Figure 4A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs ( CAF110 ) grown for 24 h in EMM medium containing THI and TSA as indicated . DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe . Marker molecular weights are on the left in kilobases . Numbers at the bottom indicate gel lanes . B Genomic DNA from cells carrying two tiTELs and deleted for trt1 + ( CAF113 ) was analyzed as in ( A ) . Marker molecular weights are on the left in kilobases . Numbers at the bottom indicate gel lanes . C ter1 + cells carrying two tiTELs ( CAF110 , left panel ) or trt1Δ cells carrying one tiTEL ( MKSP2104 , central panel ) were cultured in EMM medium in presence of THI for 24 h ( THI + ) or in absence of THI for 24 , 48 or 72 h . In the right panel , cells carrying two tiTELs ( CAF110 ) were treated with TSA for 24 h in the presence or absence of THI . TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform . The statistical analysis for each set of conditions is displayed above each panel . P values for each comparison were derived from the Student ' s t test between each condition and the THI + sample using Welch ' s correction when appropriate . The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red ( center panel ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A Telomere restriction fragment analysis of genomic DNA from wt cells and cells carrying two tiTELs (CAF110) grown for 24 h in EMM medium containing THI and TSA as indicated. DNA was digested with HindIII and hybridized first with a nmt1 probe and successively with a telomeric probe. Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.B Genomic DNA from cells carrying two tiTELs and deleted for trt1+ (CAF113) was analyzed as in (A). Marker molecular weights are on the left in kilobases. Numbers at the bottom indicate gel lanes.C ter1+ cells carrying two tiTELs (CAF110, left panel) or trt1Δ cells carrying one tiTEL (MKSP2104, central panel) were cultured in EMM medium in presence of THI for 24 h (THI+) or in absence of THI for 24, 48 or 72 h. In the right panel, cells carrying two tiTELs (CAF110) were treated with TSA for 24 h in the presence or absence of THI. TiTEL telomeric sequences were amplified from genomic DNA and sequenced using a PacBio platform. The statistical analysis for each set of conditions is displayed above each panel. P values for each comparison were derived from the Student's t test between each condition and the THI+ sample using Welch's correction when appropriate. The new population of longer tiTELs appearing in trt1Δ cells upon prolonged transcription induction is indicated in red (center panel).


Figure 5A qRT - PCR analysis of G - rich and polyA + tiTERRA in cells carrying one tiTEL and expressing Trt1 - myc ( CAF610 ) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h . Values are normalized to ACT1 mRNA and expressed as fold increase over THI + TSA - samples . Bars and error bars are averages and SD from 4 independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ( relative to THI + TSA - ; two - tailed Student ' s t test ) . B Telo - PCR analysis of tiTELs using genomic DNA extracted from cells as in ( A ) . Marker ( m ) molecular weights are on the right in base pairs . C RIP experiments performed using anti - myc antibodies and extracts from cells as in ( A ) followed by qRT - PCR analysis of G - rich and polyA + tiTERRA . Values correspond to fraction of input RNA expressed as fold increase over an untagged ( unt ) control tiTEL strain ( CAF545 ) . Bars and error bars are averages and SD from three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to unt ; two - tailed Student ' s t test ) . D ChIP experiments performed using anti - myc antibodies and extracts from cells as in ( A ) followed by qPCR using tiTEL specific oligonucleotides . Values correspond to fraction of input DNA expressed as fold increase over THI + TSA - . Unt : untagged strain ( CAF545 ) . Bars and error bars are averages and SD from at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 ( relative to THI + TSA - ; two - tailed Student ' s t test ) .

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

Figure 5A qRT-PCR analysis of G-rich and polyA+ tiTERRA in cells carrying one tiTEL and expressing Trt1-myc (CAF610) cultured in EMM medium with the indicated combinations of THI and TSA for 24 h. Values are normalized to ACT1 mRNA and expressed as fold increase over THI+ TSA- samples. Bars and error bars are averages and SD from 4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, (relative to THI+ TSA-; two-tailed Student's t test).B Telo-PCR analysis of tiTELs using genomic DNA extracted from cells as in (A). Marker (m) molecular weights are on the right in base pairs.C RIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qRT-PCR analysis of G-rich and polyA+ tiTERRA. Values correspond to fraction of input RNA expressed as fold increase over an untagged (unt) control tiTEL strain (CAF545). Bars and error bars are averages and SD from three independent experiments. * P < 0.05, ** P < 0.01 (relative to unt; two-tailed Student's t test).D ChIP experiments performed using anti-myc antibodies and extracts from cells as in (A) followed by qPCR using tiTEL specific oligonucleotides. Values correspond to fraction of input DNA expressed as fold increase over THI+ TSA-. Unt: untagged strain (CAF545). Bars and error bars are averages and SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (relative to THI+ TSA-; two-tailed Student's t test).


Figure 1mRNA and protein expression of CiTLR5a - HA ( D ) , CiTLR5b - HA ( D ) , CiTLR5a - GFP ( E ) , and CiTLR5b - GFP ( E ) plasmids in FHM cells were examined by qRT - PCR ( upper panel ) with EF1α gene as an internal control and by immunoblotting ( IB ) ( lower panel ) with β - tubulin as a reference ( n = 4 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( D ) and ( E ) . n represents biological replicates . * * * P < 0 . 001 . FHM cells in 10 cm2 dishes were transfected with the indicated plasmids for 24 h . Co - IP was performed with anti - HA monoclonal antibody . Mouse IgG was used as control . Immunoblotting was performed with anti - HA and anti - GFP antibodies , respectively .

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 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

Figure 1mRNA and protein expression of CiTLR5a-HA (D), CiTLR5b-HA (D), CiTLR5a-GFP (E), and CiTLR5b-GFP (E) plasmids in FHM cells were examined by qRT-PCR (upper panel) with EF1α gene as an internal control and by immunoblotting (IB) (lower panel) with β-tubulin as a reference (n = 4). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (D) and (E). n represents biological replicates. ***P < 0.001.FHM cells in 10 cm2 dishes were transfected with the indicated plasmids for 24 h. Co-IP was performed with anti-HA monoclonal antibody. Mouse IgG was used as control. Immunoblotting was performed with anti-HA and anti-GFP antibodies, respectively.


