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Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity

Nature Immunology volume 20, pages 1299–1310 (2019)Cite this article

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Abstract

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

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Fig. 1: Denisovan, modern human and mouse alleles substituting non-catalytic residues of the A20 OTU domain.
Fig. 2: The I325N OTU variant confers heightened immunity in mice.
Fig. 3: Subclinical inflammatory and metabolic consequences of the I325N allele.
Fig. 4: Alleles with OTU domain variants decrease A20 phosphorylation and NF-κB inhibition.
Fig. 5: Immunoblot and RT–PCR analysis of peripheral blood mononuclear cells from healthy individuals with the Denisovan T108A;I207L TNFAIP3 haplotype.
Fig. 6: C243Y causes spontaneous inflammation and early lethality in homozygous mice.
Fig. 7: Introduction of human T108A;I207L and C243Y alleles in mice tunes immunity.
Fig. 8: Shift to detrimental effects in response to environmental stressors and increased susceptibility to autoimmunity.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. PDB accession codes are 5V3P, 5V3B and 5DQ6.

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Acknowledgements

This manuscript is dedicated to the memory of Susan Watson, who led the isolation of the I325N mouse. We thank the National Computational Infrastructure, Xenon systems, Nvidia and the Multi-modal Australian ScienceS Imaging and Visualization Environment (MASSIVE; http://www.massive.org.au/) for computing resources. We thank O. Venn, I. Mathieson, B. Llamas, Y. Souilmi, R. Tobler and A. Cooper for stimulating discussions. C.N.J. thanks J. Cyster for mentorship. We thank K. Newton and V. Dixit for reagents (Genentech). We thank M. Flodström-Tullberg (Karolinska Institutet) for kindly providing coxsackievirus B4 strain E2. This study makes use of data generated by the Telethon Kids Institute. A full list of the investigators who contributed to the generation of the data is available from http://bioinformatics.childhealthresearch.org.au/AGHS. We thank CIRCA for collecting and coordinating human samples (http://www.garvan.org.au/research/collaborative-programs/circa/about-circa). Funding for the project was provided by the National Health and Medical Research Council of Australia (NHMRC) under award 634301. N.W.Z. was supported by an Australian postgraduate award and is supported by the International Pancreas and Islet Transplant Association Derek Gray Scholarship. A.M.B. was an NHMRC Senior Research Fellow (1022688). S.G.T. was a Principal Research Fellow (1042925) of the NHMRC. C.C.G. was supported by the Bill and Patricia Ritchie Chair and by an NHMRC Senior Principal Research Fellowship. S.T.G. was supported by an NHMRC Senior Research Fellowship. The research was supported by grants to C.C.G. from the NIH (AI52127, AI054523, AI100627) and NHMRC (1016953, 585490, 1081858) and to S.T.G. from the NIH (DK076169) and NHMRC (1130222, 1140691). CIRCA was supported by the Jeffrey Modell Foundation, the John Cook Brown Foundation and the Sydney Children’s Hospital Network.

Author information

Author notes

  1. Stephen R. Daley

    Present address: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

  2. Geeta Chaudhri

    Present address: Research School of Population Health, The Australian National University, Canberra, Australian Capital Territory, Australia

  3. Gunasegaran Karupiah

    Present address: School of Medicine, The University of Tasmania, Hobart, Tasmania, Australia

  4. Craig N. Jenne

    Present address: Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada

  5. Matthew H. Spitzer

    Present address: Departments of Otolaryngology and Microbiology & Immunology and the Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA

  6. Frank Schmitz

    Present address: Celgene, Seattle, WA, USA

  7. These authors contributed equally: Nathan W. Zammit, Owen M. Siggs, Christopher C. Goodnow, Shane T. Grey.

  8. Deceased: Susan R. Watson.

Authors and Affiliations

  1. Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia

    Nathan W. Zammit, Owen M. Siggs, David B. Langley, Stacey N. Walters, Claudia Loetsch, Joanna Warren, Jin Yan Yap, Daniele Cultrone, Amanda Russell, Elisabeth K. Malle, Jeanette E. Villanueva, Mark J. Cowley, Velimir Gayevskiy, Marcel E. Dinger, Robert Brink, David Zahra, Stuart G. Tangye, Cecile King, Daniel Christ, Christopher C. Goodnow & Shane T. Grey

