-β as a key mediator of viral latency in vivo through metabolic suppression.
IntroductionHIV and SIV establish persistent viral reservoirs that represent major obstacles to cure strategies (1, 2). These reservoirs consist of infected cells harboring proviruses that persist despite antiretroviral therapy (ART) (3). Viral transcription continues from both defective and intact proviruses, with the immune system playing a crucial role in containing these on-ART reactivation events and shaping reservoir composition (4). This dynamic interplay, alongside heterogeneous latency mechanisms, complicates reservoir elimination efforts.
Elevated TGF-β levels have been documented in people living with HIV (PLWH) since the early epidemic (5), and remain high despite prolonged ART, correlating with immune dysfunction and disease progression (6–8). While TGF-β’s immunosuppressive and fibrogenic properties have been well characterized in the context of HIV (9, 10), TGF-β’s direct impact on HIV infection and transcription remains under-characterized. This knowledge gap is significant considering TGF-β’s central role in primary CD4+ T cell latency models (11–13) and its regulation of immune processes influencing viral persistence (14, 15).
Our previous work demonstrated that TGF-β signaling contributes to the maintenance of viral latency and that inhibition of this pathway using galunisertib (LY2157299), a small, safe, orally available drug that reached phase II clinical development with Eli Lilly (16, 17), can effectively reverse latency in SIV-infected rhesus macaques on ART (18, 19). Specifically, we showed that TGF-β blockade induced latency reversal ex vivo in cells from PLWH and in vivo in SIV-infected, ART-treated rhesus macaques (18, 19). Moreover, we connected the in vivo latency reversal with the induction of a transitional effector phenotype in CD4+ T cells characterized by an increase in some, but not all, canonical activation markers at both the transcriptional and the protein level (19). Finally, in vivo galunisertib treatment also led to enhanced SIV-specific responses and attrition of the viral reservoir (19).
Notably, in previous studies, we consistently observed a substantial enrichment of metabolic pathways in T cells from blood and lymph nodes. Pathways linked to mitochondrial biosynthesis and respiration were significantly upregulated following galunisertib treatment in vivo in both infected and uninfected macaques (18, 19).
The relationship between HIV latency and cellular metabolism has emerged as a critical determinant of viral persistence (20–22). HIV preferentially infects metabolically active CD4+ T cells (22, 23), and upon ART initiation, infected cells that survive likely transition to a quiescent metabolic state, contributing to the establishment of the long-lived reservoir (22, 24). This metabolic quiescence is characterized by reduced glycolysis, oxidative phosphorylation, and overall decreased energy production — an environment unfavorable for viral transcription and production (25).
Recent studies have demonstrated that manipulating specific metabolic pathways can effectively reverse HIV latency (26). For instance, activators of mTOR signaling stimulate glycolysis and anabolic metabolism, leading to increased viral transcription (21, 27). Similarly, compounds that enhance mitochondrial function have shown promise in reversing latency in various models (28, 29). TGF-β signaling itself is intimately connected to cellular metabolism, as TGF-β promotes metabolic quiescence in various immune cells, particularly T cells, by suppressing glycolysis and mitochondrial activity (30–32).
Here, we hypothesized that the main mechanism underlying the latency reversal effect seen in vivo by TGF-β blockade would be connected to its impact on cellular metabolism. To test this hypothesis, we developed a highly sensitive way to characterize SIV-infected cells producing viral transcripts on ART before and after treatment.
Conventional approaches to detect viral RNA–positive cells within single-cell transcriptome studies have typically been based on the identification of cells expressing viral transcripts bioinformatically, leading to potential biases toward high-expressing cells and dependence on sequencing depth (33, 34). We developed a sensitive SIV transcripts capture assay (SIV Transcripts Capture Assay with Parse Biosciences [SCAP]) that enabled a less biased identification and characterization of SIV RNA–positive cells within single-cell RNA sequencing (scRNA-seq) data from lymph node cells before and after galunisertib treatment. Using SCAP, accompanied by metabolomics and Seahorse studies on bulk CD4+ T cells, we demonstrated that galunisertib-mediated latency reversal is predominantly driven by enhanced cellular metabolism. Moreover, high-dimensional flow cytometry analyses revealed that this metabolic reprogramming extended beyond CD4+ T cells to include other immune cell populations in tissues, such as macrophages and NK cells.
ResultsIn vivo TGF-β blockade increases lymph node CD4+ T cell metabolism. This study builds on previously published in vivo research (Figure 1A) wherein eight SIVmac239M2-infected, ART-treated rhesus macaques received four 2-week cycles of galunisertib (20 mg/kg, orally, twice daily). The original investigation demonstrated that galunisertib induced a transitional effector phenotype in CD4+ T cells, leading to on-ART latency reversal, enhanced immune responses, and reduced SIV reservoirs. Latency reversal was evidenced by increased plasma and cell-associated viral RNA, as well as by immunoPET/CT, which revealed substantial viral reactivation in deep tissue compartments (19).
