Glycolysis inhibition functionally reprograms T follicular helper cells and reverses lupus

Seung Chul Choi; Yong Ge; Ahmed Elshikha; Yuk Pheel Park; Cenxiao Fang; Milind V Joshi; Maria Montes de Oca Arena; Lauren Padilla; Yanan Zhu; William L Clapp; Eric S Sobel; Mansour Mohamadzadeh; Laurence Morel

doi:10.1016/j.celrep.2025.116600

. Author manuscript; available in PMC: 2026 Jan 18.

Published in final edited form as: Cell Rep. 2025 Nov 22;44(12):116600. doi: 10.1016/j.celrep.2025.116600

Glycolysis inhibition functionally reprograms T follicular helper cells and reverses lupus

Seung Chul Choi ^1,⁵, Yong Ge ^1,⁵, Ahmed Elshikha ², Yuk Pheel Park ¹, Cenxiao Fang ¹, Milind V Joshi ¹, Maria Montes de Oca Arena ¹, Lauren Padilla ¹, Yanan Zhu ¹, William L Clapp ², Eric S Sobel ³, Mansour Mohamadzadeh ^1,^4,^*, Laurence Morel ^1,^6,^*

PMCID: PMC12812013 NIHMSID: NIHMS2132729 PMID: 41275496

SUMMARY

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the production of pathogenic autoantibodies depends on T follicular helper (T_FH) cells. This study investigated the mechanisms by which the glycolysis inhibitor 2-deoxy-d-glucose (2DG) reduces the expansion of T_FH cells and the associated production of autoantibodies in lupus-prone mice. Integrated cellular, transcriptomic, epigenetic, and metabolic analyses showed that 2DG reversed the enhanced cell expansion and effector functions, as well as mitochondrial and lysosomal defects in lupus T_FH cells, including increased expression of chaperone-mediated autophagy (CMA) markers associated with Toll-like receptor 7 activation. Importantly, adoptive transfer of 2DG-reprogrammed T_FH cells protected lupus-prone mice from disease progression. The orthologs of genes responsive to 2DG in murine lupus T_FH cells were overexpressed in the T_FH cells of SLE patients, suggesting a therapeutic potential for targeting glycolysis to eliminate aberrant T_FH cells and curb the production of autoantibodies that induce tissue damage.

Graphical Abstract

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In brief

Choi et al. show that pharmacologically restricting glycolysis improves the cellular, transcriptional, and metabolic machinery in murine lupus T_FH cells. Transcript overlaps between T_FH cells from lupus patients and experimental mice indicate glycolysis-sensitive mechanisms, thus demonstrating that uncontrolled glycolysis exacerbated T_FH cell functions and autoantibody production in lupus.

INTRODUCTION

T follicular helper (T_FH) cells critically regulate the production of high-affinity class-switched autoantibodies, the main pathogenic effectors in Systemic lupus erythematosus (SLE), regardless of whether they differentiate through the germinal center (GC) or the extrafollicular route.¹ The frequency of T_FH cells correlates with the disease activity in SLE patients,^2,3 and the pharmacological inhibition of interleukin-21 (IL-21), a cytokine produced by T_FH cells, ameliorated disease in mice.⁴ T_FH cells from the triple congenic (TC) B6.Sle1.Sle2.Sle3 lupus mice present activated transcriptomic effector programs, including increased T cell receptor (TCR) signaling as well as cytokine and co-receptor expression, compared to T_FH cells from B6 congenic healthy controls.⁵ In addition, naive TC CD4⁺ T cells (T_N) are poised to differentiate into T_FH cells. These results indicated that lupus genetic susceptibility skews CD4⁺ T cells toward differentiation into highly active T_FH cells.

T cell metabolism is altered in SLE patients and mice, and the combined inhibition of glycolysis and oxidative phosphorylation (OXPHOS) reverses the disease in multiple models of SLE in correlation with decreased CD4⁺ T cell activation, suggesting that metabolic alterations may point to therapeutic targets.^6,7 Healthy T_FH cells are sustained by both glycolysis and OXPHOS.^8,9 Inhibiting glycolysis through the production of glucose-6-phosphate (G6P) by HK2 with 2-deoxy-d-glucose (2DG)¹⁰ or PKM2 with TEPP-46,¹¹ impaired T_FH cell polarization in a cell-intrinsic manner. The inhibition of glycolysis in vivo with CG-5, an inhibitor of the glucose transporter GLUT1, with 2DG, or TEPP-46 abrogated the expansion of T_FH cells and the production of autoantibodies in several strains of lupus-prone mice.^11-13 In contrast, T_FH cell expansion induced by immunization with a foreign antigen or influenza infection was not affected by 2DG.¹² Thus, these results suggest that autoreactive lupus T_FH cells may depend more on glycolysis than T_FH cells induced by foreign antigens.

In the present study, we elucidated some of the mechanisms by which the inhibition of glycolysis reduces the frequency and reprograms the functions of T_FH cells in lupus. We used the (NZW × BXSB.Yaa) F1 mice, herein referred to as W.Yaa, in which the Yaa duplication of Toll-like receptor 7 (Tlr7) on the Y chromosome directs a highly penetrant disease through TLR7/type I interferon (IFN) signaling,¹⁴ a critical pathway in SLE.¹⁵ W.Yaa mice present a high frequency of age-related B cells (ABCs) and extrafollicular T helper (T_EXFH) cells, corresponding to DN2 B cells¹⁶ and Tph cells² that are expanded in SLE patients. In addition, the mechanisms of T_FH cell expansion¹⁷ and GC dynamics¹⁸ have been investigated in the related B6.Sle1.Yaa model. 2DG completely reversed the autoimmune manifestations and renal pathology in W.Yaa mice¹⁹ to a greater extent than in other models of lupus in which the addition of metformin was necessary.⁷ The W.Yaa model is thus highly relevant to elucidating the mechanisms leading to the rewiring of cellular glycolysis to potentially control the autoreactive T_FH cells and thus mitigate disease progression. Here, we demonstrate that 2DG reprograms T_FH cell activation and metabolism and normalizes the corresponding gene expression and DNA methylation. Particularly, 2DG restored mitochondrial homeostasis and the autophagolysosomal flux, which were impaired in association with increased expression of markers of CMA and TLR7 activation. The functional reprogramming of lupus T_FH cells by 2DG was demonstrated by their protective effect upon adoptive transfers as well as by their reduced ability to activate B cells in vitro. Finally, the transcript signature affected by 2DG in W.Yaa T_FH cells overlaps with the T_FH cell signature in SLE patients. Thus, these results suggest that uncontrolled glycolysis drives dysfunctional T_FH cells, and its inhibition reprograms the mitochondria and their autophagolysosomal machinery to attenuate disease progression.

RESULTS

Glycolysis controls T_FH cell expansion in W.Yaa mice

We confirmed (Figures S1A-S1C) that a 2DG treatment initiated in anti-dsDNA immunoglobulin G (IgG)-positive W.Yaa mice prevented the development of renal pathology and reversed autoantibody production.¹⁹ 2DG reduced the frequency of W.Yaa T_FH cells and, to a lesser extent, follicular regulatory (T_FR) cells to B6 levels, thereby reducing the high T_FH/T_FR ratio associated with SLE²⁰ (Figures 1A-1C). W.Yaa mice develop a robust T_EXFH cell expansion, which was also reduced by 2DG (Figure 1D). 2DG also decreased the proliferation of T_FH, T_FR, and T_EXFH cells (Figure S1D). Inducible T cell costimulator (ICOS) induces Bcl6 expression early in T_FH cell differentiation and maintains their phenotype.²¹ Here, 2DG reduced ICOS expression by W.Yaa T_N, T_FH, T_EXFH cells, and B6 T_FH cells (Figure 1E). Icos transcription is triggered by TCR activation,²² which is stronger in lupus T_FH cells. Accordingly, the higher Icos expression in W.Yaa T_FH cells decreased by 2DG (Figure 1F). ICOS is degraded through ubiquitination by CBL/CBL-b, which is expressed at lower levels by CD4⁺ T cells from SLE patients.²³ Cbl expression was lower in W.Yaa than B6 T_FH cells without change by 2DG (Figure S1E). CBL and CBL-b proteins were expressed at lower levels in CD44⁺CD4⁺ T (surrogates for T_FH cells) than T_N cells from both B6 and W.Yaa mice. However, 2DG increased CBL and CBL-b expression in W.Yaa CD44⁺CD4⁺ T cells (Figures 1G and 1H), suggesting that it may increase ICOS degradation.

Figure 1. — (A and B) Frequency of T_FH (A) and T_FR (B) cells.

(C) T_FH/T_RF cell ratio.

(D) Frequency of T_EXFH cells.

(E) ICOS mean fluorescence intensity (MFI) in T_FH, T_FR, and T_EXFH cells measured by flow cytometry.

(F) *Icos* expression in T_FH cells measured by RNA-seq (n = 3–7).

(G and H) CBL and CBL-b Western blot analysis with representative images of T_N and CD44⁺CD4⁺ T cells from W.Yaa mice treated with 2DG or control (G) and band intensity quantitation relative to β-ACTIN in CD44⁺CD4⁺ T cells from B6 and W.Yaa mice (H) (n = 3–5).

(I–Q) Oxygen consumption rate (OCR) (I) and extracellular acidification rate (ECAR) (J) in CD44⁺CD4⁺ T cells. Vertical lines indicate the injection of oligomycin (oligo), FCCP, and rotenone + antimycin A (rot + ant). Basal OCR (K), non-mitochondrial OCR (L), basal ECAR (M), and glycolytic ATP production (N). Frequency of GC B cells (O), ABCs (P), and plasma cells (PC) (Q). Dunnett’s T3 multiple comparison tests. Differences between W.Yaa B6 groups were highly significant in (A)–(E) and (L)–(Q) and not indicated for clarity. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. 2DG-treated and control W.Yaa mice, and B6 mice (A)–(E) and (O)–(Q): n = 10–21; (I)–(M): n = 4–5.

