Mechanistic target of rapamycin activation triggers IL-4 production and necrotic death of double-negative T cells in patients with systemic lupus erythematosus

Abstract

The mechanistic target of rapamycin (mTOR) is recognized as a sensor of mitochondrial dysfunction and effector of T cell lineage development; however, its role in autoimmunity, including systemic lupus erythematosus, remains unclear. In this study, we prospectively evaluated mitochondrial dysfunction and mTOR activation in PBLs relative to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) during 274 visits of 59 patients and 54 matched healthy subjects. Partial least square-discriminant analysis identified 15 of 212 parameters that accounted for 70.2% of the total variance and discriminated lupus and control samples (p < 0.0005); increased mitochondrial mass of CD3(+)/CD4(-)/CD8(-) double-negative (DN) T cells (p = 1.1 × 10(-22)) and FOXP3 depletion in CD4(+)/CD25(+) T cells were top contributors (p = 6.7 × 10(-7)). Prominent necrosis and mTOR activation were noted in DN T cells during 15 visits characterized by flares (SLEDAI increase ≥ 4) relative to 61 visits of remission (SLEDAI decrease ≥ 4). mTOR activation in DN T cells was also noted at preflare visits of SLE patients relative to those with stable disease or healthy controls. DN lupus T cells showed increased production of IL-4, which correlated with depletion of CD25(+)/CD19(+) B cells. Rapamycin treatment in vivo blocked the IL-4 production and necrosis of DN T cells, increased the expression of FOXP3 in CD25(+)/CD4(+) T cells, and expanded CD25(+)/CD19(+) B cells. These results identify mTOR activation to be a trigger of IL-4 production and necrotic death of DN T cells in patients with SLE.

Fig. 1

Expansion of DN T cells with MHP and increased mitochondrial mass in PBL of patients with SLE. Frequency of annexin V⁻ cell subsets (panel A) and subpopulations with increased ΔΨ_m (panel B: DiOC6 fluorescence; panel C: TMRM fluorescence) and mitochondrial mass (panel D, MTG fluorescence) were assessed in PBL during 274 visits of 59 patients relative to 214 PBL samples of 54 healthy subjects. p < 0.05 reflects unpaired two-tailed t-test.

Fig. 2

Partial Least Squares - Discriminant Analysis (PLS-DA) of 212 metabolic biomarkers in 274 lupus and 214 healthy control PBL samples. A, Coefficient-based importance measures of the top 15 contributors to components 1, 2, and 3. B, 3-dimensional score plot of PLS-DA using components 1, 2, and 3, accounting for 28.3% and 29.2%, and 12.7% of total variance, respectively. C, Validation of PLS-DA by permutation tests.

Fig. 3

mTOR activation in DN T cells correlates with contraction of Tregs in SLE. A, Upper panel: Detection of increased mTOR activity via phosphorylation of S6 ribosomal protein (pS6RP) in T-cell subsets from lupus and matched control donors. %pS6RP^hi cells are indicated for control (blue histograms) and lupus T cells (red histograms), respectively. Lower panel: Dot plot and histogram of FoxP3 expression within CD25⁺ T cells, gating on CD3⁺/CD4⁺ T cells in control and lupus PBL. B, Cumulative analysis of mTOR activity in T-cell subsets in 274 lupus and 214 control PBL samples. C, Cumulative analysis of CD4⁺ T cells by expression of FoxP3 and CD25. D, Correlation of mTOR activity in DN T cells with expression of FoxP3 in CD4⁺/CD25⁺ T cells of 264 lupus PBL samples.

Fig. 4

Correlation of SLEDAI and traditional biomarkers of disease activity, such as C3, C4, and anti-DNA, with 212 metabolic, cell surface, and gene expression biomarkers during 274 visits of 59 patients with SLE. Due to missing data, the actual number of available data pairs are indicated for each comparison (n = 228–255). Correlation r values were considered significant at p < 0.000236 when corrected for multiple comparisons (0.05/212). Correlation p values above and below 0.000236 are indicated in blue and red symbols, respectively.

Fig. 5

Activation of mTOR, depletion of Tregs, and expansion of necrotic DN T cells distinguish SLE patients in flare. A, Detection of necrotic cells by PI staining in healthy (Control), remitting (SLE remission) and flaring SLE donors (SLE flare). B, Cumulative analyses of necrotic T cells during 214 healthy subject visits, 61 remitting SLE patient visits, and 15 flaring SLE patient visits. C, ΔΨ_m (DiOC6 and TMRM), mitochondrial mass (MTG), and mTOR activity (pS6RP) in healthy subjects, remitting SLE patients, and flaring SLE patients. D, Frequency of FoxP3⁻/CD25⁺ cells within CD4⁺ T cells and frequency of FoxP3⁺ cells within CD25⁺/CD4⁺ T cell compartments of healthy subjects, remitting SLE patients, and flaring SLE patients. p < 0.05 reflect unpaired two-tailed t-test.