Figure 2Lysates of FHM cells overexpressing CiTLR5a were respectively incubated with FlaA , FlaB , FlaC , at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads or streptavidin agarose beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA or anti - His antibodies . Lysates of FHM cells overexpressing CiTLR5b were respectively incubated with FlaA , FlaB , FlaC , at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads or streptavidin agarose beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA or anti - His antibodies . Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with biotin - poly ( I : C ) , biotin - ssRNA ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 for 1 h at 4 ° C , mixed with HA Ab - coupled beads for 2 h at 4 ° C , then analyzed by immunoblotting ( IB ) with anti - HA antibodies . N , O CiUNC93B1 - Myc and CiTLR5a - HA ( M ) or CiTLR5b - HA ( N ) were transfected into FHM cells . Co - IP and immunoblotting were conducted to test the interactions between CiUNC93B1 - Myc and CiTLR5a - HA or CiTLR5b - HA .

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 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 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

Figure 2Lysates of FHM cells overexpressing CiTLR5a were respectively incubated with FlaA, FlaB, FlaC, at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.Lysates of FHM cells overexpressing CiTLR5b were respectively incubated with FlaA, FlaB, FlaC, at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads or streptavidin agarose beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA or anti-His antibodies.Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b were respectively incubated with biotin-poly(I:C), biotin-ssRNA (1 μg/mL) at pH 5.0 or pH 7.4 for 1 h at 4 °C, mixed with HA Ab-coupled beads for 2 h at 4 °C, then analyzed by immunoblotting (IB) with anti-HA antibodies.N, O CiUNC93B1-Myc and CiTLR5a-HA (M) or CiTLR5b-HA (N) were transfected into FHM cells. Co-IP and immunoblotting were conducted to test the interactions between CiUNC93B1-Myc and CiTLR5a-HA or CiTLR5b-HA.


Figure 3O , P Protein expression of IFN1 ( O ) and NF - κB1 ( P ) in spleen tissue were investigated by immunoblotting ( IB ) after RNAi in vivo for 24 h , and A . hydrophila or GCRV challenge for 24 h ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 3O, P Protein expression of IFN1 (O) and NF-κB1 (P) in spleen tissue were investigated by immunoblotting (IB) after RNAi in vivo for 24 h, and A. hydrophila or GCRV challenge for 24 h (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 5Lysates of FHM cells overexpressing mutants were incubated with FlaC for pull - down assays and analyzed by immunoblotting ( IB ) . Vector and wild type ( WT ) were used as controls . Relative band intensity ratios were analyzed by ImageJ ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

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

Figure 5Lysates of FHM cells overexpressing mutants were incubated with FlaC for pull-down assays and analyzed by immunoblotting (IB). Vector and wild type (WT) were used as controls. Relative band intensity ratios were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for n represents biological replicates. *P < 0.05, **P < 0.01.


Figure 6Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b ( including region deletants ) were incubated with biotin - poly ( I : C ) . Vector was used as a control . Pull - down and immunoblotting ( IB ) were performed . H - K Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b site mutants were incubated with biotin - poly ( I : C ) . Vector and wild type were used as controls . Pull - down and immunoblotting were carried out ( H , J ) . ( I , K ) Relative band intensity ratios of ( H ) and ( J ) were analyzed by ImageJ , respectively ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( I ) , and ( K ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

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 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

Figure 6Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b (including region deletants) were incubated with biotin-poly(I:C). Vector was used as a control. Pull-down and immunoblotting (IB) were performed.H-K Lysates of FHM cells overexpressing CiTLR5a and CiTLR5b site mutants were incubated with biotin-poly(I:C). Vector and wild type were used as controls. Pull-down and immunoblotting were carried out (H, J). (I, K) Relative band intensity ratios of (H) and (J) were analyzed by ImageJ, respectively (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (I), and (K). n represents biological replicates. *P < 0.05, **P < 0.01.


Figure 7Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting ( IB ) with anti - HA antibody . Mutants with the predicted N - glycosylation sites were transfected into FHM cells and examined via immunoblotting to show the change of molecular weight . Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin - poly ( I : C ) ( I ) Vector and wild type were used as controls . Pull - down and immunoblotting assays were conducted ( I , ( J , Relative band intensity ratios of ( I ) were analyzed by ImageJ ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( J ) , n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with FlaC ( K ) . Vector and wild type were used as controls . Pull - down and immunoblotting assays were conducted K ) . L ) Relative band intensity ratios of ( K ) were analyzed by ImageJ ( n = 3 ) . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( L ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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 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 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 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

Figure 7Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b were respectively treated with Endo H and analyzed by immunoblotting (IB) with anti-HA antibody.Mutants with the predicted N-glycosylation sites were transfected into FHM cells and examined via immunoblotting to show the change of molecular weight.Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with biotin-poly(I:C) (I) Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted (I, (J, Relative band intensity ratios of (I) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (J), n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.Lysates of FHM cells overexpressing CiTLR5a or CiTLR5b mutants were respectively incubated with FlaC (K). Vector and wild type were used as controls. Pull-down and immunoblotting assays were conducted K). L) Relative band intensity ratios of (K) were analyzed by ImageJ (n = 3). Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (L). n represents biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.


Figure 8Lysates of FHM cells overexpressing LoTLR5 ( A ) , DrTLR5a ( B ) , DrTLR5b ( B ) , IpTLR5 - 1 ( C ) , IpTLR5 - 2 ( C ) , IpTLR5S ( C ) , OmTLR5M ( D ) , OmTLR5S ( D ) , were respectively incubated with FlaC - His ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by Anti - GFP antibody coupling beads and analyzed by immunoblotting ( IB ) with anti - His antibody . Lysates of FHM cells overexpressing HsTLR5 ( E ) were respectively incubated with FlaC - His ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by Anti - GFP antibody coupling beads and analyzed by immunoblotting ( IB ) with anti - His antibody . Lysates of FHM cells overexpressing LoTLR5 ( F ) , DrTLR5a ( G ) , DrTLR5b ( G ) , IpTLR5 - 1 ( H ) , IpTLR5 - 2 ( H ) , IpTLR5S ( H ) , were respectively incubated with biotin - poly ( I : C ) ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti - GFP antibody . Lysates of FHM cells overexpressing OmTLR5M ( I ) , OmTLR5S ( I ) , and HsTLR5 ( J ) were respectively incubated with biotin - poly ( I : C ) ( 1 μg / mL ) at pH 5 . 0 or pH 7 . 4 . Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti - GFP antibody . Lysates of FHM cells overexpressing DrTLR5a and DrTLR5b mutants were incubated with biotin - poly ( I : C ) and analyzed by immunoblotting ( N ) , and relative band intensity ratios were analyzed by ImageJ ( O ) ( n = 3 ) . Vector and wild type ( WT ) were used as controls . Data information : All data are presented as mean ± SD . The two - tailed Mann - Whitney U - test was used for ( O ) . n represents biological replicates . * P < 0 . 05 , * * P < 0 . 01 .

