Figure 4Saline and 2G8 - treated MiaPaca - 2 and Colo375 xenografts were subjected to RNA - seq analysis for mouse gene expression changes . A heat map was generated with gene clustering ( A ) . Saline and 2G8 - treated MiaPaca - 2 and Colo375 xenografts were subjected to RNA - seq analysis for mouse gene expression changes . Molecules most significantly by 2G8 treatment . Y - axis values shown are log10 ( FPKM + 1 ) of the transcript levels ( D - F ) . An in vitro NK cell cytotoxicity assay was performed . Human NKL cells were used as effector cells , and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM ( control ) , conditioned medium collected from TGFβ ( 30 ng / ml ) - treated human pancreatic CAFs ( CAF - PC2 ; CAF TGFβ - CM ) , CAF TGFβ - CM + IL - 6 antibody ( 100 ng / ml ) or normal DMEM + TGFβ ( TGFβ ) . Living cells were labeled with CFSE , and dead cells were labeled with 7 - AAD . Samples were analyzed by flow cytometry . Cytotoxicity percentage was calculated using the formula ( 7 - AAD positive cells % ) / ( 7 - AAD positive cells % + CFSE positive cells % ) x 100 % . n = 4 / group , P values by t test are shown . Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice . After tumor establishment , mice were randomized to receive rat IgG Mac84 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 6 / group ) for 3 weeks . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H & E - stained liver tissue . Ten sections of the anterior lobe of the liver ( n = 6 per group ) were scored for lesions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Saline and 2G8-treated MiaPaca-2 and Colo375 xenografts were subjected to RNA-seq analysis for mouse gene expression changes. A heat map was generated with gene clustering (A).Saline and 2G8-treated MiaPaca-2 and Colo375 xenografts were subjected to RNA-seq analysis for mouse gene expression changes. Molecules most significantly by 2G8 treatment. Y-axis values shown are log10(FPKM+1) of the transcript levels (D-F).An in vitro NK cell cytotoxicity assay was performed. Human NKL cells were used as effector cells, and mouse pancreatic cancer BMFA3 cells were used as target cells in normal DMEM (control), conditioned medium collected from TGFβ (30 ng/ml)-treated human pancreatic CAFs (CAF-PC2; CAF TGFβ-CM), CAF TGFβ-CM + IL-6 antibody (100 ng/ml) or normal DMEM + TGFβ (TGFβ). Living cells were labeled with CFSE, and dead cells were labeled with 7-AAD. Samples were analyzed by flow cytometry. Cytotoxicity percentage was calculated using the formula (7-AAD positive cells %) / (7-AAD positive cells % + CFSE positive cells %) x 100%. n = 4/group, P values by t test are shown.Human pancreatic cancer cell line Colo357 was orthotopically implanted into NOD SCID mice. After tumor establishment, mice were randomized to receive rat IgG Mac84 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 6/group) for 3 weeks. For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Tumors were harvested for analysis and metastatic burden was determined by histologic evaluation of H&E-stained liver tissue. Ten sections of the anterior lobe of the liver (n = 6 per group) were scored for lesions.


Figure 5KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Immune landscape changes were detected by immunohistochemistry for macrophage makers F4 / 80 ( total , A - C ) , iNOS ( M1 , D - F ) , ARG1 ( M2 , G - I ) , CD11b ( myeloid - derived suppressor cells , J - L ) and T cell markers CD3 ( M - O ) , CD8 ( P - R ) and FOXP3 ( regulatory T cells , S - U ) in KIC and KPC mice . Scale bars = 50 μm . n = 4 / group , P values by t test are shown . Spatial distribution analysis of CD8 - positive T cells after 2G8 treatment . Details are described in Appendix Fig S7 . Sample images of KPC tumors are shown ( V ) . Substantial cytotoxic T cell infiltration was observed after 2G8 treatment . Histological score was calculated using the following formula ( W - X ) : low IHC cells % + 2x ( medium IHC cells % ) + 3x ( high IHC cells % ) . Scale bars = 500 μm . n = 4 / group , P values vs . control by t test are shown .

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Figure 5KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Immune landscape changes were detected by immunohistochemistry for macrophage makers F4/80 (total, A-C), iNOS (M1, D-F), ARG1 (M2, G-I), CD11b (myeloid-derived suppressor cells, J-L) and T cell markers CD3 (M-O), CD8 (P-R) and FOXP3 (regulatory T cells, S-U) in KIC and KPC mice. Scale bars = 50 μm. n = 4/group, P values by t test are shown.Spatial distribution analysis of CD8-positive T cells after 2G8 treatment. Details are described in Appendix Fig S7. Sample images of KPC tumors are shown (V). Substantial cytotoxic T cell infiltration was observed after 2G8 treatment. Histological score was calculated using the following formula (W-X): low IHC cells % + 2x (medium IHC cells %) + 3x (high IHC cells %). Scale bars = 500 μm. n = 4/group, P values vs. control by t test are shown.


Figure 6KIC mice were treated in a survival study with rat IgG Mac48 ( control ) or 2G8 . Median overall survival of rat IgG treatment was 72 days , while median overall survival of 2G8 treatment was 63 days ( B ) . Tissues from ( A ) were stained for Ki67 . Scale bars = 50 μm . Overall survival of PDA patients from TCGA with and without TGFBR2 mutation . Loss - of - function mutation of Tgfbr2 was generated by CRISPR with two different gRNAs ( mut1 and mut2 ) in the mouse PDA cell line BMFA3 derived from KPfC model . Control cell line ( Tgfbr2wt ) and two Tgfbr2 - mutant cell lines ( Tgfbr2mut1 and Tgfbr2mut2 ) were treated with TGFβ ( 30 ng / ml ) for 5 hours . Cell lysates were harvested and western blotting for P - SMAD2 , SMAD2 , SMAD4 , P - ERK1 / 2 , ERK1 / 2 , P - P38 , P38 , and actin was performed . Immunofluorescence was performed to study the expression and localization of E - cadherin and SMAD4 after TGFβ treatment ( 30 ng / ml for 24 hours ) in control cell line and two Tgfbr2 - mutant cell lines . Scale bars = 50 μm ( fluorescent ) , scale bars = 100 μm ( brightfield ) .

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Figure 6KIC mice were treated in a survival study with rat IgG Mac48 (control) or 2G8. Median overall survival of rat IgG treatment was 72 days, while median overall survival of 2G8 treatment was 63 days (B).Tissues from (A) were stained for Ki67. Scale bars = 50 μm.Overall survival of PDA patients from TCGA with and without TGFBR2 mutation.Loss-of-function mutation of Tgfbr2 was generated by CRISPR with two different gRNAs (mut1 and mut2) in the mouse PDA cell line BMFA3 derived from KPfC model. Control cell line (Tgfbr2wt) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) were treated with TGFβ (30 ng/ml) for 5 hours. Cell lysates were harvested and western blotting for P-SMAD2, SMAD2, SMAD4, P-ERK1/2, ERK1/2, P-P38, P38, and actin was performed.Immunofluorescence was performed to study the expression and localization of E-cadherin and SMAD4 after TGFβ treatment (30 ng/ml for 24 hours) in control cell line and two Tgfbr2-mutant cell lines. Scale bars = 50 μm (fluorescent), scale bars = 100 μm (brightfield).


