a defect in copi-mediated transport of sting causes immune ... · 1 title: a defect in copi...

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Title: A defect in COPI-mediated transport of STING causes immune 1 dysregulation in COPA syndrome 2

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Authors: Zimu Deng1†, Zhenlu Chong1†, Christopher S. Law1, Kojiro Mukai2, Frances O. Ho1, 4 Tereza Martinu3, Bradley J. Backes1, Walter L. Eckalbar1, Tomohiko Taguchi2, Anthony K. 5

Shum1,4,* 6

Affiliations: 7

8 1Department of Medicine, University of California San Francisco, San Francisco, CA. 9

2Laboratory of Organelle Pathophysiology, Department of Integrative Life Sciences, Graduate 10

School of Life Sciences, Tohoku University, Sendai, Japan 11

3Toronto Lung Transplant Program, University Health Network, University of Toronto, Toronto, 12

ON, Canada 13

4Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA. 14

*Corresponding author: [email protected] 15

† equal contribution 16

was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 20, 2020. . https://doi.org/10.1101/2020.05.20.106500doi: bioRxiv preprint

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Pathogenic COPA variants cause a Mendelian syndrome of immune dysregulation 17

with elevated type I interferon signaling1,2. COPA is a subunit of coat protein complex I 18

(COPI) that mediates Golgi to ER transport3. Missense mutations that disrupt the COPA 19

WD40 domain impair binding and sorting of proteins targeted for retrieval to the ER but 20

how this causes disease remains unknown1,4. Given the importance of COPA in Golgi-ER 21

transport, we speculated that type I interferon signaling in COPA syndrome involves 22

missorting of STING. Here we show that a defect in COPI transport due to mutant COPA 23

causes ligand-independent activation of STING. Furthermore, SURF4 is an adapter 24

molecule that facilitates COPA-mediated retrieval of STING at the Golgi. Activated STING 25

stimulates type I interferon driven inflammation in CopaE241K/+ mice that is rescued in 26

STING-deficient animals. Our results demonstrate that COPA maintains immune 27

homeostasis by regulating STING transport at the Golgi. In addtion, activated STING 28

contributes to immune dysregulation in COPA syndrome and may be a new molecular target 29

in treating the disease. 30

COPA syndrome is a genetic disorder of immune dysregulation caused by missense 31

mutations that disrupt the WD40 domain of COPA1. COPA is a subunit of coat protein complex I 32

(COPI) that mediates retrograde movement of proteins from the Golgi apparatus to the 33

endoplasmic reticulum (ER)3. Prior studies have shown that alterations to the COPA WD40 34

domain lead to impaired binding and sorting of proteins bearing a Carboxyl-terminal dilysine motif 35

as well as a defect in retrograde Golgi to ER transport1,4. To date, the molecular mechanisms of 36

COPA syndrome remain unknown including whether missorted proteins are critical for initiating 37

the disease. 38


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A clue to the pathogenesis of COPA syndrome recently arose with the observation that 39

type I interferon signaling appears to be highly dysregulated in the disease2. This led us to 40

investigate whether COPA syndrome shares features with any of the well-described Mendelian 41

interferonopathy disorders5. COPA syndrome manifests similarly to the type I interferonopathy 42

STING-associated vasculopathy with onset in infancy (SAVI). Both diseases present at early age 43

with interstitial lung disease, activation of type I interferon stimulated genes (ISG) and evidence 44

of capillaritis6,7. STING is an ER-localized transmembrane protein involved in innate immune 45

responses to cytosolic nucleic acids. After binding cyclic dinucleotides STING becomes activated 46

as it translocates to the ER-Golgi intermediate compartment (ERGIC) and Golgi. At the 47

ERGIC/Golgi, STING forms multimers and activates the kinase TBK1 which subsequently 48

phosphorylates the transcription factor IRF3 to induce expression of type I interferons and other 49

cytokines8. In SAVI, gain of function mutations cause STING to aberrantly exit the ER and traffic 50

to the Golgi and become activated9. Prior work has suggested that COPI may be involved in 51

STING transport at the Golgi, but this is not well established and the molecular interactions 52

between COPI and STING remain unknown8,10. Because COPA plays a critical role in mediating 53

Golgi to ER transport, we hypothesized that activation of type I interferon signaling in COPA 54

syndrome involves missorting of STING. 55

To examine this, we assessed lung fibroblasts from a COPA syndrome patient to determine 56

if there was evidence of STING activation. We measured mRNA transcript levels of several 57

interferon stimulated genes and found they were significantly elevated in comparison to healthy 58

control lung fibroblasts in the presence or absence of a STING agonist (Fig. 1a and Extended Data 59

Fig. 1a, b). Confocal microscopy of COPA syndrome fibroblasts revealed prominent co-60

localization of STING with the ERGIC (Fig. 1b). Western blots of cellular protein lysates showed 61


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an increase in STING multimerization (Fig. 1c) consistent with localization of STING at the 62

ERGIC/Golgi and also higher levels of phosphorylated TBK-1 (pTBK1) and phosphorylated 63

STING (pSTING) (Fig. 1d, Extended Data Fig. 1c), indicative of STING activation11. These data 64

suggest that the elevated type I ISGs observed in COPA syndrome patients may be caused by 65

spontaneous activation of STING. 66

To establish the specific role of mutant COPA in triggering STING pathway activation, we 67

transduced cells with retroviral vectors encoding EGFP-STING and then transfected them with 68

plasmids encoding wild-type or E241K mutant COPA. We performed confocal microscopy to 69

examine whether mutant COPA caused STING to localize on the ERGIC/Golgi, similar to what 70

we observed in patient fibroblasts. In cells expressing wild-type COPA, EGFP-STING was 71

normally distributed throughout the cytoplasm8 whereas in cells with E241K mutant COPA, 72