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 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 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 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 O O O O O O O O O O O O O O O O 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 8Lysates of FHM cells overexpressing LoTLR5 (A), DrTLR5a (B), DrTLR5b (B), IpTLR5-1 (C), IpTLR5-2 (C), IpTLR5S (C), OmTLR5M (D), OmTLR5S (D), were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.Lysates of FHM cells overexpressing HsTLR5 (E) were respectively incubated with FlaC-His (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by Anti-GFP antibody coupling beads and analyzed by immunoblotting (IB) with anti-His antibody.Lysates of FHM cells overexpressing LoTLR5 (F), DrTLR5a (G), DrTLR5b (G), IpTLR5-1 (H), IpTLR5-2 (H), IpTLR5S (H), were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.Lysates of FHM cells overexpressing OmTLR5M (I), OmTLR5S (I), and HsTLR5 (J) were respectively incubated with biotin-poly(I:C) (1 μg/mL) at pH 5.0 or pH 7.4. Complexes were pulled down by streptavidin beads and analyzed via immunoblotting with anti-GFP antibody.Lysates of FHM cells overexpressing DrTLR5a and DrTLR5b mutants were incubated with biotin-poly(I:C) and analyzed by immunoblotting (N), and relative band intensity ratios were analyzed by ImageJ (O) (n = 3). Vector and wild type (WT) were used as controls. Data information: All data are presented as mean ± SD. The two-tailed Mann-Whitney U-test was used for (O). n represents biological replicates. *P < 0.05, **P < 0.01.


Figure 1Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type ( WT ) or Nlrc3 - / - mice and treated with LPS ( 100 ng / ml ) for 48 hours . ELISA of cytokines in culture supernatants of DCs treatedThe intracellular production of IFN - γ and IL - 17 by CD4 + T cells among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treatedcytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treatedthymidine incorporation proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treatedCFSE proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

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Figure 1Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type (WT) or Nlrc3-/- mice and treated with LPS (100 ng/ml) for 48 hours.ELISA of cytokines in culture supernatants of DCs treatedThe intracellular production of IFN-γ and IL-17 by CD4+ T cells among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treatedcytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treatedthymidine incorporation proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treatedCFSE proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


Figure 2WT and Nlrc3 - / - mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized WT ( n = 10 ) and Nlrc3 - / - mice ( n = 10 ) . Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells from the spinal cord and brain of WT or Nlrc3 - / - mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel . Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3 - / - mice 26 d after EAE induction . Expression of Nlrc3 , Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3 - / - mice 26 d after EAE induction , presented relative to that of Gapdh .

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Figure 2WT and Nlrc3-/- mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized WT (n= 10) and Nlrc3-/- mice (n= 10).Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells from the spinal cord and brain of WT or Nlrc3-/- mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3-/- mice 26 d after EAE induction.Expression of Nlrc3, Il12, Il6, Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3-/- mice 26 d after EAE induction, presented relative to that of Gapdh.


Figure 3DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Recall response to MOG ( 35 - 55 ) by splenocytes isolated from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . CFSE proliferation assay of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . cytokine secretion of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

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 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 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 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 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

Figure 3DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Recall response to MOG(35-55) by splenocytes isolated from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6, Il23 and Il27 mRNA in DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. CFSE proliferation assay of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. cytokine secretion of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


Figure 4DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for phosphorylated p65 ( p - p65 ) , total p65 , p - AKT , AKT , p - p38 , p38 , p - ERK , ERK , p - JNK , JNK and GAPDH . DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Concentrations of IL - 12 , IL - 6 , IL - 23 and IL - 27 in supernatants were detected by ELISA . DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control . DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control .

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 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 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 4DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH.DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA.DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.


Figure 5Activity phosphorylation of p38 were detected in DCs in the spleens from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . Pooled data are presented in the right panel . DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) . DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Frequencies of CD4 + T cells that express IFN - γ and IL - 17A in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel . DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 and Il23 mRNA in DCs sorted from spleens of DC ( WT ) , DC ( NLRC3 - KO ) , DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh .

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Figure 5Activity phosphorylation of p38 were detected in DCs in the spleens from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction. Pooled data are presented in the right panel.DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.


Figure 6DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for p - p38 , p38 , NLRC3 and GAPDH . Densitometry quantification of band intensity were presented in the right panel . DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Enzyme - linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours . DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours . DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours .

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Figure 6DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. DC lysates were probed for p-p38, p38, NLRC3 and GAPDH. Densitometry quantification of band intensity were presented in the right panel.DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Enzyme-linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours.DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.


Figure 7EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Mean clinical scores of EAE ( n = 5 mice per group ) . Arrows indicate DC vaccine administration . EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Recall proliferative to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction . EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . cytokine response ( D ) to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction .

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Figure 7EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration.EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Recall proliferative to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.


Figure 1C Representative plot showing gating strategy for CD4 + T cell phenotypes . Effector Memory RA ( TEMRA ) : CD45RA + CD62L - ; Effector Memory ( EM ) : CD45RA - CD62L - ; Central Memory ( CM ) : CD45RA - CD62L + ; Stem Cell Memory ( TSCM ) : CD45RA + CD62L + . D Percentage of GFP + cells within T cell subpopulations 17 days after CD40LG editing of healthy male donor ( HD ; n = 11 ) or patient ( Pt ; n = 1 ) derived CD4 + T cells , measured by FACS analysis . Median ± IQR . E Percentage of GFP + cells or HDR in T cells from ( D ) . Median ± IQR . F Population composition in male HD or patient derived untreated ( n = 9 HD UT , 1 Pt UT ) or bulk edited T cells ( Treat ) from ( D ) . For each subpopulation , the comparisons between HD Treated vs UT were performed by using LME models , with random effects defined to account for the same donor and for different number of replicates per donor within group . All p - values were adjusted with Bonferroni ' s correction to account for multiple testing . In all LME models , the percentages were used in square root scale to meet the assumption of normality of the residuals of the model . Mean ± SEM . G Percentage of TCRBV families detected by spectratyping . UT , sorted edited ( GFP + ) , sorted non - edited ( GFP - ) CD4 + T cells derived from male HD ( n = 1 ) or patient ( Pt ; n = 1 ) were analyzed at 17 days after CD40LG editing . H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by Relative Fluorescence Intensity ( RFI , normalized to T0 ; left ) and percentage ( right ) on UT ( n = 3 ) , edited ( GFP + ) or unedited ( GFP - ) HD or Pt CD4 + T cells from ( D ) ( n = 9 HD , 1 Pt ) . Longitudinal comparisons between HD GFP + vs GFP - were performed with an LME model , accounting for multiple donors and separately for RFI and % CD40L + cells ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to 8 hours time - point ( * * * * p < 0 . 0001 and * p = 0 . 0450 , respectively ) . # measured on the small fraction of CD40L + cells . Median ± IQR . I Representative plots showing CD40L and GFP expression in UT or bulk edited ( Tretated ) CD4 + T cells derived from male HD or Pt from ( D ) at 8 hr after PMA / Ionomycin stimulation . K Left : IgG positive spots resulting from B - T cell co - culture . Right : IgG + secreting B cells , evaluated by ELISPOT assay . B cells were isolated from PB of HD and co - cultured with male HD or Pt sorted GFP + , GFP - and UT T cells , resting ( R ) or stimulated with beads ( B ) or PMA / Ionomycin ( PI ) . B cells cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative and positive controls , respectively ( n = 1 for each group ) . L Left : Histograms representing proliferation results from B - T cell co - culture . Right : Analysis of B cell proliferative capacity by Cell Trace dilution assay in allogeneic sorted B cells isolated from PB of HD and co - cultured with HD or Pt T cells from ( K ) . B cells cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative and positive controls , respectively ( n = 2 for each group ) . Mean ± SEM .

