Mechanisms governing the bioenergetics of naive and effector CD4+ T cells as a means of controlling autoimmunity

CD4+ T cells are a critical component of the adaptive immune system as they generate large amounts of cytokines that help shape the immune milieu. Additionally, they are the primary contributors to the immunopathology exhibited in Type 1 Diabetes. The field of immunometabolism has elucidated that the cellular metabolic profile of immune cells has a significant impact on their function, and ultimately the fate of the overall response. Naïve CD4+ T cells rely primarily on mitochondrial oxidative phosphorylation, but upon antigen encounter, reprogram their metabolism to aerobic glycolysis. Understanding the mechanisms that govern these programs could be critical in developing new therapies for limiting aberrant T cell responses in autoimmunity. Here we examined the contributions of two different molecules, Lymphocyte Activation Gene 3 (LAG-3) and reactive oxygen species (ROS) in controlling T cell metabolism. LAG-3 is an inhibitory receptor expressed on the surface of CD4+ T cells, and deficiency in naïve T cells leads to enhanced homeostatic expansion. Our results indicate that LAG-3 expression on naïve CD4+ T cells serves to restrain cellular metabolism and mitochondrial biogenesis as a means of maintaining quiescence. These results are compelling as loss of LAG-3 expression in a model of Type 1 Diabetes results in accelerated disease progression, potentially due to T cell metabolic enhancements, as our data would suggest. Single nucleotide polymorphisms in the LAG-3 gene have also been linked to autoimmune disease susceptibility. With regards to ROS, Type 1 Diabetes is known to be highly driven by oxidative stress, and CD4+ T cells require acute doses of ROS to drive optimal activation. Therefore, we sought to understand if ROS signaling contributes to the metabolic transition that occurs during T cell activation. Indeed, ROS inhibition resulted in reduced mTOR signaling and aerobic glycolysis. Altering metabolism in this manner also delayed Type 1 Diabetes progression in an adoptive transfer model of disease. Collectively, this work demonstrates that both LAG-3 and ROS regulate CD4+ T cell metabolism, which, in turn, greatly impacts T cell activation potential and ability to drive disease

Reactive oxygen species are required for driving efficient and sustained aerobic glycolysis during CD4+ T cell activation.

The immune system is necessary for protecting against various pathogens. However, under certain circumstances, self-reactive immune cells can drive autoimmunity, like that exhibited in type 1 diabetes (T1D). CD4+ T cells are major contributors to the immunopathology in T1D, and in order to drive optimal T cell activation, third signal reactive oxygen species (ROS) must be present. However, the role ROS play in mediating this process remains to be further understood. Recently, cellular metabolic programs have been shown to dictate the function and fate of immune cells, including CD4+ T cells. During activation, CD4+ T cells must transition metabolically from oxidative phosphorylation to aerobic glycolysis to support proliferation and effector function. As ROS are capable of modulating cellular metabolism in other models, we sought to understand if blocking ROS also regulates CD4+ T cell activation and effector function by modulating T cell metabolism. To do so, we utilized an ROS scavenging and potent antioxidant manganese metalloporphyrin (MnP). Our results demonstrate that redox modulation during activation regulates the mTOR/AMPK axis by maintaining AMPK activation, resulting in diminished mTOR activation and reduced transition to aerobic glycolysis in diabetogenic splenocytes. These results correlated with decreased Myc and Glut1 upregulation, reduced glucose uptake, and diminished lactate production. In an adoptive transfer model of T1D, animals treated with MnP demonstrated delayed diabetes progression, concurrent with reduced CD4+ T cell activation. Our results demonstrate that ROS are required for driving and sustaining T cell activation-induced metabolic reprogramming, and further support ROS as a target to minimize aberrant immune responses in autoimmunity

Mn porphyrin regulation of aerobic glycolysis: Implications on the activation of diabetogenic immune cells