Fig. 6

mTOR activation in DN T cells and discordant expression FoxP3 and CD25 in CD4⁺ T cells predict flare in SLE patients with stable disease. Pre-flare visits were defined as visits preceding flare characterized by ≥4 increase of SLEDAI on subsequent visit in 129 ± 29 days (n=15). Pre-flare visits were compared to visits of patients with stable disease, defined as having SLEDAI change < 4 relative to preceding and follow-up visit (n=124). Within the stable visit group, treatment regimens included mTOR inhibitors, rapamycin or NAC, in 75 cases; for the remaining 49 cases, patients were treated without mTOR blockade. A, Frequency of live (% AnnV⁻/PI⁻) cells in T-cell compartments. B, Assessment of necrosis (% PI⁺ cells) in CD3, CD4, CD8 and DN T-cell compartments. p values < 0.05 reflect comparison to flaring patients using two-tailed t-test. C, Assessment of mTOR activity (% pS6RP^hi cells) in CD3, CD4, CD8 and DN T-cell compartments. p values < 0.05 reflect comparison to pre-flare patients using two-tailed t-test. D, Detection of FoxP3⁺CD25⁻CD4⁺ T cells following CD3/CD28 activation. p values < 0.05 reflect comparison to pre-flare patients using two-tailed t-test.

Fig. 7

Increased IL-4 production by DN T cells correlates with skewing of B-cell subsets and anti-DNA in patients with SLE. A, Intracellular production of IL-4 by CD3⁺, CD4⁺, CD8⁺, and DN T cells in 26 patients with SLE and 26 matched healthy subjects. Percentage of IL-4-producing cells was determined by flow cytometry. %IL-4⁺ cells were increased among DN T cells relative to other T-cell subsets both in lupus and control PBL (<0.0001). p < 0.05 reflects paired two-tailed t-test comparing lupus and healthy subjects. B, Intracellular production of IL-17 by CD3⁺, CD4⁺, CD8⁺, and DN T cells in SLE and matched healthy subjects. %IL-17⁺ cells were increased among CD4⁺ T cells relative to other T-cell subsets both in lupus and control PBL. There was no difference in %IL-17⁺ cells between lupus and control subjects using paired t-test. C, Production of IL-4 and IL-17 by MFI of CD3⁺, CD4⁺ and DN T cells of lupus patients normalized to matched healthy controls set at 1.0 for each analysis and expressed as fold changes. p < 0.05 reflects comparison of lupus and matched healthy subjects with paired two-tailed t-test. D, Correlation of %IL-4⁺ DN T cells with anti-DNA levels in SLE. E, Positive correlation of IL-4⁺ DN T cell and CD25⁻ B cell frequencies. F, Negative correlation of IL-4⁺ DN T cell and CD25⁺ B cell frequencies. G, Correlation analysis of %IL-17⁺ DN T cells and anti-DNA levels in SLE. H, Correlation analysis of IL-17⁺ DN T cell and CD25⁻ B cell frequencies. I, Correlation analysis of IL-17⁺ DN T cell and CD25⁺ B cell frequencies.

Fig. 8

Effect of rapamycin on biomarkers of disease activity in patients with SLE. A, SLEDAI and BILAG disease activity scores in 14 SLE patients before and during rapamycin treatment of 126±18 days monitored by therapeutic plasma levels of 8.7±1.2 ng/ml; p values reflect paired t-test. B, Effect of rapamycin treatment on necrosis monitored by the prevalence of PI⁺ cells in CD3⁺, CD4⁺, CD8⁺, and DN T-cell subsets. C, Effect of rapamycin treatment on necrosis monitored by the prevalence of PI⁺ cells in CD3⁺, CD4⁺, CD8⁺, and DN T-cell subsets following CD3/CD28 co-stimulation. D, Effect of rapamycin on IL-4 expression in CD3⁺, CD4⁺, CD8⁺, and DN T-cell subsets assessed by relative fluorescence intensity (RFI) in comparison to matched healthy controls. E, Effect of rapamycin on FoxP3 expression in CD4⁺/CD25⁺ T cells assessed by relative fluorescence intensity (RFI) in comparison to matched healthy controls. F, Effect of rapamycin on the frequency of CD25⁻/CD19⁺ and CD25⁺/CD19⁺ B cells in SLE patients.