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 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 B-PANEL_START 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 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 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 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 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 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 1C Representative plot showing gating strategy for CD4+ T cell phenotypes. Effector Memory RA (TEMRA): CD45RA+ CD62L-; Effector Memory (EM): CD45RA-CD62L-; Central Memory (CM): CD45RA-CD62L+; Stem Cell Memory (TSCM): CD45RA+ CD62L+.D Percentage of GFP+ cells within T cell subpopulations 17 days after CD40LG editing of healthy male donor (HD; n=11) or patient (Pt; n=1) derived CD4+ T cells, measured by FACS analysis. Median ± IQR.E Percentage of GFP+ cells or HDR in T cells from (D). Median ± IQR.F Population composition in male HD or patient derived untreated (n=9 HD UT, 1 Pt UT) or bulk edited T cells (Treat) from (D). For each subpopulation, the comparisons between HD Treated vs UT were performed by using LME models, with random effects defined to account for the same donor and for different number of replicates per donor within group. All p-values were adjusted with Bonferroni's correction to account for multiple testing. In all LME models, the percentages were used in square root scale to meet the assumption of normality of the residuals of the model. Mean ± SEM.G Percentage of TCRBV families detected by spectratyping. UT, sorted edited (GFP+), sorted non-edited (GFP-) CD4+ T cells derived from male HD (n=1) or patient (Pt; n=1) were analyzed at 17 days after CD40LG editing.H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by Relative Fluorescence Intensity (RFI, normalized to T0; left) and percentage (right) on UT (n=3), edited (GFP+) or unedited (GFP-) HD or Pt CD4+ T cells from (D) (n=9 HD, 1 Pt). Longitudinal comparisons between HD GFP+ vs GFP- were performed with an LME model, accounting for multiple donors and separately for RFI and %CD40L+ cells (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to 8 hours time-point (****p<0.0001 and *p=0.0450, respectively). #measured on the small fraction of CD40L+ cells. Median ± IQR.I Representative plots showing CD40L and GFP expression in UT or bulk edited (Tretated) CD4+ T cells derived from male HD or Pt from (D) at 8 hr after PMA/Ionomycin stimulation.K Left: IgG positive spots resulting from B-T cell co-culture. Right: IgG+ secreting B cells, evaluated by ELISPOT assay. B cells were isolated from PB of HD and co-cultured with male HD or Pt sorted GFP+, GFP- and UT T cells, resting (R) or stimulated with beads (B) or PMA/Ionomycin (PI). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=1 for each group).L Left: Histograms representing proliferation results from B-T cell co-culture. Right: Analysis of B cell proliferative capacity by Cell Trace dilution assay in allogeneic sorted B cells isolated from PB of HD and co-cultured with HD or Pt T cells from (K). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=2 for each group). Mean ± SEM.


Figure 2B Percentage of NGFR + cells or HDR at 17 days after CD40LG editing of male HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T cells ( n = 7 for each group ) . Median ± IQR . C Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by RFI ( normalized to T0 ) on UT ( n = 2 ) , edited ( NGFR + ) or unedited ( NGFR - ) HD or Pt derived CD4 + T cells ( n = 4 HD , 1 Pt ) . Median ± IQR . D Representative plots showing CD40L and NGFR expression in UT or bulk edited ( Treated ) CD4 + T cells derived from male HD or Pt from ( C ) before and 8 hr after Pma / Ionomycin stimulation . E Population composition in male HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T cells from ( B ) ( n = 7 for each group ) . Friedman test ( to account for the same donor ) with p - values adjusted with Bonferroni ' s correction to account for multiple testing . Mean ± SEM . F Human CD45 + cell engraftment in PB after transplantation of male HD UT ( n = 5 ) , bulk edited ( NGFR bulk ; n = 5 ) or sorted NGFR + ( n = 5 ) CD4 + T cells . Longitudinal comparisons were performed with an LME model , followed by an appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups . Median ± IQR . G Percentage of HDR within human cells over time , measured by ddPCR in PB of mice from ( F ) ( n = 5 for each group , measured in engrafted mice with sufficient blood material ) . Longitudinal comparisons were performed with an LME model with time treated as a continuous variable ( see Appendix Supplementary Statistical Methods ) . UT group was not included in the analysis . Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection , highlighting a constant ( and significantly different , * * * * p < 0 . 0001 ) behavior of the groups over time . Median ± IQR . H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by MFI on pooled CD4 + T cells retrieved from spleens of mice from ( F ) ( n = 1 for each group , except for NGFR - unedited group , n = 3 ) . Median ± IQR . I Dot plot depicting the ratio between expression of CD40LG edited mRNA ( codon - usage optimized , CO ) and CD40LG wild - type mRNA ( WT ) in cells edited with three different donor templates from Fig EV2H . Three independent experiments ( n = 3 EF1a . GFP , 1 IRES . NGFR , 3 IRES . GFP ) . Median ± IQR . J Time course of expression of WT - CD40LG mRNA or CO - CD40LG mRNA , measured as fold change ( FC ) on IPO8 housekeeping gene and normalized to T0 . Sorted edited ( + ) and sorted unedited ( - ) CD4 + T cells were analyzed before and 3 , 6 , 12 hr after Actinomycin D treatment ( n = 1 for each group ) .