EGFP-STING co-localized with the Golgi marker GM130 (Fig. 2a). We next reconstituted 73

HEK293T cells (which lack endogenous STING) with retroviral vectors encoding EGFP-STING 74

and then transfected cells with wild-type or E241K mutant COPA. We found that even in the 75

absence of a STING agonist, cells demonstrated a significant increase in pTBK1 (Fig. 2b) and 76

higher levels of mRNA transcripts encoding IFNB1 and type I ISGs (Fig. 2c). Importantly, all of 77

these increases were abolished in HEK293T cells without EGFP-STING, indicating that mutant 78

COPA activates TBK1 and type I interferon signaling specifically through STING rather than other 79

innate immune pathways such as TRIF or MAVS (Fig 2b, c)12. Taken together, these data show 80

that mutant COPA causes ligand-independent activation of STING on the Golgi with upregulation 81

of type I interferon signaling. 82

We hypothesized that retention of STING on the Golgi might reflect a failure of STING to 83

be taken up into mutant COPA containing COPI complexes for transport to the ER. To evaluate 84


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this, we analyzed the protein-protein interaction between COPA and STING. We performed co-85

immunoprecipitation experiments and found that although we could pull-down STING with wild-86

type COPA the amount of STING that co-immunoprecipitated with E241K mutant COPA was 87

substantially reduced (Fig. 2d). We previously showed that disease-causative COPA mutations 88

cause a defect in binding between the COPA WD40 domain and C-terminal dilysine motif (e.g. 89

KKxx and KxKxx) of proteins targeted for retrieval to the ER1. Because STING lacks a dilysine-90

tag, we hypothesized that an adaptor protein mediates the interaction between STING and COPA. 91

A review of published studies uncovered seven STING interacting partners that contain a C-92

terminal dilysine motif (Extended Table 1)13-16. Among these, SURF4 was the only protein shown 93

to cycle between the ER and Golgi via COPI3,17 and function as a cargo receptor18. Thus, we 94

speculated that SURF4 was a likely candidate for mediating an interaction between COPA and 95

STING. We confirmed through co-immunoprecipitation assays that SURF4 associates with 96

STING and COPA (Fig. 2e and Extended Data Fig. 2a) and then mutated the C-terminal dilysine 97

motif of SURF4 by replacing the lysines at positions -3, -4 and -5 from the C-terminus with serines 98

(SURF4-SSS). The amount of SURF4-SSS pulled down with COPA was significantly less in 99

comparison to wild-type SURF4, demonstrating the importance of the dilysine motif to the 100

SURF4-COPA interaction (Fig. 2e). Consistent with this, we found that the association between 101

SURF4 and mutant COPA was markedly reduced in comparison to wild-type COPA, reflecting a 102

defect in binding between the mutant COPA WD40 domain and SURF4 di-lysine tag (Fig. 2e). 103

Finally, as further evidence that SURF4 functions as an adapter molecule for STING and COPA, 104

loss of SURF4 led to a reduction in the amount of STING that co-immunoprecipitated with wild-105

type COPA (Extended Data Fig. 2b) and also led to an increase in transcript levels of Ifnb1 and 106

type I ISGs (Extended Data Fig. 2 c). In aggregate, our data suggests that SURF4 functions as a 107


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cargo receptor for STING and that mutant COPA is unable to bind SURF4 and incorporate STING 108

into COPI vesicles. This results in retention of STING on the Golgi where it becomes 109

spontaneously activated and triggers type I interferon signaling. 110

We next turned to a mouse model of COPA syndrome to understand how mutant COPA-111

mediated STING activation causes immune dysregulation in vivo. We previously reported that 112

CopaE241K/+ mice which express one of the same disease causative mutations as patients 113

spontaneously develop activated cytokine-secreting T cells and T cell-mediated lung disease19. 114

Through bone marrow chimera and thymic transplant experiments, we showed that mutant COPA 115

within thymic epithelial cells perturbs thymocyte development and leads to a defect in immune 116

tolerance. Because STING is highly expressed in thymic tissue20, we wondered how missorting of 117

STING due to mutant COPA might contribute to the T cell phenotypes we observed in CopaE241K/+ 118

mice. 119

To evaluate this, we first confirmed that CopaE241K/+ mice demonstrate retention of 120

activated STING on the Golgi with elevated type I interferon signaling, similar to what we 121

observed in patient cells and our transfection assays (Fig. 3a-c). We next performed bulk RNA 122

sequencing to identify gene expression programs that were altered in thymic epithelial cells. Genes 123

involved in response to viruses were highly enriched in the set of genes differentially expressed 124

between CopaE241K/+ and wild-type mice in medullary thymic epithelial cells (mTEC) (Extended 125

Data Fig. 3). Among the differentially expressed genes, we found significant upregulation of Ifnb 126

and several type I ISGs, consistent with STING activation (Fig. 4a). Thymocytes migrating 127

through the thymic epithelium were impacted by higher Ifnb levels because they exhibited an 128

elevated type I interferon signature (Fig. 4b) and higher levels of Qa2 (Fig. 4c), a cell surface 129

marker expressed in response to IFN-b21. We examined thymocyte populations using a staining 130


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protocol that subsets increasingly mature single positive (SP) cells into semi-mature (SM), mature 131

