( F ) Western blot of ubiquitinated using an anti - multi - ubiquitin antibody in 6 months old WT p32cKO heart ( n = 4 mice per group ) . Error bars are presented as mean ± SD . Student ' s t - was performed on WT vs p32cKO , * * * p < 0 . 005 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( B ) Autofluorescence showing lipofuscin localization around nucleus in 6 months old p32cKO heart . Tissues were excited at a wavelength of 540 ( upper panel ) or 470 ( lower panel ) emission spectra were collected with a confocal microscope at wavelengths ( band path ) of 580 - 630 nm ( upper panel ) or 510 - 560 nm ( lower panel ) . Enlarged view of squares is shown right . Scale bars , 20 µm . of of autofluorescence of DAPI is presented in plot in right panel as mean ± ( three months old mice , n = 11 ; 6 months old mice , n = 9 - 14 , 9 months old mice , n = 9 - 15 , 12 months old mice , n = 11 - 21 ) . Statistical significance was assessed by Student ' s t - , * * * p < 0 . 005 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005.


( D ) Western blot of Lamp2 in 6 months old WT p32cKO hearts . is shown in lower panel ( n = 4 mice per group ) . GAPDH was used as an internal control . Error bars are presented as mean ± SD . Student ' s t - was performed on WT vs p32cKO , * * * p < 0 . 005 .

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

(D) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts. Quantification is shown in the lower panel (n = 4 mice per group). GAPDH was used as an internal control. Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( A ) LC - MS / MS metabolomic of NAD + , NADH , NADP + , nicotinamide NAAD in 6 months old WT p32cKO hearts . NAD + NADP + showed significantly different between WT p32cKO hearts ( n = 12 ) . Error bars are presented as mean ± SD . Student ' s t - was performed on WT vs p32cKO , * * p < 0 . 001 , * * * p < 0 . 0002 .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) LC-MS/MS metabolomic analysis of NAD+, NADH, NADP+, nicotinamide and NAAD in the 6 months old WT and p32cKO hearts. NAD+ and NADP+ showed significantly different levels between WT and p32cKO hearts (n = 12). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.001, ***p < 0.0002.


( B ) Real - time PCR of expression of NAD - synthesizing enzymes in 6 months old WT p32cKO hearts . Error bars are presented as mean ± SD . Student ' s t - was performed on WT mice vs p32cKO mice ( n = 6 ) , * * * p < 0 . 005 , * p < 0 . 05 . right panel shows NAD synthesis pathway mRNA expression are indicated by red arrows . NAM : nicotinamide , NA : nicotinic acid , QA : quinolic acid , NMN : nicotinamide mononucleotide .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real-time PCR analysis of RNA expression of NAD-synthesizing enzymes in the 6 months old WT and p32cKO hearts. Error bars are presented as mean ± SD. Student's t-test was performed on WT mice vs p32cKO mice (n = 6), ***p < 0.005, *p < 0.05. The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows. NAM: nicotinamide, NA: nicotinic acid, QA: quinolic acid, NMN: nicotinamide mononucleotide.


( G ) Nmnat3 mRNA expression in 3T3 - L1 after 1 mM CAP treatment for 72 h . HIF1α inhibitor ( 20 or 50 µM ) was added 2 h before CAP treatment . Error bars are presented as mean ± SD of three independent experiments . Student ' s t - was performed on WT vs WT treated CAP ( or ) HIF1α inhibitor , * * p < 0 . 01 , * p < 0 . 05 .

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

(G) Nmnat3 mRNA expression in 3T3-L1 cells after 1 mM CAP treatment for 72 h. The HIF1α inhibitor (20 or 50 µM) was added 2 h before the CAP treatment. Error bars are presented as mean ± SD of three independent experiments. Student's t-test was performed on WT cells vs WT cells treated CAP and (or) HIF1α inhibitor,**p < 0.01, *p < 0.05.


( I ) Nmnat3 mRNA expression in 3T3 - L1 after 150 µM CoCl2 treatment for 72 h ( n = 3 ) . Error bars are presented as mean ± SD of three independent experiments . Statistical significance was assessed by Student ' s t - , * * p < 0 . 01 .

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

(I) Nmnat3 mRNA expression in 3T3-L1 cells after 150 µM CoCl2 treatment for 72 h (n = 3). Error bars are presented as mean ± SD of three independent experiments. Statistical significance was assessed by Student's t-test, **p < 0.01.


( A ) Lysosomal acidification is impaired in p32KO MEFs . Representative of WT p32KO MEFs , stained with dextran - Oregon Green ( 488 ) dextran - TMRM . Scale bar , 5 µm . Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification , 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification . Dextran - Oregon Green is quenched under acidic pH , thus an increased green : red denotes impaired lysosomal acidification . average ± of green : red in at least 30 in two independent experiments is presented in plot ( normalized to WT green / red = 1 ) . Statistical significance was assessed by Student ' s t - , * p < 0 . 05 , * * * p < 0 . 001 .

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

(A) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001.


( E ) Nmnat3 ( v1 ) overexpression in p32KO MEFs rescued lysosomal function . LysoTracker Red - stained p32KO MEFs were quantified by flow cytometry . Error bars are presented as mean ± of 4 independent experiments . Statistical significance was assessed by Student ' s t - , * * * p < 0 . 001 .

