An Essential Role of UBXN3B in B Lymphopoiesis Tingting Geng et al. This file contains 9 supplemental figures and legends. a Viral load (relative) load Viral Serum TNF-α b +/+ Serum IL-6 Ubxn3b Ubxn3b-/- Ubxn3b+/+ 100 100 ** Ubxn3b-/- ** 10 10 IL-6 IL-6 (pg/ml) GM-CSF (pg/ml) GM-CSF 1 1 0 3 8 14 35 0 3 8 14 35 Time post infection (Days) Time post infection (Days) Serum IL-10 Serum CXCL10 Ubxn3b+/+ Ubxn3b-/- 10000 1000 +/+ -/- * Ubxn3b Ubxn3b 1000 100 IFN-γ (pg/ml) CXCL10 (pg/ml) CXCL10 100 10 0 3 8 14 35 0 3 8 14 35 Time post infection (Days) Time post infection (Days) IL-1β IL-1β (pg/ml) Supplemental Fig.s1 UBXN3B is essential for controlling SARS-CoV-2 pathogenesis. Sex- and-age matched littermates were administered 2x105 plaque forming units (PFU) of SARS-CoV-2 intranasally. a) Quantitative RT-PCR (qPCR) quantification of SARS-CoV-2 loads in the lung at days 3 and 10 post infection (p.i). Each symbol= one mouse, the small horizontal line: the median of the result. *, p<0.05; **, p<0.01, ***, p<0.001 (non-parametric Mann-Whitney test) between Ubxn3b+/+ and Ubxn3b-/- littermates at each time point. Ubxn3b+/+ Ubxn3b-/- Live Live 78.0 83.6 CD45+ CD45+ UV UV CD45 94.1 CD45 90.9 FSC-A FSC-A FSC-A FSC-A Mac Mac 23.0 38.1 MHC II MHC II CD11b Eso CD11b Eso 5.81 7.44 CD19, MHCII subset F4_80, CD11b subset CD19, MHCII subset F4_80, CD11b subset Myeloid panel Myeloid 70.1 65.6 94.2 51.1 CD19 F4/80 CD19 F4/80 Neu DC Neu DC 4.87 1.02 16.2 2.24 CD11b, Ly-6G subset CD11b, Ly-6G subset 94.9 83.3 Ly-6G Ly-6G MHC II MHC II CD11b CD11c CD11b CD11c Live Live 82.3 76.5 CD45+ CD45+ 91.1 91.3 UV UV CD45 CD45 FSC-A FSC-A FSC-A FSC-A Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 Lymphoid panel Lymphoid 20.1 0.29 54.1 1.17 4.27 0.060 39.2 2.55 CD4 CD4 CD19 CD19 Q4 Q3 Q4 Q3 Q4 Q3 Q4 Q3 47.0 32.6 7.21 37.5 67.7 28.0 7.27 51.0 CD3 CD8 CD3 CD8 Supplemental Fig.s2 Dysregulated immune compartmentalization in Ubxn3b-/- lung. The graphs illustrate the gating strategy of different immune populations in the lung at day 35 post SARS- CoV-2 infection (related to Fig.2f,g). Mac: macrophage, Neu: neutrophil, DC: dendritic cell, Eso: eosinophil. CD3+ T, CD8+ T, CD4+ T, CD19+ B. Ubxn3b+/+ Ubxn3b-/- 40x T WP RP 200x A MZ MS MS Supplemental Fig.s3 Splenic atrophy in Ubxn3b-/- mice. The figures show H&E staining of spleens on day 35 after SARS-CoV-2 infection. The red arrow head indicates reduced cell density in the marginal zones of white pulps. The green circles likely show the myeloid clumps in red pulps. WP: white pulp, RP: red pulp, A: central arteriole, MS: marginal sinus, MZ: marginal zone, T: trabecula. Sting +/+ Sting-/- a Q1 Q2 Q1 Q2 28.1 0.38 29.4 0.62 Q4 