  2. Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia

    Nathan W. Zammit, David B. Langley, Stacey N. Walters, Joanna Warren, Daniele Cultrone, Mark J. Cowley, Robert Brink, David Zahra, Stuart G. Tangye, Cecile King, Daniel Christ, Christopher C. Goodnow & Shane T. Grey

  3. Department of Ophthalmology, Flinders University, Bedford Park, South Australia, Australia

    Owen M. Siggs

  4. Department of Immunology and Infectious Diseases, Sydney Children’s Hospital, Randwick, New South Wales, Australia

    Paul E. Gray & John B. Ziegler

  5. School of Women’s and Children’s Health, University of New South Wales, Sydney, New South Wales, Australia

    Paul E. Gray, John B. Ziegler & Maria E. Craig

  6. John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia

    Keisuke Horikawa, Stephen R. Daley, Geeta Chaudhri, Gunasegaran Karupiah, Belinda Whittle, Carla Roots, Edward Bertram, Michiko Yamada, Yogesh Jeelall & Anselm Enders

  7. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia

    Marcel E. Dinger

  8. Research School of Chemistry, The Australian National University, Canberra, Australian Capital Territory, Australia

    Benjamin E. Clifton, Peter D. Mabbitt & Colin J. Jackson

  9. Department of Microbiology & Immunology and the Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA

    Susan R. Watson, Craig N. Jenne & Lewis L. Lanier

  10. Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA

    Tim Wiltshire

  11. Department of Microbiology & Immunology, Stanford University, Stanford, CA, USA

    Matthew H. Spitzer & Garry P. Nolan

  12. Seattle Biomedical Research Institute, Seattle, WA, USA

    Frank Schmitz & Alan Aderem

  13. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

    Benjamin T. Porebski & Ashley M. Buckle

  14. Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA

    Derek W. Abbott

  15. Department of Haematology, The Children’s Hospital at Westmead, Westmead, New South Wales, Australia

    Maria E. Craig, Paul Benitez-Aguirre, Juliana Teo & Melanie Wong

  16. School of Fundamental Sciences, Massey University, Palmerston North, New Zealand

    Murray P. Cox

  17. Genentech Inc., South San Francisco, CA, USA

    Wilson Phung, Jia Tang, Wendy Sandoval & Ingrid E. Wertz

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Contributions

Clinical recruitment and genome sequence analyses were conducted by P.G., O.M.S., A.R., J.B.Z., M.E.C., P.B.-A., J.T., M.W., M.J.C., V.G., M.E.D. and C.C.G. Tracing of Denisovan haplotypes in human populations was conducted by O.M.S., P.G., A.R., M.P.C. and C.C.G. Peripheral blood analysis was conducted by N.W.Z., A.R., J.Y.Y., S.G.T., C.C.G. and S.T.G. Identification of I325N mice was conducted by S.R.W., C.N.J., L.L.L., T.W., C.R., B.W. and C.C.G. Immune cell analysis was conducted by N.W.Z., K.H., S.R.D., Y.J., M.Y., E.B., A.E., M.H.S., G.P.N., F.S., A.A., I.E.W., S.N.W., S.T.G. and C.C.G. Coxsackievirus studies were carried out by N.W.Z., C.L., J.W., C.K. and S.T.G. Ectromelia studies were conducted by G.C. and G.K. Body weight, islet, LPS and inflammation studies were conducted by N.W.Z., S.N.W., E.K.M., J.E.V. and S.T.G. Protease activity and structural studies were conducted by N.W.Z., D.B.L., K.H., D.C., W.P., I.E.W., B.E.C., P.D.M., C.J.J., B.T.P., A.M.B., S.T.G. and C.C.G. C243Y and I207L mouse strain generation and analysis were conducted by N.W.Z., R.B., D.Z., S.N.W., D.C., J.W., C.C.G. and S.T.G. A20 phosphorylation and NF-κB luciferase analyses were performed by N.W.Z., D.C., D.W.A., W.P., W.S., J.T., I.E.W., C.C.G. and S.T.G.

Corresponding authors

Correspondence to Christopher C. Goodnow or Shane T. Grey.

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Competing interests

W.P., J.T., W.S. and I.E.W. were employees of Genentech. All other authors declare no competing interests.