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

(E) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.


( A ) WT MEFs were stained with dextran - Oregon Green dextran - TMRM to examine lysosomal acidification after 10 nM FK866 treatment . FK866 decreased lysosomal acidification , which was rescued by 1 mM NMN pretreatment . stained MEFs were quantified using a microscope ( BZ - X800 , KEYENCE ) . Error bars are presented as mean ± of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

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

(A) WT MEFs were stained with dextran-Oregon Green and dextran-TMRM to examine lysosomal acidification after 10 nM FK866 treatment. FK866 decreased lysosomal acidification, which was rescued by 1 mM NMN pretreatment. The stained MEFs were quantified using a microscope (BZ-X800, KEYENCE). Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( F ) To examine autophagosome autolysosome formation , we used DAPRed , which indicates autophagosomes , DALGreen , which indicates autolysosomes . After starvation , co - localization of both agents was observed upon FK866 or bafilomycin A treatment in WT MEF . Scale bars , 10 µm . is shown on right . Error bars are presented as mean ± of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

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

(F) To examine autophagosome and autolysosome formation, we used DAPRed, which indicates autophagosomes, and DALGreen, which indicates autolysosomes. After starvation, co-localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells. Scale bars, 10 µm. Quantification is shown on the right. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( D ) After adding various concentrations of NaCl to lysosomal fraction of WT MEFs , pellet ( bound ) supernatant ( free ) were subjected to immunoblotting with GAPDH PGK1 antibodies .

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

(D) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs, the membrane pellet (bound) and supernatant (free) were subjected to immunoblotting with GAPDH and PGK1 antibodies.


( I ) After addition of GAP , NAD Pi , ATP production in lysosomal fraction isolated by MAG10 was measured using a luciferin / luciferase kit . Iodoacetate ( IA ; 4 µM ) was used to inhibit GAPDH activity . Three independent experiments were performed . Error bars are presented as mean ± of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * p < 0 . 002 .

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

(I) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, **p < 0.002.


C Representative plot showing gating strategy for CD4 + T . Effector Memory RA ( TEMRA ) : CD45RA + CD62L - ; Effector Memory ( EM ) : CD45RA - CD62L - ; Central Memory ( CM ) : CD45RA - CD62L + ; Memory ( TSCM ) : CD45RA + CD62L + .

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

C 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 of GFP + within T subpopulations 17 days after CD40LG editing of healthy donor ( HD ; n = 11 ) or ( Pt ; n = 1 ) derived CD4 + T , measured by FACS . Median ± IQR .

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

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 of GFP + or HDR in T from ( D ) . Median ± IQR .

E Percentage of GFP+ cells or HDR in T cells from (D). Median ± IQR.


F Population composition in HD or derived untreated ( n = 9 HD UT , 1 Pt UT ) or bulk edited T ( Treat ) from ( D ) . For each subpopulation , comparisons between HD Treated vs UT were performed by using LME models , with random effects defined to account for same donor for different number of replicates per donor within group . p - values were adjusted with Bonferroni ' s correction to account for multiple testing . In LME models , percentages were used in square root scale to meet assumption of normality of residuals of model . Mean ± .

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

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 of TCRBV families detected by spectratyping . UT , sorted edited ( GFP + ) , sorted non - edited ( GFP - ) CD4 + T derived from HD ( n = 1 ) or ( Pt ; n = 1 ) were analyzed at 17 days after CD40LG editing .

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

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 ( RFI , normalized to T0 ; left ) ( right ) on UT ( n = 3 ) , edited ( GFP + ) or unedited ( GFP - ) HD or Pt CD4 + T from ( D ) ( n = 9 HD , 1 Pt ) . Longitudinal comparisons between HD GFP + vs GFP - were performed with an LME model , accounting for multiple donors separately for RFI % CD40L + ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to 8 hours time - point ( * * * * p < 0 . 0001 * p = 0 . 0450 , respectively ) . # measured on small fraction of CD40L + . Median ± IQR .

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

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 GFP expression in UT or bulk edited ( Tretated ) CD4 + T derived from HD or Pt from ( D ) at 8 hr after PMA / Ionomycin stimulation .

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

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 co - culture . Right : IgG + secreting B , evaluated by ELISPOT . B were isolated from PB of HD co - cultured with HD or Pt sorted GFP + , GFP - UT T , resting ( R ) or stimulated with beads ( B ) or PMA / Ionomycin ( PI ) . B cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative positive controls , respectively ( n = 1 for each group ) .

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

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 co - culture . Right : of B proliferative capacity by Trace dilution in allogeneic sorted B isolated from PB of HD co - cultured with HD or Pt T from ( K ) . B cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative positive controls , respectively ( n = 2 for each group ) . Mean ± .

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

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.


B of NGFR + or HDR at 17 days after CD40LG editing of HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T ( n = 7 for each group ) . Median ± IQR .

O O O O O O O O O O O O O O O O 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 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 ( n = 4 HD , 1 Pt ) . Median ± IQR .