Figure 7Control cell line ( Tgfbr2wt ) ( A ) and two Tgfbr2 - mutant cell lines ( Tgfbr2mut1 and Tgfbr2mut2 ) ( B - C ) were treated with TGFβ at different time points , and cell growth ( MTT ) assays were performed . P value by t test is shown . NS , not significant . Subcutaneous tumors established from control cell line ( D ) and two Tgfbr2 - mutant cell lines ( E - F ) in C57Bl / 6 mice received rat IgG Mac48 ( control ) or 2G8 ( each 30 mg / kg 2x / week , n = 4 / group ) . Therapy started at day 12 post tumor cell injection ; mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are shown . Control cell line ( Tgfbr2wt ; G & I ) and the Tgfbr2 - mutant cell line ( Tgfbr2mut1 ; H & J ) were orthotopically implanted in C57Bl / 6 mice . Mice received rat IgG Mac48 or 2G8 , n = 9 - 10 / group for survival study ( each 30 mg / kg 2x / week ) . Control cell line ( Tgfbr2wt ; G & I ) and the Tgfbr2 - mutant cell line ( Tgfbr2mut1 ; H & J ) were orthotopically implanted in C57Bl / 6 mice . Mice were sacrificed when they became moribund . Tumors were harvested and weighed ( K - L ) . Immunohistochemistry with tumor samples from ( G - H ) for Ki67 ( M - O ) , P - STAT3 ( P - R ) , ZAP70 , and CD3 ( S - X ) was performed . ZAP70 is a common marker for NK cells and T cells and was stained with a brown chromogen , and CD3 is a specific T - cell marker and was stained with a blue chromogen . Brown signal was converted to red signal and blue signal was converted to green by ImageJ ( V ) ; therefore NK cells are highlighted as red ( ZAP70 + CD3 - ) and T cells are highlighted as yellow ( ZAP70 + CD3 + ) . Scale bars outside the magnification boxes = 50 μm , scale bars inside the magnification boxes = 10 μm . n = 4 / group , P values vs . control by t test are shown .

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Figure 7Control cell line (Tgfbr2wt) (A) and two Tgfbr2-mutant cell lines (Tgfbr2mut1 and Tgfbr2mut2) (B-C) were treated with TGFβ at different time points, and cell growth (MTT) assays were performed. P value by t test is shown. NS, not significant.Subcutaneous tumors established from control cell line (D) and two Tgfbr2-mutant cell lines (E-F) in C57Bl/6 mice received rat IgG Mac48 (control) or 2G8 (each 30 mg/kg 2x/week, n = 4/group). Therapy started at day 12 post tumor cell injection; mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are shown.Control cell line (Tgfbr2wt; G&I) and the Tgfbr2-mutant cell line (Tgfbr2mut1; H&J) were orthotopically implanted in C57Bl/6 mice. Mice received rat IgG Mac48 or 2G8, n = 9-10/group for survival study (each 30 mg/kg 2x/week).Control cell line (Tgfbr2wt; G&I) and the Tgfbr2-mutant cell line (Tgfbr2mut1; H&J) were orthotopically implanted in C57Bl/6 mice. Mice were sacrificed when they became moribund. Tumors were harvested and weighed (K-L).Immunohistochemistry with tumor samples from (G-H) for Ki67 (M-O), P-STAT3 (P-R), ZAP70, and CD3 (S-X) was performed. ZAP70 is a common marker for NK cells and T cells and was stained with a brown chromogen, and CD3 is a specific T-cell marker and was stained with a blue chromogen. Brown signal was converted to red signal and blue signal was converted to green by ImageJ (V); therefore NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 4/group, P values vs. control by t test are shown.


Figure 8Control shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2 - mutant cell line Tgfbr2mut1 . Cell lysates were harvested and western blotting for IL6RA and tubulin . 3D culture : control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . 3D culture : control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . Control IgG , 2G8 and IL - 6 neutralizing antibody ( each 100 ng / ml , n = 5 / group ) . Scale bars = 50 μm . P values by t test are shown ( C ) . Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2 - mutant cell lines in C57Bl / 6 mice received rat IgG Mac48 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 5 / group ) . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are indicated . Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2 - mutant cell lines in C57Bl / 6 mice received rat IgG Mac48 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 5 / group ) . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are indicated . Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2 - mutant cell lines in C57Bl / 6 mice received rat IgG Mac48 ( control ) , 2G8 or anti - mouse IL - 6 antibody MP5 - 20F3 ( each 30 mg / kg 2x / week , n = 5 / group ) . For NK cell depletion , prior to therapies , mice received 50 μg of control rabbit IgG or anti - Asialo - GM1 three days in a row . For maintenance , 25 μg of control rabbit IgG or anti - Asialo - GM1 were given 2X / week throughout the whole study . Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days . Tumor volume was measured twice per week . P values by t test are indicated . Immunohistochemistry with tumor samples from ( D ) for Ki67 ( G and K ) , P - STAT3 ( H and L ) , ZAP70 , and CD3 ( I - J and M - N ) was performed . NK cells are highlighted as red ( ZAP70 + CD3 - ) and T cells are highlighted as yellow ( ZAP70 + CD3 + ) by ImageJ ( N ) . Scale bars outside the magnification boxes = 50 μm , scale bars inside the magnification boxes = 10 μm . n = 5 / group , P values vs . control by t test are shown .

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Figure 8Control shRNA or two different shRNAs against IL6RA were used to knock down IL6RA in the Tgfbr2-mutant cell line Tgfbr2mut1. Cell lysates were harvested and western blotting for IL6RA and tubulin.3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture).3D culture: control or IL6RA knockdown Tgfbr2mut1 cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). Control IgG, 2G8 and IL-6 neutralizing antibody (each 100 ng/ml, n = 5/group). Scale bars = 50 μm. P values by t test are shown (C).Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.Subcutaneous tumors established from control and IL6RA knockdown Tgfbr2-mutant cell lines in C57Bl/6 mice received rat IgG Mac48 (control), 2G8 or anti-mouse IL-6 antibody MP5-20F3 (each 30 mg/kg 2x/week, n = 5/group). For NK cell depletion, prior to therapies, mice received 50 μg of control rabbit IgG or anti-Asialo-GM1 three days in a row. For maintenance, 25 μg of control rabbit IgG or anti-Asialo-GM1 were given 2X/week throughout the whole study. Therapy started at day 12 post tumor cell injection and mice were on therapy for 16 days. Tumor volume was measured twice per week. P values by t test are indicated.Immunohistochemistry with tumor samples from (D) for Ki67 (G and K), P-STAT3 (H and L), ZAP70, and CD3 (I-J and M-N) was performed. NK cells are highlighted as red (ZAP70+CD3-) and T cells are highlighted as yellow (ZAP70+CD3+) by ImageJ (N). Scale bars outside the magnification boxes = 50 μm, scale bars inside the magnification boxes = 10 μm. n = 5/group, P values vs. control by t test are shown.