Figure 1Galunisertib upregulates metabolic pathways in lymph node CD4+ T cells. (A) Schematic of the study: Eight macaques were infected with SIVmac239M2 intravenously, and ART was started at week 6 post-infection. The red rectangle indicates the time points analyzed by scRNA-seq: before (BC1; week 35 post-infection) and after the first galunisertib (Gal) cycle (AC1; week 37 post-infection) when the lymph nodes were collected. LN BX, lymph node biopsy; R BX, rectal biopsy; ATI, Antiretroviral therapy interruption; FNA, fine-needle aspiration; Nivo, nivolumab. (B) UMAP projection of scRNA-seq data from lymph node cells, showing annotation of distinct cell subsets. (C) Volcano plot showing differentially expressed genes (DEGs) resulting from the comparison of CD4+ T cells by MAST hurdle model at BC1 versus AC1. Labeled genes that reached significance (BH FDR–adjusted q ≤ 0.1) and log2FC ≥ 1 are shown in the bubble plot. (D and E) Enriched hallmark (D) and KEGG metabolic (E) pathways in total CD4+ T cells based on GSEA (BC1 vs. AC1). Upregulated pathways (red) and downregulated pathways (blue) are shown with their respective normalized enrichment scores (BH FDR *q ≤ 0.1).
To elucidate the mechanistic impact of TGF-β signaling blockade in vivo with galunisertib, we conducted a targeted reanalysis of scRNA-seq data generated from lymph node cells of the 8 macaques collected before (BC1) and after (AC1) the first 2-week galunisertib cycle (Figure 1A). The scRNA-seq data were clustered using the Louvain algorithm and visualized with uniform manifold approximation and projection (UMAP) (35) to reduce dimensionality and display transcriptional heterogeneity. Clusters were annotated with identified cell types (Figure 1B) based on canonical marker gene expression (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.198810DS1).
Differential expression analysis of the CD4+ T cell cluster before (BC1) and after (AC1) galunisertib revealed 7 significantly upregulated genes with no downregulated genes (Benjamini-Hochberg [BH] FDR–adjusted q < 0.1; Figure 1C). These differentially expressed genes (DEGs) are implicated in cellular activation (EGR1, ZNF165, FLYWCH1) (36, 37), differentiation, migration, and immune recognition (RARB, CLEC6A, ASTL) (38, 39), confirming the ability of TGF-β blockade to drive immune activation and T cell differentiation. Gene set enrichment analysis (GSEA) further confirmed robust upregulation of hallmark activation and inflammatory pathways, lipid homeostasis, and stress responses (Figure 1D).
Notably, consistent with previous studies in which oxidative phosphorylation (OXPHOS) was among the most enriched pathways after galunisertib (18, 19), we observed significant upregulation of genes involved in OXPHOS and mitochondrial respiration, along with overall enrichment in metabolic pathways (Figure 1D and Supplemental Figure 1B). To characterize the specific impact of TGF-β blockade on CD4+ T cell metabolism, we performed GSEA on a curated set of metabolic pathways within the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Supplemental Table 1). Corroborating the hallmark results, this analysis revealed strong upregulation of OXPHOS following galunisertib, reinforcing the observation of enhanced mitochondrial activity. Additionally, proinflammatory lipid metabolism pathways were upregulated, including linoleic, arachidonic, and α-linolenic acid pathways (Figure 1E and Supplemental Figure 1C).
Conversely, several amino acid metabolic pathways — glycine, serine, threonine, tryptophan, tyrosine, and glutamate — trended toward downregulation, indicating a shift away from biosynthetic maintenance typically associated with quiescent T cells. This metabolic profile suggests a transition toward a transcriptionally engaged, metabolically active state rather than one primed for proliferation. Supporting this increased energetic demand in the absence of enhanced proliferation, we observed downregulation of one-carbon pool by folate and reduced glycosphingolipid biosynthesis, processes typically upregulated during proliferation to support cell division and expansion (40, 41). Collectively, these findings demonstrate comprehensive metabolic reprogramming favoring effector function, stress adaptation, and transcriptional readiness in CD4+ T cells following galunisertib treatment.