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

C 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 NGFR expression in UT or bulk edited ( Treated ) CD4 + T derived from HD or Pt from ( C ) before 8 hr after Pma / Ionomycin stimulation .

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

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 HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T from ( B ) ( n = 7 for each group ) . Friedman ( to account for same donor ) with p - values adjusted with Bonferroni ' s correction to account for multiple testing . Mean ± .

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

E 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 + engraftment in PB after transplantation of HD UT ( n = 5 ) , bulk edited ( NGFR bulk ; n = 5 ) or sorted NGFR + ( n = 5 ) CD4 + T . Longitudinal comparisons were performed with an LME model , followed by an appropriate post - hoc ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to overall difference among groups . Median ± IQR .

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 of HDR within human 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 . Effect of time eventual different effect of time in groups were not retained in final model after backward variable selection , highlighting a constant ( significantly different , * * * * p < 0 . 0001 ) behavior of groups over time . Median ± IQR .

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

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 retrieved from spleens of mice from ( F ) ( n = 1 for each group , except for NGFR - unedited group , n = 3 ) . 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 between expression of CD40LG edited mRNA ( codon - usage optimized , CO ) CD40LG wild - type mRNA ( WT ) in edited with three different donor templates from Fig EV2H . Three independent experiments ( n = 3 EF1a . GFP , 1 IRES . NGFR , 3 IRES . GFP ) . Median ± IQR .

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

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 normalized to T0 . Sorted edited ( + ) sorted unedited ( - ) CD4 + T were analyzed before 3 , 6 , 12 hr after Actinomycin D treatment ( n = 1 for each group ) .

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

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).


B Representative plots showing hEGFRt expression in bulk edited CD4 + T derived from HD before 8 hr after Pma / Ionomycin stimulation . were edited with three constructs depicted in ( A ) .

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

B 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 derived from HD at 3 days after treatment with immunotoxin ( left ) , antibody ( middle ) or toxin ( right ) .

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 of EGFR + T at 3 days after treatment with 5 nM or 1 nM of immunotoxin , antibody or toxin , measured by FACS . Friedman with Dunn ' s multiple comparisons . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * * p = 0 . 0052 * * p = 0 . 0024 for immunotoxin vs antibody * * p = 0 . 0010 * * p = 0 . 0024 for immunotoxin vs toxin , referring to EGFRmod1 EGFRmod2 respectively ) . Different dose - conditions were used as a unified group for statistical ( n = 10 for each group ) . Median ± IQR .

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

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.


B of HDR in HSPC subpopulations at 4 days after CD40LG editing , measured by molecular ( ddPCR ) . were edited with donor template either comprising only corrective cDNA ( NR ; n = 4 ) , or carrying selector cassette IRES . NGFR ( NGFR ; n = 5 ) sorted according to surface markers : CD34 - ; CD34 + CD133 - CD90 - ; CD34 + CD133 + CD90 - ; CD34 + CD133 + CD90 + . CD34 + CD133 + CD90 + / CD90 - CD34 + CD133 - / CD34 - conditions were used as unified groups for statistical . comparisons between groups were performed with LME models accounting for different subpopulations included in with random effects defined to account for same donor . In comparison about CD34 + CD133 + CD90 + / CD90 - , percentages were used in square root scale to meet assumption of normality of residuals of model . For CD34 + CD133 + CD90 + / CD90 - , * * p = 0 . 0035 , for CD34 + CD133 - / CD34 - p = 0 . 2051 . Median ± IQR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 from NGFR group , n = 7 ) . Paired Wilcoxon ' s , to account for same donor , with p - values adjusted with Bonferroni ' s correction to account for multiple testing . was performed separately for NR NGFR groups . Mean ± .

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

C 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 + engraftment in PB after transplantation of HSPC edited with two donor templates described in ( B ) 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 ) . reported statistical comparisons refer only to first time - point ( i . e . 6 ; * p = 0 . 0213 * * p = 0 . 0069 ) . Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching final plateau with respect to both GSE56 groups ( p = 0 . 0034 for NGFR + GSE56 p = 0 . 0080 for NR + GSE56 ) . Median ± IQR .

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

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 of HDR within human 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 , followed by an appropriate post - hoc ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to last time - point ( i . e . 20 ; * * * * p < 0 . 0001 in both comparisons ) . NR + GSE56 group shows a constant behavior over time ( p = 0 . 0588 ) . slope of 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 .

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

E 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.


of HDR within human in ( F ) PB , bone marrow , spleen , thymus measured by ddPCR in mice from ( D ) . Median .

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

Percentage of HDR within human cells in (F) PB, bone marrow, spleen, thymus measured by ddPCR in mice from (D). Median.


of HDR within human within BM - sorted human subpopulations ( CD19 + , CD33 + , CD3 + , CD34 + ) , measured by ddPCR in mice from ( D ) . Median .

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

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 retrieved from spleen of mice from ( D ) ( n = 4 NGFR + , 4 NGFR - ) . Mean ± .

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

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.


B Total of recipient CD3 + gated within CD45 . 2 + 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 ) . reported statistical comparisons refer only to day 1 ( * * * * p < 0 . 0001 ) . Mean ± .

