The common TMEM173 HAQ, AQ alleles rescue CD4 T cellpenia, restore T-regs, and prevent SAVI (N153S) inflammatory disease in mice

Abstract

The significance of STING (encoded by the TMEM173 gene) in tissue inflammation and cancer immunotherapy has been increasingly recognized. Intriguingly, common human TMEM173 alleles R71H-G230A-R293Q (HAQ) and G230A-R293Q (AQ) are carried by ~60% of East Asians and ~40% of Africans, respectively. Here, we examine the modulatory effects of HAQ, AQ alleles on STING-associated vasculopathy with onset in infancy (SAVI), an autosomal dominant, fatal inflammatory disease caused by gain-of-function human STING mutations. CD4 T cellpenia is evident in SAVI patients and mouse models. Using STING knock-in mice expressing common human TMEM173 alleles HAQ, AQ, and Q293, we found that HAQ, AQ, and Q293 splenocytes resist STING-mediated cell death ex vivo, establishing a critical role of STING residue 293 in cell death. The HAQ/SAVI(N153S) and AQ/SAVI(N153S) mice did not have CD4 T cellpenia. The HAQ/SAVI(N153S), AQ/SAVI(N153S) mice have more (~10-fold, ~20-fold, respectively) T-regs than WT/SAVI(N153S) mice. Remarkably, while they have comparable TBK1, IRF3, and NFκB activation as the WT/SAVI, the AQ/SAVI mice have no tissue inflammation, regular body weight, and normal lifespan. We propose that STING activation promotes tissue inflammation by depleting T-regs cells in vivo. Billions of modern humans have the dominant HAQ, AQ alleles. STING research and STING-targeting immunotherapy should consider TMEM173 heterogeneity in humans.

Fig. 1.. Splenocytes from HAQ, AQ, and Q293 mice are resistant to STING-mediated cell death ex vivo.

(A). C57BL/6N splenocytes were treated directly (no transfection) with diABZI (100ng/ml), RpRpss-Cyclic di-AMP (5 μg/ml) or 2′3′-cGAMP (10μg/ml), DMXAA (25μg/ml) for 24hrs in culture. CD4, CD8 T cells and CD19 B cells death were determined by PI staining. (B). Splenocytes from C57BL/6N mice were pre-treated with indicated small molecules, GSK2334470 (1.25μM), GSK8612 (2.5μM), Bx-795 (0.5μM), QVD-OPH (25μM) for 2hrs. diABZI (100ng/ml) was added in culture for another 24hrs. Dead cells were determined by PI staining. (C-D). Flowcytometry of HAQ, AQ, IFNAR1^−/− or C57BL/6N splenocytes treated with diABZI (100ng/ml) for 24hrs. Cell death was determined by PI staining. (E-F). Q293 or the WT littermates splenocytes were treated with diABZI (100ng/ml), RpRpss-Cyclic di-AMP (5 μg/ml) or 2′3′-cGAMP 10μg/ml) for 24h. Cell death was determined by PI staining. (G). WT/HAQ, WT/AQ, or WT/WT littermates splenocytes were treated with DMXAA (10, 25 or 100μg/ml) for 24h. Cell death was determined by PI staining. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. p values are determined by one-way ANOVA Tukey’s multiple comparison test (A, E, G) or unpaired student T-test (B, D). * p<0.05. n.s: not significant.

Fig. 2.. HAQ, AQ, and Q293 human cells are resistant to STING agonists-induced death.

(A-B). Total human Lung cells from WT/WT individuals were treated with diABZi (100ng/ml) for 24hrs. Cell death in CD4, CD8 T cells and CD19 B cells were determined by PI staining. (C). Total lung cells from a WT/HAQ (2 individuals) and a WT/WT (3 individuals) were treated with diABZi (25, 100ng/ml) for 24h. Cell death in CD4 T cells was determined by PI staining. (D-E). STING-KO THP-1 cells (Invivogen,, cat no. thpd-kostg) were stably reconstituted with human WT (R232), HAQ, AQ, Q293. Cells were treated with diABZI (200ng/ml) in culture for 24 hrs. Dead cells were determined by Annexin V staining. (F). STING-KO THP-1 cells stably reconstituted with human WT (R232), Q293 were treated with indicated dose of diABZI for 24hs in culture. Dead cells were determined by Annexin V staining. (G). STING-KO THP-1 cells stably reconstituted with human WT (R232), Q293 were treated with indicated dose of diABZI for 2hs in culture. STING activation was detected by anti-STING antibody (Proteintech, #19851–1-AP). (H). STING-KO THP-1 cells stably reconstituted with human WT (R232), HAQ, AQ, Q293 were treated with 50ng/ml diABZI in culture for 24hrs. ISG-54 reporter luciferase activity was determined in cell supernatant and normalized to 10ng/ml IFNβ-stimulated ISG-54 luciferase activity. (I). STING-KO THP-1 cells stably reconstituted with human WT (R232), HAQ, AQ were treated with 50ng/ml diABZI in culture for 2hrs. STING and IRF3 activation were determined by anti-STING antibody and anti-p IRF3 antibody (CST, Ser396, clone 4D4G). Densitometry was determined by ImageLab 5. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. p values determined by one-way ANOVA Tukey’s multiple comparison test (B, C, F, H, G) or unpaired student T-test (E, I). * p<0.05, n.s: not significant.