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 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 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 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 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 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 O O O O O O O

Figure 2B Percentage of NGFR+ cells or HDR at 17 days after CD40LG editing of male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells (n=7 for each group). Median ± IQR.C Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by RFI (normalized to T0) on UT (n=2), edited (NGFR+) or unedited (NGFR-) HD or Pt derived CD4+ T cells (n=4 HD, 1 Pt). Median ± IQR.D Representative plots showing CD40L and NGFR expression in UT or bulk edited (Treated) CD4+ T cells derived from male HD or Pt from (C) before and 8 hr after Pma/Ionomycin stimulation.E Population composition in male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells from (B) (n=7 for each group). Friedman test (to account for the same donor) with p-values adjusted with Bonferroni's correction to account for multiple testing. Mean ± SEM.F Human CD45+ cell engraftment in PB after transplantation of male HD UT (n=5), bulk edited (NGFR bulk; n=5) or sorted NGFR+ (n=5) CD4+ T cells. Longitudinal comparisons were performed with an LME model, followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups. Median ± IQR.G Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (F) (n=5 for each group, measured in engrafted mice with sufficient blood material). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable (see Appendix Supplementary Statistical Methods). UT group was not included in the analysis. Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection, highlighting a constant (and significantly different, ****p<0.0001) behavior of the groups over time. Median ± IQR.H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleens of mice from (F) (n=1 for each group, except for NGFR- unedited group, n=3). Median ± IQR.I Dot plot depicting the ratio between expression of CD40LG edited mRNA (codon-usage optimized, CO) and CD40LG wild-type mRNA (WT) in cells edited with three different donor templates from Fig EV2H. Three independent experiments (n=3 EF1a.GFP, 1 IRES.NGFR, 3 IRES.GFP). Median ± IQR.J Time course of expression of WT-CD40LG mRNA or CO-CD40LG mRNA, measured as fold change (FC) on IPO8 housekeeping gene and normalized to T0. Sorted edited (+) and sorted unedited (-) CD4+ T cells were analyzed before and 3, 6, 12 hr after Actinomycin D treatment (n=1 for each group).


Figure 3B Representative plots showing hEGFRt expression in bulk edited CD4 + T cells derived from male HD before and 8 hr after Pma / Ionomycin stimulation . Cells were edited with the three constructs depicted in ( A ) . C Representative plots showing EGFR expression in bulk edited CD4 + T cells derived from male HD at 3 days after treatment with immunotoxin ( left ) , antibody ( middle ) or toxin ( right ) . D Bar plot showing percentage of EGFR + T cells at 3 days after treatment with 5 nM or 1 nM of immunotoxin , antibody or toxin , measured by FACS Analysis . Friedman test with Dunn ' s multiple comparisons . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * * p = 0 . 0052 and * * p = 0 . 0024 for immunotoxin vs antibody and * * p = 0 . 0010 and * * p = 0 . 0024 for immunotoxin vs toxin , referring to EGFRmod1 and EGFRmod2 respectively ) . Different dose - conditions were used as a unified group for statistical analysis ( n = 10 for each group ) . Median ± IQR .

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 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 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

Figure 3B Representative plots showing hEGFRt expression in bulk edited CD4+ T cells derived from male HD before and 8 hr after Pma/Ionomycin stimulation. Cells were edited with the three constructs depicted in (A).C Representative plots showing EGFR expression in bulk edited CD4+ T cells derived from male HD at 3 days after treatment with immunotoxin (left), antibody (middle) or toxin (right).D Bar plot showing percentage of EGFR+ T cells at 3 days after treatment with 5 nM or 1 nM of immunotoxin, antibody or toxin, measured by FACS Analysis. Friedman test with Dunn's multiple comparisons. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (**p=0.0052 and **p=0.0024 for immunotoxin vs antibody and **p=0.0010 and **p=0.0024 for immunotoxin vs toxin, referring to EGFRmod1 and EGFRmod2 respectively). Different dose-conditions were used as a unified group for statistical analysis (n=10 for each group). Median ± IQR.


Figure 4B Percentage of HDR in HSPC subpopulations at 4 days after CD40LG editing , measured by molecular analysis ( ddPCR ) . Cells were edited with donor template either comprising only the corrective cDNA ( NR ; n = 4 ) , or carrying the selector cassette IRES . NGFR ( NGFR ; n = 5 ) and sorted according to surface markers : CD34 - ; CD34 + CD133 - CD90 - ; CD34 + CD133 + CD90 - ; CD34 + CD133 + CD90 + . CD34 + CD133 + CD90 + / CD90 - and CD34 + CD133 - / CD34 - conditions were used as unified groups for statistical analysis . The comparisons between the groups were performed with LME models accounting for the different subpopulations included in the analysis and with random effects defined to account for the same donor . In comparison about CD34 + CD133 + CD90 + / CD90 - , the percentages were used in square root scale to meet the assumption of normality of the residuals of the model . For CD34 + CD133 + CD90 + / CD90 - , * * p = 0 . 0035 , for CD34 + CD133 - / CD34 - p = 0 . 2051 . Median ± IQR . C Population composition in UT or bulk edited HSPC from ( B ) ( n = 8 for each group , except UT cells from NGFR group , n = 7 ) . Paired Wilcoxon ' s test , to account for the same donor , with p - values adjusted with Bonferroni ' s correction to account for multiple testing . Analysis was performed separately for NR and NGFR groups . Mean ± SEM . D Human CD45 + cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in ( B ) and with ( n = 4 ) or without ( n = 5 ) GSE56 . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the first time - point ( i . e . 6 weeks ; * p = 0 . 0213 and * * p = 0 . 0069 ) . Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups ( p = 0 . 0034 for NGFR + GSE56 and p = 0 . 0080 for NR + GSE56 ) . Median ± IQR . E Percentage of HDR within human cells over time , measured by ddPCR in PB of mice from ( D ) . Longitudinal comparisons were performed with an LME model with time treated as a continuous variable , and followed by an appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the last time - point ( i . e . 20 weeks ; * * * * p < 0 . 0001 in both comparisons ) . NR + GSE56 group shows a constant behavior over time ( p = 0 . 0588 ) . The slope of the linear trajectory over time is not significantly different between GSE56 groups ( p = 0 . 1624 ) , indicating gene editing stability of both groups over time . Mice transplanted with HSPC treated without GSE56 shows significantly lower slope than GSE56 groups ( p < 0 . 0001 in respect of NGFR group , p = 0 . 0054 in respect of NR group ) , thus highlighting decreased gene editing efficiency over time . Median ± IQR . Percentage of HDR within human cells in ( F ) PB , bone marrow , spleen , thymus measured by ddPCR in mice from ( D ) . Median . Percentage of HDR within human cells within BM - sorted human subpopulations ( CD19 + , CD33 + , CD3 + , CD34 + ) , measured by ddPCR in mice from ( D ) . Median . H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by MFI on pooled CD4 + T cells retrieved from spleen of mice from ( D ) ( n = 4 NGFR + , 4 NGFR - ) . Mean ± SEM .