Q3 Q4 Q3 Comp-APC-A :: CD19 Comp-APC-A :: CD19 28.9 42.7 17.2 52.8 Comp-PE-Dazzle594-A :: CD3 Comp-PE-Dazzle594-A :: CD3 Neu Neu 61.5 46.2 Blood cell Blood CD11b, Ly-6G subset CD11b, Ly-6G subset 37.6 52.5 Comp-Alexa Fluor 700-A ::Ly-6G Comp-Alexa Fluor 700-A ::Ly-6G Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b Mono Mono 15.9 13.0 CD11b, CD115 subset CD11b, CD115 subset 82.9 85.6 Comp-BVio711-A ::CD115 Comp-BVio711-A ::CD115 Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b b c + % of CD45 Cell counts / ml / counts Cell Supplemental Fig.s4 STING is dispensable for steady-state hematopoietic homeostasis. a) The gating strategy, b) The percentage and c) counts of each blood cell type in Sting+/+ and Sting-/- littermates, quantitated by flow cytometry. Each symbol=one animal. The horizontal line indicate the mean of the results. RBC: red blood cell, WBC: white blood cell, CD19+: B cell, CD3+ : T cell, Neu: neutrophil, Mono: monocyte. a B cell T cell Neu Mono Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 0.015 4.51E-3 7.54 0.058 0 0 0 0 BM > WT >BM +/+ Comp-PE-A45_1:: Q4 Q3 Comp-PE-A::45_1 Q4 Q3 Comp-PE-A::45_1 Q4 Q3 Comp-PE-A45_1:: Q4 Q3 0 100.0 3.04E-3 92.4 0 100 0 100 Ubxn3b Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Q1 Q2 Q1 Q2 Q1 Q2 Q1 Q2 0.052 0.10 17.0 0.23 0 0 0 0.016 BM >WT BM -/- Ubxn3b Comp-PE-A45_1 :: Q4 Q3 Comp-PE-A45_1 :: Q4 Q3 Comp-PE-A45_1 :: Q4 Q3 Comp-PE-A45_1 :: Q4 Q3 0 99.8 0 82.8 0 100 0 100.0 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 Comp-PE-Cy7-A :: 45_2 b Q1 Q2 Neu Mono 57.0 1.17 48.3 53.6 BM > WT >BM +/+ Q4 Q3 CD11b, Ly-6G subset Comp-APC-A CD19:: 23.3 18.6 51.0 Comp-BVio711-A :: CD115 Comp-Alexa Fluor 700-A:: Ly-6G Comp-PE-Dazzle594-A :: CD3 Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b Ubxn3b Q1 Q2 Neu Mono 6.26 0.17 52.7 67.2 BM >WT BM -/- Q4 Q3 CD11b, Ly-6G subset Comp-APC-A :: CD19 60.5 33.0 46.7 Comp-BVio711-A CD115:: Comp-Alexa Fluor 700-A Ly-6G:: Ubxn3b Comp-PE-Dazzle594-A :: CD3 Comp-APC-Fire750-A :: CD11b Comp-APC-Fire750-A :: CD11b Supplemental Fig.s5. UBXN3B plays a cell-intrinsic role in B lymphopoiesis. Irradiated wild type (WT, CD45.1 ) mice were transplanted with Cre+Ubxn3bf/f (CD45.2) bone marrow . The mice were then treated with tamoxifen (TMX) to delete Ubxn3b (designated Ubxn3b−/− BM–WT) or not (designated Ubxn3b+/+ BM–WT) in hematopoietic cells. a) The percentage of blood CD45.1 and CD45.2 cells 1.5 month after bone marrow transplantation (BMT). Over 99% B/Neu/Mono are CD45.2+, over 82% T cell are CD45.2+, indicating successful irradiation and reconstitution. b) The gating strategy of blood immune cells in BMT mice (related to Fig.4). B: