Cell death analysis by Celigo of PI - positive A375 treated with the indicated agents for 12 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 .

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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death analysis by Celigo of PI-positive A375 treated with the indicated agents for 12 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


Cell viability analysis using CellTiter - Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell viability analysis using CellTiter-Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL, **** P ≤ 0.0001.


Cell death analysis by Celigo of PI - positive MeWo ( G ) , DO4 ( H ) or SW - 872 ( I ) cells . Cells were treated with the indicated agents for 24 hrs ( MeWO and SW - 872 ) or 12 hrs ( DO4 ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death analysis by Celigo of PI-positive MeWo (G), DO4 (H) or SW-872 (I) cells. Cells were treated with the indicated agents for 24 hrs (MeWO and SW-872) or 12 hrs (DO4). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


Cell death analysis by Celigo of PI - positive HS - ES - 2R Cells were treated with the indicated agents for 48 hrs ( HS - ES - 2R ) DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * P ≤ 0 . 001 .

O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death analysis by Celigo of PI-positive HS-ES-2R Cells were treated with the indicated agents for 48 hrs (HS-ES-2R) DMSO (Unt), zVAD (10 µM), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.


Cell death analysis by Celigo of PI - positive HS - SY - II cells . Cells were treated with the indicated agents for 24 hrs ( HS - SY - II ) . DMSO ( Unt ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * P ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell death analysis by Celigo of PI-positive HS-SY-II cells. Cells were treated with the indicated agents for 24 hrs (HS-SY-II). DMSO (Unt), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.


Cell Death analysis by IncuCyte Zoom of CellTox Green - positive MET1 ( L ) , MET2 ( M ) and IC8 ( N ) cells . Cells were treated with the indicated agents for 40 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 6 , and statistical analysis was performed with a one - way Anova , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell Death analysis by IncuCyte Zoom of CellTox Green-positive MET1 (L), MET2 (M) and IC8 (N) cells. Cells were treated with the indicated agents for 40 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=6, and statistical analysis was performed with a one-way Anova, *** P ≤ 0.001, **** P ≤ 0.0001.


Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, * P ≤ 0.05, *** P ≤ 0.001.


The addition of SM to the standard - of - care treatment ILP - TNF / Mel significantly prolonged survival compared to either modality alone ( n = 6 rats per cohort ) . Statistical analysis was performed with a log - rank test , * * P ≤ 0 . 01 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The addition of SM to the standard-of-care treatment ILP-TNF/Mel significantly prolonged survival compared to either modality alone (n = 6 rats per cohort). Statistical analysis was performed with a log-rank test, ** P ≤ 0.01.


Median TNF / Mel is 0 . 4472 and TNF / Mel / SM is 1 . 154 . TNF / Mel 25th percentile limit 0 . 02887 and 75th percentile limit 0 . 7627 . TNF / Mel / SM 25th percentile limit 1 . 136 and 75th percentile limit 1 . 67 . TNF / Mel Min error bar - 0 . 2389 and Max 0 . 7687 . TNF / SM / Mel Min error bar 1 . 12 and Max 1 . 813 . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * P ≤ 0 . 01 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Median TNF/Mel is 0.4472 and TNF/Mel/SM is 1.154. TNF/Mel 25th percentile limit 0.02887 and 75th percentile limit 0.7627. TNF/Mel/SM 25th percentile limit 1.136 and 75th percentile limit 1.67. TNF/Mel Min error bar -0.2389 and Max 0.7687. TNF/SM/Mel Min error bar 1.12 and Max 1.813. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ** P ≤ 0.01.


DEVDase activity assay of parental BN175 cells and drug - tolerant tumour cell lines isolated from ILP - TNF / Mel / SM treated animals ( 124 , 133 , 136 ) , treated with the indicated drugs for 6 hrs ( n = 4 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

DEVDase activity assay of parental BN175 cells and drug-tolerant tumour cell lines isolated from ILP-TNF/Mel/SM treated animals (124, 133, 136), treated with the indicated drugs for 6 hrs (n = 4 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml).