1 (M1) and mature 2 (M2) populations (Fig. 4d). Prior studies found that type I interferons are 132

required during the transition of thymocytes from SM to M2 cells21. In CopaE241K/+ mice, we found 133

a significant increase in the proportion of M2 cells (Fig. 4d), suggesting that higher IFN-b levels 134

promoted an expansion of late stage thymocytes. To determine the role of STING in these changes, 135

we crossed CopaE241K/+ mice to STING-deficient Sting1gt/gt mice. We measured mRNA transcripts 136

of thymic epithelial cells for Ifnb1 and type I ISGs and found that all of the increases returned to 137

wild-type levels in CopaE241K/+xSting1gt/gt mice (Fig. 4e). In addition, loss of STING abrogated all 138

of the thymocyte phenotypes caused by mutant COPA including the elevated type I IFN signature, 139

increased Qa2 expression and higher proportion of M2 cells (Fig. 4c-e). Taken together, these data 140

indicate that activated STING in the thymus of CopaE241K/+ mice perturbs T cell development by 141

elevating type I interferons to promote late stage thymocyte maturation. 142

We next focused on peripheral lymphoid organs to examine whether mutant COPA-143

mediated STING-activation contributed to systemic inflammation in CopaE241K/+ mice. We 144

reasoned that if STING were to have a significant role in systemic disease, this would provide an 145

opportunity to test whether targeting STING activation dampens systemic inflammation in COPA 146

syndrome patients. Splenocytes from CopaE241K/+ mice had a significant increase in type I ISGs 147

that completely reverted to wild-type levels in CopaE241K/+xSting1gt/gt mice (Extended Data Fig. 148

4a). An analysis of peripheral T cell populations revealed that STING deficiency reversed the 149

significant increase in activated effector memory cells and cytokine-secreting T cells caused by 150

mutant COPA (Fig. 5a and Extended Data Fig. 4b-d). Finally, in strong support of STING being a 151

critical mediator of disease in COPA syndrome pathogenesis, we found that loss of STING rescued 152

embryonic lethality of homozygous CopaE241K/E241K mice (Extended Table 2). In aggregate, these 153


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data indicate that STING contributes to immune dysregulation in CopaE241K/+ mice and that 154

targeting STING in COPA syndrome may be an important therapy for patients. 155

Activation of STING requires palmitoylation at the Golgi22 and recent studies report that 156

inhibition of activation-induced palmitoylation of STING with small molecules prevents 157

multimerization of the protein and recruitment of downstream signaling partners23. We treated 158

splenocytes from CopaE241K/+ mice with C-176 mouse STING inhibitor and found that this was 159

highly effective at dampening activation of type I ISGs (Fig. 5b). We then took peripheral blood 160

mononuclear cells (PBMCs) from a COPA syndrome patient or healthy control subject and treated 161

them with the small molecule human STING inhibitor H-151. In parallel, we treated PBMCs with 162

a JAK-STAT inhibitor since drugs in this class have recently been reported as a clinical therapy 163

for COPA syndrome24. Although both the JAK-STAT inhibitor and STING inhibitor reduced type 164

I ISGs, only the STING inhibitor was able to cause a decrease in the levels of the upstream cytokine 165

IFN-b (Fig. 5c, d). Our results suggest that targeting STING activation with small molecule 166

inhibitors might be an effective treatment for COPA syndrome patients with increased efficacy 167

over current therapies. 168

This work establishes COPA as a critical regulator of STING transport that maintains 169

immune homeostasis by mediating retrieval of STING from the Golgi. Defects in COPA function 170

cause STING to multimerize and become spontaneously activated at the Golgi even in the absence 171

of STING ligand, suggesting that at steady state low levels of STING continuously cycle between 172

the ER and Golgi. In support of our findings, Mukai et. al used cultured cells to show that 173

retrograde transport by COPI is essential to maintaining STING in its dormant state independent 174

of cGAMP synthase (cGAS)28. Although we used a candidate approach to identify SURF4 as a 175

putative cargo receptor that engages STING for incorporation into COPI vesicles by COPA, Mukai 176


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et. al directly tested 18 STING-binding proteins containing C-terminal dilysine motifs and found 177

that knockdown of SURF4 was the only one that caused STING to localize on the Golgi28. Taken 178

together, our data demonstrates an important role for SURF4 in mediating the retrieval of STING 179

from the Golgi by COPI and provides new insight into the steady state dynamics of STING 180

transport in resting cells. 181

Missense mutations in COPA that lie in the WD40 domain impair retrograde transport of 182

a broad range of COPI cargo proteins containing C-terminal dilysine motifs1. Despite this, our data 183

suggests that impaired trafficking of STING in particular is key to the pathogenesis of COPA 184

syndrome, since not only does loss of STING reverse many of the immunological derangements 185

in our mouse model, it also strikingly rescues embryonic lethality of homozygous mutant mice. 186

Further study should be undertaken to determine whether other COPA cargo contribute to COPA 187

syndrome pathogenesis independent of STING. 188

The ubiquitous expression of COPA has made it difficult to identify the cell types that are 189

responsible for causing disease particularly since COPA is not enriched in any specific immune or 190

lung cell. The tissue specificity of STING may provide additional insight to understanding COPA 191

syndrome and the organs most affected. One unexpected outcome of our work was the finding that 192

STING has a functional role in thymic stromal tissue. Although thymic epithelial cells are known 193

to be a significant source of type I interferons21,25, the mechanisms regulating interferon secretion 194

in the thymus remain largely unexplored. Interestingly, STING also modulates autophagy8 which 195

in thymic epithelial cells is essential for processing self-antigen peptides for presentation to 196

thymocytes26. Additional research might address whether activated STING alters autophagic 197

function in thymic epithelial cells and if so, whether this impacts T cell selection. 198


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Our work indicates that COPA syndrome belongs to a category of diseases defined by 199

STING activation5. Going forward, clinicians may want to compare and contrast clinical features 200

and treatment approaches in COPA syndrome and SAVI to establish optimal care of patients. 201

