Type I interferon and mitochondrial dysfunction are associated with dysregulated cytotoxic CD8+ T cell responses in juvenile systemic lupus erythematosus

Abstract

Juvenile systemic lupus erythematosus (JSLE) is an autoimmune condition which causes significant morbidity in children and young adults and is more severe in its presentation than adult-onset SLE. While many aspects of immune dysfunction have been studied extensively in adult-onset SLE, there is limited and contradictory evidence of how cytotoxic CD8+ T cells contribute to disease pathogenesis and studies exploring cytotoxicity in JSLE are virtually non-existent. Here, we report that CD8+ T cell cytotoxic capacity is reduced in JSLE versus healthy controls, irrespective of treatment or disease activity. Transcriptomic and serum metabolomic analysis identified that this reduction in cytotoxic CD8+ T cells in JSLE was associated with upregulated type I interferon (IFN) signalling, mitochondrial dysfunction, and metabolic disturbances when compared to controls. Greater interrogation of the influence of these pathways on altered cytotoxic CD8+ T cell function demonstrated that JSLE CD8+ T cells had enlarged mitochondria and enhanced sensitivity to IFN-α leading to selective apoptosis of effector memory (EM) CD8+ T cells, which are enriched for cytotoxic mediator-expressing cells. This process ultimately contributes to the observed reduction in CD8+ T cell cytotoxicity in JSLE, reinforcing the growing evidence that mitochondrial dysfunction is a key pathogenic factor affecting multiple immune cell populations in type I IFN-driven rheumatic diseases.

Keywords: CD8+ T cells; cytotoxicity; interferon; juvenile systemic lupus erythematosus.

Graphical Abstract

Figure 1.

Reduction in cytotoxic CD8⁺ T cell capacity in JSLE. Representative flow plots and box plots showing in ex vivo PBMCs the percentage of CD8⁺ T cells (a) expressing perforin (HC n = 57, JSLE n = 42) in HC and JSLE patients. Representative flow plots and box plots showing percentage of CD8⁺ T cells expressing (b) IFN-γ (HC n = 37, JSLE n = 27), and (c) TNF-α (HC n = 31, JSLE n = 27) in PBMCs cultured with PMA/ionomycin in the presence of brefeldin and monensin for 4 h. Boxplots quantifying levels of (d) perforin, (e) IFN-γ, and (f) TNF-α in serum of HC (n = 18) and JSLE patients (n = 17). Analytes were measured using a flow cytometry-based bead multiplex assay. All boxplots show median ± IQR. Flow plots show percentage of cells within each gate. P-values calculated using unpaired Mann–Whitney U test (a–c, e–f) or unpaired t-test as appropriate (d). HC: healthy controls; IFN: interferon; IQR: interquartile range; JSLE: juvenile systemic lupus erythematosus; PBMCs: peripheral blood mononuclear cells; PMA: phorbol 12-myristate 13-acetate; TNF: tumour necrosis factor.

Figure 2.

JSLE CD8⁺ T cells are functional and do not exhibit an exhausted or senescent phenotype. PBMCs from JSLE patients and HC were cultured in the presence or absence of CEFX (a pool of synthetic peptides of known MHC-I restricted epitopes) for 6 h. (a) Representative flow plots and (b) graph quantifying the percentage of CD107a⁺IFN-γ⁺ CD8⁺ T cells in unstimulated (DMSO only) and CEFX peptide treated HC (n = 9) and JSLE (n = 9) PBMCs are shown. (c) Boxplots quantifying differences in percentage of CD107a⁺IFN-γ⁺ CD8⁺ T cells in HC vs JSLE across all experimental conditions. Representative flow diagrams and boxplots showing (d) frequencies of PD-1 expressing CD8⁺ T cells in JSLE (n = 27) and HC (n = 37) in PBMCs stimulated with PMA/ionomycin and (e) ex vivo frequencies of terminally differentiated CD27⁻CD28⁻ EMRA cells (highlighted in red in bottom flow panel) in JSLE (n = 21) and HC (n = 31). (f) KLRG1 expression in EMRA and CD27⁻CD28⁻ EMRA cells JSLE (n = 21) and HC (n = 31). Line plots show KLRG1 MFI in indicated subsets. Numbers in gates and quadrants in flow plots indicate percentage of cells. All boxplots show median ± IQR. P-values calculated using unpaired Mann–Whitney U test (c, d, e), paired Mann–Whitney U test (b) or unpaired t-test (f, g) as appropriate to data distribution. DMSO: dimethyl sulfoxide; EMRA: effector memory cells re-expressing CD45RA; HC: healthy controls; IFN: interferon; IQR: interquartile range; JSLE: juvenile systemic lupus erythematosus; KLRG1: killer cell lectin-like receptor G1; MFI: mean fluorescence intensity; MHC-I: major histocompatibility complex type I; PBMCs: peripheral blood mononuclear cells; PD-1: programmed cell death protein-1; PMA: phorbol 12-myristate 13-acetate.

Figure 3.

