Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 8;6(19):e143245.
doi: 10.1172/jci.insight.143245.

Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice

Christopher S Wilson  1 , Blair T Stocks  2 , Emilee M Hoopes  1 , Jillian P Rhoads  2 , Kelsey L McNew  2 , Amy S Major  2   3 , Daniel J Moore  1   2
Affiliations

Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice

Christopher S Wilson et al. JCI Insight. .

Abstract

Autoimmune disease has presented an insurmountable barrier to restoration of durable immune tolerance. Previous studies indicate that chronic therapy with metabolic inhibitors can reduce autoimmune inflammation, but it remains unknown whether acute metabolic modulation enables permanent immune tolerance to be established. In an animal model of lupus, we determined that targeting glucose metabolism with 2-deoxyglucose (2DG) and mitochondrial metabolism with metformin enables endogenous immune tolerance mechanisms to respond to tolerance induction. A 2-week course of 2DG and metformin, when combined with tolerance-inducing therapy anti-CD45RB, prevented renal deposition of autoantibodies for 6 months after initial treatment and restored tolerance induction to allografts in lupus-prone mice. The restoration of durable immune tolerance was linked to changes in T cell surface glycosylation patterns, illustrating a role for glycoregulation in immune tolerance. These findings indicate that metabolic therapy may be applied as a powerful preconditioning to reinvigorate tolerance mechanisms in autoimmune and transplant settings that resist current immune therapies.