Aims: The immune system is critical for protection against infections and cancer, but requires scrupulous regulation to limit self-reactivity and autoimmunity. Our group has utilized a manganese porphyrin catalytic antioxidant (MnTE-2-PyP5+, MnP) as a potential immunoregulatory therapy for type 1 diabetes. MnP has previously been shown to modulate diabetogenic immune responses through decreases in proinflammatory cytokine production from antigen-presenting cells and T cells and to reduce diabetes onset in nonobese diabetic mice. However, it is unclear whether or not MnP treatment can act beyond the reported inflammatory mediators. Therefore, the hypothesis that MnP may be affecting the redox-dependent bioenergetics of diabetogenic splenocytes was investigated. Results: MnP treatment enhanced glucose oxidation, reduced fatty acid oxidation, but only slightly decreased overall oxidative phosphorylation. These alterations occurred because of increased tricarboxylic acid cycle aconitase enzyme efficiency and were not due to changes in mitochondrial abundance. MnP treatment also displayed decreased aerobic glycolysis, which promotes activated immune cell proliferation, as demonstrated by reduced lactate production and glucose transporter 1 (Glut1) levels and inactivation of key signaling molecules, such as mammalian target of rapamycin, c-myc, and glucose-6-phosphate dehydrogenase. Innovation: This work highlights the importance of redox signaling by demonstrating that modulation of reactive oxygen species can supplant complex downstream regulation, thus affecting metabolic programming toward aerobic glycolysis. Conclusion: MnP treatment promotes metabolic quiescence, impeding diabetogenic autoimmune responses by restricting the metabolic pathways for energy production and affecting anabolic processes necessary for cell proliferation. Antioxid. Redox Signal. 19, 1902-1915. © Mary Ann Liebert, Inc

Mechanism for the effect of ROS inhibition on CD4⁺ T cells during activation-induced metabolic reprogramming.

<p>Upon T cell receptor–MHC interaction, ROS are generated by a functional NOX expressed by T cells. These ROS serve as signaling molecules to help propagate mTOR signaling resulting in Myc upregulation and progression to aerobic glycolysis. Treatment with the ROS-scavenging and potent antioxidant results in inhibition of ROS and maintains potent AMPK activation; thereby, inhibiting mTOR via a two-pronged approach, stabilizing OXPHOS, and limiting T cell proliferation.</p

Alteration in redox status of CD4⁺ T cells maintains AMPK activation.

<p>(a) Protein lysates were probed with antibodies for phosphorylated AMPK-1α (Thr172; activated), total AMPK, and β actin (loading control). Data are a representative of n = 5 independent experiments.</p

Reducing glycolytic capacity by redox modulation inhibits diabetogenic potential of CD4⁺ T cells.

<p>(a) Schematic of adoptive transfer model of T1D. (b) Survival curve of diabetes free animals following adoptive transfer. Animals were deemed diabetic following 2 consecutive blood glucose readings of >350 mg/dL. Statistical significance of disease progression was calculated using a Kaplan-Meier test (* = p<0.05). (c) T cell activation and prediction of diabetes was measured by serum levels of sLAG-3 by ELISA. Statistical significance was calculated using a Two-way ANOVA with Bonferroni post-hoc analysis. (** = p<0.01). (d) Normalized percent of LAG-3⁺CD25⁺CD4⁺ T cells from spleens of control diabetic and MnP-treated non-diabetic animals (n = 6–8 animals per group; p = 0.0761). (e) Mean fluorescence intensity of pS6 in peripheral blood CD4⁺ T cells from animals at day 4 post-transfer. (f) Representative histograms of S6 phosphorylation from peripheral blood CD4⁺ T cells at day 4 post-transfer from control and MnP treated animals. (g-h) Quantification of frequency of pS6^hi and pS6^lo CD4⁺ T cells. (i) Forward scatter analysis of pS6^lo and pS6^hi CD4⁺ T cells.</p