Cell viability analysis using CellTiter - Glo of BN175 parental and 124 , 133 and 136 cells treated with the indicated drugs for 24 hrs ( n = 3 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Statistical analysis was performed with a one - way Anova , ns = not significant .

O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Cell viability analysis using CellTiter-Glo of BN175 parental and 124, 133 and 136 cells treated with the indicated drugs for 24 hrs (n = 3 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Statistical analysis was performed with a one-way Anova, ns=not significant.


Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 KO cells treated with the indicated agents for 24 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , SM ( 100 ng / ml ) and Riboxxol ( 1 µg / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 KO cells treated with the indicated agents for 24 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), SM (100 ng/ml) and Riboxxol (1 µg/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP with TNF / Mel / SM significantly increased infiltration by CD3 + lymphocytes ( B ) and CD3 - CD161 + NK cells ( C ) .

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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C).


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . While ILP treatment with TNF / Mel significantly reduced Granzyme B positive NK cells ( D ) , the numbers of Granzyme B positive NK cells were restored to normal levels upon co - treatment with SM ( TNF / Mel / SM ) .

O O 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-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM).


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . Importantly , ILP - mediated treatment with TNF / Mel / SM significantly increased the numbers of CD3 + CD8 + cytotoxic T cells ( E ) .

O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E).


Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP - TNF / Mel / SM significantly upregulated Granzyme B in CD3 + CD8 + CD161 - cytoxtoxic T

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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O

Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cells


ILP - TNF / Mel / SM significantly reduced infiltration by CD3 + CD4 + Foxp3 - T cells whilst ( G ) increasing infiltrations by CD3 + CD4 + Foxp3 + T regulatory cells ( H ) .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H).


A non - significant increase in T regulatory cell ICOS expression was also noted ( I ) .

A non-significant increase in T regulatory cell ICOS expression was also noted (I).


Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O

Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.


The addition of either anti - CTLA - 4 or anti - PD - 1 antibodies prolonged survival in comparison to ILP - TNF / Mel / SM alone ( n = 6 animals per cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 .

O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

The addition of either anti-CTLA-4 or anti-PD-1 antibodies prolonged survival in comparison to ILP-TNF/Mel/SM alone (n = 6 animals per cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.


Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - CTLA - 4 antibodies led to a significant increase in CD4 + Foxp3 - helper T cells ( C ) accompanied by a depletion of CD4 + Foxp3 + regulatory T cells ( D ) . SM represents Birinapant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). SM represents Birinapant.


Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - PD - 1 antibodies led to an increase in Granzyme B ( GzmB ) expression on NK cells ( E ) . SM represents Birinapant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-PD-1 antibodies led to an increase in Granzyme B (GzmB) expression on NK cells (E). SM represents Birinapant.


Tumour volumes at the time of surgery following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * * * , P ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Tumour volumes at the time of surgery following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ****, P ≤ 0.0001.


Individual growth curves following surgery indicated improved control following ILP - TNF / Mel / SM ( n = 6 animals per cohort ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Individual growth curves following surgery indicated improved control following ILP-TNF/Mel/SM (n=6 animals per cohort).


Tumour volume on re - challenge with BN175 sarcoma on contralateral limb of long - term survivors following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . Unless otherwise stated , SM represents Birinapant . Error bars represent S . D .

O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O

Tumour volume on re-challenge with BN175 sarcoma on contralateral limb of long-term survivors following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). Unless otherwise stated, SM represents Birinapant. Error bars represent S.D.


ILP - TNF / Mel / SM significantly delayed growth leading to prolonged survival on re - challenge ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

ILP-TNF/Mel/SM significantly delayed growth leading to prolonged survival on re-challenge (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.


Westernblot of producer cell lysates and concentrated reporter particles .

Westernblot of producer cell lysates and concentrated reporter particles.