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

Figure 4B Percentage of HDR in HSPC subpopulations at 4 days after CD40LG editing, measured by molecular analysis (ddPCR). Cells were edited with donor template either comprising only the corrective cDNA (NR; n=4), or carrying the selector cassette IRES.NGFR (NGFR; n=5) and sorted according to surface markers: CD34-; CD34+CD133-CD90-; CD34+CD133+CD90-; CD34+CD133+CD90+. CD34+CD133+CD90+/CD90- and CD34+CD133-/CD34- conditions were used as unified groups for statistical analysis. The comparisons between the groups were performed with LME models accounting for the different subpopulations included in the analysis and with random effects defined to account for the same donor. In comparison about CD34+CD133+CD90+/CD90-, the percentages were used in square root scale to meet the assumption of normality of the residuals of the model. For CD34+CD133+CD90+/CD90-, **p= 0.0035, for CD34+CD133-/CD34- p=0.2051. Median ± IQR.C Population composition in UT or bulk edited HSPC from (B) (n=8 for each group, except UT cells from NGFR group, n=7). Paired Wilcoxon's test, to account for the same donor, with p-values adjusted with Bonferroni's correction to account for multiple testing. Analysis was performed separately for NR and NGFR groups. Mean ± SEM.D Human CD45+ cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in (B) and with (n=4) or without (n=5) GSE56. Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the first time-point (i.e. 6 weeks; *p=0.0213 and **p=0.0069). Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups (p=0.0034 for NGFR+GSE56 and p=0.0080 for NR+GSE56). Median ± IQR.E Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (D). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable, and followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the last time-point (i.e. 20 weeks; ****p<0.0001 in both comparisons). NR+GSE56 group shows a constant behavior over time (p=0.0588). The slope of the linear trajectory over time is not significantly different between GSE56 groups (p=0.1624), indicating gene editing stability of both groups over time. Mice transplanted with HSPC treated without GSE56 shows significantly lower slope than GSE56 groups (p<0.0001 in respect of NGFR group, p=0.0054 in respect of NR group), thus highlighting decreased gene editing efficiency over time. Median ± IQR.Percentage of HDR within human cells in (F) PB, bone marrow, spleen, thymus measured by ddPCR in mice from (D). Median.Percentage of HDR within human cells within BM-sorted human subpopulations (CD19+, CD33+, CD3+, CD34+), measured by ddPCR in mice from (D). Median.H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleen of mice from (D) (n=4 NGFR+, 4 NGFR-). Mean ± SEM.


Figure 5B Total counts of recipient CD3 + cells gated within CD45 . 2 + cells in PB of experimental mice ( n = 4 CPA 300 , 9 NC ) . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 1 ( * * * * p < 0 . 0001 ) . Mean ± SEM . C Total counts of engrafted donor CD45 . 1 . 2 + cells in PB of mice from ( B ) . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to plateau ( * * * p = 0 . 0001 ) . Mean ± SEM . D , E Percentage of donor CD4 + and CD8 + cells gated within CD3 + CD45 . 1 . 2 + cells in PB of CPA treated ( D ) or NC mice ( E ) from ( B ) . Mean ± SEM . F TNP - KLH specific IgG in sera of transplanted mice collected at the times indicated in ( A ) ( n = 7 CPA 300 , 13 NC for first boost and 4 CPA 300 , 9 NC for second boost ) . Sera from vaccinated WT ( n = 5 ) and CD40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . Two independent experiments . Comparisons of first boost data were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * * p < 0 . 0001 in all comparisons ) . Median ± IQR . G Average area of PNA + foci in splenic sections of experimental mice from ( B ) , calculated as ratio between total PNA + area and number of PNA + foci / cells . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test ( n = 9 CD40LG , 6 WT , 18 NC , 8 CPA300 ) . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0149 , * * p = 0 . 0065 and * * * * p < 0 . 0001 ) . Mean ± SEM . H Detection of splenic TNP - KLH specific IgM ( left ) and IgG ( right ) - secreting cells by ELISPOT assay ( n = 7 NC , 4 CPA300 , 3 WT , 2 CD40LG ) . Spots were counted by an ELISPOT Reader using a size range of 0 . 005 - 1 mm . Median ± IQR .

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 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 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 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 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

Figure 5B Total counts of recipient CD3+ cells gated within CD45.2+ cells in PB of experimental mice (n=4 CPA 300, 9 NC). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 1 (****p<0.0001). Mean ± SEM.C Total counts of engrafted donor CD45.1.2+ cells in PB of mice from (B). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to plateau (***p=0.0001). Mean ± SEM.D, E Percentage of donor CD4+ and CD8+ cells gated within CD3+CD45.1.2+ cells in PB of CPA treated (D) or NC mice (E) from (B). Mean ± SEM.F TNP-KLH specific IgG in sera of transplanted mice collected at the times indicated in (A) (n=7 CPA 300, 13 NC for first boost and 4 CPA 300, 9 NC for second boost). Sera from vaccinated WT (n=5) and CD40lg-/- (n=5) mice were used as positive and negative controls, respectively. Two independent experiments. Comparisons of first boost data were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.G Average area of PNA+ foci in splenic sections of experimental mice from (B), calculated as ratio between total PNA+ area and number of PNA+ foci/cells. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test (n=9 CD40LG, 6 WT, 18 NC, 8 CPA300). P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0149, **p=0.0065 and ****p<0.0001). Mean ± SEM.H Detection of splenic TNP-KLH specific IgM (left) and IgG (right) -secreting cells by ELISPOT assay (n=7 NC, 4 CPA300, 3 WT, 2 CD40LG). Spots were counted by an ELISPOT Reader using a size range of 0.005-1 mm. Median ± IQR.