Figure 1A . ( Top ) USP44 transcript levels across CD4 + T cell populations . Suspension of the lymph node and spleen cells of 6 - 8 week old female C57BL / 6 mice ( n = 5 experiments ) were obtained , and naïve ( CD62Lhigh / CD25 - ) CD4 + T cells and nTregs ( CD4 + / CD25high ) were obtained by FACS . The indicated effector T cell populations were generated by activating 1x10 ^ 6 naïve CD4 + T precursors with anti - CD3 and anti - CD28 antibodies ( 1 and 4 ug / ml , respectively ) for 4 days under the indicated in vitro skewing conditions ( described in the Methods section ) . After harvesting RNA by Trizol reagent and generating cDNA , the Usp44 mRNA levels expressed by the resulting Teff and iTreg , as well as freshly isolated nTreg cells were determined by qRT - PCR . ( Bottom ) Suspension of lymph node and spleen cells of 6 - 8 week old female C57BL / 6 mice were obtained , and naïve ( CD62LhighCD25 - ) CD4 + T cells and nTregs ( CD4 + / CD25high ) were sorted by FACS . iTregs were generated by activating 1x10 ^ 6 naïve CD4 + T cells with anti - CD3 and anti - CD28 antibodies ( 1 and 4 ug / ml , respectively ) for 4 days in the presence of IL - 2 ( 100u / ml ) and TGF - β ( 5ng / ml ) before the cells were harvested for SDS - PAGE . Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH . B . Naïve murine CD4 + T cells were obtained and differentiated into the iTreg lineage as in A . Expression levels of Usp44 and Foxp3 mRNA were assessed daily over time by qRT - PCR . C . Expression of USP44 and FOXP3 protein by T cells during in vitro differentiation was also measured . After 1 - 4 days of iTreg skewing , cell lysates were resolved by SDS PAGE following by immunoblotting with the indicated antibodies . D . Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα , IL - 6 , or IL - 1β ( each at a 10 ng / ml concentration ) , or LPS ( 1µg / ml ) for 24 hours . Treated Tregs were then harvested and USP44 protein levels were determined as above .

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Figure 1A. (Top) USP44 transcript levels across CD4+ T cell populations. Suspension of the lymph node and spleen cells of 6-8 week old female C57BL/6 mice (n= 5 experiments) were obtained, and naïve (CD62Lhigh/CD25-) CD4+ T cells and nTregs (CD4+/CD25high) were obtained by FACS. The indicated effector T cell populations were generated by activating 1x10^6 naïve CD4+ T precursors with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days under the indicated in vitro skewing conditions (described in the Methods section). After harvesting RNA by Trizol reagent and generating cDNA, the Usp44 mRNA levels expressed by the resulting Teff and iTreg, as well as freshly isolated nTreg cells were determined by qRT-PCR. (Bottom) Suspension of lymph node and spleen cells of 6-8 week old female C57BL/6 mice were obtained, and naïve (CD62LhighCD25-) CD4+ T cells and nTregs (CD4+/CD25high) were sorted by FACS. iTregs were generated by activating 1x10^6 naïve CD4+ T cells with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days in the presence of IL-2 (100u/ml) and TGF-β (5ng/ml) before the cells were harvested for SDS-PAGE. Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH.B. Naïve murine CD4+ T cells were obtained and differentiated into the iTreg lineage as in A. Expression levels of Usp44 and Foxp3 mRNA were assessed daily over time by qRT-PCR.C. Expression of USP44 and FOXP3 protein by T cells during in vitro differentiation was also measured. After 1-4 days of iTreg skewing, cell lysates were resolved by SDS PAGE following by immunoblotting with the indicated antibodies.D. Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα, IL-6, or IL-1β (each at a 10 ng/ml concentration), or LPS (1µg/ml) for 24 hours. Treated Tregs were then harvested and USP44 protein levels were determined as above.


Figure 2A . HEK293T cells were transfected with MYC - FOXP3 and FLAG - USP44 encoding expression constructs using Polyethylenimine . 48hrs post - transfection , cells were harvested , lysed , and anti - FLAG or anti - MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner . Co - IP ' ed proteins were subjected to SDS PAGE followed by immunoblot analysis . Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3 , respectively . B . Endogenous co - IP of USP44 and FOXP3 in murine iTregs . iTregs were generated as in Fig . 1 from naïve CD4 + T cells FACS isolated from pooled suspensions of the lymph node and spleen cells of wild type C57BL / 6 mice ( n = 2 - 3 / experiment ) . iTregs were lysed and key proteins were immunoprecipitated using either anti - USP44 ( right panel ) or anti - FOXP3 ( left panel ) antibody . Proteins pulled - down in this experiment were then resolved and analyzed by immunoblot using anti - FOXP3 or anti - USP44 antibodies . C . Endogenous co - IP of USP44 and FOXP3 in murine nTregs . nTregs ( CD4 + CD25high ) isolated by FACS were activated by anti - CD3 and anti - CD28 ( 1 and 4 ug / ml , respectively ) overnight in the presence of IL - 2 ( 100 U / ml ) . The cells were lysed and proteins were immunoprecipitated using either anti - Foxp3 ( left panel ) or anti - Usp44 ( right panel ) . Proteins pulled down in this experiment were then resolved and identified with the indicated antibodies . D . Naïve murine CD4 + T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl / fl CD4Cre + mice and that of their wild type littermates ( USP44fl / fl CD4Cre - mice ; n = 2 - 3 / group / experiment ) . iTreg cells were generated from these mice as described for Fig . 1 before incubation on a microscope slide pre - coated with poly - L lysine for 1h . Adhered cells were then fixed by PFA for 0 . 5 followed by blocking with 1 % BSA for 1h , then incubation with the specified antibodies . Representative confocal microscopy images ( 40X ) were visualized for endogenous USP44 ( red ) and FOXP3 Baxter et al ( ) . DAPI was used to visualize cell nuclei ( blue ) ; scale bar 50μm .

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Figure 2A. HEK293T cells were transfected with MYC-FOXP3 and FLAG-USP44 encoding expression constructs using Polyethylenimine. 48hrs post-transfection, cells were harvested, lysed, and anti-FLAG or anti-MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner. Co-IP'ed proteins were subjected to SDS PAGE followed by immunoblot analysis. Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3, respectively.B. Endogenous co-IP of USP44 and FOXP3 in murine iTregs. iTregs were generated as in Fig. 1 from naïve CD4+ T cells FACS isolated from pooled suspensions of the lymph node and spleen cells of wild type C57BL/6 mice (n=2-3/experiment). iTregs were lysed and key proteins were immunoprecipitated using either anti-USP44 (right panel) or anti-FOXP3 (left panel) antibody. Proteins pulled-down in this experiment were then resolved and analyzed by immunoblot using anti-FOXP3 or anti-USP44 antibodies.C. Endogenous co-IP of USP44 and FOXP3 in murine nTregs. nTregs (CD4+CD25high) isolated by FACS were activated by anti-CD3 and anti-CD28 (1 and 4 ug/ml, respectively) overnight in the presence of IL-2 (100 U/ml). The cells were lysed and proteins were immunoprecipitated using either anti-Foxp3 (left panel) or anti-Usp44 (right panel). Proteins pulled down in this experiment were then resolved and identified with the indicated antibodies.D. Naïve murine CD4+ T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl/fl CD4Cre+ mice and that of their wild type littermates (USP44fl/fl CD4Cre- mice; n=2-3/group/experiment). iTreg cells were generated from these mice as described for Fig. 1 before incubation on a microscope slide pre-coated with poly-L lysine for 1h. Adhered cells were then fixed by PFA for 0.5 followed by blocking with 1% BSA for 1h, then incubation with the specified antibodies. Representative confocal microscopy images (40X) were visualized for endogenous USP44 (red) and FOXP3 Baxter et al(). DAPI was used to visualize cell nuclei (blue); scale bar 50μm.