BHK cells were transfected with either human ACE2 or empty vector , subsequently infected with VSV - reporter particles pseudotyped with chimeric spikes , luciferase was measured and normalized to no pseudotype as a readout for cell entry . Shown are the data for 3 replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BHK cells were transfected with either human ACE2 or empty vector, subsequently infected with VSV-reporter particles pseudotyped with chimeric spikes, luciferase was measured and normalized to no pseudotype as a readout for cell entry. Shown are the data for 3 replicates.


Primate cells , were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primate cells, were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.


human cells were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

human cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.


bat cells were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates .

bat cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.


Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated . Shown are data from 3 replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated. Shown are data from 3 replicates.


Expression and pseudotype incorporation of SARS - S - 2019 - nCoV RBD chimeras .

Expression and pseudotype incorporation of SARS-S-2019-nCoV RBD chimeras.


Pseudotypes were used to infect cells expressing hACE2 , hDPP4 , hAPN , or empty vector , without protease treatment .

O O O O O O O O O O O O O O O O O O O O O

Pseudotypes were used to infect cells expressing hACE2, hDPP4, hAPN, or empty vector, without protease treatment.


VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector . Shown are data for 3 replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector. Shown are data for 3 replicates.


Westernblot of producer cell lysates and concentrated pseudotyped particles .

Westernblot of producer cell lysates and concentrated pseudotyped particles.


Westernblot of producer cell lysates and concentrated pseudotypes particles .

Westernblot of producer cell lysates and concentrated pseudotypes particles.


Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh - 7 . 5 cells . Shown are data for 3 replicates .

Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh-7.5 cells. Shown are data for 3 replicates.


Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2 . Shown are data for 3 replicates .

Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2. Shown are data for 3 replicates.


c , Log normalised expression of markers for epithelial ( EPCAM ) , mature luminal epithelial ( ESR1 ) , myoepithelial ( KRT5 , KRT14 and ACTA2 ) and proliferating cancer cells ( MKI67 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, Log normalised expression of markers for epithelial (EPCAM), mature luminal epithelial (ESR1), myoepithelial (KRT5, KRT14 and ACTA2) and proliferating cancer cells (MKI67).


e , Log normalised expression of markers for fibroblasts ( PDGFRB , THY1 , COL1A1 , ITGB1 and S100A4 ) , endothelial cells ( PECAM1 ) , T - cells ( CD3D ) , CD8 T cells ( CD8A ) , T - regulatory cells ( FOXP3 ) , B - cells ( MS4A1 ) , myeloid cells ( CD68 ) and plasma cells ( JCHAIN ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Log normalised expression of markers for fibroblasts (PDGFRB, THY1, COL1A1, ITGB1 and S100A4), endothelial cells (PECAM1), T-cells (CD3D), CD8 T cells (CD8A), T-regulatory cells (FOXP3), B-cells (MS4A1), myeloid cells (CD68) and plasma cells (JCHAIN).


c , Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells .

c, Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells.


b , Candidate transcriptional drivers of each CAF and PVL subset . Violin plots showing the log normalised gene expression ( left ) of the TF and its respective AUC regulon activity ( right ) . TFs ZEB1 and FOXP1 enriched in myofibroblast - like CAFs , EGR2 and TCF7L2 enriched in inflammatory - like CAFs , MEF2C enriched in PVL cells and NR2F2 enriched in immature - PVL cells .

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b, Candidate transcriptional drivers of each CAF and PVL subset. Violin plots showing the log normalised gene expression (left) of the TF and its respective AUC regulon activity (right). TFs ZEB1 and FOXP1 enriched in myofibroblast-like CAFs, EGR2 and TCF7L2 enriched in inflammatory-like CAFs, MEF2C enriched in PVL cells and NR2F2 enriched in immature-PVL cells.


c - d , Immunofluorescence of cultured human CAFs ( c ) and PVL cells ( d ) from passage 8 , staining for CD34 ( CAFs ) , ⍺ - SMA ( myCAFs and PVL cells ) , CD146 ( PVL cells ) and CD36 ( imPVL cells ) .