Understanding the mechanisms by which STING causes interstitial lung disease has the potential 202

to substantially improve outcomes in both disorders6,7. For those with COPA syndrome, the 203

dramatic reversal of immune dysregulation we observed in CopaE241K/+xSting1gt/gt mice provides 204

some hope that small molecule STING inhibition can be an effective molecularly targeted 205

approach for treating this highly morbid disease. 206


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10. Ablasser, A. & Hur, S. Regulation of cGAS- and RLR-mediated immunity to nucleic acids. 226

Nature Immunology 21, 17–29 (2020). 227

11. Srikanth, S. et al. The Ca2+ sensor STIM1 regulates the type I interferon response by retaining 228

the signaling adaptor STING at the endoplasmic reticulum. Nature Immunology 20, 152–162 229

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12. Liu, S. et al. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF 231

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and retroviral infection by an integrative approach. Nature Immunology 14, 179–185 (2012). 234

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Regulator of SeV-Induced Innate Immunity. Proteomics 18, 1700403–13 (2018). 238

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to maintain the architecture of ERGIC and Golgi. Mol. Biol. Cell 19, 1976–1990 (2008). 244


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18. Emmer, B. T. et al. The cargo receptor SURF4 promotes the efficient cellular secretion of 245

PCSK9. Elife 7, 154 (2018). 246

19. Deng, Z. et al. A Defect in Thymic Tolerance Causes T Cell–Mediated Autoimmunity in a 247

Murine Model of COPA Syndrome. The Journal of Immunology 204, ji2000028–2373 (2020). 248

20. Manils, J. et al. Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation 249

of DNA in lingual cornifying keratinocytes without activating inflammatory responses. Sci Rep 7, 250

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thymus involve NF-[kappa]B and tonic type I interferon signaling. Nature Immunology 17, 565–253

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22. Mukai, K. et al. Activation of STING requires palmitoylation at the Golgi. Nat Commun 7, 255

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year-old girl. Eur J Rheumatol 1–4 (2019). doi:10.5152/eurjrheum.2019.18177 260

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26. Nedjic, J., Aichinger, M., Emmerich, J., Mizushima, N. & Klein, L. Autophagy in thymic 264

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28. Mukai, K. et al. Homeostatic regulation of STING by Golgi-to-ER membrane traffic. In 268

preparation. 269

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Figure 1 271

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Fig. 1. COPA syndrome patient fibroblasts demonstrate spontaneous STING activation. 273

(A) Real-time PCR was performed for ISG expression in primary lung fibroblasts from a healthy 274 control and COPA syndrome subject. (B) Primary lung fibroblasts from a healthy control and 275 COPA syndrome subject were reconstituted with wild-type EFGP-STING retrovirus. The 276 reconstituted cells were stained with indicated antibody and localization of STING observed using 277 a confocal microscope. ERGIC 53 is an ERGIC marker. (C) Immunoblots of endogenous STING 278 in primary lung fibroblasts from 2 healthy controls and a COPA syndrome subject under non-279 reducing SDS-PAGE conditions with and without cGAMP (0.5µg/ml) stimulation. (D) 280 Immunoblots of indicated antibodies in primary lung fibroblasts with cGAMP (2µg/ml) 281 stimulation for indicated time (n=2 per group). Data in (A) from two independent experiments 282 represent means ± SD. *p<0.05, **p<0.01, (two-tailed Mann-Whitney U test). Scale bar, 20µm in 283 (B). 284

285 286


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Figure 2 287

Fig. 2. Activated STING is retained on the Golgi after failed retrieval of SURF4 by mutant 288 COPA. (A) HeLa cells reconstituted with wild-type EGFP-STING retrovirus and transfected with 289 wild-type COPA or mutant COPA (E241K) for 24 hours. Cells were stained with indicated 290


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antibodies and localization of STING observed using a confocal microscope. GM130 is a Golgi 291 marker. (B) HEK293T cells with and without EGFP-STING were transfected with wild-type or 292 mutant COPA (E241K) for 24 hours and then immunoblot performed with indicated antibodies. 293 (C) HEK293T cells with and without EGFP-STING were transfected with wild-type or mutant 294 COPA (E241K) for 24 hours and then real-time PCR performed for ISG expression. (D) FLAG 295 immunoprecipitates (IP) from lysates of HEK293 cells overexpressing FLAG-tagged COPA 296 (WT/E241K) were immunoblotted for indicated antibodies. (E) FLAG immunoprecipitates from 297 lysates of HEK293T cells overexpressing FLAG-tagged COPA (WT/E241K) and HA-tagged 298 SURF4 (WT/mutant) immunoblotted for indicated antibodies. Data in (C) from 2 independent 299 experiments represent means ± SD. **p<0.01 (two-tailed Mann-Whitney U test). Scale bar, 20µm 300 in (A). 301 302


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Figure 3 303

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Fig. 3 CopaE241K/+ mice exhibit STING activation with elevated type I interferon signaling. 305 (A) Real-time PCR was performed for ISG expression in immortalized MEF cells derived from 3 306 WT and 3 CopaE241K/+ mice. (B) Immortalized MEF cells from WT, CopaE241K/+ and 307 CopaE241K/E241K mice were reconstituted with wild-type EGFP-STING retrovirus. The 308 reconstituted cells were stained with the indicated antibodies and localization of STING 309 observed using a confocal microscope. GM130 is a Golgi marker. (C) Immunoblot was 310 performed with the indicated antibodies in splenocytes from WT or CopaE241K/+ mice (n=3 for 311 each genotype). Data in (A, C) were means ± SD. *p<0.05, **p<0.01(unpaired two-tailed 312 Student’s t tests). Scale bar, 20µm in (B). 313 314