Figure 3A . HEK293T cells were transfected with expression constructs encoding MYC - FOXP3 , His - Ubiquitin , and either a wild type or catalytically inactive ( C282S , " cs " ) version of USP44 tagged with FLAG . Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis . Pull - down of His - labeled proteins using Ni - NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC . B . Effect of USP44 knock down on FOXP3 ubiquitination . Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells . After selected for 7d , cells were transfected with MYC - FOXP3 and His - Ubiquitin , then treated with 20 μM MG132 for 4 hrs before harvest and lysis . Ubiquitinated FOXP3 proteins were visualized as in A . C . Levels of polyubiquitinated FOXP3 in the presence or absence of USP44 . As in Fig 2b , murine iTregs were generated from naïve CD4 + precursors isolated from wild type and mice globally deficient in USP44 - / - mice ( n = 3 / group / experiment ) . Ubiquitinated proteins were extracted from iTreg lysates with anti - ubiquitin antibodies ( anti - Ubi ) prior to resolution by SDS PAGE and immunoblot analysis , probing for FOXP3 . D , E . HEK293T cells were transfected with plasmids encoding MYC - FOXP3 , FLAG - USP44 ( either wild type USP44 , " wt " or the C282S mutant ) . These cells were subsequently treated with cycloheximide ( CHX ; 5μg / ml ) for the indicated time points before harvested and cell lysis . FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti - MYC antibodies . F , USP44 knock down in human Tregs . Naïve CD4 + T cells ( CD4 + CD25 - CD45RA + ) were isolated from the peripheral blood of healthy donors by FACS , and iTreg were generated after 7 days of in vitro skewing conditions . As in Fig . 3b , shRNA constructs targeting USP44 were delivered by lentivirus . Here human iTregs received either shCK ( control ) , or one of two different shUSP44 constructs . Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot ( F ) G . USP44 knock down in human Tregs . Naïve CD4 + T cells ( CD4 + CD25 - CD45RA + ) were isolated from the peripheral blood of healthy donors by FACS , and iTreg were generated after 7 days of in vitro skewing conditions . As in Fig . 3b , shRNA constructs targeting USP44 were delivered by lentivirus . Here human iTregs received either shCK ( control ) , or one of two different shUSP44 constructs . FOXP3 expression was assessed by intracellular staining followed by flow cytometry ( G ) . H . Murine naïve CD4 T cells ( CD62L + / CD25 - / CD4 + ) from WT and USP44 - / - mice ( n = 2 - 3 / group / experiment ) were FACS purified and activated in vitro for four days by CD3 / CD28 cross - linking antibodies ( 1ug and 4ug / ml , respectively ) in the presence of 100 U / ml IL - 2 and the indicated dose of TGF - β . Down - regulation of FOXP3 was observed in USP44 - / - mice by intracellular immunostaining and flow cytometry .

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Figure 3A. HEK293T cells were transfected with expression constructs encoding MYC-FOXP3, His-Ubiquitin, and either a wild type or catalytically inactive (C282S, "cs") version of USP44 tagged with FLAG. Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis. Pull-down of His-labeled proteins using Ni-NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC.B. Effect of USP44 knock down on FOXP3 ubiquitination. Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells. After selected for 7d, cells were transfected with MYC-FOXP3 and His-Ubiquitin, then treated with 20 μM MG132 for 4 hrs before harvest and lysis. Ubiquitinated FOXP3 proteins were visualized as in A.C. Levels of polyubiquitinated FOXP3 in the presence or absence of USP44. As in Fig 2b, murine iTregs were generated from naïve CD4+ precursors isolated from wild type and mice globally deficient in USP44-/- mice (n=3/group/experiment). Ubiquitinated proteins were extracted from iTreg lysates with anti-ubiquitin antibodies (anti-Ubi) prior to resolution by SDS PAGE and immunoblot analysis, probing for FOXP3.D, E. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, FLAG-USP44 (either wild type USP44, "wt" or the C282S mutant). These cells were subsequently treated with cycloheximide (CHX; 5μg/ml) for the indicated time points before harvested and cell lysis. FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti-MYC antibodies.F, USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot (F)G. USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. FOXP3 expression was assessed by intracellular staining followed by flow cytometry (G).H. Murine naïve CD4 T cells (CD62L+/CD25-/CD4+) from WT and USP44-/- mice (n=2-3/group/experiment) were FACS purified and activated in vitro for four days by CD3/CD28 cross-linking antibodies (1ug and 4ug/ml, respectively) in the presence of 100 U/ml IL-2 and the indicated dose of TGF-β. Down-regulation of FOXP3 was observed in USP44-/- mice by intracellular immunostaining and flow cytometry.


Figure 4A . Reciprocal Co - IP of USP44 and USP7 . HEK293T cells were transfected with constructs encoding MYC - USP44 and FLAG - USP7 . These cells were lysed and pertinent factors were immunoprecipitated using either anti - FLAG or anti - MYC . Pulled - down proteins were observed by immunoblot analysis using either anti - MYC or anti - FLAG antibodies . B . Effect of USP44 on USP7 levels . HEK293T cells were transfected with MYC - FOXP3 . Cells also received combinations of FLAG - USP7 and varying doses of pLVX - USP44 encoding plasmids . FOXP3 was immunoprecipitated using anti - MYC , and immunoblots were analyzed using anti - FLAG , anti - USP44 or anti - MYC antibodies . C . Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination . HEK293T cells were transfected with plasmids encoding MYC - FOXP3 , either FLAG - USP44 or FLAG - USP7 or both , His - ubiquitin . Cells were treated with 20 μM MG132 for 4 hrs before being harvested , lysed , and incubated on Ni - NTA beads to pull down ubiquitinated FOXP3 , which was visualized by immunoblotting using antibodies specific for MYC . D . ( left panel ) iTreg were polarized from human naïve CD4 + T cells over 7 days ( n = 3 donors / experiment ) receiving control ( shCK ) , or shRNA knockdown constructs targeting USP44 ( shUSP44 - 1 , - 2 ) , or USP7 ( shUSP7 - 1 , - 2 ) , or both shRNA constructs . Freshly isolated nTregs were treated with either control or shRNA constructs . After transduction with lentivirus for 48 hours , cells were harvested and run SDS - PAGE gels . Protein levels were visualized by immunoblotting using antibodies specific for FOXP3 , USP44 , USP7 and GAPDH . Shown are representative findings from 3 independent experiments ( biological replicates ) .

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Figure 4A. Reciprocal Co-IP of USP44 and USP7. HEK293T cells were transfected with constructs encoding MYC-USP44 and FLAG-USP7. These cells were lysed and pertinent factors were immunoprecipitated using either anti-FLAG or anti-MYC. Pulled-down proteins were observed by immunoblot analysis using either anti-MYC or anti-FLAG antibodies.B. Effect of USP44 on USP7 levels. HEK293T cells were transfected with MYC-FOXP3. Cells also received combinations of FLAG-USP7 and varying doses of pLVX-USP44 encoding plasmids. FOXP3 was immunoprecipitated using anti-MYC, and immunoblots were analyzed using anti-FLAG, anti-USP44 or anti-MYC antibodies.C. Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, either FLAG-USP44 or FLAG-USP7 or both, His-ubiquitin. Cells were treated with 20 μM MG132 for 4 hrs before being harvested, lysed, and incubated on Ni-NTA beads to pull down ubiquitinated FOXP3, which was visualized by immunoblotting using antibodies specific for MYC.D. (left panel) iTreg were polarized from human naïve CD4+ T cells over 7 days (n=3 donors /experiment) receiving control (shCK), or shRNA knockdown constructs targeting USP44 (shUSP44-1, -2), or USP7 (shUSP7-1, -2), or both shRNA constructs. Freshly isolated nTregs were treated with either control or shRNA constructs. After transduction with lentivirus for 48 hours, cells were harvested and run SDS-PAGE gels. Protein levels were visualized by immunoblotting using antibodies specific for FOXP3, USP44, USP7 and GAPDH. Shown are representative findings from 3 independent experiments (biological replicates).