Transcriptomic analysis reveals upregulation of IFN-α responses and potential metabolic and mitochondrial disturbances in CD8⁺ T cells in JSLE. (a) Volcano plot showing differences in gene expression from RNA sequencing of CD8⁺ T cells from JSLE (n = 26) vs HC (n = 29). Blue and red points represent statistically significant differentially expressed genes below the FDR adjusted P-value threshold of 0.05. Blue and red arrows indicate number of statistically significant downregulated and upregulated genes, respectively. (b) Bar plot showing −log10p values and enrichment ratios (ER) of summary enriched pathway GO BP ontology terms in CD8⁺ T cells in JSLE vs HC using the 147 significantly upregulated and 91 significantly downregulated genes (FDR adjusted P < 0.05). Statistical significance of enrichment was determined using a P-value cut-off of 0.01 and a minimum enrichment score of 1.5. Terms highlighted in red and blue represent pathways of potential interest, derived from upregulated (red) and downregulated (blue) genes in JSLE vs HC. DEG: differentially expressed genes; ER: enrichment ratio; FDR: false discovery rate; GO BP: gene ontology biological process; HC: healthy controls; ISG-15: interferon-stimulated gene 15; JAK-STAT: Janus kinase/signal transducers and activators of transcription; JSLE: juvenile systemic lupus erythematosus; NF-kappaB: nuclear factor kappa-light-chain-enhancer of activated B cells; NIK: NF-kappaB-inducing kinase; RNA: ribonucleic acid.

Figure 4.

Type I IFN gene scores are increased and correlate negatively with mitochondrial gene expression in CD8⁺ T cells in JSLE. (a) Boxplots displaying transcript per million (TPM) gene counts of 13 mitochondrially expressed genes in HC (n = 29) and JSLE (n = 26) CD8⁺ T cells from RNA-seq. FDR corrected P-values are shown. (b) Boxplot quantifying mitochondrial score, calculated by taking expression of all genes in (a) into account, in JSLE (n = 26) vs HC (n = 29). (c) Boxplots displaying transcript per million (TPM) gene counts of 15 type I IFN-stimulated genes in HC (n = 29) and JSLE (n = 26) CD8⁺ T cells from RNA-seq. FDR corrected P-values are shown. (d) Box plot of interferon score in HC (n = 29) and JSLE (n = 26) samples. All boxplots show median ± IQR. P-values calculated using unpaired t-test (a, b) or Mann–Whitney U test (a, c, d) as appropriate to data distribution. Scatter plots showing (e) correlations between type I IFN score and mitochondrial score (HC: n = 29, JSLE: n = 26) and (f) correlations between mitochondrial score and frequencies of perforin-expressing CD8⁺ T cells (HC n = 16, JSLE n = 18). Spearman’s rho correlation coefficients and the associated P-values are shown (e, f). FDR: false discovery rate; HC: healthy controls; IFN: interferon; IQR: interquartile range; JSLE: juvenile systemic lupus erythematosus; RNA-seq: RNA sequencing; TPM: transcript per million.

Figure 5.

Alterations in CD8⁺ T cell mitochondrial morphology and non-lipid serum metabolome in JSLE. (a) Representative images of CD8⁺ T cells from JSLE patients (n = 3) and HC (n = 4) stained with MitoTracker (MT) Green and 3D modelling images showing mitochondrial volume and surface area. White bars represent 1µm. (b) Violin plot showing the distribution of total mitochondrial volume (µm³) per cell in JSLE CD8⁺ T cells (n = 3) compared with HC (n = 4). (c) Violin plot showing the distribution of total mitochondrial surface area (µm²) from all JSLE CD8⁺ T cells (n = 3) compared with HC (n = 4). P-values for HC vs JSLE comparisons calculated using unpaired Mann–Whitney U test (b, c). (d) Volcano plot showing fold changes and FDR adjusted P-values obtained from individual Mann–Whitney tests of non-lipid metabolites in JSLE compared to HC. The significance threshold (FDR P-value < 0.05) is indicated by the horizontal dashed line. Significantly altered metabolites are highlighted in colour according to metabolite group. (e) Boxplots plotting serum levels of metabolites altered in JSLE showing median ± IQR. FDR adjusted P-values calculated using unpaired Mann–Whitney U test are shown. (f) Forest plot summarizing results of linear regression analysis for each metabolite (log transformed and scaled), showing which metabolites are significantly altered in JSLE when accounting for sex, age, and ethnicity. Regression results highlighted in colour by metabolite group and filled circles indicate significantly altered metabolites (FDR P-value <0.05). FDR: false discovery rate; HC: healthy controls; IQR: interquartile range; JSLE: juvenile lupus erythematosus; MT: MitoTracker.

Figure 6.

EM CD8⁺ T cells in JSLE are susceptible to IFN-α-induced apoptosis. (a) Representative flow plots showing Annexin V and PI staining. (b) Stacked bar plot quantifying frequencies of early apoptotic, late apoptotic, and live EM CD8⁺ T cells in HC (n = 9) and JSLE patients (n = 10) in PBMCs with and without stimulation with IFN-α for 48 h. Bar plot displays means ± SD. P-values comparing total apoptotic cells (early apoptotic + late apoptotic) were calculated using unpaired t-test in HC vs JSLE comparisons and paired t-test for unstimulated vs IFN-α stimulated comparisons. (d) Representative flow plots and boxplots showing percentage of CM, EM, effector, and naïve CD8⁺ T cell populations in HC (n = 65) and JSLE (n = 42). Numbers in all flow plot quadrants indicate percentage of cells. All boxplots show median ± IQR. P-values for HC vs JSLE comparisons calculated using Mann–Whitney U test. (d) Correlation matrix showing strength of correlation between frequency of EM CD8⁺ T cells and differentially expressed metabolites and mitochondrial score in HC and JSLE. Spearman correlations are shown. *P < 0.05, **P < 0.01. EM: effector memory; CM: central memory; GlycA: glycoprotein acetyls; HC: healthy controls; IFN: interferon; IQR: interquartile range; JSLE: juvenile systemic lupus erythematosus; PBMCs: peripheral blood mononuclear cells; PI: propidium iodide; SD: standard deviation.