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Peer review information Laurie Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Evolutionary conservation of A20 OTU alleles amongst jawed vertebrates and global distribution of the T108A;I207L TNFAIP3 haplotype.

(a) Aligned amino acid sequence and secondary structure elements of the A20 OTU domain, from40. T108, I207, C243 and I325 residues are boxed in red (b) gnomAD r2.0.2 population allele frequencies for the T108A and I207L missense alleles in TNFAIP3, with allele counts above each bar (total alleles as denominator). Populations were defined by principal component clustering of samples with individuals of known ancestry: those which do not cluster fall into the OTH group. AFR, African; AMR, Latino; ASJ, Ashkenazi Jew; EAS, East Asian; FIN, Finnish European; NFE, non-Finnish European; OTH, other; SAS, South Asian. The OTH population includes one homozygote, while all other alleles are heterozygous. (c) Frequency of the T108A;I207L TNFAIP3 haplotype within 279 individual genomes from the Simons Genome Diversity Project22.

Supplementary Figure 2 Population genetics of the Denisovan TNFAIP3 haplotype.

(a) Geographic distribution of 514 samples used for population genetic analyses. Wallace Line (red)31, Lydekker Line (yellow). Eastern Oceania was defined here as islands east of the Bismarck Archipelago (green line). (b) Principal component analysis (PCA) of the samples presented in (a), with the proportion of variance explained by each PC indicated on each axis (c) Genome-wide FST values between indigenous populations east and west of the Wallace Line. (d) Maximum FST values spanning the TNFAIP3 locus on chromosome 6. Shaded regions correspond to peak FST values between populations east and west of the Wallace Line (grey, 6:137881500-138448062), a previously described haplotype of elevated Denisovan ancestry in Oceanians (blue, 6:137800000-138300000,25), and a putatively adaptively introgressed haplotype in Papuans (red, 6:138160925-138246514,29). (e) Phred-scaled CADD scores of all PASS variants with gnomAD allele frequency <0.01 across the extended Denisovan haplotype27. Shading as per panel d. (f) Relative abundance of Denisovan T108A;I207L and reference TNFAIP3 mRNA from heterozygous leukocytes. Peripheral blood mononuclear cells from five healthy T108A;I207L heterozygous donors were cultured with (+) or without (-) TNF for 2 hours, mRNA isolated and converted to cDNA and amplified with primers in exon 3 and 4 on either side of the I207 codon. The products were deep sequenced on an Illumina MiSeq. Shown are the fraction and percentage (above bars) of reads derived from the Denisovan T108A;I207L allele. (g) RT-PCR analysis of TNFIAP3 gene expression in peripheral blood mononuclear cells from individuals heterozygous (green circles; n=5) or homozygous (purple circle; n=1) for the Denisovan TNFAIP3 haplotype (hap) and 15 healthy control donors (HCs; blue squares), comparing induction kinetics following stimulation with recombinant human TNF for 0, 2 or 4 h. Fold change was determined by comparing stimulated values against non-stimulated PBMC preparations within the same group. Significance between groups was determined by area under the curve analysis (AUC) and data shown as mean ± s.d. P values represent two-tailed Student’s t-test.

Supplementary Figure 3 Reduced IκBα protein in immune cell subpopulations with prolonged NF-κB signaling and cell autonomous activation in T cells extracted from Tnfaip3I325N C57BL/6 mice.