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

B 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 of engrafted donor CD45 . 1 . 2 + in PB of mice from ( B ) . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to plateau ( * * * p = 0 . 0001 ) . Mean ± .

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

C 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 of donor CD4 + CD8 + gated within CD3 + CD45 . 1 . 2 + in PB of CPA treated ( D ) or NC mice ( E ) from ( B ) . Mean ± .

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

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 times indicated in ( A ) ( n = 7 CPA 300 , 13 NC for first boost 4 CPA 300 , 9 NC for second boost ) . Sera from vaccinated WT ( n = 5 ) CD40lg - / - ( n = 5 ) mice were used as positive 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 ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to overall difference among groups ( * * * * p < 0 . 0001 in comparisons ) . Median ± IQR .

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

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 of PNA + foci in splenic sections of experimental mice from ( B ) , calculated as between total PNA + number of PNA + foci / . Kruskal - Wallis followed by post - hoc with Dunn ' s ( 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 * * * * p < 0 . 0001 ) . Mean ± .

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

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 ) IgG ( right ) - secreting by ELISPOT ( n = 7 NC , 4 CPA300 , 3 WT , 2 CD40LG ) . Spots were counted by an ELISPOT Reader using a range of 0 . 005 - 1 mm . Median ± IQR .

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

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.


B Total of recipient CD3 + CD45 . 2 + 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 ( see Appendix Supplementary Statistical Methods ; * * * p = 0 . 0009 * * * * p < 0 . 0001 in comparisons ) . Mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 of engrafted donor CD45 . 1 + 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 ( see Appendix Supplementary Statistical Methods ; * * * p = 0 . 0002 * * * * p < 0 . 0001 in comparisons ) . CPA200 was not included in because n = 4 . Mean ± .

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

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 of relationship between total of recipient CD3 + gated within CD45 . 2 + at day 3 , 4 engrafted donor CD45 . 1 + T at 18 \ 24 days . This relationship was modeled with an asymptotic NLME model ( Appendix Supplementary Statistical Methods ) , accounting for different groups multiple experiments . From estimated model , a lower value of recipient CD3 + T corresponds to a higher value of engrafted donor CD4 + T ( p = 0 . 0006 ) .

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

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 in sera of mice from ( B ) collected before ( day 35 ; pre ) after ( day 49 ; post ) first boost ( n = 13 NC , 19 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ; three independent experiments ) at day 81 ( pre ) 89 ( post ) for second boost ( n = 4 NC , 4 CPA 300 , 4 CPA 200 , 5 ALS , 6 ANTI - CD4 ) . Sera from vaccinated WT ( n = 5 ) Cd40lg - / - ( n = 5 ) mice were used as positive negative controls , respectively . For early challenge data , comparisons were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to overall difference among groups ( * * * p = 0 . 0002 * * * * p < 0 . 0001 in comparisons ) . CPA200 was not included in because n = 4 . WT group , not indicated in figure , is significantly different from groups ( p < 0 . 0001 in comparisons ) . Of note , regarding comparisons between time - points , groups CPA 300 WT show a significant increase between pre post values ( p < 0 . 0001 for both ) , while other groups show a significant decrease between pre post values ( p < 0 . 0001 for except p = 0 . 0022 for ANTI - CD4 ) . Median ± IQR .

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

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 of recipient CD3 + gated within CD45 . 2 + after transfer of primed donor T 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 ( Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to day 4 ( * * * * p < 0 . 0001 in comparisons ) . Mean ± .

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

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 of engrafted donor CD45 . 1 + in PB of mice from ( F ) . Longitudinal comparisons were performed with an LME model followed by an appropriate post - hoc ( Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to day 25 . At day 215 a significant difference was observed between CPA 300 vs NC CPA 300 vs CPA 200 ( * * * * p < 0 . 0001 for both ) . Mean ± .

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

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 in sera of mice from ( F ) collected before ( day 36 , pre ) after ( day 50 , post ) first boost ( n = 6 NC , 11 CPA 300 , 11 CPA 200 ) at day 212 ( pre ) 219 ( post ) for second boost ( n = 6 NC , 11 CPA 300 , 9 CPA 200 ) . Sera from vaccinated WT ( n = 11 ) Cd40lg - / - ( n = 5 ) mice were used as positive negative controls , respectively . Comparisons were performed with an LME model followed by an appropriate post - hoc , separately for each challenge data ( Appendix Supplementary Statistical Methods ) . Reported statistical comparisons refer to overall difference among groups , for early challenge data ( * p = 0 . 0282 * * p = 0 . 0037 ) , while to differences between time - points within each group , for late challenge data ( * * p = 0 . 0018 * * * * p < 0 . 0001 in both comparisons ) . Of note , in late challenge data , no significant overall differences were observed among groups . Median ± IQR .

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

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.


B Total of WT donor CD4 + T 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 ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 in sera of mice from ( A ) collected seven days before ( pre ) 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 ( Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to overall difference among groups ( * * * * p < 0 . 0001 in comparisons ) . Median ± IQR .

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

C 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 of PNA + GL7 + GC B within spleen of mice from ( A ) ( n = 16 100 % WT , 6 25 % WT , 16 10 % WT , 11 1 % WT , 18 0 % WT ) . Kruskal - Wallis followed by post - hoc with Dunn ' s . 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 * * * * p < 0 . 0001 in both comparisons ) . Median ± IQR .