Figure 5A . Jurkat T cells were transfected with the indicated combinations and relative doses of expression constructs encoding MYC - FOXP3 , FLAG - USP44 along with an IL - 2 promoter - driven firefly luciferase reporter . After 8 hours stimulation with PMA / Inomycin , cells were lysed and luciferase activity , which was observed and normalized to renilla luciferase activity . B . Tregs ( CD4 + / CD25High ) were recovered from the lymph nodes and spleens of wild type C57BL / 6 ( WT ) and USP44 - / - mice by FACS purification . Cells were activated with anti - CD3 / CD28 agonist antibodies overnight , and RNA was isolated , and cDNA was generated from these samples for the measurement of several signature Treg - associated transcripts by qRT - PCR . The relative expression level of these by WT and KO - derived Tregs is shown . C , D . Analysis of the in vitro suppressive function of Tregs isolated from WT mice or age - and sex - matched USP44 - / - mice ( n = 3 / group / experiment ) . Tregs were mixed with CFSE - labeled responder naïve T cells from CD45 . 1 + C57BL / 6 mice ( n = 3 / experiment ) for three days . CFSE dilution in CD45 . 1 + T cells was observed by flow cytometry and the percent suppression of responder cell proliferation by the co - cultured Tregs was determined .

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Figure 5A. Jurkat T cells were transfected with the indicated combinations and relative doses of expression constructs encoding MYC-FOXP3, FLAG-USP44 along with an IL-2 promoter-driven firefly luciferase reporter. After 8 hours stimulation with PMA/Inomycin, cells were lysed and luciferase activity, which was observed and normalized to renilla luciferase activity.B. Tregs (CD4+/CD25High) were recovered from the lymph nodes and spleens of wild type C57BL/6 (WT) and USP44-/- mice by FACS purification. Cells were activated with anti-CD3/CD28 agonist antibodies overnight, and RNA was isolated, and cDNA was generated from these samples for the measurement of several signature Treg-associated transcripts by qRT-PCR. The relative expression level of these by WT and KO-derived Tregs is shown.C, D. Analysis of the in vitro suppressive function of Tregs isolated from WT mice or age- and sex-matched USP44-/- mice (n=3/group/experiment). Tregs were mixed with CFSE-labeled responder naïve T cells from CD45.1+ C57BL/6 mice (n=3/experiment) for three days. CFSE dilution in CD45.1+ T cells was observed by flow cytometry and the percent suppression of responder cell proliferation by the co-cultured Tregs was determined.


Figure 6A . Colitis was induced by intravenous coinjection of CD4 + CD25 - CD62Lhigh T cells and congenically distinct ( CD45 . 2 ) CD4 + CD25high Treg cells into Rag2 - / - mice ( n = 8 / group ; 1x106 and 2x105 cells per Rag2 - / - recipient , respectively ) . Changes in body weight over time were monitored and are expressed as a percentage of the original weight . B . Representative photomicrographs of the distal colon of Rag2 - / - mice after T cell transfer . 10 weeks after transfer , mice were euthanized , and colons were harvested , fixed in 10 % buffered formalin , and processed for standard H & E staining prior to histological analysis . ( i ) - ( iv ) present bright - field micrographs ( 100x ) . C . H & E slides were scored in a blinded fashion , and colon pathology was scored as described in the Methods Section . Shown are mean scores for each treatment group . D . Spleen , mesenteric and lamina propria lymph node cells were isolated from the mice 10 weeks after adoptive T cell transfer , and the number of Teff cells was determined by flow cytometry . E . Expression of FOXP3 protein and mRNA by adoptively transferred Tregs . Mesenteric lymph nodes were excised from the recipients of WT and USP44 - / - Tregs in A . FOXP3 protein was detected by intracellular immunostaining and flow cytometry ( left ) . Shown are events within the gate for transferred Tregs ( CD4 + / CD45 . 1 + ) . Transferred Tregs were also recovered from mesenteric lymph node cell suspensions by staining for the congenic marker and CD4 followed by FACS , and Foxp3 mRNA was measured in these recovered Tregs by RT - PCR ( right ) . F . Cell numbers of injected ( CD45 . 2 + ) Treg cells and FOXP3 + cells were determined by flow cytometry . G . CD4 + T cells were recovered from suspensions of lamina propria - infiltrating leukocytes . These lymphocytes were stimulated ex vivo by PMA / Ionomycin cocktail in the presence of brefeldin A and IFN - γ and IL - 17 production by these lymphocytes was analyzed by ELISA .

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Figure 6A. Colitis was induced by intravenous coinjection of CD4+CD25-CD62Lhigh T cells and congenically distinct (CD45.2) CD4+CD25high Treg cells into Rag2-/- mice (n=8/group; 1x106 and 2x105 cells per Rag2-/- recipient, respectively). Changes in body weight over time were monitored and are expressed as a percentage of the original weight.B. Representative photomicrographs of the distal colon of Rag2-/- mice after T cell transfer. 10 weeks after transfer, mice were euthanized, and colons were harvested, fixed in 10% buffered formalin, and processed for standard H&E staining prior to histological analysis. (i)- (iv) present bright-field micrographs (100x).C. H&E slides were scored in a blinded fashion, and colon pathology was scored as described in the Methods Section. Shown are mean scores for each treatment group.D. Spleen, mesenteric and lamina propria lymph node cells were isolated from the mice 10 weeks after adoptive T cell transfer, and the number of Teff cells was determined by flow cytometry.E. Expression of FOXP3 protein and mRNA by adoptively transferred Tregs. Mesenteric lymph nodes were excised from the recipients of WT and USP44-/- Tregs in A. FOXP3 protein was detected by intracellular immunostaining and flow cytometry (left). Shown are events within the gate for transferred Tregs (CD4+/CD45.1+). Transferred Tregs were also recovered from mesenteric lymph node cell suspensions by staining for the congenic marker and CD4 followed by FACS, and Foxp3 mRNA was measured in these recovered Tregs by RT-PCR (right).F. Cell numbers of injected (CD45.2+) Treg cells and FOXP3+ cells were determined by flow cytometry.G. CD4+ T cells were recovered from suspensions of lamina propria-infiltrating leukocytes. These lymphocytes were stimulated ex vivo by PMA/Ionomycin cocktail in the presence of brefeldin A and IFN-γ and IL-17 production by these lymphocytes was analyzed by ELISA.