Fig. 3.. HAQ and AQ rescue the lymphopenia and suppress myeloid cell expansion in SAVI (N153S) mice.

(A). The size and weight of spleens from WT/HAQ, HAQ/SAVI, AQ/SAVI, WT/AQ, WT/SAVI. (B-D). Spleen CD19⁺ B cells, CD4, CD8 T cells were determined in the indicated mice by Flow. (E-H). Spleen Ly6G⁺ neutrophils, Ly6C^hi monocytes and F4/80⁺ macrophage was determined in the indicated mice by Flow. Data are representative of three independent experiments. n=3~5 mice/group. Graphs represent the mean with error bars indication s.e.m. P values are determined by one-way ANOVA Tukey’s multiple comparison test. * p<0.05, n.s: not significant.

Fig. 4.. HAQ and AQ alleles prevent SAVI(N153S) disease in mice.

(A, B, G). The size and body weight of HAQ/SAVI, AQ/SAVI, WT/SAVI and their littermates WT/HAQ, WT/AQ mice. (D, E, H, I). Airway resistance, and pulmonary artery pressure were determined as described in Materials and Methods. (C, F). HAQ/SAVI, AQ/SAVI, WT/SAVI (10 mice/group), were monitored for survival by Kaplan-Meier. (J, K). Representative hematoxylin and eosin (H&E) staining of lung, liver sections from indicated mice. n=3~5 mice/group. Data are representative of 3 independent experiments. Graphs represent the mean with error bars indication s.e.m. p values are determined by one-way ANOVA Tukey’s multiple comparison test. * p<0.05, **p<0.01. n.s.: not significant. WBC: white blood cells; H: hepatocytes; K: Kupper cells; PV: portal vein; CV: central vein.

Fig. 5.. AQ/SAVI(N153S) cells had similar TBK1-IRF3, NFκB activation and STING degradation as the WT/SAVI(N153S) cells.

(A, C). BMDM from WT/WT, WT/SAVI, HAQ/SAVI and AQ/SAVI were treated with 100ng/ml diABZi in culture for 2 hrs. Cells were lysed and run on a 4~20% Mini-PROTEAN TGX Stain-Free Precast Gel. The blot was probed for phospho-Thr172-TBK1 antibody (CST, clone D52C2), phosphor-Ser 396-IRF3 (CST, clone 4D4G), STING (Proteintech, #19851–1-AP) and IκBα (CST, clone 44D4) antibody. (E). BMDM from WT/WT, WT/SAVI, HAQ/SAVI and AQ/SAVI were treated with 100ng/ml diABZi in culture for 24 hrs. Cells were lysed and run on a 4~20% Mini-PROTEAN TGX Stain-Free Precast Gel. The blot was probed for STING antibody (Proteintech, 19851–1-AP). (B, D). IFNβ and TNF were determined by ELISA in the cell supernatant from E. (F). BMDM from WT/WT, WT/SAVI, HAQ/SAVI and AQ/SAVI were treated with 400μM cleavable chemical crosslinker DSP (Pierce^™, cat no: PG82081) in PBS for 1 hr at 4°C. Cells were washed with PBS and lysed in RIPA buffer. Whole cell lysate was mixed with 4x Laemmli Sample Buffer (BioRad, cat no 1610747) containing 5% 2-mercaptoethanol, heated at 95°C for 10 min and, run on a 4~20% Mini-PROTEAN TGX Stain-Free Precast Gel. The blot was probed for STING antibody (Proteintech, 19851–1-AP). Densitometry was determined by ImageLab 5. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. p values are determined by unpaired student T-test (A-E) or one-way ANOVA Tukey’s multiple comparison test (D, B). * p<0.05, **p<0.01, ***p<0.001. n.s.: not significant; n.d: not detected.

Fig. 6.. HAQ/SAVI(N153S) and AQ/SAVI(N153S) cells had 10-fold and 20-fold increased spleen T-regs compared to WT/SAVI mice.

(A). Flow cytometry analysis of IFNγ producing spleen CD4⁺ T cells from WT/HAQ, WT/AQ, WT/SAVI, HAQ/SAVI and AQ/SAVI mice. (B). Flow cytometry analysis of CD4⁺ FoxP3⁺ spleen T-regs. n=3~5 mice/group. Data are representative of 3 independent experiments. Graphs represent the mean with error bars indication s.e.m. p values are determined by one-way ANOVA Tukey’s multiple comparison test. * p<0.05, **p<0.01, ***p<0.001. n.s.: not significant.