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

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 ( n = 8 100 % WT , 7 25 % WT , 7 10 % WT , 8 0 % WT ) infected with pathogen . Results are expressed in P . murina / HPRT copies . Kruskal - Wallis followed by post - hoc with Dunn ' s . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only groups 100 % WT 0 % WT resulted to be significantly different ( * * p = 0 . 0028 ) . Median ± IQR .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 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 of mice from ( E ) . Kruskal - Wallis followed by post - hoc with Dunn ' s . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only groups 100 % WT 0 % WT resulted to be significantly different ( * p = 0 . 0254 ) . Median ± IQR .

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

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 antibody ( upper left : negative sample ; lower left : positive sample ) . Brown areas represent intra - alveolar 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 ) .

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

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 ) or not ( n = 8 CD40LG ) with in vivo primed CD4 + T in absence of conditioning infected with pathogen . Results are expressed in P . Murina / HPRT copies . Mann - Whitney . * * * P - value of comparison = 0 . 0003 . Median ± IQR .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of experimental mice from ( H ) . Mann - Whitney . * * P - value of comparison = 0 . 0055 . Median ± IQR .

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

I Lung weight of experimental mice from (H). Mann-Whitney test. **P-value of the comparison = 0.0055. Median ± IQR.


( A ) Livedo reticularis in left abdominal lower quadrant after first infusion in Pt 01 .

(A) Livedo reticularis in the left abdominal lower quadrant after the first infusion in Pt 01.


( B ) Small clump of MABs observed in first preparation of MP before infusion of Pt 01 .

(B) Small clump of MABs observed in the first preparation of MP before the infusion of Pt 01.


( C ) Livedo reticularis in left of Pt02 after first infusion .

(C) Livedo reticularis in the left hand of Pt02 after the first infusion.


( D ) Brain MRI acquired one day after MAB infusion showing acute small thalamic stroke in Pt03 . Axial diffusion - weighted imaging ( left ) fluid - attenuated inversion recovery ( FLAIR ; right ) show a focal spot of hyperintensity within right thalamus consistent with acute stroke . ( E ) FLAIR MRI axial image obtained in Pt 03 one month after acute stroke , showing expected evolution of right thalamic lesion .

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

(D) Brain MRI acquired one day after the MAB infusion showing acute small thalamic stroke in Pt03. Axial diffusion-weighted imaging (left) and fluid-attenuated inversion recovery (FLAIR; right) images show a focal spot of hyperintensity within the right thalamus consistent with acute stroke.(E) FLAIR MRI axial image obtained in Pt 03 one month after the acute stroke, showing the expected evolution of the right thalamic lesion.


( A ) Haematoxilin Eosin of muscle biopsies from Pt 01 , Pt 02 Pt 03 performed 2 months after last MAB infusion . show diffuse increase of connective tissue , atrophic hypertrophic , degenerating , diffuse centralization of nuclei .

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

(A) Haematoxilin and Eosin staining of muscle biopsies from Pt 01, Pt 02 and Pt 03 performed 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei.


( B ) Haematoxilin Eosin of muscle biopsies from Pt 05 Pt 06 performed one month before first MAB infusion 2 months after last MAB infusion . show diffuse increase of connective tissue , atrophic hypertrophic , degenerating , diffuse centralization of nuclei . No evident differences were observed between biopsy performed after before treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Haematoxilin and Eosin staining of muscle biopsies from Pt 05 and Pt 06 performed one month before the first MAB infusion and 2 months after the last MAB infusion. Images show diffuse increase of connective tissue, atrophic and hypertrophic fibers, degenerating fibers, and diffuse centralization of nuclei. No evident differences were observed between biopsy performed after and before treatment.


( C ) Immunohistochemistry showing myosin expression in muscle biopsy of DMD performed 2 months after last MAB infusion . was performed in tibialis anterior muscle for Pt 01 , Pt 02 Pt 03 in gastrocnemius muscle for Pt 05 Pt 06 . A higher number of positive ( brownish ) ( representing regenerating ) were observed in Pt 02 , Pt 05 Pt 06 .

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

(C) Immunohistochemistry showing fetal myosin expression in muscle biopsy of DMD patients performed 2 months after the last MAB infusion. The staining was performed in tibialis anterior muscle for Pt 01, Pt 02 and Pt 03 and in gastrocnemius muscle for Pt 05 and Pt 06. A higher number of positive (brownish staining) fibers (representing regenerating fibers) were observed in Pt 02, Pt 05 and Pt 06.


( A ) Confocal immunofluorescence of muscle biopsy from Pt 01 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( which recognizes fragment encoded by exons 26 - 30 , green signal ) anti laminin - 2 ( to delineate muscle , red signal ) ; dapi identifies nuclei ( blue signal ) . In left image only anti dystrophin is shown . No dystrophin positive were observed . ( B ) Confocal immunofluorescence of muscle biopsy from Pt 02 ( tibialis anterior post - treatment ) stained with anti - dystrophin dys1 antibody ( green signal ) anti lamininin - 2 ( red signal ) ; dapi identifies nuclei ( blue signal ) . In left image only anti dystrophin is shown . Some fiber shows mild discontinuous dystrophin .

