Activated STING in the thymus alters T cell development and selection leading to autoimmunity

Abstract

Classifying systemic inflammatory disorders as autoinflammatory or autoimmune provides insight into disease pathogenesis and whether treatment should target innate molecules and their signaling pathways or the adaptive immune response. COPA syndrome is a monogenic disorder of immune dysregulation that leads to interstitial lung disease and high-titer autoantibodies. Studies show constitutive activation of the innate immune molecule STING is centrally involved in disease. However, the mechanisms by which STING results in loss of T cell tolerance and autoimmunity in COPA syndrome or more common autoimmune diseases is not understood. Using Copa ^E241K/+ mice, we uncovered a functional role for STING in the thymus. Single cell data of human thymus demonstrates STING is highly expressed in medullary thymic epithelial cells (mTECs) involved in processing and presenting self-antigens to thymocytes. In Copa ^E241K/+ mice, activated STING in mTECs triggered interferon signaling, impaired macroautophagy and caused a defect in negative selection of T cells. Wild-type mice given a systemic STING agonist phenocopied the selection defect and showed enhanced thymic escape of a T cell clone targeting a self-antigen also expressed in melanoma. Our work demonstrates STING activation in TECs shapes the T cell repertoire and contributes to autoimmunity, findings important for settings that activate thymic STING.

Figure 1.. Activated STING in thymic stroma upregulates interferon signaling.

(A) Representative immunoblot of phosphorylated STING (pSTING) in thymic epithelial cells (TECs) versus thymocytes with relative band density quantification. Data represents 2 independent experiments. (B) Immunofluorescence stain of keratin 5 (KRT5) and pSTING expression on thymic sections from Copa^E241K/+ mice. (C) Volcano plot of RNA sequencing analysis of sorted medullary TECs (mTECs) from WT and Copa^E241K/+ mice (n = 3 per genotype). (D) Immunofluorescence stain of KRT5 and keratin 8 (KRT8) on thymic sections from Copa^E241K/+ and WT littermates. (E) Top left: flow cytometry of Ly51 vs EpCAM on TECs; top right: percentages of mTECs and cortical TECs (cTECs); bottom left: mTEC expression of CD80 and MHC-II; bottom right: ratio of mTEC^high versus mTEC^low. Data are mean ± SD. Unpaired, parametric, two-tailed Student’s t-test was used for statistical analysis. p < 0.05 is considered statistically significant. ns: not significant.

Figure 2.. Activated STING in thymic stroma increases single positive thymocytes.

(A) Left: representative flow plots of CD4 and CD8 on reconstituted thymocytes in bone marrow chimeras. Right: percentages of CD4 SP and CD8 SP thymocytes among the reconstituted thymocytes (WT→WT, n = 6; WT→Copa^E241K/+, n = 6; WT→Sting^gt/gt, n = 5; WT→Copa^E241K/+ × Sting^gt/gt, n = 5). (B) Left: representative flow analysis of CD69 and TCRβ on reconstituted thymocytes in bone marrow chimeras. Right: percentages of CD69^highTCRβ^high and CD69^lowTCRβ^high among the reconstituted thymocytes (C) Percentages of CD69^lowMHCI^high (mature stage 2: M2) among the reconstituted CD4 and CD8 single positive thymocytes. Flow strategy in Supplementary Fig. 1D. (WT→WT, n = 7; WT→Copa^E241K/+, n = 6; Copa^E241K/+→WT, n = 6; Copa^E241K/+→Copa^E241K/+, n = 4; WT→Sting^gt/gt, n = 4; WT→Copa^E241K/+ × Sting^gt/gt, n = 3). Data are mean ± SD. Unpaired, parametric, two-tailed Student’s t-test was used for statistical analysis. p < 0.05 is considered statistically significant. ns: not significant. HET: Copa^E241K/+, Sting KO: Sting^gt/gt.

Figure 3.. Constitutive activation of STING in the thymus impairs autophagic flux.