(a) Mass cytometric analysis of intracellular IκBα in immune cells. Unstimulated spleen cells from Tnfaip3+/+ or Tnfaip3I325N/I325N mice (n=4 per genotype from two independent experiments) were individually labeled with mass-barcodes, mixed, permeabilized and stained with mass-labeled antibodies to a panel of cell surface markers and intracellular proteins including IκBα, and analysed by mass cytometry66,67. Spanning-tree Progression Analysis of Density-normalized Events (SPADE;68) analysis was used to resolve leukocyte lineages and subsets, and the relative intensity of IκBα in each subset was compared between Tnfaip3I325N/I325N and wild-type cell counterparts. Shown by color and numbers is the mean hyperbolic arcsine (arcsinh) ratio in minor and major leukocyte subsets. Note that IκBα levels are reduced in B cell subsets indicating increased NF-κB activation. Significant differences indicated by two-tailed Student’s t-test comparison of major subsets are marked: *P < 0.05; **P < 0.01; ***P < 0.001. (b) Bone marrow macrophages derived from conditionally Hoxb8-immortalized bone marrow progenitor cells were derived from two separate pairs of wild-type and I325N homozygous mice, stimulated with LPS (10ng/ml) for the indicated times, and Cxcl1 and Cxcl11 mRNA determined by qPCR relative to Ef1a expression. Data shown is representative of three independent experiments, each with two different mouse donors of each genotype. (c) Representative immunoblot analysis (IB; n=3 independent experiments) of canonical NF-κB components following immunoprecipitation (IP) of TNFR1 from lysates of Tnfaip3+/+ or Tnfaip3I325N/I325N thymocytes treated with 200 U/ml hTNF for the indicated times. Following TNF stimulation RIPK1 was recruited to the TNFR1 complex, with increased high molecular weight polyubiquitinated forms of RIPK1 (UbRIP1) and auto-phosphorylated transforming growth factor β activated kinase-1 (TAK1) in Tnfaip3I325N/I325N thymocytes. Mutant thymocytes exhibited a modest increase in IκBα degradation relative to wild-type, and increased JNK phosphorylation (lysate blots). A similar pattern of enhanced TAK1 activation and JNK phosphorylation was seen in murine embryonic fibroblasts and bone marrow derived macrophages expressing catalytically dead A20 OTU or ZnF4 mutants18. (d-f) Two B6.CD45.1+ wild-type mice were transplanted with a congenic bone marrow mixture from Tnfaip3+/+ CD45.1+ donors, to provide wild-type lymphocytes as an internal control, or CD45.2+ donors of Tnfaip3I325N/I325N, Tnfaip3C103A/C103A or Tnfaip3+/+ genotypes. (d) Pairwise comparison of the percentage of HELIOS+ cells among the indicated CD45.2+ and CD45.1+ subsets of CD4+ CD8- CCR7+ CD24+ FOXP3- thymocytes from the same chimera, pooled from two independent experiments. Analysis by paired Student’s t-test; ****P < 0.0001. Compared to the C103A mutation, the I325N mutation exaggerated thymic formation of FOXP3+ CD4+ cells and their Helios+ FOXP3- precursors, which depend on TCR signaling through CARD11 to NF-κB. (e, f) Representative profiles gated on CD45.2+ cells of the indicated Tnfaip3 genotypes. (e) Representative, analysis of CD4+ CD8- CCR7+ CD24+ FOXP3- thymocytes, showing the percentage of immature medullary CD4 T cells induced by strong self-reactivity to express high levels of HELIOS and BIM. (f) Percentage of FOXP3+ CD44+ cells among CD4+ splenic T cells. (d-f) were performed two independent times.

Supplementary Figure 4 B cell lymphocytosis and cell autonomous exaggeration of B cell activation by Tnfaip3I325N more than C103A mutations.

(a, b) Representative flow cytometry from four independent experimetns. Plots from mice of the indicated genotypes showing frequencies and absolute numbers of (a) splenic and (b) peritoneal B cells, based upon their expression of CD23 and CD21, IgD+, and IgM+ (a) or CD23, CD11b, IgM, and CD5 (b). The number of splenocyte B cell subsets in (a) is increased in Tnfaip3I325N/I325N mice (Fig. 2c), as is the number of B cells in the peritoneum of individual Tnfaip3 mice (c). (d) Wild-type (+/+) and I325N homozygous (I325N/I325N) Tnfaip3 splenic B lymphocytes were stimulated for the indicated times with anti-IgM, or lipopolysaccharide (LPS) and lysates analysed by immunoblotting for A20, IκBα and β-actin (loading control). Data represent three independent experiments. (e-g) Two B6.CD45.1+ wild-type mice were transplanted with a congenic bone marrow mixture from each Tnfaip3+/+ CD45.1+ donors, to provide wild-type lymphocytes as an internal control, or CD45.2+ donors of Tnfaip3I325N/I325N, Tnfaip3C103A/C103A or Tnfaip3+/+ genotypes. Chimeric sets were set up two independent times. (e) Representative flow cytometric histograms of CD25 and CD44 expression on B cells from chimeric mice cultured for 1 day and CFSE dilution after 3 days with 0.1 ug/ml LPS or anti-IgM. Black or colored open histograms, CD45.2+ B cells of the indicated Tnfaip3 genotype; grey line open histograms, CD45.1+ control B cells in the same stimulated culture; filled histograms, B cells in a parallel unstimulated culture. (f, g) Data from independent (n=2) mixed chimeric mice (n=4) showing relative mean fluorescence intensity (MFI) of CD25 or CD44 on CD45.2+ B cells of the indicated genotypes (red, I325N; blue, C103A; black, WT) compared to the co-cultured CD45.1+ wild-type B cells. Statistical analysis by ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Figure 5 Increased resistance to Coxsackievirus in Tnfaip3I325N mice.