See also Figures S1 and S2.

W.Yaa CD44⁺CD4⁺ T cells showed enhanced respiration, glycolysis, non-mitochondrial respiration, as well as ATP generated from glycolysis. 2DG reduced all these parameters to B6 levels without affecting B6 T cells (Figures 1I-1N). Glycolytic rate assay confirmed the enhanced basic glycolysis in W.Yaa CD44⁺CD4⁺ T cells (Figure S1H). 2DG drastically reduced the frequency and the number of ABCs and plasma cells, as well as the frequency of GC B cells (Figures 1O-Q and S1I). However, 2DG did not reduce respiration and glycolysis of W.Yaa B cells (Figure S1J), suggesting that its effect on B cells may be secondary to CD4⁺ T cells. We thus focused this study on T_FH cells, although we cannot rule out that the inhibition of glycolysis also impacted W.Yaa B cells, as we have previously shown in the TC lupus model.²⁴

To assess the specificity of the inhibition of glycolysis established for 2DG or CG-5,¹³ we treated W.Yaa mice with PFK15, a second-generation PFKFB3 inhibitor that inhibited glycolysis in dendritic cells in a melanoma model.²⁵ PFK15 did not reduce glycolysis in CD4⁺CD44⁺ T cells, but increased their respiration (Figure S2A). However, none of the 2DG effects were obtained in the PFK15-treated mice (Figure S2BI). Overall, these results demonstrate that inhibiting the first two steps of glycolysis, i.e., glucose import or G6P production, curtailed the expansion of T_FH cells as well as the related T_FR and T_EXFH cells in lupus-prone W.Yaa mice, in association with reduced ICOS expression and normalization of their glycolysis and respiration.

Glycolysis alters TCR signaling and mitochondria homeostasis in lupus T_FH cells

Next, we focused on the effect of 2DG on activation and metabolic markers in W.Yaa T_FH cells. We evaluated the differences in T_FH relative to T_N cells in both W.Yaa and B6 mice to establish T_FH specificity relative to lupus vs. control differences. We then compared T_FH cells from 2DG-treated and untreated W.Yaa mice, with B6 T_FH cells as a reference. As expected, W.Yaa T cells express more TLR7 than B6, but only W.Yaa T_FH cells expressed higher levels of TLR7 than T_N cells, which were reduced by 2DG (Figure 2A). TCR-dependent activation of RelA/p65 increases cMyc expression, which triggers glycolysis.²⁶ TCRβ expression was lower on T_FH than on T_N cells in both strains and unaltered by 2DG (Figure 2B), and consistently, RelA and cMyc were downregulated in B6 T_FH compared to T_N cells (Figures 2C and 2D). However, W.Yaa T_N cells expressed higher levels of RelA and cMyc, which were further increased in W.Yaa T_FH cells and reduced by 2DG. These results suggest a stronger TCR signaling linked to glycolysis in W.Yaa T_FH cells.

Contrary to B6, W.Yaa T_FH cells increased their mitochondrial mass (mMASS) relative to T_N cells (Figure 2E), with a strong correlation with their cell proliferation (Figure 2F). Consistent with high oxidation in T_FH cells,²⁷ B6 T_FH cells increased mitochondrial reactive oxygen species (mROS) production relative to T_N cells (Figure 2G). This was not the case for W.Yaa T_FH cells despite their increased mMASS. 2DG normalized mMASS and partially restored mROS levels in W.Yaa T_FH cells (Figures 2E and 2G). Reduced mROS correlated with ICOS expression by CD4⁺ T cells from untreated W.Yaa mice, as well as with serum anti-dsDNA IgG in the entire W.Yaa cohort (Figures 2H and 2I). The expression of NDUFS1, a key subunit of complex I, which is a major source of mROS,²⁸ strongly increased in T_FH compared to T_N cells, but from a lower level in W.Yaa T_N cells (Figure 2J). 2DG had no effect on NDUFS1 expression. As expected from increased complex I levels in T_FH cells, the NAD⁺/NADH ratio increased from T_N cells to CD44⁺CD4⁺ T cells in B6 but not in W.Yaa mice unless they were treated with 2DG (Figure 2K). An increased expression of the NAD⁺ degrading enzyme CD38, as in CD4⁺ T cells from SLE patients,²⁹ could reduce NAD⁺ levels. CD38 levels increased in T_FH compared to T_N cells in both strains, although lower in W.Yaa than in B6 T_FH cells, and were restored by 2DG (Figure 2L). This suggests that CD38 is unlikely to account for lower NAD⁺ levels. Finally, NAMPT, the rate-limiting enzyme in the NAD⁺ salvage pathway, is overexpressed in CD4⁺ T cells from SLE patients and lupus-prone MRL/lpr mice.³⁰ Nampt expression was also higher in W.Yaa than in B6 T_FH cells (false discovery rate = 0.0015), but it was not altered by 2DG. These results suggest that the reduced NAD⁺ level in W.Yaa T_FH cells restored by 2DG is not regulated at the level of production by complex 1, degradation by CD38, or biosynthesis by NAMPT. Overall, W.Yaa T_FH cells present glycolysis-dependent defective mitochondrial functions associated with a decreased NAD⁺/NADH ratio and mROS production, which correlates with ICOS expression and autoantibody production.

T_FH cell differentiation requires both mTORC1 and mTORC2 activation, at least in part through glycolysis.⁸ W.Yaa and B6 T_FH cells showed a similar increased expression of pEBP1 and pS6 (mTORC1 targets) and pAKT Ser473 (mTORC2 target) and it was minimally affected by 2DG (Figures S1K-S1M). These results suggest that the glucose-dependent expansion of T_FH cells in W.Yaa mice does not depend on mTOR activation.

Inhibiting glycolysis normalized the transcriptome of W.Yaa T_FH cells

To elucidate the mechanisms involved in the glycolysis-dependent expansion of W.Yaa T_FH cells, we first performed bulk RNA sequencing (RNA-seq) on T_N and T_FH cells from W.Yaa and B6 mice. T_FH cells were sorted as CD4⁺CD44⁺PD-1⁺PSGL1^lo cells, which include T_FR and T_EXFH cells. This is a gating strategy that maximizes cell viability by not including CXCR5 and FOXP3 and has been used by others to compare gene expression between T_FH and T_H1 cells.³¹ Further, we have shown an extensive overlap in gene expression between CD4⁺CD44⁺PD-1⁺PSGL1^lo and CD4⁺PD1⁺CXCR5⁺ cells.⁵ Moreover, as for the T_FH, T_FR, and T_EXFH subsets (Figures 1A-1D), 2DG decreased the frequency of CD4⁺CD44⁺PD-1⁺PSGL1^lo cells in W.Yaa but not in B6 mice (Figures S1N and S1O).

W.Yaa T_FH cells showed many differentially expressed genes (DEGs) with B6 T_FH cells (Figures S2A and S2B). The top upregulated genes in W.Yaa T_FH cells included Fcnb, which encodes for Ficolin-1, a disease biomarker in SLE patients,³² as well as Eno1b, a retrotransposon-encoded homolog of the gene encoding for enolase, a key glycolytic enzyme.³³ The expression of the glucose and lactate transporters, Slec2a1 and Slc16a3, as well as most glycolytic enzymes, was higher in W.Yaa T_FH cells (Figure S3A), supporting the observed higher glycolytic activity. The higher expression of GLUT1 in W.Yaa T_FH cells was validated by flow cytometry (Figure S4C). The expression of the lactate importer Slc16a1, as well as that of Pdha1 and Pdk1, favoring mitochondrial pyruvate utilization, was downregulated in W.Yaa T_FH cells, suggesting that anaerobic glycolysis is the main utilization of glucose. In addition, W.Yaa T_FH cells expressed higher levels of G6PD, the rate-limiting entry enzyme in the oxidative pentose phosphate pathway (PPP), at both transcriptional and protein levels (Figures S4B and S4D). The expression of the next oxidative PPP enzyme, PGLS, as well as that of Tkt, the rate-limiting enzyme of the non-oxidative PPP, was decreased in W.Yaa T_FH cells (Figures S4B and S4C).

In addition to these metabolic alterations, cell cycle and cytokine-cytokine receptor interaction were the two main pathways enriched in W.Yaa T_FH cells, consistent with high proliferation, differentiation, and effector functions (Figures S3C and S3D). The lysosome pathway was also overexpressed in W.Yaa T_FH cells, including lysosome-associated membrane protein 2 (Lamp2) as well as cathepsin genes, such as Ctse, which is highly expressed by CD4⁺ T cells in lupus-prone MRL/lpr mice.³⁴ As reported in TC mice,⁵ the majority of DEGs between W.Yaa and B6 T_FH cells were shared with their respective T_N cells (Figure S3E). Indeed, gene expression in T_N cells differed between the two strains, including the lysosome and several metabolic pathways (Figure S5). The cytokine-cytokine receptor interaction pathway dominated the T_FH-specific DEGs between W.Yaa and B6 mice (Figure S3F).

2DG markedly altered gene expression in W.Yaa T_FH cells (Figures 3A and 3B), and the DEGs between 2DG-treated vs. control W.Yaa T_FH cells extensively overlapped the DEGs between W.Yaa T_FH and B6 T_FH cells (Figures S4D and S4E), suggesting that 2DG normalizes the expression of many W.Yaa genes to B6 levels. 2DG reversed the overexpression of GLUT1 and G6PD at both transcriptional and protein levels, as well as that of most glycolytic enzymes (Figures S4A-S4D). This, combined with the upregulation of the OXPHOS pathway, with an enrichment of both mitochondrial and chromosomally encoded respiratory genes, strongly suggests that 2DG reprogrammed W.Yaa Tfh cells away from a glycolytic to a mitochondrial metabolism. Moreover, 2DG reduced the lysosome, cytokine-cytokine receptor interaction, and cell cycle pathways (Figures 3C and 3D), which were differentially expressed between W.Yaa and B6 T_FH cells (Figures S3C and S3D). The cytokine pathway responded to 2DG by the inhibition of T_FH and T_H1-related cytokines, chemokines, and chemokine receptors (e.g., Il21, Il10, Ifng, Ccl3, Ccl4, Ccr5, and IL-10-associated Ccr8) combined with increased expression of T_H2-related cytokine and cytokine receptors (e.g., Il4, Il13ra1, and Il9r). This cytokine signature was validated in an independent cohort of mice (Figure 3C). In addition, W.Yaa T_FH cells secreted higher amounts of IFNγ than B6 T_FH cells, and this production was reduced by 2DG (Figure S4E). Overall, these results suggest that W.Yaa T_FH cells were transcriptionally reprogrammed by 2DG to normalize their metabolism and function.