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

(A) Confocal immunofluorescence of muscle biopsy from Pt 01 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (which recognizes protein fragment encoded by exons 26-30, green signal) and anti laminin-2 (to delineate muscle fibers, red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. No dystrophin positive fibers were observed.(B) Confocal immunofluorescence of muscle biopsy from Pt 02 (tibialis anterior post-treatment) stained with anti-dystrophin dys1 antibody (green signal) and anti lamininin-2 (red signal); dapi identifies nuclei (blue signal). In the left image only anti dystrophin staining is shown. Some fiber shows mild and discontinuous dystrophin staining.


( C ) Immunofluorescence of muscle biopsy from Pt 05 taken before ( left , gastrocnemius ) after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody ( which recognizes exon 77 - 79 ) . number of dystrophin - positive is increased in post - treatment biopsy . ( D ) Immunofluorescence of muscle biopsy from Pt 06 taken before ( left , gastrocnemius ) after treatment ( right , gastrocnemius ) stained with anti dystrophin dys2 antibody . Few show scattered dystrophin , without obvious differences between pre post - treatment samples .

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

(C) Immunofluorescence of muscle biopsy from Pt 05 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody (which recognizes exon 77-79). The number and intensity of dystrophin-positive fibers is increased in the post-treatment biopsy.(D) Immunofluorescence of muscle biopsy from Pt 06 taken before (left, gastrocnemius) and after treatment (right, gastrocnemius) stained with anti dystrophin dys2 antibody. Few fibers show scattered dystrophin staining, without obvious differences between pre and post-treatment samples.


( E ) Results of western blot involving dystrophin antibodies dys1 , Mandys18 Manex46e ( recognizing respectively exon 26 - 30 , 17 - 35 , 46 ) , of total extracts ( 20 µg ) obtained from post - treatment biopsy specimens of Pt 01 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . No bands corresponding to full dystrophin were observed . A schematic representation of deleted portion of dystrophin region recognized by used antibodies is depicted above western blot . ( F ) Results of western blot involving dystrophin antibodies Mandys18 Manex46e , of total extracts ( 10 - 20 µg ) obtained from pre - treatment ( performed at time of diagnosis ) post - treatment biopsy specimens of Pt 02 ( tibialis anterior muscle ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . One faint band corresponding to full dystrophin is observed only in post - treatment samples with both Mandys18 Manex46e antibodies . A schematic representation of deleted portion of dystrophin region recognized by used antibodies is depicted above western blot . ( G ) Results of western blot involving dystrophin antibodies Mandys106 ( recognizing exon 43 ) dys1 , of total extracts ( 80 µg , following by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment post - treatment biopsy specimens of Pt 05 ( gastrocnemius ) ; Ct was used as positive dystrophin control , CtDmd was used as dystrophin negative control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full dystrophin are observed in pre post - treatment samples with both antibodies However , bands in post - treatment sample appeared higher in amount . A schematic representation of dystrophin point mutation ( black vertical bar ) region recognized by used antibodies is depicted above western blot . ( H ) Results of western blot involving dystrophin antibodies Mandys106 dys1 , of total extracts ( 80 µg , following by Amicon Ultra - 0 . 5 centrifugal filter devices , Millipore ) obtained from pre - treatment post - treatment biopsy specimens of Pt 06 ( gastrocnemius ) ; Ct was used as positive dystrophin control . Below , bands corresponding to myosin heavy chain are shown as loading control . Bands corresponding approximately to full dystrophin are observed in pre post - treatment samples with both antibodies Bands in pre - treatment sample appeared higher in amount as compared to post - treatment sample . A schematic representation of dystrophin point mutation ( black vertical bar ) region recognized by used antibodies is depicted above western blot .

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

(E) Results of western blot analysis involving dystrophin antibodies dys1, Mandys18 and Manex46e (recognizing respectively exon 26-30, 17-35, and 46), of total protein extracts (20 µg) obtained from post-treatment biopsy specimens of Pt 01 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. No bands corresponding to full length dystrophin were observed. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(F) Results of western blot analysis involving dystrophin antibodies Mandys18 and Manex46e, of total protein extracts (10-20 µg) obtained from pre-treatment (performed at time of diagnosis) and post-treatment biopsy specimens of Pt 02 (tibialis anterior muscle); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. One faint band corresponding to full length dystrophin is observed only in the post-treatment samples with both Mandys18 and Manex46e antibodies. A schematic representation of the deleted portion of the dystrophin and the region recognized by used antibodies is depicted above the western blot.(G) Results of western blot analysis involving dystrophin antibodies Mandys106 (recognizing exon 43) and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 05 (gastrocnemius); Ct was used as positive dystrophin control, CtDmd was used as dystrophin negative control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies However, bands in post-treatment sample appeared higher in amount. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.(H) Results of western blot analysis involving dystrophin antibodies Mandys106 and dys1, of total protein extracts (80 µg, following protein concentration by Amicon Ultra-0.5 centrifugal filter devices, Millipore) obtained from pre-treatment and post-treatment biopsy specimens of Pt 06 (gastrocnemius); Ct was used as positive dystrophin control. Below, bands corresponding to myosin heavy chain are shown as loading control. Bands corresponding approximately to full length dystrophin are observed in pre and post-treatment samples with both antibodies Bands in pre-treatment sample appeared higher in amount as compared to post-treatment sample. A schematic representation of the dystrophin point mutation (black vertical bar) and the region recognized by used antibodies is depicted above the western blot.