Infection of C57BL/6 female mice of the indicated Tnfaip3 genotypes with 20 plaque forming units (PFU) of Coxsackievirus by intraperitoneal injection. (a) Kaplan-Meier survival curve showing a mean survival time of 6.5 and 12.5 days for Tnfaip3+/+ and Tnfaip3+/I325N mice, respectively. No Tnfaip3I325N/I325N mice succumbed to infection. Tnfaip3+/+ (n=15), Tnfaip3I325N/+ (n=14) or Tnfaip3I325N/I325N (n=10). Significance determined by Log-rank test. (b) Percent change in body weight after Coxsackievirus infection and (c) Blood glucose levels of mice with indicated Tnfaip3 genotype, compared to wild-type by area under the curve analysis. Tnfaip3+/+ (n=29), Tnfaip3I325N/+ (n=34) or Tnfaip3I325N/I325N (n=20). Cumulative data from five independent experiments. (d) Coxsackievirus mRNA (CVB4) abundance on indicated days (D0-3) post infection. (e) Hematoxylin & eosin stained sections of pancreas from Tnfaip3+/+ and Tnfaip3I325N/I325N mice at post-infection day 9. Note better preserved pancreatic architecture in Tnfaip3I325N/I325N mice. Scale bar = 200 µm. (f, g) RTPCR analysis for inflammatory genes Il1b (f) or Ifnb1 (G) at post-infection day 0, 1 and 3. Representative of two independent experiments (n=2). Columns represent mean ± s.e.m and two-tailed Student t-test used for significance analysis unless otherwise stated, *P < 0.05; ***P < 0.001; ****P < 0.0001.

Supplementary Figure 6 Histological analysis of tissues from Tnfaip3I325N mice.

(a) Colon length (cm) and (b) hematoxylin and eosin (H&E) stained sections from Tnfaip3I325N mice colon (20x magnification; scale bar = 200 µm) and (c) power-mosaic image of whole colon cross section of indicated genotypes (R=rectum; C=Caecum; m=muscularis; mu=mucosa; pp=Peyer’s patches; scale = 1 mm). (d) H&E sections of kidney (4× top panel, scale bar = 500 µm; 10× bottom panel, scale bar = 200 µm) and (e) liver (4× top panel, scale bar = 500 µm; 10× bottom panel, scale bar = 200 µm). (f) H&E stained sections of pancreas (10x images, scale bar = 200 µm) or, (g) insulin stained sections (20x images, scale bar = 100 µm). (h) Weight of spleens in grams (g). (i) Gross appearance of spleen and salivary glands from Tnfaip3+/+ and Tnfaip3I325N/I325N mice. Images are representative of n=4 Tnfaip3+/+ and n=4 Tnfaip3I325N/I325N 16 week-old mice from three different litters. Columns represent mean ± s.e.m, **P < 0.01.

Supplementary Figure 7 Glucose homeostasis of Tnfaip3I325N mice and isolated islets.

(a) Random blood glucose levels of Tnfaip3+/+, Tnfaip3I325N/+ or Tnfaip3I325N/I325N female (♀) or male (♂) mice at 8 or 12 weeks of age. (b, c) Blood glucose levels (mM) were monitored following an intraperitoneal injection of glucose (2 g/kg) in 12 week-old mice. (d, e) Pancreatic islets were isolated from individual mice of the indicated A20 genotypes, incubated overnight, and treated with 200 U/ml TNFα for the indicated times. (d) Representative immunoblot for phosphorylated JNK (pJNK) and total JNK (TJNK, loading control). Cumulative densitometry from 4 independent experiments is shown below. Columns represent mean ± s.e.m. Two-tailed Student t-test used for significance analysis, *P < 0.05. (e) Immunoblot for non-canonical NF-κB components NIK, p100/p52 and RelB; representative of 2 independent experiments. Tnfaip3I325N/I325N islets exhibited increased activation of the non-canonical NF-κB pathway that can alter beta cell transcriptional programs to favor reduced insulin output38.