Lymphocyte Activation Gene-3 Maintains Mitochondrial and Metabolic Quiescence in Naive CD4+ T Cells

Summary: Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics, we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo, LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3−/− CD4+ T cells, solidifying that LAG-3 controls these processes. Lag3−/− CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation, enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally, enhancement of STAT5 activation, as a result of LAG-3 deficiency, contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. : Previte et al. show that LAG-3 expression regulates the metabolic profile of naive CD4+ T cells during homeostatic expansion. They observed that Lag3-deficient CD4+ T cells are resistant to Interleukin-7 deprivation due to enhanced STAT5 activation. Increased STAT5 signaling also mediated greater activation potential in these T cells following stimulation. Keywords: LAG-3, CD4+ T cell, metabolism, mitochondria, STAT

mTOR signaling is inhibited upon MnP treatment.

<p>(a) Western blot analysis for phosphorylated mTOR (Ser4998; active), total mTOR, and the downstream mTOR target, phosphorylated 4E-BP1 (Thr70). β actin expression served as the loading control. (b) Forward scatter (FSC) of CD4⁺ T cells was measured by flow cytometry as an indication of cell blasting. Histogram is representative of n = 5 experiments. (c) IFNγ secretion was assessed by ELISA of culture supernatants. (d) Analysis of <i>Ifnγ</i> gene expression by qRT-PCR at 24 hours post-stimulation. (e) ELISA results measuring IL-2 secretion from culture supernatants. (e) Frequency of CD4⁺ T cells expressing CD25 following activation. Data displayed are combined means ± SEM of n = 6–9 experiments. Statistical significance was calculated using either a one-way or two-way ANOVA with Tukey or Bonferroni post-hoc analysis, respectively. (* = p<0.05). Media alone treated splenocytes served as negative controls.</p

Redox modulation during activation inhibits cell cycle progression of CD4⁺ T cells.

<p>NOD.BDC.2.5.TCR.Tg splenocytes were plated in complete splenocyte media and stimulated with 0.05 μM mimotope with or without 68 μM MnP (M + MnP) for 48–72 hours. (a) Prior to stimulation cells were loaded with 1 μM CFSE. Cells were stained with CD4 following harvest and analyzed by flow cytometry for CFSE dilution, indicating proliferation. Unstimulated cells (grey shaded curve) served as negative controls to set proliferation gates. CFSE tracings are representative of n = 6 independent experiments. (b) Cells were fixed, permeabilized, and stained with propidium iodide and CD4-FITC. Cells were analyzed by flow cytometry to determine cell cycle status. CD4⁺ T cells were gated on and cell cycle phases were set based upon unstimulated controls (left panel). (c) Percentages of n = 5 experiments were combined and graphed as mean ± SEM (* = p<0.05). (d) 48 hrs. post-stimulation, cells were harvested and analyzed by Western blot for p27 Kip1 and Cyclin D3 expression. β actin expression served as the loading control.</p

MnP treatment effectively scavenges NADPH oxidase and mitochondrial derived superoxide, while demonstrating no toxicity.

<p>NOD splenocytes were pre-treated with or without MnP and then loaded with either Dihydroethidium (DHE; a) or MitoSOX red (b). Splenocytes were stimulated with PMA and ionomycin and read for fluorescence by flow cytometry at the indicated time points. Data are displayed as delta mean fluorescence intensity (Δ MFI) ± SEM calculated as MFI_stimulated − MFI_unstimulated. (c and d) BDC2.5.TCR.Tg splenocyte cultures were stained for 7AAD and CD4 to assess viability of cultures due to MnP treatment. Data are displayed as percent 7AAD^- of whole splenocytes (c) and CD4⁺ T cells (d). Significance was determined by Two-way ANOVA with Bonferroni post-hoc analysis of a combined n = 3–5 mice (**** = p<0.0001; *** = p<0.001; ** = p<0.01; *p<0.05).</p