Figure 3. — (A–D) RNA-seq was performed on T_FH cells from 2DG-treated or untreated W.Yaa mice (n = 3 per group).

(A) Principal-component analysis (PCA) in T_FH cells from 2DG-treated (green) and control (Ctrl, red) W.Yaa mice.

(B) Volcano plot of DEGs upregulated (green) or downregulated (red) in 2DG-treated W.Yaa T_FH cells.

(C) Kyoto encyclopedia of genes and genomes pathways differentially enriched (green: up, red: down) in 2DG-treated W.Yaa T_FH cells.

(D) Heatmaps of DEGs in the indicated pathways.

(E) Quantitative reverse-transcription PCR validation of DEGs in the cytokine-cytokine receptor interaction pathway in T_FH cells from two independent cohorts of 2DG-treated (green) and Ctrl (red) W.Yaa mice (n = 7). Results were normalized to the Ctrl means. Mean ± SEM, compared with t tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

See also Figures S3-S6.

Inhibiting glycolysis promotes DNA methylation in W.Yaa T_FH cells

DNA hypomethylation of immune-related genes correlates with disease activity in SLE patients.³⁵ CpG methylation was higher in T_FH cells from 2DG-treated W.Yaa mice as compared to untreated controls, with 357 hyper- and 75 hypo-differentially methylated regions (DMRs) in 2DG-treated T_FH cells (Figure 4A), 14.6% of which were in promoters and enhancers (Figure 4B). 2DG neither affected the expression of DNA methyl transferases Dnmt3a and Dnmt3b nor DNA demethylases Tet1 and Tet3 in T_FH cells, suggesting that this direct mechanism is not responsible for the observed difference. Sixteen DEGs between 2DG-treated and control T_FH cells presented altered CpG methylation within their promoters, enhancers, or introns, 13 of which had both reduced gene expression and increased DNA methylation (Figure 4C). These included Ifng and Il10, both of which are involved in lupus pathogenesis,^36,37 as well as Lag3, whose expression in T_FH cells has been associated with IL-10 and IL-21 production.^38,39 T_FH signature genes, Irf4, Ikzf3, and Blimp1 were also downregulated and hypermethylated in 2DG-T_FH cells. Relevant to the 2DG-responsive mitochondrial alterations in the observed W.Yaa T_FH cells, Irf4 regulates mitochondrial homeostasis in plasma cells,⁴⁰ and IKAROS (encoded by Ikzf3) has been linked to mitochondrial metabolism and glycolysis in macrophages.⁴¹ Other hypermethylated genes include Mid1, which links mitochondrial respiration to calcineurin during autophagy,⁴² and Vmp1, which is critically implicated in autophagosome formation,⁴³ both parts of the lysosome pathway. Ubash3a modulates TCR signaling,⁴⁴ and Kcnk5 regulates the phosphorylation of numerous mitochondrial proteins.⁴⁵ Examination of specific DMRs in 4 of these genes confirmed the increased methylation levels in T_FH cells from 2DG-treated mice (Figure 4D). Overall, these results suggest that the inhibition of glycolysis reverts DNA hypomethylation that may control the expression of some genes contributing to W.Yaa T_FH cell pathogenesis.

Figure 4. — (A) Whole-genome CpG methylation ratios in T_FH cells from W.Yaa mice treated with 2DG or controls (n = 3 each) and distribution of hyper- or hypomethylated DMRs in the 2DG-treated samples. Mean ± SEM compared with a t test. **p < 0.01.

(B) Distribution of DMRs relative to protein-coding genes.

(C) DEGs (y axis) with differential CpG methylation (x axis) in their promoters (red), enhancers (blue), or introns (green).

(D) DMRs in *Mid1*, *Vmp1*, *Irf4*, and *Ifng*, shown in (C), comparing the distribution of methylated (blue) and unmethylated (red) CpGs between T_FH cells from 2DG-treated and control W.Yaa mice. DMR regions are shown by green boxes.

Inhibiting glycolysis normalized the metabolites in W.Yaa T_FH cells

Untargeted metabolomic analysis comparing W.Yaa and B6 T_FH cells demonstrated altered sialic acid metabolism, PPP, as well as ascorbate and aldarate metabolism (Figure S6A), a key carbohydrate metabolic pathway that protects cells from oxidative damage. An increased ribulose and a decreased gluconate and erythrulose levels in W.Yaa cells indicate an activated oxidative PPP flux relative to the gluconate shunt, as observed in SLE patients.⁴⁶ Moreover, the abundance of these metabolites (Figure S6C) matched the expression of PPP enzymes in the three groups of T_FH cells (Figures S4B-S4D). The PPP supports nucleotide synthesis, here shown as guanine and cytosine, in agreement with the enhanced cell cycle pathway found in the W.Yaa T_FH transcriptome (Figures 3C and 3D). 2-oxoadipate and 5-acetamidopentanoate belong to tryptophan metabolism, which is altered in SLE and associated with T cell activation in lupus-prone mice.^47,48 In addition, anti-inflammatory and antioxidant glycolate and N-acetylneuraminate^49,50 were reduced in W.Yaa T_FH cells. 2DG altered most of these pathways, and the individual metabolites showed similar abundances in T_FH cells from B6 and 2DG-treated W.Yaa mice compared to W.Yaa controls (Figures S6B and S6C). Mitochondrial metabolites (e.g., carnitine, succinate, and α-ketoglutarate) were enriched by 2DG in T_FH cells (Figure S6B), consistent with 2DG increasing the OXPHOS transcriptomic signature (Figures 3C and 3D) and improving mitochondrial functions (Figure 2).

Inhibiting glycolysis normalized autophagolysosomal defects in W. Yaa T_FH cells

We investigated the lysosomal gene signature expressed by W.Yaa T_FH cells and normalized by 2DG. The size of the lysosome as well as its activity increased in T_FH cells compared to T_N cells in both strains, but to a greater extent in W.Yaa T_FH cells, and both were reduced by 2DG (Figures 5A and 5B). However, the expression of TFEB, the transcription factor responsible for lysosome biogenesis, was similar between W.Yaa and B6 T_FH cells (Figure S7A). The autophagy vacuole content was larger in W.Yaa T_N and T_FH cells as compared to B6, and it was reduced by 2DG in W.Yaa T_FH cells (Figure 5C). The autophagic vacuole content as well as the amount of LC3, an autophagosome marker, increased with age and thus with disease development in W.Yaa T_FH cells (Figure S7B). Consistent with an expanded lysosome, T_FH cells expressed higher levels of LAMP1 than T_N cells. However, LAMP1 expression was lower in W.Yaa than in B6 T_FH cells, and it was partially rescued by 2DG (Figure 5D). LAMP2, a mediator of autophagolysosomal fusion, also increased in T_FH cells compared to T_N cells, but contrary to LAMP1, it was reduced by 2DG (Figure 5E). The resulting skewed LAMP2/LAMP1 ratio in W.Yaa T_FH cells compared to B6 T_FH cells was restored by 2DG (Figure 5F). Accordingly, W.Yaa CD4⁺CD44⁺ T cells showed large LAMP1⁺ areas with unfused LC3 autophagosomes that were normalized in T cells from 2DG-treated mice, which displayed co-localized and reduced LAMP1 and LC3 staining (Figures 5G and S7C). These results suggest that the inhibition of glycolysis rescued an impaired autophagolysosome clearance in W.Yaa T_FH cells.

Figure 5. — (A–F). Graphs on the left compare T_N and T_FH cells from untreated W.Yaa (Y) and B6 mice with paired t or Wilcoxon matched-pair signed rank tests, and t tests compare the T_N between strains. Graphs on the right compare T_FH cells between untreated Ctrl and 2DG-treated (n = 14–34) W.Yaa mice and untreated B6 (n = 5). (A) Lysotracker, (B) DQ-BSA, (C) Cyto-ID, (D) LAMP1, (E) LAMP2, and (F) LAMP2/LAMP1 ratio. Results are shown as MFI measured by flow cytometry.

(G). Representative confocal images of CD4⁺CD44⁺ T cells from 2DG-treated and control W.Yaa mice stained with LAMP2a, LAMP1, and DAPI (left) and with LC3, LAMP1, and DAPI (right). Scale bars: 5 μm for LAMP2a images on the left and 10 μm for LC3 images on the right.

(H) LAMP2a as in (A)–(E).

(I) Representative western blot analysis of CD4⁺CD44⁺ T cells from 2DG-treated, control W.Yaa, and B6 mice probed for LAMP2a and HSC70 with quantification relative to β-ACTIN. Dunnett’s T3 multiple comparison tests.

(J) CD4⁺CD44⁺ T cells from B6, control, and 2DG-treated W.Yaa mice stained with LAMP2a, LAMP1, and DAPI at a higher magnification. All images were acquired at the same magnification. Scale bars, 3 μm.

(K) Mitophagy in T_N and T_FH cells from Ctrl and 2DG-treated W.Yaa mice and B6 controls. Representative FACS histograms and MFI quantitation of the mitophagy dye (n = 5–7). Dunnett’s T3 multiple comparison tests compared the groups. Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

See also Figures S7-S9.