Figure 7MC38 colon carcinoma , B16F10 melanoma cell lines were s . c . injected into the shaved flanks of Usp44fl / flFoxp3Cre + mice and their wild type ( Usp44fl / flFoxp3Cre - ) littermates ( 1x105 cells / mouse ; n = 5 - 9 mice / group ) . Tumor dimensions were measured every 2 days , and changes in the mean tumor volume over time were observed ( lower panels ) . Upper panels depict representative photographs of excised tumors . EL4 thymoma cell lines were s . c . injected into the shaved flanks of Usp44fl / flFoxp3Cre + mice and their wild type ( Usp44fl / flFoxp3Cre - ) littermates ( 1x105 cells / mouse ; n = 5 - 9 mice / group ) . Tumor dimensions were measured every 2 days , and changes in the mean tumor volume over time were observed ( lower panels ) . Upper panels depict representative photographs of excised tumors . D . 21 days post - implantation , proportions of FOXP3 + Tregs in the CD4 + compartment of all MC38 tumor - bearing mice in each experiment were found by flow cytometry . Suspensions of cells obtained from spleen , tumor - draining lymph nodes , and tumor - infiltrating leukocytes ( TILs ) were stained for surface CD4 and intracellular FOXP3 prior to analysis by flow cytometry . E , F . Frequencies of CD4 + and CD8 + cells producing the proinflammatory cytokine IFNγ were found among the tissues of tumor - bearing mice in the MC38 modelby intracellular cytokine staining Kralovics et al ( , 2005 ) and flow cytometry after ex vivo re - stimulation of recovered leukocytes by PMA and ionomycin in the presence of brefeldin A . G , H . The capacity of CD8 + cells for effector function in MC38 tumors was further assessed by measuring ( G ) production of TNFα by ICS and ( H ) intracellular straining of the cytotoxic mediator granzyme B followed by flow cytometry .

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Figure 7MC38 colon carcinoma, B16F10 melanoma cell lines were s.c. injected into the shaved flanks of Usp44fl/flFoxp3Cre+ mice and their wild type (Usp44fl/flFoxp3Cre-) littermates (1x105 cells/mouse; n=5-9 mice/group). Tumor dimensions were measured every 2 days, and changes in the mean tumor volume over time were observed (lower panels). Upper panels depict representative photographs of excised tumors.EL4 thymoma cell lines were s.c. injected into the shaved flanks of Usp44fl/flFoxp3Cre+ mice and their wild type (Usp44fl/flFoxp3Cre-) littermates (1x105 cells/mouse; n=5-9 mice/group). Tumor dimensions were measured every 2 days, and changes in the mean tumor volume over time were observed (lower panels). Upper panels depict representative photographs of excised tumors.D. 21 days post-implantation, proportions of FOXP3+ Tregs in the CD4+ compartment of all MC38 tumor-bearing mice in each experiment were found by flow cytometry. Suspensions of cells obtained from spleen, tumor-draining lymph nodes, and tumor-infiltrating leukocytes (TILs) were stained for surface CD4 and intracellular FOXP3 prior to analysis by flow cytometry.E, F. Frequencies of CD4+ and CD8+cells producing the proinflammatory cytokine IFNγ were found among the tissues of tumor-bearing mice in the MC38 modelby intracellular cytokine staining Kralovics et al(, 2005) and flow cytometry after ex vivo re-stimulation of recovered leukocytes by PMA and ionomycin in the presence of brefeldin A.G, H. The capacity of CD8+ cells for effector function in MC38 tumors was further assessed by measuring (G) production of TNFα by ICS and (H) intracellular straining of the cytotoxic mediator granzyme B followed by flow cytometry.


Figure 1Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( A ) ZIKV titers in Sw . 71 and HPC trophoblasts . * p < 0 . 05 by One - way ANOVA . Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( B ) IFNα is not highly induced in response to ZIKV infection in trophoblast cells . IFNα mRNA expression was inhibited in the first 24 h . p . i . and maintained at low level during ZIKV infection . Difference not significant ( n . s . ) by One - way ANOVA . Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( C ) ZIKV infection induced a time - dependent increase in IFNβ mRNA expression in Sw . 71 and HPC . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA . Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . ( D ) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay . Supernatants were collected from ZIKV - infected and control trophoblast cultures at different times , and IFNβ protein secretion was quantified by ELLA assay . Note the increase of secreted IFNβ in the ZIKV - infected supernatant in a time - dependent manner . * * * * p < 0 . 0001 by Student ' s t - test against individual time point NT control .

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Figure 1Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (A) ZIKV titers in Sw.71 and HPC trophoblasts. *p < 0.05 by One-way ANOVA.Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (B) IFNα is not highly induced in response to ZIKV infection in trophoblast cells. IFNα mRNA expression was inhibited in the first 24 h.p.i. and maintained at low level during ZIKV infection. Difference not significant (n.s.) by One-way ANOVA.Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA.Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. (D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.


Figure 2Sw . 71 ( left panel ) and HPC cells ( right panel ) were treated with IFNβ to detect ISG20 mRNA expression . Data represent as mean ± SEM ; n = 3 biological replicates . NT , no treatment group ; HPC , human primary culture . ( A ) Sw . 71 and HPC cells were treated with different doses of IFNβ ( 3 , 30 , 300 ng / ml ) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT - PCR . Note the increase of ISG20 mRNA expression in a dose - dependent manner . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA . ( B ) Sw . 71 and HPC cells were treated with 300ng / ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT - PCR . Note the increase of ISG20 mRNA expression in a time - dependent manner . * p < 0 . 05 by One - way ANOVA . ( C ) Sw . 71 ( left panel ) and HPC cells ( right panel ) were treated with IFNβ to detect ISG20 protein expression . ( D ) Sw . 71 and HPC cells were treated with 300ng / ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot . Note that there was no ISG20 protein expression in the no treatment group in both Sw . 71 and HPC , only after IFNβ treatment , ISG20 protein expression exhibited a time - dependent manner . β - actin served as a loading control .

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Figure 2Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA expression. Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture. (A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA. (B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA. (C) Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.


Figure 3 ( B ) No ISG20 protein expression in ISG20 - / - Sw . 71 in ZIKV infection . Sw . 71 and ISG20 - / - Sw . 71 cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media for 48 h , and proteins were collected for western blot analysis . β - actin served as a loading control . ( C ) Higher ZIKA titer was shown in ISG20 - / - Sw . 71 cells . Sw . 71 and ISG20 - / - Sw . 71 cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media for 48 h , and RNA was collected for determining the viral titer and gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - test . ( D ) More viral shedding in ZIKV - infected ISG20 - / - Sw . 71 cell culture supernatant . Supernatant from ZIKV - infected Sw . 71 and ISG20 - / - Sw . 71 cells were collected and plaque assay was performed using Vero cells . A representative plaque assay picture is presented . Note that more plaques were formed in Vero cells by incubating with ZIKV - infected ISG20 - / - Sw . 71 cell culture supernatant . Scale bar = 130μm . ( E ) Plaques were counted and plaque forming - unit ( pfu ) ratio was calculated for the comparison of the viral titers . Data represent as mean ± SEM ; n = 3 biological replicates . * p < 0 . 05 by Student ' s t - test .

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Figure 3(B) No ISG20 protein expression in ISG20-/- Sw.71 in ZIKV infection. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and proteins were collected for western blot analysis. β-actin served as a loading control.(C) Higher ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.(D) More viral shedding in ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Supernatant from ZIKV-infected Sw.71 and ISG20-/- Sw.71 cells were collected and plaque assay was performed using Vero cells. A representative plaque assay picture is presented. Note that more plaques were formed in Vero cells by incubating with ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Scale bar=130μm. (E) Plaques were counted and plaque forming-unit (pfu) ratio was calculated for the comparison of the viral titers. Data represent as mean ± SEM; n=3 biological replicates. *p < 0.05 by Student's t-test.