C

A

B


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Figure 4 315 316

C

A B

D

E


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Fig. 4 Activated STING perturbs thymocyte development by increasing type I interferons in 317 the thymus. (A) Medullary thymic epithelial cells sorted from wild-type and CopaE241K mice were 318 used for RNA-seq analysis (n=3 each genotype). Volcano plot of the differentially affected genes 319 (FDR<0.1, red; FDR<0.1, ISG from viral response GO categories, purple). (B) Real-time PCR 320 performed for Ifit1, Rsad2 and Isg15 expression in SP thymocytes (top:CD4SP; bottom:CD8SP). 321 (n=4 mice each genotype, 2 independent experiments). (C) left: Percentages of Qa2high population 322 among SP thymocytes (top: CD4SP; bottom: CD8SP). right: Representative flow analysis of Qa2 323 expression on SP thymocytes (top: CD4SP; bottom: CD8SP). (n=5 each genotype, 3 independent 324 experiments). (D) top: CD69 versus MHCI expression on SP thymocytes (top:CD4SP; 325 bottom:CD8SP). bottom: Percentages of SM and M2 population among SP thymocytes (top: 326 CD4SP; bottom: CD8SP). (WT: n=5 each genotype, 3 independent experiments). (E) Real-time 327 PCR performed for Ifnb1, Ifit1 and Isg15 expression in thymic epithelial cells (CD45-328 ,Epcam+,Ly51-,MHC-IIhighmTEC) (n≥3 each genotype, 2 independent experiments). Data in (B-329 F) represent means ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant 330 (unpaired, parametric, two-tailed student’s t-test). 331 332


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Figure 5333

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Fig. 5 Loss of STING function dampens inflammation caused by mutant COPA (A) Top left: 335 intracellular levels of IFNγ and IL17A in splenic CD4+ T cells after PMA/Ionomycin stimulation. 336 Top right: percentages of IFNγ producing CD4+ T cells. (n≥3 each genotype, >3 independent 337 experiments). Bottom left: intracellular IFNγ production in splenic CD8+ T cells after 338 PMA/Ionomycin stimulation. Bottom right: percentages of IFNγ producing CD8+ T cells. (n≥3 339 each genotype, >3 independent experiments). (B) Real-time PCR for ISG expression in 340 splenocytes harvested from 4 WT mice and 4 CopaE241K/+mice treated with or without STING 341 inhibitor C-176 (10µM) for 24 hours. (C) Real-time PCR performed in duplicate for ISG 342 expression in PBMCs from a healthy control and COPA syndrome subject treated with or without 343

C

D

B

A


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STING inhibitor H-151 (10µM) or (D) JAK inhibitor Tofacitinib (2µM) for 24 hours. Data 344 represent means ± SD. **p<0.01, ***p<0.001, ns, not significant. (unpaired, parametric, two-345 tailed Student’s t tests). 346 347


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Methods: 348

Reagents 349

2′3′-cGAMP and CP-690550 (Tofacitinib) were purchased from InvivoGen. STING 350

inhibitors N-(4-iodophenyl)-5-nitrofuran-2-carboxamide (C176) and 1-(4-ethylphenyl)-3-(1H-351

indol-3-yl)urea (H151) were synthesized by SYNthesis med chem at or greater than 99% purity 352

by HPLC. 353

Plasmids 354

Human STING and SURF4 were subcloned from HEK293T cDNA into pCMV6-AC 355

(Origene) with a FLAG tag at the C-terminus or a HA tag at the N-terminus, respectively. Plasmids 356

expressing FLAG tagged wild-type and mutant E241K human COPA were previously generated1. 357

Retroviral plasmids expressing EGFP tagged human and mouse STING were kindly provided by 358

Dr. Tomohiko Taguchi (Tohoku University). 359

Study subjects 360

Subjects were selected on the basis of COPA syndrome diagnosis, with their written 361

informed consent, were studied via protocols approved by the Research Ethics Board of Toronto 362

General Hospital and the Institutional Review Boards for the protection of human subjects of 363

Cleveland Clinic or the University of California San Francisco. 364

Isolation of human lung fibroblasts 365

Control lung tissue was harvested from anonymous brain-dead donors from the Northern 366

California Transplant Donor Network. Screening criteria for selection of healthy lung for specimen 367

collection were previously described2. Mutant lung explants were from two COPA syndrome 368

patients receiving lung transplants. Fibroblasts were isolated as described3. In brief, tissue was 369

minced with scissors into 5 mm pieces and digested three times with 0.25% trypsin (GE Healthcare 370


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Life Sciences) for 10 minutes at 37°C. The resulting cell and tissue suspension was collected, 371

neutralized with complete media (45% Ham’s F12, 45% DMEM, 10% FBS), pelleted, plated onto 372

FBS coated 60 mm dishes and cultured in 5% CO2 at 37°C. Fibroblasts were ready to passage and 373

use one week following isolation. 374

Isolation of mouse embryonic fibroblasts 375

MEFs were isolated as described4. In brief, day 13.5 embryos were washed with PBS, 376

minced with scissors into 1-2 mm pieces and digested three times with 0.25% trypsin for 10 377

minutes at 37°C. The cell suspension was neutralized with complete media (DMEM, 10% FBS), 378

pelleted, resuspended in complete media, and cultured in 5% CO2 at 37°C. 379

To create immortal MEFs, Phoenix packaging cells (kindly gifted by Dr. Mark Anderson, 380

UCSF) were transfected with pBABE-neo largeTcDNA plasmid (a gift from Bob Weinberg; 381

Addgene plasmid # 1780; http://n2t.net/addgene:1780; RRID:Addgene_1780) 5, and viral 382

supernatant was collected two days later. Early passage MEFs were incubated in viral supernatant 383

for two days and then transformed cells were selected with G418 (Teknova). 384

siRNA knockdown 385

Predesigned siRNA oligomers (ON-TARGETplus SMARTPool) for SURF4 and 386

transfection control (siGENOME RISC-Free) were obtained from Dharmacon and resuspended in 387