Although 2DG decreased LC3 levels in W.Yaa T_FH cells, it had no effect on the classical markers of macroautophagy (Figures S7D and S7E) or activation-induced macroautophagy in isolated CD4⁺ T cells (Figure S7F). The expression of LAMP2a, the LAMP2 splice isoform that mediates CMA, increased in T_FH cells compared to T_N cells in both strains, was higher in W.Yaa than in B6 T_FH cells, and was reduced by 2DG (Figures 5H and 5I). The expression of CMA chaperone HSC70 in T_FH cells mirrored that of LAMP2a (Figure 5I). Accordingly, CD4⁺CD44⁺ and CD4⁺ W.Yaa T cells accumulated LAMP2A⁺ LAMP1⁺ lysosomes, which were reduced by 2DG (Figures 5G-5J and S6C).

We next investigated whether TLR7 signaling could promote glycolysis-dependent CMA in W.Yaa T_FH cells. As previously reported for CD8⁺ T cells,⁵¹ TLR7 agonist R848 increased glycolysis relative to respiration in both unstimulated and anti-CD3-stimulated CD4⁺ T cells (Figure S8A). In addition, the 2DG treatment reduced the expression of type I IFN-stimulated genes in W.Yaa CD4⁺ T cells (Figure S8B). Next, we validated the results obtained in W.Yaa T_FH cells using the related B6.Sle1.Yaa strain with a milder disease.⁵² T_FH cell frequency was higher in B6.Sle1.Yaa than B6.Sle1 or B6 mice (Figure S8C). LAMP2a expression increased in B6.Sle1.Yaa T_FH but not T_N cells compared to B6.Sle1 and B6 cells (Figures S8D and S8E). To directly test whether TLR7 activation induced LAMP2a expression in T_FH cells, we treated B6, B6.Sle1, and TC mice with R848 (Figure S8F). R848 increased T_FH cell frequency (Figures S8G and S8H) as well as LAMP2a expression in both T_N and T_FH cells (Figures S8I and S8J). These results were confirmed by confocal analysis of CD44⁺CD4⁺ T cells from B6.Sle1 mice treated or not with R848 (Figures S8K and S8L). The increased TLR7 expression in W.Yaa T_FH cells (Figure 2A), combined with the induction of LAMP2a expression by R848, suggests that TLR7 signaling, most likely through its downstream product type I IFN, increased glycolysis-dependent CMA in expanded T_FH cells.

As reported in TC T_FH cells,⁵ W.Yaa T_FH cells present an impaired mitochondrial and mitophagy gene signature, shown by decreased Tfam and Pink and increased Fis1 and Tomm20 expression (Figure S9A). Only Fis1 was restored to the B6 level by 2DG. However, Mpp1 expression, which degrades Pink1, was increased in W.Yaa T_FH cells and decreased by 2DG. In addition, Csnk2a1, which is essential for mitophagy, was reduced in W.Yaa T_FH cells and restored by 2DG. Bnip3 and Nix, which are regulated by Csnk2b and mediate interactions between mitochondria and LC3, showed an inverse pattern. Finally, TOMM34 is a mitochondrial membrane protein that promotes mitophagy and regulates HSP70, the obligate chaperone in CMA, by preventing its dimerization.⁵³ Therefore, a lower level of Tomm34 expression by W.Yaa T_FH cells is predicted to reduce mitophagy and increase HSP70 levels, which we have shown in W.Yaa CD44⁺CD4⁺ T cells (Figure 5I), leading to greater CMA. Using a mitophagy dye validated in human CD4⁺ T cells,⁵⁴ we showed that B6 but not W.Yaa T_FH cells have a higher mitophagy flux than T_N cells, and that a higher flux was restored in T_FH cells from 2DG-treated W.Yaa mice (Figure 5K). The same results were obtained with in vitro activated cells, which, as expected, showed an increased mitophagy as compared to ex vivo cells (Figures S9B and S9C).

Overall, our results suggest that W.Yaa T_FH cells demonstrate an altered autophagolysosomal flux associated with increased CMA induced by TLR7 activation as well as decreased mitophagy. The inhibition of glycolysis by 2DG restored the macroautophagic flux, reduced CMA, and increased mitophagy in T_FH cells, suggesting that TLR7-induced T_FH cell expansion and defective autophagolysosomal machinery may be associated with uncontrolled glycolysis in these cells.

2DG-reprogramed T_FH cells protected mice against lupus nephritis

To test whether 2DG-reprogrammed T_FH cells could mitigate disease progression in lupus-prone mice, we performed adoptive transfers of T_FH cells from 2DG-treated or control W.Yaa mice into pre-autoimmune W.Yaa mice (Figure 6A). Age-matched W.Yaa control mice received PBS. Transfers of W.Yaa 2DG-T_FH cells reduced the relative amount of anti-dsDNA IgG (Figure 6B) and ameliorated renal pathology with lower T cell and macrophage infiltrates, smaller glomeruli, and complement C3 deposits (Figures 6C-6E). Furthermore, there was a trend toward lower glomerulonephritis scores and elimination of tubulointerstitial lesions (Figures 6F-6H), which are associated with poor SLE outcomes.⁵⁵ Transfers of 2DG-T_FH cells in the TC model reduced splenocyte numbers and anti-dsDNA IgG production (Figures 6I and 6J), and alleviated renal pathology (Figures 6K-6M). Combined results from the two models showed a marked reduction in serum anti-dsDNA IgG, lower frequencies of splenic T_FH and GC T cells, and reduced mTORC1 activation in CD4⁺ T cells (Figures 6N-6Q). Since 2DG reduced the T_FR cell frequency (Figure 1B) co-transferred with T_FH cells, it is unlikely that T_FR cells were responsible for the improved outcomes of 2DG-T_FH recipients.

Figure 6. — (A) T_FH cells from 2DG-treated or control lupus-prone mice were transferred into pre-disease mice from the same strain and compared to age-matched mice that received PBS.

(B–H). W.Yaa mice evaluated after T_FH cell transfer (n = 6 PBS, 7 Ctrl, and 4 2DG from 3 cohorts). (B) Serum anti-dsDNA IgG/IgG ratio. (C) Representative kidney sections stained for C3 and CD3 (top), and IgG2a and F4/80 (bottom). Scale bars, 200 μm. Quantitation of the glomerulus surface area (D) and C3 deposits (E) (PBS: 60, Ctrl: 83, and 2DG: 34 glomeruli). Glomerulonephritis (F) and interstitial (G) scores evaluated on periodic acid-Schiff (PAS)-stained kidney sections with representative images shown in (H): a and b, Ctrl; c and d, 2DG. Scale bars: 50 μm in (a) and (c); and 20 μm in (b) and (d). Magnification, 200× (a) and (c); and 400× in (b) and (d). Arrows point to droplets in proximal tubules in (a) and normal proximal tubules in (c). In (b), the star shows a fibrocellular crescent, the plain arrow shows an immune infiltrate, and the dashed arrow points to glomerular endocapillary hypercellularity and mesangial expansion. In (d), the dashed arrow points to a mild mesangial expansion with normal glomerular cellularity.

(I–M) TC mice evaluated after T_FH cell transfer (n = 4 PBS, 6 Ctrl, and 3 2DG). (I) Splenocyte numbers. (J) Anti-dsDNA IgG/IgG ratio. Quantitation of the glomerulus surface area (K) and C3 deposits (L), with representative kidney sections shown in (M). Scale bars: 200 μm for (M).

(N–Q) Combined results of transfers in the two strains. (N) Anti-dsDNA IgG/IgG ratio. Frequency of splenic T_FH (O) and GC T (P) cells. (Q) Normalized p4EBP-1 expression in CD4⁺ T cells measured by flow cytometry.

(R–S) B6 B cells were co-cultured with T_FH cells from B6.Sle1.Yaa mice treated with or without 2DG. IgG1 (R) and CD86 (S) expression. Each data point represents the average values between the 5 T_FH-Ctrl and T_FH-2DG.

(B, D–G, and I–L): Dunnett’s T3 multiple comparison tests; (N–Q): t tests; (R and S): paired t tests. (D, E, K, and L): violin plots with medians and quartiles. All other graphs: mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Finally, to assess the ability of 2DG-T_FH cells to provide help to B cells, we co-cultured T_FH cells from B6.Sle1.Yaa mice treated or not with 2DG with B6 B cells. 2DG-T_FH cells induced less IgG1 class-switching and CD86 expression than control T_FH cells (Figures 6R and 6S). T_FH cells induce a high expression of CD86 on light zone GC B cells that, in turn, sustains T_FH activation,⁵⁶ a process that may be impaired by 2DG. Overall, these results suggest that inhibition of glycolysis by 2DG functionally rewired T_FH cells toward a less functional and immunomodulatory phenotype.

T_FH cells from SLE patients and W.Yaa mice share a transcriptome partially controlled by glycolysis

As previously reported,^2,3 SLE patients presented a higher frequency of circulating T_FH cells than healthy controls (HCs); however, there was no difference in T_PH cell frequency (Figures S9A and S9B). T_FH cells from SLE patients and HCs showed a differential gene expression (Figures 7A and 7B). LAMP3 was one of the most upregulated genes. Importantly, overexpression of LAMP3 in the salivary glands of patients with Sjögren’s syndrome was associated with lysosomal dysfunction and impaired autophagy.⁵⁷ Other overexpressed genes in SLE T_FH cells were MAFB, encoding for transcription factor MAF-BZIP, which is a part of the T_FH cell lineage program,⁵⁸ as well as ALOX5, which is strongly activated by mitochondrial dysfunction and lipid peroxidation.⁵⁹ Intracellular iron accumulation leads to lipid peroxidation and drives the expansion of lupus T_FH cells.^60,61 As in TC murine T_FH cells,⁵ anti-apoptotic BCL2 was overexpressed in SLE T_FH cells, suggesting that apoptosis resistance might sustain the expansion of SLE T_FH cells. Finally, FOXP3 expression was higher in SLE than HC T_FH cells (Figure S10C). IL-12 drives the differentiation of human T_FR cells.⁶² IL12RB2 expression was higher in SLE than HC T_FH cells, and it was positively correlated with FOXP3 expression. In addition, SLE T_FH cells expressed higher levels of IZKF2 (encoding for HELIOS) than HC T_FH cells, which was positively correlated to FOXP3 (Figure S9C). HELIOS was expressed at a higher level by T_FH cells from SLE patients than from HCs, and these HELIOS⁺ T_FH cells are high IL-21 producers.⁶³ This suggests that SLE patients may have an expanded T_FR subset, which is poorly defined in SLE, or an expanded subset of effector T_FH cells expressing FOXP3 as an activation marker.