Figure 4 ( B ) ISGs mRNA expression stimulated by IFNβ in Sw . 71 and ISG20 - / - Sw . 71 trophoblast cells . Sw . 71 and ISG20 - / - Sw . 71 cells were treated with 30 ng / ml IFNβ for 24 h , and RNA was collected for determining the ISGs gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 by Student ' s t - test against control . ( D ) IFNβ pre - treatment significantly prevented trophoblast cells from ZIKV infection , however , this protection was evidently attenuated due to lack of ISG20 . Sw . 71 and ISG20 - / - Sw . 71 cells were pre - treated with or without 30 ng / ml IFNβ for 24 h , followed by ZIKV infection ( MOI = 2 ) for 1 h and refreshed with regular media for 24 h , and RNA was collected to determine the viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - test .

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Figure 4(B) ISGs mRNA expression stimulated by IFNβ in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and RNA was collected for determining the ISGs gene expression by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test against control.(D) IFNβ pre-treatment significantly prevented trophoblast cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71 cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.


Figure 5 ( D ) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells . Supernatants from negative and positive clones were collected and added to ISG20 - / - Sw . 71 trophoblast cells together with ZIKA virus ( MOI = 2 ) for 1 h , followed by refreshing with new growth media for 48 h . RNA was then collected to determine viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 3 biological replicates ; * * p < 0 . 01 by Student ' s t - test . ( E ) Recombinant ISG20 - Fc degrades ZIKA viral RNA and HSV2 viral DNA . 50ng purified viral RNA ( ZIKV ) or DNA ( HSV - 2 ) were incubated with increasing concentrations of rISG20 - Fc ( 5 , 50 , 500 ng ) in the presence of RNase inhibitor for 90min at 37 ° C followed by quantification of viral titers by qRT - PCR , and agarose gel was used to evaluate the RNA degradation by electrophoresis . The representative picture of agarose gel is presented . rISG20 - Fc was able to degrade both ZIKV RNA and HSV - 2 DNA in a dose dependent manner ; however , rISG20 - Fc was more efficient in degrading viral RNA than DNA . Data represent as mean ± SEM ; n = 3 biological replicates ; * * p < 0 . 01 . * * * p < 0 . 001 and * * * * p < 0 . 0001 by One - way ANOVA .

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Figure 5(D) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast cells together with ZIKA virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h. RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.(E) Recombinant ISG20-Fc degrades ZIKA viral RNA and HSV2 viral DNA. 50ng purified viral RNA (ZIKV) or DNA (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA and HSV-2 DNA in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.


Figure 6 ( B ) Macroscopic evaluation of the pregnant uterus and fetus . Compare to the fetal death and reabsorptions in ZIKV - infected mice , rISG20 - Fc treatment rescued this phenotype by decreasing pregnancy loss ( resorption ) .

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Figure 6(B) Macroscopic evaluation of the pregnant uterus and fetus. Compare to the fetal death and reabsorptions in ZIKV-infected mice, rISG20-Fc treatment rescued this phenotype by decreasing pregnancy loss (resorption).


Figure 7 ( A ) rISG20 - Fc treatment alleviates maternal viral burden . Note that rISG20 - Fc treatment significantly decreased the maternal serum viral titers . Data represent as mean ± SEM ; n = 3 mice for control PBS group , and n = 4 mice for treatment groups ; * p < 0 . 05 by Student ' s t - test . ( B ) rISG20 - Fc treatment reduces ZIKV titers in the fetal brain . Although there was no difference of ZIKV titer in the placenta , at the fetal side , there was a significant decrease of viral titer in the fetal brain , suggesting rISG20 - Fc can block viral transmission from placenta to fetus . Data represent as mean ± SEM ; n = 4 for each group , and 3 - 4 placentas / fetal brains from every mouse were analyzed ; * p < 0 . 05 by Student ' s t - test . ( ( C ) rISG20 - Fc treatment decreases Eotaxin significantly in maternal circulation . Data represent as mean ± SEM ; n = 2 for PBS control group , and n = 3 for every treatment group ; * p < 0 . 05 by One - way ANOVA ; difference not significant for IL - 9 and IFNγ by One - way ANOVA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. ( (C) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA.


Figure 1A . Rabies virus infection kinetics in the central nervous system of infected mice . Tha - RABV load ( copies / µg of RNA ) detected in the thoracolumbar spinal cord , in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time - points post - infection ( n = 4 per time - point ) . nd : not detected . Horizontal lines indicate the median . B . Follow - up of the clinical signs of mice under different treatments . Heatmaps were established based on a progressive 0 - 7 clinical score scale ( 0 : no apparent changes | 1 : ruffled fur | 2 : slow movement , hind limb ataxia | 3 : apathy | 4 : monoplegia | 5 : hind limb paralysis , tremors | 6 : paralysis , conjunctivitis / keratitis , urine staining of the haircoat of the perineum | 7 : death ) . Each line represents one animal throughout time . C . Body weight progression of mice under different treatments . Mice were weighted on a daily basis during the treatment administration , and then twice a week up to 100 days post - infection . All the mice were equipped with iPrecio pumps weighting ca . 3 . 3 g . Each line represents one animal throughout time . D - E . Mouse behavioral testing . The tested animals were non - infected ( n = 3 mice with iPrecio pump + n = 4 age - related mice without iPrecio pump ) , infected non - treated ( n = 5 ) , infected and treated at 7 dpi ( n = 5 , mice # 9 to # 13 ) , and infected and treated at 8 dpi ( n = 5 , mice # 24 to # 28 ) . ( D ) Open field test performed at 7 dpi ( prodromal phase ) and after the remission of clinical signs for the survivors ( 80 dpi ) . ( E ) Rotarod motor performance at 7 dpi ( prodromal phase ) , 8 dpi ( symptomatic phase ) and after the remission of clinical signs for the survivors ( 20 , 30 and 60 dpi ) . Horizontal lines indicate the median . Kruskal - Wallis followed by the Dunn ' multiple comparisons test ( 7 and 8 days post - infection ) and Mann - Whitney test ( 20 , 30 and 60 days post - infection ) . The rotarod performance presented a negative correlation ( Spearman r = - 0 . 768 ) with the clinical score at 8 dpi . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = NI : non - infected ( n = 5 ) ; NT : infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/µg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time-points post-infection (n=4 per time-point). nd: not detected. Horizontal lines indicate the median.B. Follow-up of the clinical signs of mice under different treatments. Heatmaps were established based on a progressive 0-7 clinical score scale (0: no apparent changes | 1: ruffled fur | 2: slow movement, hind limb ataxia | 3: apathy | 4: monoplegia | 5: hind limb paralysis, tremors | 6: paralysis, conjunctivitis/ keratitis, urine staining of the haircoat of the perineum | 7: death). Each line represents one animal throughout time.C. Body weight progression of mice under different treatments. Mice were weighted on a daily basis during the treatment administration, and then twice a week up to 100 days post-infection. All the mice were equipped with iPrecio pumps weighting ca. 3.3 g. Each line represents one animal throughout time.D-E. Mouse behavioral testing. The tested animals were non-infected (n=3 mice with iPrecio pump + n=4 age-related mice without iPrecio pump), infected non-treated (n=5), infected and treated at 7 dpi (n=5, mice #9 to #13), and infected and treated at 8 dpi (n=5, mice #24 to #28). (D) Open field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase) and after the remission of clinical signs for the survivors (20, 30 and 60 dpi). Horizontal lines indicate the median. Kruskal-Wallis followed by the Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann-Whitney test (20, 30 and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman r = -0.768) with the clinical score at 8 dpi. *P<0.05, **P<0.01, ***P<0.001. Treatments description= NI: non-infected (n=5); NT: infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).