( A ) Axial T1 - weighted of right thighs obtained in immediately before ( upper panel ) 18 months after MAB infusion . Please note progression of fatty degeneration atrophy except for Pt 05 where no evident modifications can be detected . ( B ) Representative percentile of MRI quantitative parameters in transplanted compared to untreated . SIR ( left image ) MVI ( right image ) of quadriceps . Red : Pt 01 ; dotted green : Pt 02 ; dotted purple : Pt 03 ; dotted blue : Pt 05 ; dotted blue : Pt 06 ) . Empty dots correspond to pre - treatment measures . Orange dots correspond to post - transplantation measurement . black corresponds to median , while gray dotted lines to 75 ° , 90 ° 95 ° percentile . Please note modification of trend of Pt 05 after treatment .

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

(A) Axial T1-weighted images of the right thighs obtained in patients immediately before (upper panel) and 18 months after MAB infusion. Please note the progression of fatty degeneration and atrophy except for Pt 05 where no evident modifications can be detected. (B) Representative percentile of MRI quantitative parameters in transplanted patients compared to untreated patients. SIR (left image) and MVI (right image) of the quadriceps. Red line: Pt 01; dotted green line: Pt 02; dotted purple line: Pt 03; dotted dark blue line: Pt 05; dotted light blue line: Pt 06). Empty dots correspond to pre-treatment measures. Orange dots correspond to post-transplantation measurement. The black line corresponds to the median, while gray dotted lines to 75°, 90° and 95° percentile. Please note the modification of the trend of Pt 05 after treatment.


( C ) Representative quantile spline regressions of post - transplant trend amelioration of MRI quantitative parameters in Pt 05 . Upper left : SIR of quadriceps . Lower left : SIR of thigh flexor . Upper right : MVI of quadriceps . Lower right : MVI of semitendinosus . Please note significant modification of trend ( red lines ) compared to median measurements obtained with 85 MRI examinations of untreated . Empty dots correspond to pre - treatment measures . Full dots correspond to post - transplantation measurement . grey dotted corresponds to time of first intra - arterial transplantation .

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

(C) Representative quantile spline regressions of post-transplant trend amelioration of MRI quantitative parameters in Pt 05. Upper left: SIR of the quadriceps. Lower left: SIR of thigh flexor muscles. Upper right: MVI of the quadriceps. Lower right: MVI of the semitendinosus muscles. Please note the significant modification of the patient trend (red lines) compared to median measurements obtained with 85 MRI examinations of untreated patients. Empty dots correspond to pre-treatment measures. Full dots correspond to post-transplantation measurement. The grey dotted line corresponds to the time of the first intra-arterial transplantation.


( A ) North Star scale measurement ( NSAA score ) plotted against of ambulant DMD before , throughout after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted indicates time period in which Pt05 spontaneously suspended steroids treatment without communication to parents clinicians . Pt 02 Pt 05 showed score stabilization throughout eight months of MAB infusions ; Pt05 showed stabilization even in subsequent period , whereas Pt02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion lost ambulation soon after end of trial .

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

(A) North Star scale measurement (NSAA score) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout eight months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.


( B ) Six minute walking ( meters ) plotted against of ambulant DMD before , throughout after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted indicates time period in which Pt05 spontaneously suspended steroids treatment without communication to parents clinicians . Pt 02 Pt 05 showed score stabilization throughout 8 months of MAB infusions ; Pt05 showed stabilization even in subsequent period , whereas Pt 02 showed progressive deterioration until lost of ambulation . Pt 03 showed progressive deterioration throughout MAB infusion lost ambulation soon after end of trial .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Six minute walking test (meters) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed score stabilization throughout 8 months of MAB infusions; Pt05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive deterioration until lost of ambulation. Pt 03 showed progressive deterioration throughout MAB infusion and lost ambulation soon after the end of the trial.


( C ) Time to run 10 meters ( time ) plotted against of ambulant DMD before , throughout after MAB clinical trial . Arrows indicate time points of MAB infusion . Dotted indicates time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents clinicians . Pt 02 Pt 05 showed time stabilization throughout 8 months of MAB infusions ; Pt 05 showed stabilization even in subsequent period , whereas Pt 02 showed progressive increase of time until lost of ambulation . Pt 03 showed progressive increase of time throughout MAB infusion lost ambulation soon after end of trial .

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

(C) Time to run 10 meters (time) plotted against age of ambulant DMD patients before, throughout and after MAB clinical trial. Arrows indicate time points of MAB infusion. Dotted line indicates the time period in which Pt 05 spontaneously suspended steroids treatment without communication to parents and clinicians. Pt 02 and Pt 05 showed time stabilization throughout 8 months of MAB infusions; Pt 05 showed stabilization even in the subsequent period, whereas Pt 02 showed progressive increase of time until lost of ambulation. Pt 03 showed progressive increase of time throughout MAB infusion and lost ambulation soon after the end of the trial.