Figure 6B Total counts of recipient CD3 + CD45 . 2 + cells in PB of mice NC ( n = 13 ) or pre - conditioned with 300 mg / kg CPA ( n = 19 ) , 200 mg / kg CPA ( n = 6 ) , anti - lymphocyte serum ( ALS ; n = 5 ) or anti - CD4 antibody ( n = 6 ) . Three independent experiments . Comparisons at day 3 \ 4 were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ; * * * p = 0 . 0009 and * * * * p < 0 . 0001 in all comparisons ) . Mean ± SEM . C Total counts of engrafted donor CD45 . 1 + cells in PB of mice from ( B ) . Three independent experiment . Comparisons at day 18 \ 24 were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ; * * * p = 0 . 0002 and * * * * p < 0 . 0001 in all comparisons ) . CPA200 was not included in the analysis because n = 4 . Mean ± SEM . D Analysis of the relationship between total counts of recipient CD3 + cells gated within CD45 . 2 + cells at day 3 , 4 and engrafted donor CD45 . 1 + T cells at 18 \ 24 days . This relationship was modeled with an asymptotic NLME model ( Appendix Supplementary Statistical Methods ) , accounting for the different groups and multiple experiments . From the estimated model , a lower value of recipient CD3 + T cells corresponds to a higher value of engrafted donor CD4 + T cells ( p = 0 . 0006 ) . E TNP - KLH specific IgG concentration in sera of mice from ( B ) collected before ( day 35 ; pre ) and after ( day 49 ; post ) the first boost ( n = 13 NC , 19 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ; three independent experiments ) and at day 81 ( pre ) and 89 ( post ) for the second boost ( n = 4 NC , 4 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ) . Sera from vaccinated WT ( n = 5 ) and Cd40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . For early challenge data , comparisons were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * p = 0 . 0002 and * * * * p < 0 . 0001 in all comparisons ) . CPA200 was not included in the analysis because n = 4 . WT group , not indicated in the figure , is significantly different from all groups ( p < 0 . 0001 in all comparisons ) . Of note , regarding the comparisons between time - points , the groups CPA 300 and WT show a significant increase between pre and post values ( p < 0 . 0001 for both ) , while all other groups show a significant decrease between pre and post values ( p < 0 . 0001 for all except p = 0 . 0022 for ANTI - CD4 ) . Median ± IQR . F Total counts of recipient CD3 + cells gated within CD45 . 2 + cells after transfer of primed donor T cells in PB of mice treated with 300 mg / kg CPA ( n = 11 ) , 200 mg / kg CPA ( n = 11 ) or NC ( n = 6 ) . Longitudinal comparisons were performed by LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 4 ( * * * * p < 0 . 0001 in all comparisons ) . Mean ± SEM . G Total counts of engrafted donor CD45 . 1 + cells in PB of mice from ( F ) . Longitudinal comparisons were performed with an LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 25 . At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 ( * * * * p < 0 . 0001 for both ) . Mean ± SEM . H TNP - KLH specific IgG concentration in sera of mice from ( F ) collected before ( day 36 , pre ) and after ( day 50 , post ) the first boost ( n = 6 NC , 11 CPA 300 , 11 CPA 200 ) and at day 212 ( pre ) and 219 ( post ) for the second boost ( n = 6 NC , 11 CPA 300 , 9 CPA 200 ) . Sera from vaccinated WT ( n = 11 ) and Cd40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . Comparisons were performed with an LME model followed by an appropriate post - hoc analysis , separately for each challenge data ( Appendix Supplementary Statistical Methods ) . Reported statistical comparisons refer to the overall difference among groups , for early challenge data ( * p = 0 . 0282 and * * p = 0 . 0037 ) , while to differences between time - points within each group , for late challenge data ( * * p = 0 . 0018 and * * * * p < 0 . 0001 in both comparisons ) . Of note , in the late challenge data , no significant overall differences were observed among the groups . Median ± IQR .

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

Figure 6B Total counts of recipient CD3+ CD45.2+ cells in PB of mice NC (n=13) or pre-conditioned with 300 mg/kg CPA (n=19), 200 mg/kg CPA (n=6), anti-lymphocyte serum (ALS; n=5) or anti-CD4 antibody (n=6). Three independent experiments. Comparisons at day 3\4 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0009 and ****p<0.0001 in all comparisons). Mean ± SEM.C Total counts of engrafted donor CD45.1+ cells in PB of mice from (B). Three independent experiment. Comparisons at day 18\24 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. Mean ± SEM.D Analysis of the relationship between total counts of recipient CD3+ cells gated within CD45.2+ cells at day 3, 4 and engrafted donor CD45.1+ T cells at 18\24 days. This relationship was modeled with an asymptotic NLME model (Appendix Supplementary Statistical Methods), accounting for the different groups and multiple experiments. From the estimated model, a lower value of recipient CD3+ T cells corresponds to a higher value of engrafted donor CD4+ T cells (p=0.0006).E TNP-KLH specific IgG concentration in sera of mice from (B) collected before (day 35; pre) and after (day 49; post) the first boost (n=13 NC, 19 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4; three independent experiments) and at day 81 (pre) and 89 (post) for the second boost (n=4 NC, 4 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4). Sera from vaccinated WT (n=5) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. For early challenge data, comparisons were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. WT group, not indicated in the figure, is significantly different from all groups (p<0.0001 in all comparisons). Of note, regarding the comparisons between time-points, the groups CPA 300 and WT show a significant increase between pre and post values (p<0.0001 for both), while all other groups show a significant decrease between pre and post values (p<0.0001 for all except p=0.0022 for ANTI-CD4). Median ± IQR.F Total counts of recipient CD3+ cells gated within CD45.2+ cells after transfer of primed donor T cells in PB of mice treated with 300 mg/kg CPA (n=11), 200 mg/kg CPA (n=11) or NC (n=6). Longitudinal comparisons were performed by LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 4 (****p<0.0001 in all comparisons). Mean ± SEM.G Total counts of engrafted donor CD45.1+ cells in PB of mice from (F). Longitudinal comparisons were performed with an LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 25. At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 (****p<0.0001 for both). Mean ± SEM.H TNP-KLH specific IgG concentration in sera of mice from (F) collected before (day 36, pre) and after (day 50, post) the first boost (n=6 NC, 11 CPA 300, 11 CPA 200) and at day 212 (pre) and 219 (post) for the second boost (n=6 NC, 11 CPA 300, 9 CPA 200). Sera from vaccinated WT (n=11) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. Comparisons were performed with an LME model followed by an appropriate post-hoc analysis, separately for each challenge data (Appendix Supplementary Statistical Methods). Reported statistical comparisons refer to the overall difference among groups, for early challenge data (*p=0.0282 and **p=0.0037), while to differences between time-points within each group, for late challenge data (**p=0.0018 and ****p<0.0001 in both comparisons). Of note, in the late challenge data, no significant overall differences were observed among the groups. Median ± IQR.