Figure 2A . Cumulative Kaplan - Meier survival curves of mice treated at different time - points post - infection . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) . B . Cumulative Kaplan - Meier survival curves of all the treated animals that presented with clinical signs ( n = 31 , all treatments combined ) . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * * * P < 0 . 0001 . C . Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups . D . Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi ( LOD : limit of detection = 0 . 02 µg / mL ) . E . Viral load in the brain of mice from different experimental groups . The samples were either collected at the time of death , or at 100 dpi for the survivors . Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : ( F ) IFN - β1 , ( G ) ISG15 , ( H ) Mx1 , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene . Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : , ( I ) IFN - γ , ( J ) CXCL10 , ( K ) CCL5 , ( L ) IL - 6 , ( M ) TNF - α and ( N ) IL - 1β , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Cumulative Kaplan-Meier survival curves of mice treated at different time-points post-infection. Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. **P<0.01, ***P<0.001. Treatments description= infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).B. Cumulative Kaplan-Meier survival curves of all the treated animals that presented with clinical signs (n=31, all treatments combined). Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. ****P<0.0001.C. Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups.D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors.Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: , (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.


Figure 3A - R . Histopathological analysis of the brain in Tha - RABV - infected non - treated mice ( at 11 dpi ) , versus surviving mice ( at 100 dpi ) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail . The detection of Tha - RABV , using immunohistochemistry ( IHC ) on brain sections , revealed a heterogeneous invasion between the non - treated mice , ranging from extensive ( A ) to multifocal ( B ) . Higher magnification of extensive invasion in the cortex ( F ) , hippocampus ( G ) , brain stem ( H ) and cerebellum ( I ) . ( C , J , K , L , M ) In contrast , even if a non - specific IHC signal was sometimes observed , no virus was detected on the brain sections of treated mice at 100 days post - infection . Only rare signs of neuro - inflammation were observed , as perivascular cuffing ( D ) ( 2 / 7 mice ) and minimal meningitis ( E ) ( 3 / 7 mice ) . Data information : A - C and F - M : Tha - RABV IHC D - E : HE staining . F - I are high magnifications of A ; J - M are high magnifications of C ( N , O ) Microglial cells were in general not reactive , but ( P ) small foci of reactive microglia ( hyperplasia with thicker cell processes ) could randomly be detected . As for control non - treated mice , reactive microglial cells ( Q ) ( 2 / 7 mice ) or resting microglia ( R ) were observed in treated mice . Representative images . Data information : N - R : Iba1 IHC to detect microglial cells O - P are high magnifications of N .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C(N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images. Data information: N-R: Iba1 IHC to detect microglial cells O-P are high magnifications of N.


Figure 1 ( B ) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . unparasitized hosts . Day - 4 of last instar larvae were used for this assay . Test larvae were parasitized by the wasp on day 0 of last instar . ( C ) Survival of armyworm larvae injected with PBS , unparasitized , and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . * * P < 0 . 01 vs . PBS . Test larvae were exposed to the heat stress 4 h after injection of each sample . ( D ) Time - dependent survival of armyworm larvae injected with 0 . 8 µmol / larva N - acetyl - L - tyrosine ( NAT ) : post NAT - injection 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . * * P < 0 . 01 vs . zero time . ( E ) Dose - dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . Injection of more than 10 nmol NAT / larva caused a significant difference compared with zero ( log rank test using R version 3 . 2 . 2 ) . ( F ) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . PBS . Each chemical ( 0 . 5 µmol / larva ) was dissolved in PBS ; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. unparasitized hosts. Day-4 of last instar larvae were used for this assay. Test larvae were parasitized by the wasp on day 0 of last instar.(C) Survival of armyworm larvae injected with PBS, unparasitized, and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. PBS. Test larvae were exposed to the heat stress 4 h after injection of each sample.(D) Time-dependent survival of armyworm larvae injected with 0.8 µmol/larva N-acetyl-L-tyrosine (NAT): post NAT-injection 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. zero time.(E) Dose-dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min (data are means ± SEM; n=12). Injection of more than 10 nmol NAT/larva caused a significant difference compared with zero (log rank test using R version 3.2.2).(F) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. PBS. Each chemical (0.5 µmol/larva) was dissolved in PBS; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine.


Figure 2 ( A ) HPLC chromatogram and L - MS spectrogram of N - acetyltyrosine purified from pooled human serum ( # 12181201 , Cosmo Bio Co . , Japan ) . The peak fraction purified by HPLC was confirmed to be N - acetyltyrosine by LC - MS ( inserted spectrogram ) . ( B ) NAT concentrations in the serum of mice ( Slc : ddY , 18 wks old , male ) exposed to heat ( 40oC ) and restraint stress ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time . ( C ) Effects of NAT on corticosterone concentrations in the serum of mice ( Slc : ddY , 9 - 10 wks old , male ) exposed to restraint stress for 60 min ( data are means ± SEM ; n = 6 ) . * * P < 0 . 01 vs . without stress . Test mice drank 1 . 5 mg / ml NAT solution for 24 h before exposure to stress . ( D ) Effect of NAT on stress - induced peroxidized lipid in the serum of mice ( Slc : ddY , 9 wks old , male ) forced to swim for 60 min ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . PBS .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) HPLC chromatogram and L-MS spectrogram of N-acetyltyrosine purified from pooled human serum (#12181201, Cosmo Bio Co., Japan). The peak fraction purified by HPLC was confirmed to be N-acetyltyrosine by LC-MS (inserted spectrogram).(B) NAT concentrations in the serum of mice (Slc:ddY, 18 wks old, male) exposed to heat (40oC ) and restraint stress (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. zero time.(C) Effects of NAT on corticosterone concentrations in the serum of mice (Slc:ddY, 9-10 wks old, male) exposed to restraint stress for 60 min (data are means ± SEM; n=6). ** P<0.01 vs. without stress. Test mice drank 1.5 mg/ml NAT solution for 24 h before exposure to stress.(D) Effect of NAT on stress-induced peroxidized lipid in the serum of mice (Slc:ddY, 9 wks old, male) forced to swim for 60 min (data are means ± SEM; n=10). *P<0.05 vs. PBS.


Figure 3 ( A ) Survival of Drosophila melanogaster ( W1118 ) larvae fed with 2 µmol NAT / g - diet 2 d after heat stress at 38 . 5oC for 60 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . zero time . ( B ) Effects of NAT feeding on expression of antioxidant enzymes , catalase , SOD1 , and SOD2 ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . zero time . ( C ) Representative images of nuclei ( left ) and FoxO ( middle ) in the fat body of Drosophila larvae before and after NAT feeding for 12 h . White signals in the merged image ( right ) show the overlap of both signals . Scale bar : 50 µm . ( D ) Relative whiteness intensities before and after NAT feeding ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . zero time . ( E ) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae ( data are means ± SEM ; n = 8 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time . ( F ) Effects of N - acetyltransferase ( Dat ) knockdown in gut enteroendocrine cells ( TK - Gut - Gal4 > UAS - dsDat ) on survival of Drosophila larvae ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . without NAT .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm. (D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time. (E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.