RNase-free water at 10 µM. siRNAs were transfected in Opti-Mem media (Life Technologies) for 388

48 h with Lipofectamine RNAiMAX (Life Technologies) according to manufactuer’s protocol. 389

Immunoblotting and antibodies 390

Cells were lysed in Cold Spring Harbor NP40 lysis buffer (150 mM NaCl, 50 mM Tris pH 391

8.0, 1.0% Nonidet-P40) supplemented with protease and phosphatase inhibitors (PMSF, NaF, 392

Na3VO4, Roche PhosSTOP) and then centrifuged at 12,000g for 15 mins at 4°C to get cellular 393


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lysate. Equal amounts of protein were loaded and size separated on an SDS–PAGE gel and wet 394

transfered onto PVDF membrane. The membrane was blocked in TBS-T buffer with 5% milk for 395

1 h at room temperature, followed by overnight incubation with primary antibodies diluted in TBS-396

T with 5% BSA. Membrane was washed 3 times with TBS-T buffer for 10 mins, incubated at room 397

temperature with HRP-conjugated IgG secondary antibody (Jackson Immunoresearch), washed 3 398

times with TBS-T for 10 mins followed once with TBS buffer for 10 mins, and then developed 399

with SuperSignal West Femto Chemiluminescent Substrate (Life Technologies). 400

Rabbit antibodies against TBK1, p-TBK1 (Ser172), STING, p-STING (Ser365), p-STING 401

(Ser366), FLAG, HA and GFP were from Cell Signaling Technology. Rabbit antibody against 402

SURF4 was from Novus. Mouse antibodies against GAPDH and β-Tubulin were from Santa Cruz 403

Biotechnology. Mouse antibody against GM130 was from BD Biosciences. 404

Co-immunoprecipitation 405

HEK293 or HEK293T cells transfected with indicated plasmids were collected, washed 406

once with PBS, lysed in NP40 lysis buffer supplemented with protease and phosphatase inhibitors 407

and centrifuged at 12,000g for 15 min at 4°C. The supernatant was mixed with FLAG-M2 beads 408

(Sigma) and incubated overnight at 4°C. Ten percent of lysate was saved as input. The following 409

day, the beads were washed 3 times with IP washing buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 410

150 mM NaCl and 1% Triton X-100) for 10 mins. 2× SDS loading buffer (100 mM Tris pH 6.8, 411

4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.1% bromophenol blue) was added into the IP 412

complex and boiled at 95 °C for 5 min. Samples were analyzed by immunoblot as described above. 413

Confocal microscopy 414

Cells were seeded onto glass coverslips and treated as indicated. Cells then were fixed with 415

4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Slides 416


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were incubated with primary antibody overnight at 4°C, incubated with fluorescent-conjugated 417

secondary antibody for 1 h at room temperature and followed with DAPI incubation for 10 mins 418

at room temperature. Slides were then mounted with FluorSave Reagent (Millipore) and kept at 419

4°C in the dark. Images were captured with a Leica TCS SPE microscope. 420

Mouse antibody against ERGIC-53 was from Santa Cruz Biotechnology. 421

Mice strains 422

CopaE241K knock in mice were generated in our lab6. C57BL/6J-Sting1gt/J (Stinggt/gt) mice 423

were purchased from the Jackson Laboratory. All mice were maintained in the specific pathogen 424

free (SPF) facility at UCSF, and all protocols were approved by UCSF’s Institutional Animal Care 425

and Use Committee (IACUC). 426

Flow cytometry and antibodies 427

Single cell suspension of thymocytes and splenocytes were prepared by mechanically 428

disrupting the thymus and spleen. Cells were filtered through 40 µm filters (Genesee Scientific) 429

into 15 ml conical tubes and maintained in RPMI 1640 containing 5% FBS on ice. Splenocytes 430

were further subjected to red blood cells lysis (Biolegend). For evaluation of surface receptors, 431

cells were blocked with 10 µg/ml anti-CD16/32 for 15 minutes at room temperature and then 432

stained with indicated antibodies in FACS buffer (PBS, 2% FBS) on ice for 40 minutes. 433

For intracellular cytokine detection, freshly isolated splenocytes were stimulated by PMA 434

(Sigma) and ionomycin (Sigma) in the presence of brefeldin A (Biolegend) for four hours. Cells 435

were collected and stained with Ghost Dye Violet 450 (Tonbo Biosciences) followed by surface 436

staining. Cells were then washed, fixed at room temperature for 15 minutes (PBS, 1% neutral 437

buffered formalin), permeabilized and stained with antibodies against cytokines on ice (PBS, 2% 438


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FBS, 0.2% saponin). All samples were acquired on LSRFortessa or FACSVerse (BD Bioscience) 439

and analyzed using FlowJo software V10. 440

Antibodies to the following were purchased from Biolegend: CD8 (53-6.7), IFNγ 441

(XMG1.2), Epcam (G8.8); from eBioscience: IL17A (eBio17B7), IL13 (eBio13A), MHCII (I-A) 442

(NIMR-4), Qa2 (69H1-9-9); from Tonbo Biosciences: CD4 (GK1.5), MHCII (I-A/I-E) 443

(M5/114.15.2); The CD16/CD32 (2.4G2) antibody was obtained from UCSF’s Monoclonal 444

Antibody Core. 445

Thymic epithelial cell isolation 446

Thymic epithelial cells were isolated as described7. Briefly, thymi from 4-week-old mice 447

were minced with razor blades into 1-2 mm3 pieces. The tissue fragments were collected and 448

enzymatically digested three times (DMEM, 2% FBS, 100 µg/ml DNase I and 100 µg/ml 449