To better understand the metabolic reprogramming driven by TGF-β blockade, we sorted CD4+ T cells from the remaining lymph node cells of 5 of the 8 macaques before and after the first galunisertib cycle and tested their metabolic output with a Seahorse T cell metabolic profiling assay. As suggested by the transcriptomics data, CD4+ T cells after galunisertib were more metabolically active than those from before treatment. Specifically, following galunisertib, we observed a significant reduction in the cells’ spare and maximal respiratory capacity (RC), confirming a reduced ability to respond to increased energy demands by increasing their mitochondrial RC (Figure 2, A–C). Reduced spare and maximal RC accompanied by a tendency toward reduced basal oxygen consumption rate (Supplemental Figure 1D) is typical of effector cell subsets, confirming our previous description of these cells as being in a transitional effector state. However, to reconcile the transcriptional increase in OXPHOS genes, typically associated with quiescence in T cells (34), with the increased energy metabolism and effector phenotype of these cells, we focused our transcriptional analysis on key genes involved in mitochondrial biosynthesis included in the OXPHOS hallmark and KEGG pathways (Supplemental Table 2). We observed increased expression of all genes specifically involved in mitochondrial biosynthesis and assembly, including PPARGC1A, NRF1, NFE2L2, TFAM, and GABPA (42–44), and several associated with mitochondrial fission and fusion, including OPA1, FIS1, DNM1L (DRP1) (45) and ESRRA (46) (Figure 2D). This analysis suggests that the observed increase in the OXPHOS pathway reflects cellular adaptation to increased energetic demand rather than a direct increase in OXPHOS, which is usually associated with T cell memory maintenance and stemness (47, 48).
Figure 2Galunisertib impacts lymph node CD4+ T cell metabolism. (A) Representative Seahorse T cell metabolic profiling of CD4+ T cells isolated from the lymph nodes of a monkey (08M156) before (BC1; week 35 post-infection) and after galunisertib (AC1; week 37 post-infection), showing basal oxygen consumption rate (OCR) and changes in OCR due to response to oligomycin (complex V inhibitor), BAM15 (mitochondrial uncoupler), and rotenone and antimycin A (inhibitors of mitochondrial complex I/III). (B and C) Spare and maximal respiratory capacity calculated from Seahorse T cell metabolic profiling of CD4+ T cells isolated from the lymph nodes of 5 macaques before (BC1) and after (AC1) galunisertib. Data were normally distributed (Shapiro-Wilk test) and compared by paired 2-tailed t test (**P ≤ 0.01). (D) The relative expression before and after galunisertib of selected genes associated with mitochondrial biogenesis and function (Supplemental Table 2).
Collectively, these findings suggest that in CD4+ T cells, the primary effect of TGF-β blockade was metabolic reprogramming, with pronounced upregulation of metabolic pathways associated with cellular activation and effector function. By contrast, while CD8+ T cells also exhibited enhanced energetic metabolism following galunisertib, their transcriptional response was more robust (higher number of DEGs at q < 0.1) and predominantly driven by upregulation of inflammatory and effector function genes rather than metabolic pathway genes (Supplemental Figure 2).
Higher metabolic signature explains post–TGF-β blockade viral transcription. To determine how galunisertib’s transcriptional and metabolic reprogramming in CD4+ T cells connected with SIV latency reversal, we focused our transcriptomic analysis on cells expressing viral transcripts. To identify cells expressing SIV transcripts in our scRNA-seq dataset in a specific and sensitive manner, we developed a novel technique termed SIV Transcripts Capture Assay with Parse Biosciences (SCAP; Supplemental Figure 3A).
We designed a panel of 314 probes targeting the SIV coding region, covering each coding DNA sequence (CDS) with 4 different, overlapping probes. Each probe was designed to avoid exon boundaries to prevent issues with differential splicing. For shorter CDS regions, like tat and rev, we designed 2 full-length probes bookended from the start or stop and extended up- and downstream. After probe hybridization, SIV transcripts were amplified and sequenced, generating an SIV transcript–enriched library that retained cell- and sample-specific barcodes and could be integrated with the whole-transcriptome library. This technique successfully captured viral transcripts with excellent coverage, particularly within the gag region (Supplemental Figure 3B). In contrast, a negligible number of SIV reads were identified in the whole-transcriptome library before SCAP (Supplemental Figure 3B).
Integration of whole-transcriptome and SCAP data revealed 552 candidate SIV RNA–positive (vRNA+) cells. In contrast, no vRNA+ cells were detected in our negative control sample (uninfected PBMCs) subjected to the same SCAP pipeline. Hence, all 552 SIV-RNA+ cells from SCAP of SIV-infected samples were considered. After quality control (QC) filtering to remove low-quality cells, 127 high-confidence SIV-RNA+ cells were retained (Figure 3A).
Figure 3Lymph node cells transcribing SIV on ART display a higher metabolic signature after galunisertib. (A) Schematic process for the identification and filtering of vRNA-positive cells from merged lymph node whole-transcriptome library (98,000 cells) and the SCAP analysis (which yielded 706 cells). Five hundred fifty-two cells had a corresponding barcode in the whole-transcriptome library analyzed with standard Parse pipeline. Only 127 cells passed QC (features between 50 and 8,000 and total UMI between 450 and 200,000). (B and C) UMAP projection of the CD4+ T cell cluster from Figure 1 showing manual annotation of distinct cell subsets (B) and highlighted SIV transcript–positive cells (vRNA+) (C). (D and E) The frequencies of cells, CD4+ T cells (D) and vRNA+ (E), in each CD4+ T cell subset before (BC1) and after (AC1) galunisertib are shown compared by negative bino
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