Keywords: Autoimmune diseases; Autoimmunity; Lupus; Tolerance; Transplantation.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Tolerance-inducing anti-CD45RB mobilizes regulation in both the B and T lymphocyte compartments in B6 and SLE123 mice.
(A) A 7-day course of aCD45RB was administered to B6 and SLE123 mice. Flow cytometry analysis of B6 and SLE123 mice (n = 4 per group, 9- to 10-week-old females) demonstrated a slight reduction in splenic B cells in both strains. (B) Subset analysis of the B cell compartment in these mice revealed a reduction in the marginal zone (MZ) while the Follicular (FO) and Transitional (Tr.) did not show substantial changes. (C) A cartoon diagram demonstrates the flow cytometry subsetting strategy, with quantification at right. Analyzed using a 1-way ANOVA followed by Tukey’s multiple comparison test. (D and E) Flow cytometry analysis showed B6 mice experienced an expansion of CD4+CD25+Foxp3+ Tregs when treated with aCD4RB (top panel). The SLE123 mice experienced a similar increase in the cells (bottom panel). This increase is quantified in E. Analyzed using a 1-way ANOVA followed by Tukey’s multiple comparison test. Analysis was carried out in 9- to 12-week-old female mice, n = 5 per group.
Figure 2
Figure 2. Effector CD4+ T cells respond inappropriately to anti-CD45RB, accelerating pathologic GC formation in young SLE mice.
(A and B) A 7-day course of aCD45RB was administered to B6 and SLE123 mice. SLE123 mice experienced an increase in Tfh cells in response to aCD45RB. Quantified in B. (C and D) We hypothesized that this increase would also drive expansion of GC B cells, responsible for producing anti-nuclear antibodies in SLE. Flow cytometric analysis revealed aCD45RB-treated SLE123 mice expanded GC B cells, indicating an inappropriate response to aCD45RB therapy. Quantified in D. Analyzed using a 1-way ANOVA followed by Tukey’s multiple comparison test. All mice in A–D were female and 9–12 weeks of age, n = 5 per group.
Figure 3
Figure 3. Anti-CD45RB drives transcriptional changes in metabolism in B6 CD4+ T cells, which are resisted by SLE123 mice.
(A) CD4+ T cells were isolated from B6 and SLE123 mice that had been treated with a standard 7-day course of aCD45RB or left untreated (n = 3, 9- to 12-week-old female SLE or B6 mice). RNA-Seq was performed and analyzed for differential gene expression. The volcano plot demonstrates regulation of a subset of genes in response to aCD45RB only in B6 mice, while SLE123 mice largely resisted transcriptional changes. (B) KEGG analysis of the downregulated genes in B6 mice revealed metabolic pathways to have the highest gene ratio of regulated pathways. (C) Genes in this KEGG term could be classified broadly into 2 categories that impact both glucose metabolism and mitochondrial processes. (Full gene list available in Supplemental Figure 1B.) (D and E) Analysis of glucose uptake using a fluorescent glucose analog, 2NBDG, revealed a decrease in glucose uptake in CD4+ T cells only from B6 mice treated with CD45RB, which was resisted by SLE123 mice. This corroborated the observation of downregulation of genes associated with glucose metabolism. The percentage of CD4+ T cells with high glucose uptake is quantified in E. (F and G) Mitochondrial membrane potential, a measure of mitochondrial activity, was assessed in both B6 and SLE123 CD4+ T cells by flow cytometry utilizing MitoredCMXRos. This analysis revealed increased mitochondrial membrane potential (Δψ) in CD4+ T cells from B6 mice treated with aCD45RB. SLE123 mice resisted these changes and demonstrated a decrease in membrane potential. The median fluorescence intensity of MitoredCMXRos from F is quantified in G. The analyses in D–G were repeated at least 7 times with 3 or more 9- to 12-week-old female SLE123 or B6 mouse replicates in each group. Analyzed using a 1-way ANOVA and Tukey’s multiple comparison test.
Figure 4
Figure 4. A cyclosporine sensitive element modulates immune changes seen with aCD45RB and is defective in SLE123 CD4+ T cells.
(A) TRANSFAC analysis of the metabolic genes regulated by aCD45RB in B6 mice revealed ATF-1 and CREBP likely regulated these genes. (B) As ATF-1 and CREB share similar binding motifs, we assessed the nuclear import of both CREB and ATF-1 in aCD45RB-treated and untreated CD4+ T cells from B6 and SLE. Briefly, nuclei from purified CD4+ T cells were extracted and stained for total CREB and ATF-1. The nuclear localized amount of each protein was measured by flow cytometry. No significant difference was noted in any of the conditions regardless of strain or treatment. n = 8 per group, 9- to 12-week-old female mice. (C and D) The transcriptional activity of CREB can be modified by translocation of CRTCs. Nuclear staining demonstrated translocation of CRTC2 to the nucleus following 15 minutes of aCD45RB stimulation only in isolated nuclei from B6 CD4+ T cells. SLE123 mice did not demonstrate the same response. Quantified in D. Representative of 3 repeats with at least 3 biologic replicates per group, utilizing 9- to 12-week-old female mice. (E) The translocation of CRTC proteins is controlled by the phosphatase calcineurin and the SIK, which control nuclear import and export respectively. (F and G) Pretreatment of B6 CD4+ T cells with calcineurin inhibitor, cyclosporine A, but not the NFAT-specific inhibitor, VIVIT (a major target of calcineurin), prevented the metabolic changes induced by aCD45RB. Cyclosporine prevented the downregulation of glucose uptake and changes in mitochondrial dynamics. The NFAT inhibitor VIVIT had no effect on the metabolic changes in glucose uptake or mitochondrial Δψ induced by aCD45RB. Analyzed by 2-way ANOVA and Tukey’s multiple comparison test. n = 5 per each treatment group, 9- to 12-week-old female mice.
Figure 5
Figure 5. CD4 surface expression of CD45RB and global glycosylation are altered in SLE123 mice.