(A) Left: Flow analysis of autophagosome-associated LC3 (LC3II) in mTEC^high from GFP-LC3 and GFP-LC3 × Copa^E241K/+ mice. Right: percentage of LC3II-GFP+ population among total mTEC^high and GFP MFI in mTEC^high. (GFP-LC3 × WT, n = 4, GFP-LC3 × Copa^E241K/+, n = 4). (B) Representative immunoblot and densitometric analysis of LC3II following transient transfection of WT or E241K COPA in HEK293T cells that stably express STING. (C) Quantitation of autophagic flux in mTECs of CAG-RFP-GFP-LC3 tandem reporter mice. Left: flow cytometry of autophagosome-(RFP≈GFP) and autolysosome-associated (GFP<RFP) LC3 in mTECs. Right: percentage of mTEC^high with reduced autophagic flux (RFP-GFP-LC3 × WT, n = 5, RFP-GFP-LC3 × Copa^E241K/+, n = 5; RFP-GFP-LC3 × WT × Sting^gt/gt, n = 3, RFP-GFP-LC3 × Copa^E241K/+ × Sting^gt/gt, n = 3). (D) Left: ratio of RFP/GFP fluorescence histogram in mTECs expressing LC3 tandem reporter; and right: mean RFP/GFP ratio in mTECs (RFP-GFP-LC3 × WT, n = 5, RFP-GFP-LC3 × Copa^E241K/+, n = 5; RFP-GFP-LC3 × WT × Sting^gt/gt, n = 3, RFP-GFP-LC3 × Copa^E241K/+ × Sting^gt/gt, n = 3). Data are mean ± SD. Unpaired, parametric, two-tailed Student’s t-test was used for statistical analysis. p < 0.05 is considered statistically significant. ns: not significant HET: Copa^E241K/+, Sting KO: Sting^gt/gt.

Figure 4.. Activated STING impairs negative selection of T cells and alters the T cell repertoire.

(A) Left: representative flow analysis of CD4 and CD8 on reconstituted thymocytes in bone marrow chimeras. Right: percentage of CD4 single positive thymocytes among the reconstituted thymocytes (OTII→Rip-mOVA × WT, n = 11; OTII→Rip-mOVA × Copa^E241K/+, n = 5; WT→Rip-mOVA × WT × Sting^gt/gt, n = 10; WT→Rip-mOVA × Copa^E241K/+ × Sting^gt/gt, n = 4). (B) Left: flow analysis of Vβ5 and Vα2 in CD4 SP thymocytes in the bone marrow chimeras shown in (A). Right: ratio of TCR^high versus TCR^low among CD4 SP thymocytes. (C) Left: flow analysis of TCRβ and Nur77 expression in the reconstituted CD4 SP in the bone marrow chimeras. Right: percentage of Nur77+ population among CD4 SP thymocytes. (OTII→Rip-mOVA × WT, n = 7; OTII→Rip-mOVA × Copa^E241K/+, n = 5; WT→Rip-mOVA × WT × Sting^gt/gt, n = 8; WT→Rip-mOVA × Copa^E241K/+ × Sting^gt/gt, n = 4). Data are mean ± SD. Unpaired, parametric, two-tailed Student’s t-test was used for statistical analysis. p < 0.05 is considered statistically significant. ns: not significant. HET: Copa^E241K/+, Sting KO: Sting^gt/gt, all host mice are Rip-mOVA background.

Figure 5.. A systemic STING agonist increases autoreactive T cells in the thymus.

(A) Left: CD4 and CD8 profile of thymocytes and CD3 and Vβ14 expression of CD4 single positive thymocytes in Rag1^−/− Tyrp1^B-w/wt TCR mice treated with diABZI STING agonist or vehicle. Right: change in percentage of total thymic CD4 SP and Vβ14⁺ CD4 SP following STING agonist treatment (vehicle n = 14; agonist n = 21). (B) Left: cleaved caspase 3 on CD5^high TCRβ^high thymocytes in vehicle and agonist treated mice. Right: percentage of thymocytes undergoing clonal deletion in agonist treated mice relative to vehicle treatment (vehicle n = 13, agonist n = 14). Unpaired, parametric, two-tailed Student’s t-test was used for statistical analysis. (C) Left: flow analysis of splenic CD4 SP Vβ14⁺ autoreactive T cells in vehicle and agonist treated mice. Right: absolute number of CD4 SP Vβ14⁺ autoreactive T cells. (D) Absolute number of inguinal CD4 SP Vβ14⁺ autoreactive T cells in vehicle and agonist treated mice (vehicle n = 14, agonist n = 21). (E) B16 melanoma growth in Rag1^−/− mice that received X-ray radiation, PD-1 antibody and adoptively transferred splenocytes from Rag1^−/− Tyrp1^B-w/wt TCR mice treated with STING agonist or vehicle (vehicle n = 4, agonist n = 5). Locally estimated scatterplot smoothing with 95% confidence interval of B16 tumor growth over time. Data are mean ± SD. Two-tailed Mann-Whitney U-test was used for statistical analysis unless indicated above. p < 0.05 is considered statistically significant. ns: not significant. (F) Mean expression of STING1 transcript in select cell types in human thymus.