Liberase™). The single-cell suspensions from each digestion were pooled into 20 ml of MACS 450

buffer (PBS, 0.5% BSA, 2 mM EDTA) on ice. Total cells were washed and centrifugated using a 451

three-layer Percoll gradient with specific gravities of 1.115, 1.065 and 1.0. Thymic stromal cells 452

were enriched and collected from the Percoll-light fraction, between the 1.065 and 1.0 layers. 453

Thymic stromal cells were washed, stained and sorted to isolate MHC-IIhighCD80high mTEC 454

(mTEChigh) by FACS. 455

RNA isolation and quantitative real-time PCR analysis 456

RNA was isolated with the EZNA Total RNA kit (Omega Bio-tek) was reverse transcribed 457

to cDNA with SuperScript III Reverse Transcriptase and oligo d(T)16 primers (Invitrogen). 458

Quantitative real-time PCR was performed on Bio-Rad CFX thermal cyclers with TaqMan Gene 459

Expression assays from Life Technologies (GAPDH, Hs02786624_g1; IFNB1, Hs01077958_s1; 460

IFI6, Hs00242571_m1; IFI27, Hs01086373_g1; IFI44L, Hs00915292_m1; ISG15, 461


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Hs01921425_s1; IFIT1, Hs03027069_s1; IFITM1, Hs00705137_s1; RSAD2, Hs00369813_m1; 462

Gapdh, Mm99999915_g1; Ifnb1, Mm00439552_s1; Ifit1, Mm00515153_m1; Ifi44l, 463

Mm00518988_m1; Isg15, Mm01705338_s1; Rsad2, Mm00491265_m1; S1pr1: Mm00514644). 464

RNA-Seq and data analysis: 465

RNA-sequencing libraries by first using the Nugen Ovation method (Tecan 7102-A01) to 466

create cDNA from the isolated RNA, the sequencing library was then created from cDNA using 467

the Illumina Nextera XT method (Illumina FC-131-1096). All libraries were combined and 468

sequenced on Illumina HiSeq4000 lanes, yielding approximately 300 million single end, 50bp 469

reads. Sequencing reads were then aligned to the mouse reference genome and the ensemble 470

annotation build (GRCm38.78) using STAR8 (v2.4.2a). Read counts per gene were used as input 471

to DESeq29 (v1.26.0) to test for differential gene expression between conditions using a Wald test. 472

Genes passing a multiple testing correct p-value of 0.1 (FDR method) were considered significant. 473

Pathway analysis was carried out using DAVID10 and the R/Bioconductor package 474

RDAVIDWebService11 (v1.24.0). 475

Statistical analysis 476

All statistical analysis was performed using Prism 7 (GraphPad Software) or R 4.0.0 (R 477

Foundation for Statistical Computing). Where indicated, two-tailed Mann-Whitney U-test and 478

unpaired, parametric, two-tailed student’s t-test were used to evaluate the statistical significance 479

between two groups, and p <0.05 was considered statistically significant. 480

Methods References: 481

1. Watkin, L. B. et al. COPA mutations impair ER-Golgi transport and cause hereditary 482

autoimmune-mediated lung disease and arthritis. Nature genetics 47, 654–660 (2015). 483


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2. Lee, J. W., Fang, X., Gupta, N., Serikov, V. & Matthay, M. A. Allogeneic human 484

mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in 485

the ex vivo perfused human lung. Proceedings of the national academy of Sciences 106, 486

16357–16362 (2009). 487

3. Ramos, C. et al. Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in 488

growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir 489

Cell Mol Biol 24, 591–598 (2001). 490

4. Durkin, M. E., Qian, X., Popescu, N. C. & Lowy, D. R. Isolation of Mouse Embryo 491

Fibroblasts. Bio Protoc 3, (2013). 492

5. Hahn, W. C. et al. Enumeration of the simian virus 40 early region elements necessary for 493

human cell transformation. Mol. Cell. Biol. 22, 2111–2123 (2002). 494

6. Deng, Z. et al. A Defect in Thymic Tolerance Causes T Cell–Mediated Autoimmunity in a 495

Murine Model of COPA Syndrome. The Journal of Immunology 204, ji2000028–2373 496

(2020). 497

7. Miller, C. N. et al. Thymic tuft cells promote an IL-4-enriched medulla and shape 498

thymocyte development. Nature 559, 627–631 (2018). 499

8. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 500

(2013). 501

9. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion 502

for RNA-seq data with DESeq2. Genome Biol. 15, 550–21 (2014). 503

10. Huang, D. W. et al. The DAVID Gene Functional Classification Tool: a novel biological 504

module-centric algorithm to functionally analyze large gene lists. Genome Biol. 8, R183–505

16 (2007). 506


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11. Fresno, C. & Fernández, E. A. RDAVIDWebService: a versatile R interface to DAVID. 507

Bioinformatics 29, 2810–2811 (2013). 508

Acknowledgements: Suzy A. A. Comhair and Serpil C. Erzurum from Cleveland Clinic 509

Foundation for help providing lung explants. Paul Wolters, Tien Peng, Chaoqun Wang and N. 510

Soledad Reyes de Mochel at UCSF for help with providing lung explants. David J. Erle for 511

discussions and assistance with RNA-sequencing. Mark S. Anderson for discussions and review. 512

Funding: UCSF Program for Breakthrough Biomedical Research, funded in part by the Sandler 513