CD19 + B cell, T : CD3+ T cell, Neu: neutrophil, Mono: monocyte. a B cell T cell Neu Mono +/+ WT BM > Ubxn3b > WTBM -/- WT BM >Ubxn3b WTBM b +/+ WT BM > Ubxn3b > WTBM -/- WT BM >Ubxn3b WTBM c WT BM→Ubxn3b−/− −/− WT BM→Ubxn3b + % of CD45 Cell counts / ml / counts Cell Supplemental Fig.s6. UBXN3B plays a cell-intrinsic role in B lymphopoiesis. Irradiated Cre+Ubxn3bf/f and Ubxn3bf/f (CD45.2) mice were transplanted with wild type (WT, CD45.1) bone marrow . The mice were then treated with tamoxifen (TMX) to delete Ubxn3b (designated WT BM–Ubxn3b−/−) or not (designated WT BM–Ubxn3b+/+) in non-hematopoietic cells. a) The blood CD45.1 and CD45.2 cells 1.5 month after bone marrow transplantation (BMT). Over 99% B/Neu/Mono are CD45.1+, over 92% T cell are CD45.1+, indicating successful irradiation and reconstitution. b) The gating strategy of blood immune cells in BMT mice. c) The count and percentage (relative to CD45+) of each blood cell type in BMT mice at 15 days after induction of Ubxn3b deletion by tamoxifen (TMX), quantitated by flow cytometry. *, P<0.05; **, P<0.01, ***, P<0.001 (two-tailed Student’s t-test). Each symbol=one animal. The horizontal line indicate the mean of the results. RBC: red blood cell, WBC: white blood cell, B: B cell, T : T cell, Neu: neutrophil, Mono: monocyte. +/+ a Ubxn3b Ubxn3b-/- CD19+ B CD19+ B 14.1 16.8 2.19 3.28 Comp-APC-A :: CD19 CD3, CD19 subset T Comp-APC-A :: CD19 CD3, CD19 subset T Comp-APC-A :: CD19 Comp-APC-A :: CD19 83.6 1.65 96.4 0.78 Comp-PE-Alexa 594-A :: CD3 Comp-APC-Cy7-A :: IgD Comp-PE-Alexa 594-A :: CD3 Comp-APC-Cy7-A :: IgD Neu Mono Neu Mono 56.3 24.1 53.8 35.2 BM Mature cell Mature BM CD11b, Ly6G subset CD11b, Ly6G subset Comp-BV711-A:: CD115 40.8 Comp-BV711-A :: CD115 42.4 Comp-Alexa Fluor 700-A :: Ly6G Comp-Alexa Fluor 700-A :: Ly6G Comp-FITC-A :: CD11b Comp-FITC-A :: CD11b Comp-FITC-A :: CD11b Comp-FITC-A :: CD11b b Ubxn3b+/+ Ubxn3b-/- B220 B220 68.8 40.5 Dump Dump Comp-FITC-A ::dump Comp-FITC-A ::dump 19.7 Comp-BV650-A :: B220 10.2 Comp-BV650-A :: B220 FSC-A FSC-A FSC-A FSC-A D-F A-C' C C' D-F A-C' C C' 76.2 23.6 4.2917.7 36.4 61.7 6.444.81 Comp-PE-A :: BP-1 A B Comp-PE-A BP-1:: A B Comp-BV650-A :: B220 44.5 31.9 Comp-BV650-A :: B220 69.6 17.6 BM Blinage BM cell Comp-APC-A :: CD43 Comp-PerCP-Cy5-5-A :: CD24 Comp-APC-A :: CD43 Comp-PerCP-Cy5-5-A :: CD24 E F E F 24.1 19.2 Pre-Pro B 19.3 14.9 Pre-Pro B 25.6 40.1 D D Comp-BV421-A:: IgM Comp-BV421-A:: IgM 51.3 Comp-PE-Cy7-A:: CD93 60.3 Comp-PE-Cy7-A:: CD93 Comp-APC-Cy7-A :: IgD Comp-PE-Alexa 594-A :: CD19 Comp-APC-Cy7-A :: IgD Comp-PE-Alexa 594-A :: CD19 Supplemental Fig.s7 UBXN3B is essential for early B lymphopoiesis in the bone marrow.