Figure 7B Total counts of WT donor CD4 + T cells in mice from ( A ) ( n = 13 100 % WT , 8 25 % WT , 14 10 % WT , 6 1 % , 14 0 % WT ) . Two representative experiments shown out of 3 . Mean ± SEM . C TNP - KLH specific IgG concentration in sera of mice from ( A ) collected seven days before ( pre ) and after ( post ) TNP - KLH vaccination ( n = 13 100 % WT , 13 25 % WT , 14 10 % WT , 6 1 % WT , 14 0 % WT ) . Three independent experiments . Comparisons were performed with an LME model , accounting for multiple experiments , followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * * p < 0 . 0001 in all comparisons ) . Median ± IQR . D Percentage of PNA + GL7 + GC B cells within the spleen of mice from ( A ) ( n = 16 100 % WT , 6 25 % WT , 16 10 % WT , 11 1 % WT , 18 0 % WT ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0385 for 25 % WT vs 1 % WT , * p = 0 . 0129 for 100 % WT vs 1 % WT , * * * p = 0 . 0001 and * * * * p < 0 . 0001 in both comparisons ) . Median ± IQR . E Quantitation of P . murina rRNA in lung homogenate of mice from ( A ) transplanted with different ratios of WT HSPC cells ( n = 8 100 % WT , 7 25 % WT , 7 10 % WT , 8 0 % WT ) and infected with the pathogen . Results are expressed in P . murina / HPRT RNA copies . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * * p = 0 . 0028 ) . Median ± IQR . F Lung weight of mice from ( E ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * p = 0 . 0254 ) . Median ± IQR . G Left : Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody ( upper left : negative sample ; lower left : positive sample ) . Brown areas represent intra - alveolar aggregates of P . murina organisms . Right : Representative pictures of P . murina organisms detected by immunofluorescence in lungs homogenate ( upper right : negative sample ; lower right : positive sample ) . H Quantitation of P . Murina rRNA in lung homogenate of mice adoptively transferred ( n = 9 CD40LG + T cells ) or not ( n = 8 CD40LG ) with in vivo primed CD4 + T cells in absence of conditioning and infected with the pathogen . Results are expressed in P . Murina / HPRT RNA copies . Mann - Whitney test . * * * P - value of the comparison = 0 . 0003 . Median ± IQR . I Lung weight of experimental mice from ( H ) . Mann - Whitney test . * * P - value of the comparison = 0 . 0055 . Median ± IQR .

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

Figure 7B Total counts of WT donor CD4+ T cells in mice from (A) (n=13 100% WT, 8 25% WT, 14 10% WT, 6 1%, 14 0% WT). Two representative experiments shown out of 3. Mean ± SEM.C TNP-KLH specific IgG concentration in sera of mice from (A) collected seven days before (pre) and after (post) TNP-KLH vaccination (n=13 100% WT, 13 25% WT, 14 10% WT, 6 1% WT, 14 0% WT). Three independent experiments. Comparisons were performed with an LME model, accounting for multiple experiments, followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.D Percentage of PNA+GL7+ GC B cells within the spleen of mice from (A) (n=16 100% WT, 6 25% WT, 16 10% WT, 11 1% WT, 18 0% WT). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0385 for 25% WT vs 1% WT, *p=0.0129 for 100% WT vs 1% WT, ***p=0.0001 and ****p<0.0001 in both comparisons). Median ± IQR.E Quantitation of P. murina rRNA in lung homogenate of mice from (A) transplanted with different ratios of WT HSPC cells (n=8 100% WT, 7 25% WT, 7 10% WT, 8 0% WT) and infected with the pathogen. Results are expressed in P.murina/HPRT RNA copies. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (**p=0.0028). Median ± IQR.F Lung weight of mice from (E). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (*p=0.0254). Median ± IQR.G Left: Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody (upper left: negative sample; lower left: positive sample). Brown areas represent intra-alveolar aggregates of P. murina organisms. Right: Representative pictures of P. murina organisms detected by immunofluorescence in lungs homogenate (upper right: negative sample; lower right: positive sample).H Quantitation of P. Murina rRNA in lung homogenate of mice adoptively transferred (n=9 CD40LG + T cells) or not (n=8 CD40LG) with in vivo primed CD4+ T cells in absence of conditioning and infected with the pathogen. Results are expressed in P.Murina/HPRT RNA copies. Mann-Whitney test. ***P-value of the comparison = 0.0003. Median ± IQR.I Lung weight of experimental mice from (H). Mann-Whitney test. **P-value of the comparison = 0.0055. Median ± IQR.


Figure 1Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , Xnr1 ( 50 pg ) Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) , BMP4 ( 100 pg ) . Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT - PCR . ODC serves as a loading control . - RT , control in the absence of reverse transcriptase . WE , whole embryo . Co AC , uninjected control animal caps . ( - ) , no injection of wild - type ( wt ) Wip1 or Wip1 ( D277A ) mRNA . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) ( D wt Wip1 , but not Wip1 ( D277A ) , down - regulates the activity of Activin - or BMP - responsive promoters in HEK293T cells . FAST1 , were co - transfected as indicated for efficient activation of the promoters . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test . E ) wt Wip1 , but not Wip1 ( D277A ) , down - regulates the activity of Activin - or BMP - responsive promoters in HEK293T cells . Smad1 and Smad4 were co - transfected as indicated for efficient activation of the promoters . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test . ( F - Q ) Dorsal ( F - N ) or ventral ( O - Q ) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10 . 25 as analyzed by in situ hybridization . Embryos are shown in vegetal views with dorsal to the top . Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos . Control , an embryo injected with LacZ only . The amount of mRNA injected : wt Wip1 ( 1 ng ) , Wip1 ( D277A ) ( 1 ng ) and LacZ ( 100 pg ) . Scale bar , 150 μm .

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

Figure 1Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), Xnr1 (50 pg)Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng), BMP4 (100 pg).Wip1 represses the gene responses induced by Nodal or BMP signaling in animal caps as analyzed by RT-PCR. ODC serves as a loading control. -RT, control in the absence of reverse transcriptase. WE, whole embryo. Co AC, uninjected control animal caps. (-), no injection of wild-type (wt) Wip1 or Wip1(D277A) mRNA. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng)(D wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. FAST1, were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.E) wt Wip1, but not Wip1(D277A), down-regulates the activity of Activin- or BMP-responsive promoters in HEK293T cells. Smad1 and Smad4 were co-transfected as indicated for efficient activation of the promoters. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.(F-Q) Dorsal (F-N) or ventral (O-Q) injection of wt Wip1 abrogates the in vivo expression of mesodermal markers at stage 10.25 as analyzed by in situ hybridization. Embryos are shown in vegetal views with dorsal to the top. Arrowheads denote the absence of expression of mesodermal markers in the injected region of embryos. Control, an embryo injected with LacZ only. The amount of mRNA injected: wt Wip1 (1 ng), Wip1(D277A) (1 ng) and LacZ (100 pg). Scale bar, 150 μm.