c-d, Immunofluorescence of cultured human CAFs (c) and PVL cells (d) from passage 8, staining for CD34 (CAFs), ⍺-SMA (myCAFs and PVL cells), CD146 (PVL cells) and CD36 (imPVL cells).


e - f , Quantitative analysis of collagen abundance ( e ) and orientation ( f ) using second harmonic generation ( SHG ) from cellular derived matrices from stromal subsets and representative images multiphoton SHG images ( n = 3 biological replicates ) . All SHG intensity values within each replicate were normalised to the SHG intensity of the myCAFs . Error bars represent standard deviation . Statistical significance for collagen abundance ( e ) was determined using unpaired two - tailed Student ' s t test with equal standard deviation with p ‐ values denoted by asterisks : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . After normalization of the orientation peak distributions ( f ) , statistical significant was determined using a Kruskal - Wallis test with Dunn ' s post - hoc multiple comparisons test ( p value < 0 . 0001 ) .

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e-f, Quantitative analysis of collagen abundance (e) and orientation (f) using second harmonic generation (SHG) from cellular derived matrices from stromal subsets and representative images multiphoton SHG images (n = 3 biological replicates). All SHG intensity values within each replicate were normalised to the SHG intensity of the myCAFs. Error bars represent standard deviation. Statistical significance for collagen abundance (e) was determined using unpaired two-tailed Student's t test with equal standard deviation with p‐values denoted by asterisks: *p < 0.05, **p < 0.01 and ***p < 0.001. After normalization of the orientation peak distributions (f), statistical significant was determined using a Kruskal-Wallis test with Dunn's post-hoc multiple comparisons test (p value < 0.0001).


, Immunohistochemical staining of PDGFRβ , ⍺ - SMA , CD34 and CD146 in serial sections cut 4 μm apart from matched cases ; Patient - 2 ( g ) Images were aligned using FIJI . Tumour ( T ) regions are annotated by the solid yellow line . Co - localisation of CD34 and CD146 was used to distinguish blood vessels ( BV ) , where their differential staining was used to identify CAFs and PVL cells . g , MyCAFs were found to be localised at the invasive stromal interface ( insert A ) , whilst iCAFs were located at distal regions ( insert B ) with a high abundance of tumour infiltrating lymphocytes ( TILs ) .

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, Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-2 (g) Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. g, MyCAFs were found to be localised at the invasive stromal interface (insert A), whilst iCAFs were located at distal regions (insert B) with a high abundance of tumour infiltrating lymphocytes (TILs).


Immunohistochemical staining of PDGFRβ , ⍺ - SMA , CD34 and CD146 in serial sections cut 4 μm apart from matched cases ; Patient - 4 ( h ) . Images were aligned using FIJI . Tumour ( T ) regions are annotated by the solid yellow line . Co - localisation of CD34 and CD146 was used to distinguish blood vessels ( BV ) , where their differential staining was used to identify CAFs and PVL cells . h , Case with a high abundance of perivascular - like ( PVL ) cells in regions surrounded by blood vessels .

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Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-4 (h). Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. h, Case with a high abundance of perivascular-like (PVL) cells in regions surrounded by blood vessels.


c - e , Imputed gene expression of selected candidate signalling molecules identified between the four stromal subsets and malignant ( c ) epithelial , ( d ) myeloid and ( e ) T - cells . Expression of ligands in stromal clusters are represented on the left , with cognate receptors on target cells on the right .

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c-e, Imputed gene expression of selected candidate signalling molecules identified between the four stromal subsets and malignant (c) epithelial, (d) myeloid and (e) T-cells. Expression of ligands in stromal clusters are represented on the left, with cognate receptors on target cells on the right.


b , Representative cohort ( METABRIC ) showing the prognostic value of iCAF T - cell dysfunction signature in the context of CTLs for a total of 233 patients . Kaplan - Meier ' s present two groups of patients , ' low CTL ' ( blue line ) and ' high CTL ' ( red line ) , as estimated according to the average expression of CTL - specific genes and stratified as compared to the mean . Tumours with low iCAF T - cell dysfunction signatures ( top ) show patients with high CTL levels have a better survival outcome . In contrast , this survival benefit is lost in tumours with a high iCAF T - cell dysfunction signature ( bottom ) . P - values were defined from the Cox proportional hazard ( Cox - PH ) model .