Supplementary Figure 8 Tnfaip3I325N islet grafts exhibit a reduced first-phase insulin secretory response compared to wild-type Tnfaip3 islet grafts independent to beta cell area.

(a) Islets isolated from B6 mice of the indicated genotypes were transplanted under the kidney capsule of Tnfaip3+/+ B6 recipients that had been rendered diabetic with streptozotocin. Mean and s.e.m blood glucose on the indicated days relative to islet transplantation is shown. (b-c) At post-operative day 14 when euglycemia was established, mice were challenged with an (b) intravenous injection of glucose (1 g/kg), and blood glucose monitored over time (min). (c) Blood insulin levels (ng/ml) were also measured at the same time points via an enzyme-linked immunosorbent assay from samples in (b). AUC = area under the curve. (d) Insulin immunostaining of wild-type (Tnfaip3+/+) or A20 I325N homozygous mutant (Tnfaip3I325N/I325N) islet grafts transplanted under the kidney capsule of diabetic C57BL/6 mice for 30 days (Tnfaip3+/+ donor grafts into Tnfaip3+/+ recipients, n = 5; Tnfaip3I325N/I325N donor grafts into Tnfaip3+/+ recipients, n=5). (e) Islet graft beta cell area was determined by insulin-positive area quantification in continuous serial graft sections. Tnfaip3I325/I325N grafts exhibited equivalent insulin-positive graft area, confirming that loss of glucose tolerance (b, c) was due to a defect in insulin secretion (Fig. 3h). (f) Tnfaip3+/+ or Tnfaip3I325N/I325N islet grafts from an independent cohort of recipients were isolated on post-operative day 10 and analysed for the indicated mRNAs by RT-qPCR 86. The difference in gene expression of indicated islet grafts is shown. Fold change and mean calculated using average wild-type ΔCt value. Columns represent mean ±s.e.m and ANOVA used for significance, *P < 0.05.

Supplementary Figure 9 Analysis of allele effects on A20 phosphorylation and control of NF-κB.

(a) A20 accumulation in βTC3 mouse insulinoma cells transfected with wild-type (WT) A20, C103A A20, T108A;I207L A20, I325N A20, C243Y A20, S381A A20 or S184N A20 was determined by densitometry analysis of independent experimental immunoblots. Fast migratory (A20) 98 kDa A20 was corrected to loading control and compared to WT A20 of the same immunoblot [(FM A20/β-actin)/A20]. (b) Results of mass spectrometry analysis of full length WT A20, C103A mutant A20, or I325N mutant A20 expressed in and purified from HEK293 cells and analysed by mass spectrometry for phosphorylation of the indicated residues as per references18,19. Data representative to two independent experiments. (c) βTC3 cells co-transfected with an NF-κB.luciferase reporter and a CMV.βgal expression construct alone or with PCDNA3.1 encoding human wild-type A20 (hA20 WT; blue), A20 T108A/I207L (purple), A20 C243Y (orange), A20 S184N (grey), A20 I325N (red) or A20 S381A (white). Cells were stimulated with (+) 200 U/ml hTNF for 8 h or left untreated (-). Data presented as relative luciferase units. Each column represents 3-5 independently transfected aliquots of cells within one experiment marked by circle symbols (representative of 3 independent experiments) and columns are arithmetic means ± s.e.m. Statistical comparison by two-tailed Student’s t-test against WT hA20 (blue). (d) βTC3 cells co-transfected with a NF-κB.luciferase reporter and a CMV.βgal expression construct alone or with PCDNA3.1 encoding human wild-type A20 (hA20 WT; blue), or A20 I325N (I325N; red), or A20 with serine 381 substituted to non-phosphorylatable alanine (S381A or I325N S381A), or to phosphomimetic glutamate (S381E or I325N;S381E). Cells were stimulated with (+) 200 U/ml hTNF for 8 h or left untreated (-). Data presented as relative luciferase units. Each column represents mean of 3-6 independently transfected aliquots of cells within one experiment marked by circle symbols (representative of 3 independent experiments) and columns are arithmetic means ± s.e.m. Statistical comparison by two-tailed Student’s t-test against stimulated (*) or unstimulated (^) WT hA20 (blue). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Figure 10 A20I325N subtly impacts the OTU structure.