Gene set enrichment analysis showed that type I IFN signaling dominated SLE T_FH cells (Figure 7C). The enrichment in ‘‘positive regulation of B cell proliferation’’ is consistent with an enhanced effector function. The mitochondrial electron transport pathway was also enhanced in SLE T_FH cells (Figures 7C and 7D), which might compensate for a defective mitochondrial metabolism, supported by a strong TCA cycle signature in the metabolites of SLE-T_FH cells with reduced levels of key intermediates such as succinate, isocitrate, and fumarate (Figures S10D and S10E).

Finally, we identified 108 upregulated and 11 downregulated orthologs shared between the SLE patient/HC T_FH DEGs and the W.Yaa/B6 T_FH DEGs (Figure S2), which included ALOX5, MAFBI, and IL10 (Figure 7E). The correlation between the 119 DEGs common to murine and human lupus T_FH cells and the W.Yaa T_FH genes that responded to 2DG (Figure 3) yielded 32 orthologs overexpressed in the T_FH cells of SLE patients (Figure 7F). These orthologs involved in cell cycle (CDK6, CDKN1A, and CDKN2B), B cell signaling (LILRB4A, PIRB, and PIRA2), cytokine signaling (Il10 and SOCS2), iron hemostasis (HMOX1 and HFE) and galectins (LGALS1 and LGALS3), which regulate cell activation and inflammation, highlighting the role that glycolysis may play in expanding T_FH cells in SLE patients.

DISCUSSION

The loss of immune tolerance leading to the production of autoantibodies against nucleic acid-protein complexes is at the root of SLE. In this process, the expansion of autoreactive B cells that differentiate into autoantibody-secreting cells is tightly controlled by T_FH cells.⁶⁴ Accordingly, the expansion of T_FH cells has been consistently observed in patients with SLE and in lupus-prone mice,^1,65,66 although the underlying mechanisms are largely unknown. We have reported that spontaneous T_FH cells from several lupus-prone mouse models are uniquely sensitive to the inhibition of the first step of glycolysis by 2DG, suggesting that targeting glucose metabolism could selectively reduce autoantibody production.¹² Here, we investigated the mechanisms by which 2DG restrains the expansion of T_FH cells in W.Yaa mice, a model of TLR7-driven lupus, in which 2DG completely reversed clinical disease.¹⁹ Treating W.Yaa mice with 2DG reduced T_FH cell frequency to B6 levels. Treatment with a drug that prevents the translocation of PKM2, another glycolytic enzyme, to the nucleus, where it induces the expression of glycolytic genes, also reduced the T_FH cell frequency in another model of lupus,¹¹ confirming that inhibiting glycolysis targets lupus T_FH cells. However, we showed here that the inhibition of glycolysis with PFK15, which targets PKFB3/PFK1 had no effect on T_FH cells in W.Yaa mice. Transcriptional, protein, and metabolic evidence suggest that the oxidative PPP is overactivated in W.Yaa T_FH cells, which was alleviated in mice treated with 2DG. The PPP is dysregulated in the CD4⁺ T cells of SLE patients^46,67 and TC mice,^68,69 and the PPP metabolite gluconolactone has been shown to restore the Treg/T_H17 balance with promising translational applications.⁶⁷ We propose that the downregulation of PPP may contribute to the reprogramming of lupus T_FH cells by 2DG, a hypothesis that remains to be formally validated.

Compared to B6 T_FH cells, W.Yaa T_FH cells exhibited a distinctive gene expression signature dominated by the cytokine, cell cycle, lysosome, and mitochondrial pathways. Importantly, many orthologs of these genes were also differentially expressed in the T_FH cells from SLE patients compared to healthy controls. 2DG treatment reversed these distinctive pathways in W.Yaa T_FH cells to the B6 levels, including the expression of orthologs that are overexpressed in the T_FH cells from SLE patients. These common genes control the cell cycle, cytokine signaling, and iron hemostasis, as well as galectins, all of which are critically involved in T_FH cell differentiation and functions. These results thus suggest that the inhibition of glycolysis reprograms the expression of disease-relevant genes in lupus T_FH cells that regulate their enhanced function. Notably, this conclusion was supported by reduced B cell activation in co-cultures with 2DG-T_FH cells and by adoptive transfers of T_FH cells from 2DG-treated mice, which delayed renal pathology, autoantibody production, and T_FH cell expansion in two different lupus models.

We also showed that 2DG increased DNA methylation in W.Yaa T_FH cells, including in critical genes differentially expressed between W.Yaa and B6 T_FH cells that control T_FH cell differentiation and TCR/IL-2 signaling, as well as cytokine genes that control T_FH function. Chromatin accessibility controls T_FH cell differentiation,⁷⁰ and we have reported an increased histone methylation in lupus T_FH cells.⁵ DNA methylation regulates gene expression in the glycolytic pathway.⁷¹ Further, lysine acetyltransferase KAT6A mediates an increased expression of glycolytic genes in CD4⁺ T cells of lupus-prone MRL/lpr mice, in which treatment with a KAT6A inhibitor reduced disease activity.⁷² Our results suggest that glycolysis regulates the T_FH epigenome, at least in part by reducing DNA methylation. Although the mechanism is still unknown, impaired mitochondrial homeostasis, reducing mitochondrial ATP production in lupus T_FH cells, may be involved in altered DNA methylation.⁷³

Reprogramming of mitochondrial function by 2DG in W.Yaa T_FH cells was documented by the increased expression of mitochondrial and respiratory genes, abundance of mitochondrial metabolites, and mitophagy. Defective mitochondria are a major driver of T cell dysfunction in SLE,⁷⁴ characterized by impaired mitophagy.⁷⁵ We have reported a transcriptional signature associated with defective mitochondrial functions, including mitophagy, in TC T_FH cells,⁵ suggesting that it may be a shared feature in lupus T_FH cells. We also showed that W.Yaa T_FH cells presented a lysosomal/autophagy transcriptional signature that was restored by 2DG. Several studies have shown that dysfunctional mitochondria alter the lysosome.⁷⁶ In T cells in particular, mitochondrial defects critically impair the lysosome and promote inflammatory phenotypes that can be reversed by restoring NAD⁺ levels.⁷⁷ Accordingly, W.Yaa T_FH cells showed reduced levels of mROS, which is required by the autophagy flux⁷⁸ and NAD⁺, which acts bidirectionally as a regulator and target of autophagy.⁷⁹ Canonical markers of macroautophagy remained unchanged in W.Yaa T_FH cells and by 2DG. Instead, we detected increased LAMP2A-associated accumulation of lysosomal contents in W.Yaa T_FH cells, which was reduced by 2DG. Furthermore, we also provided evidence that TLR7 activation promoted both glycolysis and LAMP2-A expression in T_FH cells, suggesting that it induces CMA. CMA is increased in B cells from MRL/lpr mice, and treatment with the P140 peptide to reduce CMA activity ameliorated clinical manifestations in this model as well as in patients with SLE.⁸⁰ More directly related to the present study, CMA promotes T cell activation by selectively deleting negative regulators of TCR signaling.⁸¹ Thus, increased CMA activity in W.Yaa T_FH cells is thought to enhance TCR activation, consistent with the transcriptomic signature of lupus T_FH cells⁵ (and this study). The functional link between CMA and mitophagy is largely unexplored except for TOMM34, which promotes the latter while inhibiting the former through HSP70. Further molecular studies will be necessary to dissect how these two autophagic processes function in opposite directions in a glycolysis-dependent manner. Overall, these preclinical results provide a model in which uncontrolled glycolysis, at least in part driven by TLR7 signaling, promotes T_FH cell expansion and enhances their function in lupus-prone mice. This model also points to the focus of future studies to elucidate the mechanisms by which 2DG may reverse this process, as summarized in Figure S13. The overlap in transcriptional signatures suggests that this model may also be relevant to human lupus T_FH cells. Our results also offer a strong rationale for the therapeutic potential of targeting glycolysis to limit the progression of lupus and subsequently improve patient health, at least in part by normalizing T_FH cell function.

Limitations of the study

A limitation of this study is that we have not determined which aspects of glycolysis are critical for lupus T_FH cells. Although we have clearly shown that 2DG reprograms lupus T_FH cells to a less pathogenic phenotype, another glycolytic inhibitor was ineffective. Thus, we have not identified which glycolytic metabolites or pathways are critical for the expansion and improved function of lupus T_FH cells. It is also possible that 2DG has additional effects besides the inhibition of the first step of glycolysis. We have also not elucidated the mechanisms by which ROS and NAD⁺ production are impaired in lupus T_FH cells and restored by 2DG, although our data suggest that it plays an important role. We further clearly showed that defective autophagosomes/lysosomes with increased LAMP2A-associated accumulation of lysosomal contents, most likely corresponding to CMA and reduced autophagy, were restored by inhibition of glycolysis in lupus T_FH cells. We have not, however, identified the molecular mechanisms linking these cellular processes besides showing that TLR7 is involved, which is highly relevant to lupus pathogenesis. Finally, the role of glycolysis in human lupus T_FH cells was not directly demonstrated. An improved polarization protocol that yields functional ‘‘in vivo-like’’ murine T_FH cells⁸² or a new humanized mouse model that supports fully functional GC responses⁸³ may provide an appropriate experimental system to close this gap.