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b, Representative cohort (METABRIC) showing the prognostic value of iCAF T-cell dysfunction signature in the context of CTLs for a total of 233 patients. Kaplan-Meier's present two groups of patients, 'low CTL' (blue line) and 'high CTL' (red line), as estimated according to the average expression of CTL-specific genes and stratified as compared to the mean. Tumours with low iCAF T-cell dysfunction signatures (top) show patients with high CTL levels have a better survival outcome. In contrast, this survival benefit is lost in tumours with a high iCAF T-cell dysfunction signature (bottom). P-values were defined from the Cox proportional hazard (Cox-PH) model.


dPVL profiles and CTL exclusion consistent in our study . g - h , Accurate quantification of CTLs ( g ) and representative immunohistochemistry staining for CD8 on matched patient tumour sections ( h ) . n = 5 stromal 1 mm2 regions were counted per tumour . The central band , boxes and whiskers represent the median , lower / upper quartile and min / max CTL counts per 1mm2 , respectively . P3 is shown as an example of a low dPVL profile with high CTLs . In contrast , P4 has a high dPVL profile with low CTLs . Statistical significance was determined using pairwise comparison with Student ' s t test with p ‐ values denoted by asterisks : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 .

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dPVL profiles and CTL exclusion consistent in our study. g-h, Accurate quantification of CTLs (g) and representative immunohistochemistry staining for CD8 on matched patient tumour sections (h). n = 5 stromal 1 mm2 regions were counted per tumour. The central band, boxes and whiskers represent the median, lower/upper quartile and min/max CTL counts per 1mm2, respectively. P3 is shown as an example of a low dPVL profile with high CTLs. In contrast, P4 has a high dPVL profile with low CTLs. Statistical significance was determined using pairwise comparison with Student's t test with p‐values denoted by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.


Mouse qPCR array analysis was performed with Colo357 and MiaPaca - 2 orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect changes of mouse stromal gene expression after treatment . A heat map was generated with ratios of gene expression ( 2G8 vs . control ) .

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Mouse qPCR array analysis was performed with Colo357 and MiaPaca-2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes of mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 vs. control).


ELISA - based Quansys mouse chemokine assay was performed with Capan - 1 ( Cap ) , MiaPaCa - 2 ( Mia ) , Colo357 ( Colo ) , and C5LM2 ( C5 ) orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect mouse chemokine changes after treatment . Change of mouse IL - 6 was shown .

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ELISA-based Quansys mouse chemokine assay was performed with Capan-1 (Cap), MiaPaCa-2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change of mouse IL-6 was shown.


KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Tumors from were collected for mouse IL - 6 ELISA ( n = 6 / group ) .

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KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL-6 ELISA (n = 6/group).


KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . The activation of SMAD2 ( P - Ser465 / 467 ) ( E and H - I ) , STAT3 ( P - Tyr705 ) ( F and J - K ) and expression of IL - 6R ( G ) was detected by immunohistochemistry ( n = 4 / group ) .

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KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group).


Single - cell RNA sequencing was performed to profile cell populations in normal mouse pancreas ( n = 2 ) , early KIC ( 40 - day - old , n = 2 ) and late KIC ( 60 - day - old , n = 3 ) pancreata . Samples from the same stage were pooled . Violin plots of expression of Il6 , Tgfbr1 and Tgfbr2 in distinct cell populations is shown .

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Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown.


Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC ( mPLRB8 , mPLRB9 ) , KPC ( KPC - M01 , KPC - M09 ) and KPfC ( BMFA3 , CT1BA5 ) mouse cancer cells , mouse macrophages ( RAW 264 . 7 ) , and mouse fibroblasts ( NIH 3T3 and pancreatic stellate cells ) . RAW 264 . 7 cells were induced into M1 ( 30 ng / ml LPS for 18 hours ) or M2 ( 20 ng / ml IL - 4 for 18 hours ) macrophages . Tubulin was used as a loading control .