(a) Structures of human I325N (orange) and wild-type (WT; grey) A20 OTU domains superposed (each with 6 molecules per asymmetric unit). The dimer partner of one of these is shown in pink. Loop 317 and 280 are indicated on the periphery. (b) The I325N substitution does not alter the fold of the putative catalytic triad (orange cartoon and sticks) compared to previously published wild-type structures (cyan cartoon and sticks; PDB entries 3DKB, 2VFJ and 3ZJD). (c) The fold is additionally unperturbed by acetamidylation of C103 (orange mesh; 2mFo-DFc composite omit map contoured at 1.2 sigma). (d-f) The I325N substituted side-chain sits at the base of the β7-β8 loop containing residue 317, in a pocket also lined by the β6-β9 loop containing residue 280 (d). Adjacent hydrophobic residues include W85, V276, F283, I312, V314 (e). The I325N substitution results in the base of the β7-β8 loop splaying apart (f, arrow), increasing β7-β8 loop disorder. (g) Structures of human I325N (orange) and wild-type (WT; grey) A20 OTU domains superposed on the wild-type OTU-ubiquitin structure 5LRX44. Conserved residues on the posterior surface are coloured orange. The β3-β4 loop containing the C243Y substitution and the structured base of the β7-β8 loop containing the I325N allele marked.

Supplementary Figure 11 Molecular characterization of A20 I325N and A20 C103A.

(a, b) Thermal stability of mouse and human wild-type (WT), C103A, and I325N variants were examined using circular dichroism, where mean residue ellipticity (MRE) was measured at 220 nm as a function of temperature as solutions were heated at a rate of 1° C/min. Human (a) or mouse (b) A20 OTU residues 1-370; WT (blue squares), C103A (red triangles), I325N (black circles). Curves were fit by linear regression. (c) Unfolding transitions temperature (Ttrs) of human A20 OTU variants (residues 1-370) and mouse variants (residues 1-360), derived from the fit of the curves in (a) and (b). Data represents three independent experiments. (d) HEK293T cells were transfected with bicistronic retrovirus vector pMXs-IRES-GFP, expressing FLAG-tagged mouse full-length TNFAIP3 wild-type or I325N. Each group of transfected cells was replated 24 h after transfection and incubated with 10 µM of cycloheximide (CHX) for the indicated times before harvesting. Lysates were probed with anti-FLAG and anti-GFP antibodies. (e) Reactions containing K48-linked tetra-ubiquitin (Ub4) and E. coli-expressed and purified WT or I325N A20 OTU domain, or the catalytically deactivated A20 OTU domain, C103A, were incubated for the indicated times. Depolymerization of polyubiquitin was detected by immunoblotting for ubiquitin and A20; representative of two independent experiments. (f) Reactions containing mixed Ub2-Ub6 K48-linked polyubiquitin chains and E. coli-expressed and purified WT or I325N A20 OTU domain ± iodoacetamide (IA) treatment were incubated at 37°C for 60 or 150 minutes. Depolymerization of polyubiquitin was detected by immunoblotting for ubiquitin and the N-terminus of A20; representative of 4 independent experiments. (g, h) Flag-tagged full length human A20 proteins (WT or I325N mutant) were expressed in HEK-293T cells and purified as previously described14. Purified A20 proteins were tested for K63-linked tetra-ubiquitin depolymerization (g) and in vitro ubiquitination assay (h). Immunoblots are representative of three (g) or two (h) independent experiments, respectively.

Supplementary Figure 12 Histological analysis of tissues from Tnfaip3I207L and Tnfaip3C243Y mice.

(a) Body weights of 8 week-old Tnfaip3I207L and Tnfaip3C243Y mice of the indicated genotype and sex. Bars represent mean ± s.e.m. Statistical analysis by one-way ANOVA: ****P < 0.0001. Hematoxylin and eosin (H&E) stained sections from Tnfaip3I207L mice of the indicated genotypes, including colon (b) (20× magnification; scale bar = 200 µm; see Fig. 6d for C243Y genotype), kidney (c) (4× top panel, scale bar = 500 µm; 10× bottom panel, scale bar = 200 µm), liver (d) (4× top panel, scale bar = 500 µm; 10× bottom panel, scale bar = 200 µm), and pancreas (e) (10× images, scale bar = 200 µm). Images are representative from 16 week old mice of the indicated genotype (n=4).