Foundation (AKS), NIH/NIAID R01AI137249 (AKS), NIH/NHLBI R01HL122533 (AKS), 514

NIH/NHLBI R00HL135403 (WLE), NIH/NHLBI PO1HL103453 (SAAC, SCE). Author 515

contributions: ZD and ZC designed and performed experiments, analyzed data. KM and TT 516

provided technical advice. CSL performed experiments and analyzed data. FOH performed 517

experiments. BJB assisted with small molecule experimental design. WLE analyzed RNA-518

sequencing data. TM provided lung explant tissue. AKS directed the study and wrote the 519

manuscript. Competing interest declaration: Authors declare no competing interests. Data and 520

materials availability: All data that support the findings of this study are available from the 521

corresponding author upon reasonable request. RNA-sequencing data will be uploaded into a 522

public respository and accession codes and unique identifiers will be provided. 523

524


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Extended Data: 525 526 Extended Data Figure 1 527 528

529 530 Extended Data Fig. 1 531

COPA syndrome patient fibroblasts demonstrate STING activation. (A) Real-time PCR was 532 performed for IFNB1 expression in primary lung fibroblasts from a healthy control and COPA 533 syndrome subject treated with cGAMP (2µg/ml) for indicated time. Data pooled from 2 534 independent experiments are means ± SD, two-tailed Mann-Whitney U test. (B) Real-time PCR 535 for ISG expression in primary lung fibroblasts from a healthy control and COPA syndrome subject 536 treated with cGAMP (2µg/ml) for indicated time. Data are means ± SD, unpaired two-tailed 537 Student’s t test. (C) Immunoblots of primary lung fibroblasts for indicated antibodies. **p<0.01, 538 ***p<0.001, ****p<0.0001. 539

540


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Extended Data Figure 2 541 542

543 544

Extended Data Fig. 2 545

SURF4 functions as a cargo receptor that mediates retrieval of STING by COPA. (A) FLAG 546 immunoprecipitates (IP) from lysates of HEK293T cells overexpressing FLAG-tagged STING and 547 HA-tagged SURF4 were immunoblotted for indicated antibodies. (B) HEK293 cells were 548 transfected with FLAG-tagged COPA and si-SURF4 for 48 hours. FLAG immunoprecipitates (IP) 549 from lysates of HEK293 cells were immunoblotted for indicated antibodies. (C) Real-time PCR 550 for ISG expression in immortalized MEF cells with EGFP-STING after si-SURF4 for 48 hours. 551 Data represent means ± SD. *p<0.05, **p<0.01 (unpaired two-tailed Student’s t tests). siNeg = 552 non-targeting siRNA control. 553 554


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Extended Data Figure 3 555 556 557

558 559 Extended Data Fig. 3 560

DAVID Gene Ontology analysis of bulk RNA sequencing in thymic epithelial cells. DAVID 561 Gene Ontology analysis comparing differentially expressed genes in MHC-IIhighCD80high mTECs 562 from WT and COPAE241K/+ mice. Top GO clusters with the key word are shown. (n=3 of each 563 genotype). 564 565


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Extended Data Figure 4 566

567 568

A

B

C

D


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Extended Data Fig. 4 569

Activated STING in CopaE241K/+ mice results in type I interferon driven inflammation. 570 (A) Real-time PCR performed for ISG expression in splenocytes from WT (n=5), CopaE241K/+ 571 (n=5), Stinggt/gt (n=5) and CopaE241K/+xStinggt/gt (n=6) mice. (B) left: Representative flow plots 572 showing expression of CD62L versus CD44 on T cells (top: CD4+ T cells; bottom: CD8+ T cells). 573 right: Percentages of naive and effector memory T cells (left: CD4+ T cells; right: CD8+ T cells). 574 (WT: n=6; CopaE241K/+: n=4; Stinggt/gt: n=5; CopaE241K/+xStinggt/gt: n = 5). (C) Left: intracellular 575 levels of IL13 in splenic CD4+ T cells after PMA/Ionomycin stimulation. right: percentages of 576 IL13 producing CD4+ T cells. (WT: n=6; CopaE241K/+: n=4; Stinggt/gt: n=5; CopaE241K/+xStinggt/gt: 577 n=5, > 3 independent experiments). (D) Left: intracellular TNFα production in splenic CD8+ T 578 cells after PMA/Ionomycin stimulation. right: percentages of TNFα producing CD8+ T cells. (WT: 579 n=4; CopaE241K/+: n=3; Stinggt/gt: n=5; CopaE241K/+xStinggt/gt: n=5, >3 independent experiments). 580 Data in (A-D) represent means ± SD. *p<0.05, **p<0.01, ****p<0.0001 (unpaired two-tailed 581 Student’s t tests). 582 583


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Extended Table 1 584 585

Protein C terminus Subcellular location Function

CCDC47 KQIKVKAM ER Calcium regulation protein DDOST MKEKEKSD ER Glycosyltransferase enzyme

HM13 KGLEKKEK ER, Plasma membrane ER-resident intramembrane protease

MATR3 ERRQKKET Nucleus RNA binding protein SAC1 LVQKEKID ER, Golgi Phosphoinositide phosphatase

STT3B KKISKKTV ER Catalytic subunit of oligosaccharyl transferase

SURF4 MDEKKKEW ER, ERGIC, Golgi Cargo receptor 586 Extended Table 1: 587 588 STING interacting partners containing a C-terminal dilysine motif. Seven STING interacting 589 partners with C-terminal dilysine motifs were identified in published studies describing STING 590 affinity purification-mass spectrometry (AP-MS) data13-16,27. Shown for each protein are its C-591 terminus sequence with dilysine motif highlighted, subcellular location and function. 592 593


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Extended Table 2 Loss of STING rescues embryonic lethality of Copa E241K/E241K mice 594 595 596 597

Stingwt/wt Stinggt/gt

Neonates from 5 litters Neonates from 5 litters

Copa +/+ 7 (24%) 11(22%)

Copa E241K/+ 22 (76%) 22(44%)

Copa E241K/E241K 0 (0%) 17 (34%)

598

599

600


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