DOI: 10.5061/dryad.m0cfxppcv
title: Data from: The common TMEM173 HAQ, AQ alleles rescue CD4 T cellpenia, restore T-regs, and prevent SAVI (N153S) inflammatory disease in mice

description: The significance of STING (encoded by the TMEM173 gene), in tissue
 inflammation and cancer immunotherapy has been increasingly
 recognized. Intriguingly, common human STING 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 STING
 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.
description: Please see the "Method" section of the manuscript.
description: # Data from: The common TMEM173 HAQ, AQ alleles rescue CD4 T cellpenia,
 restore T-regs, and prevent SAVI (N153S) inflammatory disease in mice
 [https://doi.org/10.5061/dryad.m0cfxppcv](https://doi.org/10.5061/dryad.m0cfxppcv) ## Description of the data and file structure **Figure 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&lt;0.05. n.s: not significant. **Figure 1-figure supplement 1.** Bx-795 inhibits diABZI-induced mouse splenocyte death. (A). Splenocytes from C57BL/6N mice were treated with 100ng/ml diABZI in culture for the indicated time. Dead cells were determined by PI and Annexin V staining. (B). Splenocytes from C57BL/6N mice were treated with indicated dose of diABZi in culture for 24hrs. Dead cells were determined by PI staining. (C). Splenocytes from C57BL/6N mice were pre-treated with indicated small molecules, H151 (10µg/ml), C176 (1µM), MCC950 (1.25µM), 2-BP (20µM), GSK872 (312.5nM), Liproxstatin-1 (1µM), necrostatin-1 (10µM), VX-765 (1µM), Z-DEVD-FMK (25µM), Bx-795 (0.5µM) for 2hrs. diABZi (100ng/ml) was added in culture for another 24hrs. Dead cells were determined by PI staining. (D). IFNb was determined in the cell supernatant from C by ELISA. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. n=3~5 mice/group. *p* values are determined by one-way ANOVA Tukey’s multiple comparison test (A, D) or unpaired student T-test (C). * p&lt;0.05. n.s: not significant. n.d: not detected. **Figure 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 IFNb-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*&lt;0.05, n.s: not significant. **Figure 2-figure supplement 1.** **STING activation in primary human cells and THP-1 cells reconstituted with *WT* human *STING****. (***A). Total human Lung cells from *WT/WT* individuals were treated with indicated dose of diABZi for 24hrs. Cell death in CD4, CD8 T cells and CD19 B cells were determined by PI staining. (B).** Sequencing results of human *TMEM173* gene in *WT/WT* and *WT/HAQ* individuals. (**C-D)**. STING-KO THP-1 cells stably reconstituted with human *WT (R232)* were treated with indicated dose of diABZI for indicated time in culture. ISG-54 reporter luciferase activity was determined in cell supernatant and normalized to 10ng/ml IFNb-stimulated ISG-54 luciferase activity. Data are representative of at least two 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. * *p*&lt;0.05, n.s: not significant. **Figure 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, Ly6Chi 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&lt;0.05, n.s: not significant. **Figure 3-figure supplement 1.** Splenocytes from HAQ/SAVI, AQ/SAVI partially resist to STING-activation-induced cell death ex vivo. (**A-B).** Flow cytometry of *HAQ/SAVI*, *AQ/SAVI*, *WT/WT* or *WT/HAQ* splenocytes treated with diABZI (100ng/ml) or DMXAA (20µg/ml) for 24hrs. 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. * p&lt;0.05. n.s: not significant. *Figure 3-figure supplement 2.* HAQ and *AQ* restore bone marrow monocytes in *SAVI (N153S)* mice. (**A-D).** Flow analysis of Ly6G+CD11B+ neutrophils and Ly6G-Ly6C+CD11B+ bone marrow monocytes from HAQ/SAVI, AQ/SAVI, WT/SAVI and their littermates. n=3~5 mice/group. 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.  ** p&lt;0.05, n.s: not significant. Figure 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*&lt;0.05, ***p*&lt;0.01. n.s.: not significant. **WBC**: white blood cells; **H**: hepatocytes; **K**: Kupper cells; **PV**: portal vein; **CV**: central vein. *Figure 4-figure supplement 1.* HAQ, *AQ* suppress lung myeloid cells infiltration in *SAVI(N153S)* mice. (A-C). Flow analysis of lung Ly6G+CD11B+ neutrophils and Ly6G-Ly6C+CD11B+ monocytes from *HAQ/SAVI, AQ/SAVI, WT/SAVI and their littermates WT/HAQ, WT/AQ mice*. n=3~5 mice/group. 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.  ** p&lt;0.05. **Figure 5.** ***AQ/SAVI(N153S)***** cells had similar TBK1-IRF3, NFkB 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 IkBa (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**). IFNb 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&lt;0.05, ***p*&lt;0.01, ****p*&lt;0.001. n.s.: not significant; n.d: not detected. **Figure 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 IFNg 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&lt;0.05, ***p*&lt;0.01, ****p*&lt;0.001. n.s.: not significant. ### Files and variables #### File: Figure\_5-\_source\_data\_1.xlsx **Description:** Source data for Figure 5 #### File: Figure\_6-\_source\_data\_1.xlsx **Description:**  Source data for Figure 6 #### File: Figure\_3-\_source\_data\_1.xlsx **Description:** Source data for Figure 3 #### File: Figure\_2-\_source\_data\_1.xlsx **Description:** Source data for Figure 2 #### File: Figure\_1\_-\_source\_data\_1.xlsx **Description:**  Source data for Figure 1 #### File: Figure\_2\_-source\_data\_3.pdf **Description:**  Source data for Figure 2I #### File: Figure\_2\_-source\_data\_2.pdf **Description:** Source data for Figure 2G #### File: Figure\_5\_-source\_data\_2.pdf **Description:** Source data for Figure 5 #### File: Figure\_4-\_source\_data\_1.xlsx **Description:** Source data for Figure 4