Supplementary Figure 13 Flow cytometry analysis of T cells from mice with human A20 OTU domain alleles.

(a) Cumulative data showing CD4+FOXP3+ regulatory T cells from spleens of T108A;I207L (I207L), I325N or C243Y mice. Each point indicates an individual mouse collected form 3 independent experiments (I207L and C243Y) or 4 independent experiments (I325N). Statistical analysis by 2-tailed Student’s t-test, data shown as mean ±s.e.m, **P < 0.01, ****P < 0.0001. (b, c) Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes, showing percentages of CD4+ or CD8+ T cells, and the percentage of CD44hi activated/memory cells within these subsets, and the percentage of CD25+CD44int regulatory T cells or CD25- CD44hi effector/memory T cells among CD4+ cells. Data is representative of the same 3 independent experiments conducted in (a).

Supplementary Figure 14 Flow cytometry analysis of human T108A;I207L peripheral blood mononuclear cells.

(a) Gating strategy used to analyse human peripheral blood mononuclear cell data collected by flow cytometry. Arrows used to illustrate the step wise analysis of cell populations identified on the basis of physical properties and surface marker staining. Heading titles above each plot indicates gated cell population analysed in that plot. (b-d) Peripheral blood mononuclear cells from individuals heterozygous (green circles) or homozygous (purple circle) for the Denisovan TNFAIP3 haplotype (hap) and 6 age-matched healthy control donors (HCs; blue circles), were assessed by flow cytometry for percentages of CD4+CD25hiCD127lo regulatory T cells (b), different subsets of non-regulatory CD4+CD25loCD127hi/lo T cells (c), and CD8+ T cell subsets (d). (CM=central memory; EM=effector memory; EMRA = terminally differentiated effector memory cells). Bars show mean ± s.d. Two-tailed Student’s t-test was used to test for significance, only significant differences are indicated, *P < 0.05.

Supplementary Figure 15 Diminished tolerance to self-antigen caused by the I325N A20 allele.

InsHEL-transgenic mice or non-transgenic controls, both with wild-type A20 in pancreatic islets, were transplanted with a congenic bone marrow mixture from Tnfaip3+/+ CD45.1+ TCR3A9-transgenic donors, to serve as an internal control, and CD45.2 TCR3A9-transgenic donors with either Tnfaip3I325N/I325N (black circles) or Tnfaip3+/+ (grey circles) genotype. (a) Frequency and mean of TCR3A9 positive CD4+ T cells among CD45.2+ Tnfaip3+/+ (grey circles) or Tnfaip3I325N/I325N (black circles) lymphocytes in pancreatic lymph nodes. Test recipients expressed the insHEL antigen in pancreatic islets (insHEL-transgenic), while parallel non-transgenic recipients lacked the autoantigen. (b) Percentage and mean of CD45.2+ Tnfaip3+/+ (grey circles) or Tnfaip3I325N/I325N (black circles) cells among TCR3A9+ CD4+ T cells in spleen. (c, d) Analysis of insHEL transgenic recipients. Relative mean fluorescence intensity (MFI) of staining for cell surface MHC class II or CD86 on CD45.2+ Tnfaip3+/+ (grey circles) or Tnfaip3I325N/I325N (black circles) B cells (c) or CD11c+ dendritic cells (d), compared to CD45.1+ Tnfaip3+/+ B cells or dendritic cells in the same sample. (e) Frequency of germinal center (GC) B cells among CD45.2+ B cells (black circles) compared to the frequency among CD45.1+ Tnfaip3+/+ B cells in the same animal (linked by lines). Left panel, CD45.2 Tnfaip3+/+ marrow donor; right panel, CD45.2 Tnfaip3I325N/I325N marrow donor. In (c-e), each symbol, or pair of symbols joined by a line, represents one chimeric mouse, all analyzed in a single experiment. Statistical analysis by 2-tailed Student’s t-tests: *P < 0.05; **P < 0.01; ***P < 0.001.

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Zammit, N.W., Siggs, O.M., Gray, P.E. et al. Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity. Nat Immunol 20, 1299–1310 (2019). https://doi.org/10.1038/s41590-019-0492-0

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