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

NZW/J females and BXSB/MpJ males were bred to produce F1 male progeny (W.Yaa). The B6.NZM-Sle1^NZM2410/AegSle2^NZM2410/AegSle3^NZM2410/Aeg/LmoJ (TC), B6.Sle1 and B6.Sle1.Yaa congenic mice have been previously described.^52,85 W.Yaa mice and their controls were males, and all TC mice and their controls were females. Each of these models is sex-specific with an earlier and more severe disease incidence in W.Yaa males and TC females. Cohorts of 10–12 week-old W.Yaa mice or 18–20-week-old TC mice, corresponding to an early stage of disease for each strain with anti-dsDNA IgG production without renal pathology, and age-matched control mice were orally treated or not with 2DG (6 mg/mL) in drinking water. Mice were euthanized after 4 weeks of treatment with 2DG when autoimmune phenotypes were evaluated. Some W.Yaa mice were treated with 25 mg/kg PFK15 by intraperitoneal injection 3 times a week for 3 weeks as previously described.²⁵ Control mice received PBS. For T_FH cell transfers, 1.5 × 10⁶ T_FH cells sorted from pooled 2DG-treated mice or from individual age-matched untreated mice were injected in the retro-orbital sinus of either 6-week-old W.Yaa or 2-month-old TC mice (matched strain between donor and recipients). The autoimmune phenotypes of recipients were evaluated in 14-week-old W.Yaa mice and 7-month-old TC mice. The difference in age between the two strains corresponds to the slower disease progression in TC mice. In vivo TLR7 activation was performed by treating mice with application of 100 μg resiquimod (R848) in 100 μL acetone on the ear 3 times a week for 2 weeks, as previously described.⁸⁶

Mice were maintained in SPF conditions at the University of Florida (UF) or at the University of Texas Health San Antonio (UTHSA). This study was carried out in accordance with the guidelines from the Guide for the Care and Use of Laboratory Animals of the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida and the University of Texas Health San Antonio (IACUC 202009466 and 20220032AR, respectively).

Human subjects

PBMCs were collected from SLE patients and healthy controls (HC) at the University of Florida Lupus Clinic according to an approved protocol (UF IRB 201300225). The recruitment and study were carried out in accordance with the World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects. Written informed consent was obtained from all study participants, all of whom were all females, corresponding the 9:1 female:male ration in SLE presentation. Their numbers and demographics are listed in Table S1. Inclusion criteria for SLE subjects were an SLE diagnosis at the UF Lupus Clinic and the absence of current treatment with a cytotoxic drug. The HC inclusion criteria were no current illness at the time of blood collection and the absence of a first-degree relative with an autoimmune disease diagnosis.

METHOD DETAILS

In vitro T cell assays

To measure cytokine production, sorted T_FH and T_N cells (10⁶/mL) were cultured in 96-well plates coated with 4 μg/mL anti-CD3ε for 18 h with complete RPMI medium with 1 μg/mL anti-CD28 in the presence or absence of 2DG (500 μM). IFN-γ were assayed in culture supernatants by ELISA according to following manufacturer’s protocols. Because of low cell numbers, T_FH cells were pooled from 3 B6 mice. T_N cells from both strains and W.Yaa T_FH cells were tested from individual mice.

To assess the effect of TRL7 activation on glycolysis, negatively selected B6 CD4⁺ T cells (10⁶/mL) were cultured for 16 h in RPMI in the presence of anti-CD3 (4 μg/mL), R848 (10 μg/mL) or both, as described by others for CD8⁺ T cells.⁵¹ Cell metabolism was then assessed with a Mitochondrial stress assay, and the expression of glycolysis markers was assessed by flow cytometry.

To assess the ability of T_FH cells from 2DG treated mice to provide help to B cells, T_FH cells (1.5 × 10⁵ cells/ml) sorted from Sle1.Yaa mice treated (N = 4) or not (N = 3) with 2DG for 4 weeks were co-cultured in cRPMI with B cells (2.5 × 10⁵ cells/ml) purified from B6 mice (N = 5, each cultured with the 7 Tfh samples) in presence of anti-CD3 and F(ab’)₂ anti-IgM antibodies. Flow cytometric analysis was conducted 3.5 days later.

Antibody measurement

Antinuclear antibodies (ANAs) were detected on Hep-2 cell-coated slides in sera diluted 1:40. Quantitation was performed with ImageJ. Anti-dsDNA IgG, anti-RNA IgG and total IgG were detected by ELISA in sera diluted 1:100 as previously described.^12,86

Flow cytometry and cell sorting

Flow cytometry was performed on splenocyte suspensions prepared as previously described.¹² Human PBMCs were isolated from whole blood using Ficoll-Plaque Plus. Cells were stained with fluorochrome-conjugated antibodies. Dead cells were excluded with fixable viability dye. For intracellular staining, cells were fixed and permeabilized using the FOXP3/Transcription Factor Staining Buffer. Data acquired using an LSRFortessa or FACSymphony A5 were analyzed with FlowJo V10. Gating strategies for CD4⁺ T cell and B cell subsets are shown in Figure S11A for murine and Figure S12 for human cells. Murine T_FH cells were gated as FOXP3⁻CD44⁺PD-1⁺CXCR5⁺BCL6⁺ PD-1⁺PSGL1^loCD4⁺ T cells, T_FR as FOXP3⁺ PD-1⁺CXCR5⁺BCL6⁺ PD-1⁺PSGL1^loCD4⁺ T cells, and T_EXFH as FOXP3⁻CD44⁺PD-1⁺CXCR5⁻BCL6⁻PD-1⁺PSGL1^loCD4⁺ T cells. Cell sorting was performed with a FACSARIA III cytometer. For RNASeq and metabolomic analyses, murine T_FH cells were sorted as CD4⁺CD44⁺PD-1⁺PSGL-1^lo cells and T_N cells were sorted CD4⁺CD44⁻ cells. Purity was >95% (Figure S11B). Human PD-1⁺CXCR5⁺ T_FH cells were sorted from CD4⁺ T cells isolated from PBMCs by negative selection with the RosetteSep CD4⁺ T cell enrichment cocktail. Purity after sorting was approximately 90% (Figure S12).

The Cyto-ID Autophagy Detection Kit was used on splenocytes treated with Cyto-ID diluted 1:1000 and fixed with 10% formalin. Mean fluorescence intensity (MFI) was measured by flow cytometry. Mitophagy was measured in purified CD4⁺ T cells using the Mitophagy Detection Kit according to the manufacturer’s protocol modified according to a publication in CD4⁺ T cells.⁵³ Fresh CD4⁺ T cells were stained with antibodies to identify T_FH and T_N subsets at 4°C for 30 min, washed and incubated in 100 nmol Mitophagy Dye working solution at 37°C for 30 min. Some cells were activated on plates coated with 4 μg/mL anti-CD3ε mAb in complete RPMI medium with 1 μg/mL anti-CD28 mAb for 3h. Mitophagy Dye fluorescence was detected by flow cytometry.

Metabolic measurements

Mitochondrial stress and Glycolytic Rate assays were conducted on an XF96 extracellular flux analyzer on magnetic-bead purified CD44⁺CD4⁺ T cells, CD4⁺ T cells or B cells as previously described.²⁴ OCR and ECAR values were calculated according to the manufacturer’s formulas. Data was normalized using a coupled Biotek Cytation 1 imager or to an age-matched B6 sample in each assay. The NAD⁺/NADH ratio was measured in CD44⁺CD4⁺ T cells with the NAD/NADH-Glo kit. Untargeted metabolomic profiling of sorted murine (7.5–10 × 10⁵ cells) and human T_FH cells (1 - 4 × 10⁵ cells) was performed at the University of Florida SECIM metabolomic core on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC. All samples were analyzed in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 × 2.1 mm, 2 μm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. The flow rate was 350 μL/min with a column temperature of 25°C. 4 μL were injected for negative ions and 2 μL for positive ions. Metabolites were identified by comparison to the library of purified standards or putatively annotated by their enrichment in significant pathways and by the m/z match with an accuracy of 10 ppm. These metabolites were labeled with asterisks (*) in heatmaps as previously described.¹⁹

Gene expression analyses

RNA was isolated from FACS-sorted T_FH and T_N cells using the RNeasy Plus Micro Kit. Raw sequencing reads were mapped to the reference mouse (GRCm38) or human (GRCh38.83) genomes using STAR v2.7.5c. DESeq2 was used to identify the differentially expressed genes (DEGs) based on the criteria (TPM >1, FDR <0.05, fold change >1.5) as previously described.⁵ Gene set enrichment analysis was performed using DAVID. qRT-PCR validation was performed on T_FH cells sorted from an independent cohort of 2DG-treated and control W.Yaa mice. Amplification was conducted with the SYBR Green Super-mix. Normalization to housekeeping gene Ppia was carried out with the 2ΔΔCt method. Primers are listed in Table S2.

Genome-wide DNA methylation analysis

Genomic DNA was isolated from FACS-sorted T_FH cells using the Quick-DNA Microprep Plus kit. Bisulfite-converted DNA libraries were constructed using the Next Enzymatic Methyl-seq Kit and sequenced on an Illumina NovaSeq 6000 platform. Raw sequence reads were first trimmed to remove poor quality nucleotides, and the high-quality reads were mapped to the mouse reference genome (GRCm38) using Bismark v0.22.3, yielding at least 17-fold whole genome coverage. DMRs were identified using the package DSS and genomic annotation of the DMRs was performed using BEDTools v2.29.2.⁸⁴

Western blotting

Total proteins extracted from magnetic bead-purified CD4⁺CD62L⁺ and CD4⁺CD44⁺CD62L⁻ T cells were quantified using the BCA method. Immunoblotting was performed according to a standard protocol with primary antibodies and HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies. Signal was detected using Signal-Fire-Elite. ImageJ software was used for densitometric analysis. For repeated measurements of different proteins on the same membrane, PVDF stripping buffer was applied and the membrane was re-blocked before re-probing.