(A and B) Cell surface expression of CD45RB and total CD45 were measured by flow cytometry on CD4+ T cells. SLE123 demonstrated a downregulation of CD45RB on the cell surface as compared with B6 CD4+ T cells. There was no difference in pan-CD45 expression. Quantified in B. (C) CD45 is composed of an intracellular region that controls cytoskeletal binding and its phosphatase activity. The extracellular domain is composed of a region with fibronectin repeats that is heavily N-glycosylated. An alternatively spliced region is heavily O-glycosylated, and this region imparts unique functions to each CD45 isoform. Additionally, a sialic acid residue on the B portion of this region is essential for the binding of therapeutic aCD45RB. (D and E) We utilized lectins to detect the level of α-2,3–linked sialylation (MALII), O-linked glycosylation (Jacalin), and N-linked glycosylation (PHA-L) in CD4+ T cells from B6 and SLE123 mice. We determined SLE123 CD4+ T cells had reduced levels of α-2,3–linked sialic acids and O-glycosylation and an increase in N-glycosylation. Quantified in E. (F) Utilizing an antibody that detects a desialylated form of anti-CD45RB, we determined SLE123 CD4+ T cells possessed increased binding of this antibody compared with B6. (G and H) To determine the binding of therapeutic aCD45RB to B6 and SLE123 CD4+ T cells, we incubated splenocytes from both strains with aCD45RB, followed by an anti–rat IgG2a antibody conjugated to phycoerythrin. Flow cytometry demonstrated CD4+ T cells from SLE123 mice had reduced binding of the therapeutic antibody. Quantified in H. Analyzed using a Student’s t test. Representative data of at least 4 experimental repeats, with at least 3 biologic replicates of 9- to 12-week-old mice in each group.
Figure 6
Figure 6. Metabolic intervention improves aCD45RB binding and restores responsiveness to aCD45RB therapy.
(A and B) We predicted treatment of SLE123 mice with 2DG and metformin for 1 week would improve the binding of aCD45RB to CD4+ T cells. Treatment with these metabolic therapies improved the binding of aCD45RB (dotted line) as compared with untreated controls (gray histogram). Quantified in B. Representative of 2 experimental repeats, with at least 4 biologic replicates of 9- to 12-week-old female mice in each group. (C) Treatment of SLE123 mice with 2DG/Met reduced the binding of antibody 1B11 that detects desialylated CD45RB. (D and F) B6 and SLE123 mice were treated with 2DG/Met for 1 week before starting aCD45RB and continued for an additional week concomitant with aCD45RB. At the end of this treatment, mice were sacrificed, and metabolic parameters and cell subsets were analyzed. (E) Treatment with metabolic modulators + aCD45RB restored the metabolic regulation by aCD45RB in SLE123 CD4+ T cells. (G and H) This triple therapy also reduced the expansion of Tfh and GC B cells. Analyzed using a 1-way ANOVA followed by Tukey’s multiple comparison test. In D–H, n = 5, 9- to 12-week-old female mice per group. (Control and aCD45RB-treated mice are also shown in Figure 2, B and D.)
Figure 7
Figure 7. Metabolic intervention improves lupus pathology and tolerance to transplanted islets.
(A) To determine whether a short therapeutic course of aCD45RB combined with 2DG/Met would improve the pathology of SLE123 mice, we treated 9-week-old mice with 2DG/met for 1 week before a standard course of aCD45RB with continued 2DG/Met. We also had a group that received 2DG/Met alone for 2 weeks. At that point, treatment was discontinued, and mice were allowed to age for 6 months before they were sacrificed and analyzed. (B and C) Kidney sections from SLE123 mice treated with triple therapy or 2DG/Met alone were stained with anti-IgG (red). There was a notable reduction in IgG deposition in mice treated with 2DG/Met and aCD45RB as compared with metabolic intervention alone. The area of red staining was quantified in these sections as shown in C and analyzed using a Student’s t test. (D) Additionally, we assessed the amount of circulating anti-dsDNA IgG. We noted a decreased in those mice treated with the triple therapy regime as compared with 2DG/Met alone. Analyzed using χ2 test. (E) To assess the capacity for durable tolerance with this triple therapy, we utilized an islet transplant model. Male and female mice 9–12 weeks old were placed on 2DG/Met 1 week before grafting MHC-disparate C3H islets under the kidney capsule. These mice received aCD45RB before metabolic therapy was discontinued. The blood glucose was tracked in these mice, as compared with B6 mice with aCD45RB or SLE123 mice that only received aCD45RB. Rejection was scored by 2 consecutive blood glucose readings of greater than 200 mg/dL. There is an increase in islet survival in SLE123 mice that received the triple therapy as compared with mice that only received aCD45RB. (B6 + aCD45RB, n = 13; SLE123 + aCD45RB, n = 8; and SLE123 + aCD45RB + 2DG + Metformin, n = 7). Scale bars: 100 μm.
See this image and copyright information in PMC

Comment in

References

    1. Choi S-C, Morel L. B cell contribution of the CD4 + T cell inflammatory phenotypes in systemic lupus erythematosus. Autoimmunity. 2017;50(1):37–41. doi: 10.1080/08916934.2017.1280028. - DOI - PMC - PubMed
    1. Walport MJ. Complement and systemic lupus erythematosus. Arthritis Res. 2002;suppl 3:S279–S293. - PMC - PubMed
    1. Alba P, et al. Anti-dsDNA, anti-Sm antibodies, and the lupus anticoagulant: significant factors associated with lupus nephritis. Ann Rheum Dis. 2003;62(6):556–560. doi: 10.1136/ard.62.6.556. - DOI - PMC - PubMed
    1. Contreras G, et al. Recurrence of lupus nephritis after kidney transplantation. J Am Soc Nephrol. 2010;21(7):1200–1207. doi: 10.1681/ASN.2009101093. - DOI - PMC - PubMed
    1. Monk CR, et al. MRL/Mp CD4+,CD25? T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: a novel defect of T cell regulation in systemic lupus erythematosus. Arthritis Rheum. 2005;52(4):1180–1184. doi: 10.1002/art.20976. - DOI - PubMed

Publication types

MeSH terms