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Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control.


Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA ( n = 223 ) using R .

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Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.


Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Cell lysates were harvested and blotted for P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( A ) .

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Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).


Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Conditioned media ( CM ) was collected mouse IL - 6 ELISA ( B - C ) .

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Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Conditioned media (CM) was collected mouse IL-6 ELISA (B-C).


NIH 3T3 ( D ) , pancreatic stellate cells ( PSC ) ( E ) and human CAF cell lines CAF - PC1 and CAF - PC2 ( F ) were treated with TGFβ ( 30 ng / ml ) and / or IL - 1α ( 1 ng / ml ) for 24 hours . CM was collected for mouse or human IL - 6 ELISA .

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NIH 3T3 (D), pancreatic stellate cells (PSC) (E) and human CAF cell lines CAF-PC1 and CAF-PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL-1α (1 ng/ml) for 24 hours. CM was collected for mouse or human IL-6 ELISA.


CAF - PC2 cells were treated with TGFβ ( 30 ng / ml ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . CM was collected and subjected to human IL - 6 ELISA .

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CAF-PC2 cells were treated with TGFβ (30 ng/ml) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. CM was collected and subjected to human IL-6 ELISA.


CAF - PC2 cells were treated with IL - 1α ( 1 ng / ml ) ( H ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . M was collected and subjected to human IL - 6 ELISA .

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CAF-PC2 cells were treated with IL-1α (1 ng/ml) (H) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. M was collected and subjected to human IL-6 ELISA.


KIC ( mPLRB9 ) , KPC ( KPC - M09 ) and KPfC ( BMFA3 ) cell lines were treated with normal DMEM ( CTRL ) , CM from NIH 3T3 ( CM ) , CM from TGFβ - treated NIH 3T3 ( TGFβ - CM ) , CM from TGFβ - treated NIH 3T3 + 2G8 ( TGFβ - CM + 2G8 ) normal DMEM + TGFβ ( TGFβ ) , CM from TGFβ - treated NIH 3T3 + IL - 6 neutralizing antibody ( TGFβ - CM + IL - 6 Ab ) . Cell lysates were harvested and blotted for P - STAT3 ( P - Tyr705 ) , STAT3 , P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( J ) .

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KIC (mPLRB9), KPC (KPC-M09) and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ-treated NIH 3T3 (TGFβ-CM), CM from TGFβ-treated NIH 3T3 + 2G8 (TGFβ-CM + 2G8) normal DMEM + TGFβ (TGFβ), CM from TGFβ-treated NIH 3T3 + IL-6 neutralizing antibody (TGFβ-CM + IL-6 Ab). Cell lysates were harvested and blotted for P-STAT3 (P-Tyr705), STAT3, P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (J).


3D culture : 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 ) . IL - 6 neutralizing antibody ( 100 ng / ml ) .

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3D culture: 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). IL-6 neutralizing antibody (100 ng/ml).


Saline 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 ) .

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Saline 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 ) .

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 .

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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 .

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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.


KIC 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 .

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KIC 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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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.


KIC 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 ) .

KIC 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 .

Tissues from (A) were stained for Ki67. Scale bars = 50 μm.


Overall survival of PDA patients from TCGA with and without TGFBR2 mutation .

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 .

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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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).


Control 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 .

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Control 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 .

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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 ) .

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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 ) .

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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 .

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.


Control 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 .

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Control 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 ) .

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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 ) .

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 .

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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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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.


A . ( 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 .

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A. (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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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 .

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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 )

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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 ) .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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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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).


A . 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 .

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A. 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 .

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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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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.


A . 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 .

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A. 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 ) .

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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 .

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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 .

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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 ) .

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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 .

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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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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.


MC38 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 .

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MC38 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 .

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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 .

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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 .

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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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.


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 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 .

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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 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 .

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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.