Confocal microscopy

Sorted CD4⁺CD44⁺CD62L⁻ T cells (10⁶ cells/well) were fixed with 4% paraformaldehyde, permeabilized with digitonin, then stained with primary antibodies against LAMP2a and LC3, and PE-conjugated anti-mouse LAMP-1. After washing, cells were incubated with FITC-conjugated goat anti-rabbit IgG. Stained cells transferred to cytoslides were incubated with ProLong Gold Antifade Reagent with DAPI and visualized by a Confocal Microscope equipped with a 60× oil immersion objective. A scan resolution of 2048 × 2048 was used to image acquisition.

Renal pathology

Paraformaldehyde-fixed paraffin kidney sections were stained with periodic acid Schiff (PAS). The type and extent of renal lesions were evaluated using a modification of the International Society of Nephrology and Renal Pathology Society classification of lupus nephritis and the NIH activity and chronicity indices in a blinded manner by a pathologist (WLC). Renal parenchymal components, including glomeruli, vessels, tubules, and interstitium, distributed throughout the renal sections were assessed. At least 100 glomeruli per kidney were examined for the presence of mesangial expansion, mesangial and endocapillary hypercellularity, crescents, and glomerulosclerosis. Glomerulonephritis scores were calculated as the sum of the 6 criteria divided by 100. Acute tubular injury lesions, including tubular dilatation, epithelial attenuation, and tubular casts were quantified on a scale of 1–4. Glomerular area was calculated with the Metamorph software. IgG2a and C3 immune complexes as well as T cell and macrophage infiltration were detected in frozen sections as previously described.¹⁹

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses were performed using GraphPad Prism software. All in vitro experiments consisted of three or more biological replicates per experimental group. For in vivo studies, data consisted of more than five mice per group. No data was excluded from the statistical analysis, and all results are demonstrated as individual data points. Unless otherwise stated, differences between groups were evaluated by ANOVA with correction for multiple testing, unpaired or paired t tests, as indicated in the figure legends. Specific tests used are indicated in the figure legends. The corresponding nonparametric tests were used when the data distribution deviated from normality. Results were expressed as means ± standard errors of the mean (SEM). Statistical significance levels were set at *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ****: p < 0.0001.

Supplementary Material

NIHMS2132729-supplement-1.pdf^{(2.9MB, pdf)}

NIHMS2132729-supplement-2.pdf^{(67.9KB, pdf)}

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.116600.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-mouse β-ACTIN	Cell Signaling	Cat#3700; RRID:AB_2242334
Anti-mouse ATF4	Thermo Fisher	Cat#MA5-52598; RRID:AB_3249075
Anti-mouse BCL-6-BV750	BD Biosciences	Cat#568074; RRID:AB_3146163
Anti-mouse cCBL	InVitrogen	Cat#MA5-15885; RRID:AB_11153576
Anti-mouse CBL-B	InVitrogen	Cat#PA5-102547; RRID:AB_2851949
Anti-mouse CD3ϵ-APC/Cy7	BioLegend	Cat#100329; RRID:AB_1877171
Anti-mouse CD3ϵ-APC	BD Biosciences	Cat#553066; RRID:AB_398529
Anti-mouse CD3ϵ	BD Biosciences	Cat#553066; RRID:AB_398529
Anti-mouse CD4-BUV395	BD Biosciences	Cat#563790; RRID:AB_2738426
Anti-human CD4-AF488	BioLegend	Cat#317420, RRID:AB_571939
Anti-mouse CD11c-APC	Thermo Fisher	Cat#17-0114-82; RRID:AB_469346
Anti-mouse CD16/CD32	BD Biosciences	Cat#553141; RRID:AB_394656
Anti-mouse CD19-BV786	BD Biosciences	Cat#563333; RRID:AB_2738141
Anti-mouse CD19-eFluor 450	Thermo Fisher	Cat#48-0199-41; RRID:AB_1272124
Anti-mouse CD21-BV711	BioLegend	Cat#123435; RRID:AB_2876440
Anti-mouse CD23-BV510	BioLegend	Cat#101623; RRID:AB_2563705
Anti-mouse CD28	BD Biosciences	Cat#553295; RRID:AB_394764
Anti-mouse CD44-V500	BD Biosciences	Cat#103008; RRID:AB_312959
Anti-mouse CD86-PE	BD Biosciences	Cat#553692; RRID:AB_394994
Anti-mouse CD95 (Fas)-biotin	BD Biosciences	Cat#554256; RRID:AB_395328
Anti-mouse CD107a (LAMP-1)-PE	Thermo Fisher	Cat#25–1071-82; RRID:AB_2848304
Anti-mouse CD107b (LAMP-2)-PE	BioLegend	Cat#108505; RRID:AB_313386
Anti-mouse CD107b (LAMP-2a)	Thermo Fisher	Cat#51–2200; RRID:AB_2533900
Anti-mouse CD107b (LAMP-2b)	Abcam	Cat#ab125068; RRID:AB_10971511
Anti-mouse CD138-BV605	BioLegend	Cat#142515; RRID:AB_2562336
Anti-mouse CD162 (PSGL-1)-BV605	BD Biosciences	Cat#740384; RRID:AB_2740114
Anti-mouse CD185 (CXCR5)	BD Biosciences	Cat#551961; RRID:AB_394302
Anti-human CD185 (CXCR5)-PE	BioLegend	Cat#356903; RRID:AB_2561812
Anti-mouse CD278 (ICOS)-PE	BD Biosciences	Cat#552146; RRID:AB_394349
Anti-mouse CD279 (PD-1)-eFluor 450	Thermo Fisher	Cat#48-9981-82; RRID:AB_11150068
Anti-mouse CD279 (PD-1)-BV785	BioLegend	Cat#135225; RRID:AB_2563680
Anti-human CD279 (PD-1)-AF647	BioLegend	Cat#329910, RRID:AB_940471
Anti-mouse cMyc-PE	Cell Signaling	Cat#ab169540; RRID:AB_2687932
Anti-mouse Complement C3-FITC	ThermoFisher	Cat#PA1-28933; RRID:AB_1954681
Anti-mouse F4/80-APC	eBioscience	Cat#17-4801-82; RRID:AB_2784648
Anti-mouse F(ab’)₂ anti-IgM, μ chain specific	Jackson ImmunoResearch	Cat#115-006-020; RRID:AB_2338469
Anti-mouse FOXP3-FITC	Thermo Fisher	Cat#11-5773-82; RRID:AB_465243
Anti-mouse G6PD	Cell Signaling	Cat#12263; RRID:AB_2797861
Anti-mouse GL7-eFluor 450	Thermo Fisher	Cat#48-5902-82; RRID:AB_10870775
Anti-mouse GLUT1	Abcam	Cat#ab115730; RRID:AB_10903230
Anti-mouse Hexokinase II	Cell Signaling	Cat#2867; RRID:AB_2232946
Anti-mouse HSPA8	Cell Signaling	Cat#8444; RRID:AB_10831837
Anti-mouse IgD[b]-FITC	BD Biosciences	Cat#553510; RRID:AB_394893
Anti-mouse IgG2a-FITC	South. Biotech	Cat#1080-02; RRID:AB_2794476
Anti-mouse Ki-67-PE-Cy7	Thermo Fisher	Cat#25-5698-82; RRID:AB_11220070
Anti-mouse LC3	Cell Signaling	Cat#12263; RRID:AB_2797861
Anti-mouse NDUFS1-APC	MLB	Cat#PM036; RRID:AB_2274121
Anti-mouse NFkB-P65	Abcam	Cat#ab169540; RRID:AB_2687932
Anti-mouse p4E-BP1-AF647	Cell Signaling	Cat#8242; RRID:AB_10859369
Anti-mouse pAKT-PE/Cy7	Cell Signaling	Cat#5123; RRID:AB_2097838
Anti-mouse pS6-PB	Thermo Fisher	Cat#25-9715-42; RRID:AB_2688172
Anti-mouse PGLS	Cell Signaling	Cat#8520;, RRID:AB_2797646
Anti-mouse T-bet-PE/Cy7	Thermo Fisher	Cat#P-2771MP; RRID:AB_2539845
Anti-mouse TCRβ-PE	Thermo Fisher	Cat#48-5825-80; RRID:AB_2784726
Anti-mouse TLR7-PE	Biolegend	Cat#109207, RRID:AB_313430
Goat anti-rabbit IgG H&L-FITC	Novus	Cat#NBP2-19785; RRID:AB_3264626
Anti-mouse IgG-HRP	Cell Signaling	Cat#7076; RRID:AB_330924
Goat anti-rabbit IgG H&L-AF700	Cell Signaling	Cat#86426, RRID:AB_3717489
Goat anti-rabbit IgG H&L-PE	Thermo Fisher	Cat#A-21038; RRID:AB_2535709
Goat anti-rabbit IgG-FITC	Thermo Fisher	Cat#A16118; RRID:AB_2534790
Anti-rabbit IgG-HRP	Cell Signaling	Cat#7074; RRID:AB_2099233
fixable viability dye-eFluor 780	Thermo Fisher	Cat#65-0865-14
fixable viability dye-eFluor 480	Thermo Fisher	Cat#65-0863-18
DQ^™ Red BSA	Thermo Fisher	Cat#D12051
LysoTracker^™ Deep Red	Thermo Fisher	Cat#L12492
MitoSOX^™ Red	Thermo Fisher	Cat#C10422
H2DCFDA	Thermo Fisher	Cat#D399
MitoTracker^® Red CMXRos	Thermo Fisher	Cat#M46752
MitoTracker^™ Deep Red	Thermo Fisher	Cat#M46753
Streptavidin-PE/Cy7	BD Biosciences	Cat#557598
Streptavidin-BV650	BioLegend	Cat#405231
Streptavidin-PerCP	BD Biosciences	Cat#554064
Chemicals, peptides, and recombinant proteins
2-deoxy-D-Glucose	Cayman	Cat#14325
PFK15	SelleckChem	Cat#S7289
R848 (Resiquimod)	Tocris	Cat#4536
acetone	ThermoFisher	Cat#L10407
Ficoll-Plaque Plus	Sigma	Cat#GE17-1440-02
p-nitrophenol phosphate substrate	ThermoFisher	Cat#34045
cell signaling lysis buffer	Millipore Sigma	Cat#43-040
protease inhibitor cocktail	Sigma	Cat#P8340
Restore^™ Western Blot Stripping Buffer	Thermofisher Scientific	Cat#21059
Signal-Fire-Elite.	Cell Signaling Tech	Cat#12757
digitonin	Sigma	Cat#D141
Critical commercial assays
Seahorse XF Cell Mito Stress Test Kit	Agilent	Cat#103010-100
Seahorse XF Glycolytic Rate Assay Kit	Agilent	Cat#103346-100
Mouse IFN gamma ELISA Kit	ThermoFisher	Cat#KMC4021
Mitophagy kit	Dojindo Molecular Tech.	Cat#MD0110
Fixation/Permeabilization kit	eBioscience	Cat#00-5123-43
ProLong Gold Antifade Reagent with DAPI	Cell Signaling	Cat#8961
Autophagy Antibody Sampler Kit	Cell Signaling	Cat#3700S
Cyto-ID Autophagy Detection Kit	Enzo Life Sci	Cat#ENZ-51031-K200
RosetteSep CD4⁺ T cell enrichment cocktail	Stemcell Tech.	Cat#15062
NAD/NADH-Glo^™ kit	Promega	Cat#G9071
RNeasy Plus Micro Kit	Qiagen	Cat#74034
TruSeq Stranded RNA Sample Preparation Kit	Illumina	Cat#20020596
AMPure XP beads	Beckman Coulter	Cat#A63880
Quant-iT^™ PicoGreen^™ dsDNA assay	ThermoFisher	Cat#P7589
Qick-DNA Microprep Plus kit	Zymo Research	Cat#D4074
Next Enzymatic Methyl-seq Kit	NEB	Cat#E7120
Fixation/Permeabilization kit	eBioscience	Cat#00-5123-43
CD4 T cell Isolation Kit	Miltenyi Biotec	Cat#130-104-454
B Cell Isolation Kit	Miltenyi Biotec	Cat#130-090-862
Pierce BCA protein assay kit	ThermoFisher	Cat#23225
4 - 20% SDS-PAGE gels	BioRad	Cat#4561094
Kallestad HEp-2	BioRad	Cat#26101
Deposited data
Murine Tfh cell RNASeq dataset and DNA bisulfite sequencing dataset	NCBI BioProject	PRJNA1089578
human Tfh cell RNASeq dataset	NCBI BioProject	PRJNA1089580
Untargeted metabolomics analysis of murine Tfh cells	National Metabolomics Data Repository	ST004242
Untargeted metabolomics analysis of human Tfh cells	National Metabolomics Data Repository	ST004243
Experimental models: Organisms/strains
B6.NZM-Sle1^NZM2410/AegSle2^NZM2410/AegSle3^NZM2410/Aeg/LmoJ (TC) female mice	Jackson Laboratory	RRID:IMSR_JAX:007228
C57BL6/J mice	Jackson Laboratory	RRID:IMSR_JAX:000664
NZW/J female mice	Jackson Laboratory	RRID:IMSR_JAX:001058
BXSB/MpJ male mice	Jackson Laboratory	RRID:IMSR_JAX:000740
WYaa male mice	This paper
B6.Cg-Sle1^NZM2410/Aeg Yaa/DcrJ	Jackson Laboratory	RRID:IMSR_JAX:021569
Software and algorithms
FlowJo V10	Tree Star	https://www.flowjo.com/solutions/flowjo/downloads
Prism	Graphpad	https://www.graphpad.com/scientific-software/prism/
ImageJ	NIH	https://imagej.nih.gov/ij/
Metamorph	Molecular Devices	https://www.moleculardevices.com/products/cellular-imaging-systems/high-content-analysis/metamorph-microscopy
STAR aligner v2.7	Open source	https://github.com/alexdobin/STAR
FeatureCounts (v2.0)	Bioconductor	https://subread.sourceforge.net/featureCounts.html
DESeq2	Bioconductor	https://bioconductor.org/packages/release/bioc/html/DESeq2.html
pheatmap R function (version 1.0.12)	Datacamp	https://www.rdocumentation.org/packages/pheatmap/versions/1.0.12
Matplotlib-Venn (Version 0.11.5)	MIT	https://www.piwheels.org/project/matplotlib-venn/
Bismark v0.22.3	Babraham Institute	https://www.bioinformatics.babraham.ac.uk/projects/bismark/
DSS	Ge et al.⁸⁴	https://www.bioconductor.org/packages/release/bioc/html/DSS.html
BEDTools v2.29.2	Ge et al.⁸⁴	https://bedtools.readthedocs.io/en/latest/
DAVID	NCI	https://david.ncifcrf.gov

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Highlights.

Limiting glycolysis by 2DG functionally improves T_FH cells in lupus
2DG reprograms lupus T_FH cells from glycolytic to mitochondrial transcriptome and metabolome
2DG normalized the autophagic flux in lupus T_FH cells
Human lupus T_FH cells exhibit transcripts responsive to 2DG in murine lupus T_FH cells

ACKNOWLEDGMENTS

We thank the staff from the University of Florida (UF) Lupus Clinic, the UF SECIM metabolomic core for technical support, and the UF Molecular Pathology Core for histology services, as well as the staff of the UTHSA Flow Cytometry Shared Resource. This study was supported by a grant from the National Institutes of Health (R01 AI154630) to L.M. and M.M. M.V.J. was partially supported by the South Texas Medical Scientist Training Program NIH NIGMS/T32GM113896 and T32GM145432.

Footnotes

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Laurence Morel morel@uthascsa.edu.

Materials availability

This study did not generate new, unique reagents.

Data and code availability

The murine RNA-seq dataset and the DNA bisulfite sequencing dataset have been deposited in the NCBI BioProject as PRJNA1089578 and the human RNA-seq dataset as PRJNA1089580. Raw and processed metabolomics data have been deposited in the National Metabolomics Data Repository (NMDR) via the Metabolomics Workbench under study IDs ST004242 (murine Tfh cells) and ST004243 (human Tfh cells).
No original code was generated in this paper.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

DECLARATION OF INTERESTS

The authors declare no competing interests.

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PERMALINK

Glycolysis inhibition functionally reprograms T follicular helper cells and reverses lupus

Seung Chul Choi

Yong Ge

Ahmed Elshikha

Yuk Pheel Park

Cenxiao Fang

Milind V Joshi

Maria Montes de Oca Arena

Lauren Padilla

Yanan Zhu

William L Clapp

Eric S Sobel

Mansour Mohamadzadeh

Laurence Morel

SUMMARY

Graphical Abstract

In brief

INTRODUCTION

RESULTS

Glycolysis controls TFH cell expansion in W.Yaa mice

Figure 1. 2DG treatment reduced the expansion of cells involved in autoantibody production in W.Yaa mice.

Glycolysis alters TCR signaling and mitochondria homeostasis in lupus TFH cells

Figure 2. Glycolysis altered TCR signaling and mitochondria homeostasis in lupus TFH cells.

Inhibiting glycolysis normalized the transcriptome of W.Yaa TFH cells

Figure 3. The inhibition of glycolysis normalized the W.Yaa TFH transcriptome.

Inhibiting glycolysis promotes DNA methylation in W.Yaa TFH cells

Figure 4. The inhibition of glycolysis increased DNA methylation in W.Yaa TFH cells.

Inhibiting glycolysis normalized the metabolites in W.Yaa TFH cells

Inhibiting glycolysis normalized autophagolysosomal defects in W. Yaa TFH cells

Figure 5. The inhibition of glycolysis reduced chaperone-mediated autophagy and restored mitophagy in W.Ya TFH cells.

2DG-reprogramed TFH cells protected mice against lupus nephritis

Figure 6. TFH cells from 2DG-treated mice protected from the development of lupus nephritis.

TFH cells from SLE patients and W.Yaa mice share a transcriptome partially controlled by glycolysis

Figure 7. TFH cells from SLE patients shared a 2DG-sensitive gene signature with W.Yaa TFH cells.

DISCUSSION

Limitations of the study

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Human subjects

METHOD DETAILS

In vitro T cell assays

Antibody measurement

Flow cytometry and cell sorting

Metabolic measurements

Gene expression analyses

Genome-wide DNA methylation analysis

Western blotting

Confocal microscopy

Renal pathology

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

Highlights.

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Glycolysis controls T_FH cell expansion in W.Yaa mice

Glycolysis alters TCR signaling and mitochondria homeostasis in lupus T_FH cells

Figure 2. Glycolysis altered TCR signaling and mitochondria homeostasis in lupus T_FH cells.

Inhibiting glycolysis normalized the transcriptome of W.Yaa T_FH cells

Figure 3. The inhibition of glycolysis normalized the W.Yaa T_FH transcriptome.

Inhibiting glycolysis promotes DNA methylation in W.Yaa T_FH cells

Figure 4. The inhibition of glycolysis increased DNA methylation in W.Yaa T_FH cells.

Inhibiting glycolysis normalized the metabolites in W.Yaa T_FH cells

Inhibiting glycolysis normalized autophagolysosomal defects in W. Yaa T_FH cells

Figure 5. The inhibition of glycolysis reduced chaperone-mediated autophagy and restored mitophagy in W.Ya T_FH cells.

2DG-reprogramed T_FH cells protected mice against lupus nephritis

Figure 6. T_FH cells from 2DG-treated mice protected from the development of lupus nephritis.

T_FH cells from SLE patients and W.Yaa mice share a transcriptome partially controlled by glycolysis

Figure 7. T_FH cells from SLE patients shared a 2DG-sensitive gene signature with W.Yaa T_FH cells.