Panel A : viral donor specific T - responses were measured by IFN - γELISpot at different time points : before beginning of treatment ( PRE ) , prior to each infusion 2 months after fourth infusion . Infusions are indicated by arrows . ' s peripheral blood mononuclear ( PBMC ) were challenged with irradiated autologous lymphoblastoid lines ( EBV host ) , with cytomegalovirus glycin extract ( CMV antigen ) ( Gehrz et al , 1987 ) with following irradiated harvested from donor : untreated mesoangioblasts ( MABdonor ) , MAB activated by 48 hour - exposure to 500 IU / mL IFN - γ ( γMABdonor ) , myotubes differentiated from MAB ( myotubesdonor ) PBMC ( PBMCdonor ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . Donor PBMC were challenged with autologous targets as negative controls . Results are expressed as number of specific / 105PBMC calculated according to following formula : number of spots produced by ' s PBMC - number of spots produced by donor ' s PBMC . Results for patientsPt 01 , Pt 02 , Pt 03 , Pt 05 Pt 06 are shown .

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

Panel A: viral and donor specific T-cell responses were measured by IFN-γELISpot at different time points: before the beginning of treatment (PRE), prior to each infusion and 2 months after the fourth infusion. Infusions are indicated by arrows. Patient's peripheral blood mononuclear cells (PBMC) were challenged with irradiated autologous lymphoblastoid cell lines (EBV line host), with cytomegalovirus glycin extract (CMV antigen)(Gehrz et al, 1987) and with the following irradiated cells harvested from the donor: untreated mesoangioblasts (MABdonor), MAB activated by 48 hour-exposure to 500 IU/mL IFN-γ (γMABdonor), myotubes differentiated from MAB (myotubesdonor) and PBMC (PBMCdonor). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. Donor PBMC were challenged with autologous targets as negative controls. Results are expressed as number of specific cells/105PBMC and calculated according to the following formula: number of spots produced by patient's PBMC - number of spots produced by donor's PBMC. Results for patientsPt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.


Panel B : dystrophin specific T - responses were measured by IFN - γ ELISpot at two time points : before beginning of treatment ( PRE , bar ) 2 months after fourth infusion ( black bar ) . PBMC were challenged with a library of 15 - mer overlapping peptides covering ( pool 1 - 8 ) . Polyclonal stimulation ( phytohemagglutinin , PHA ) was used as positive control . grey represents threshold calculated on a cohort of healthy donors . Results for Pt 01 , Pt 02 , Pt 03 , Pt 05 Pt 06 are shown .

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

Panel B: dystrophin specific T-cell responses were measured by IFN-γ ELISpot at two time points: before the beginning of treatment (PRE, white bar) and 2 months after the fourth infusion (black bar). PBMC were challenged with a library of 15-mer overlapping peptides covering the protein sequence (pool 1-8). Polyclonal stimulation (phytohemagglutinin, PHA) was used as positive control. The grey area represents the threshold calculated on a cohort of healthy donors. Results for Pt 01, Pt 02, Pt 03, Pt 05 and Pt 06 are shown.


( A ) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic early postnatal brain , lower in adult brain . Data are presented as mean ± . ( A ) ng PTX3 / mg of total , E15 = 5 , 702 ± 0 . 365 , P0 = 4 . 904 ± 0 . 865 , P7 = 3 . 818 ± 0 . 530 , P14 = 2 . 140 ± 0 . 107 , P28 = 3 . 125 ± 0 . 525 , P90 = 2 . 212 ± 0 . 324 . One - way ANOVA of variance followed by post hoc Tukey : E15 vs P14 * * p = 0 . 001 , E15 vs P28 * p = 0 . 022 , E15 vs P90 * * p = 0 . 002 ; P0 vs P14 $ p = 0 . 013 , P0 vs P90 $ p = 0 . 016 ; 4 for each time

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

(A) ELISA assa from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM. (A) ng PTX3/mg of total proteins, E15=5,702±0.365, P0=4.904±0.865, P7=3.818±0.530, P14=2.140±0.107, P28=3.125±0.525, P90=2.212±0.324. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P14 **p=0.001, E15 vs P28 *p=0.022, E15 vs P90 **p=0.002; P0 vs P14 $p=0.013, P0 vs P90 $p=0.016 ; 4 animals for each time point


( B ) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic early postnatal brain , lower in adult brain . Data are presented as mean ± ( B ) qPCR fold change normalized on E15 , E15 = 1 . 037 ± 0 . 154 , P0 = 0 . 711 ± 0 . 094 , P7 = 0 . 472 ± 0 . 0524 , P14 = 0 . 135 ± 0 . 007 , P28 = 0 . 168 ± 0 . 020 , P90 = 0 . 183 ± 0 . 029 . One - way ANOVA of variance followed by post hoc Tukey : E15 vs P7 * * * p = 0 . 0001 , E15 vs P14 * * * * p < 0 . 0001 , E15 vs P2 * * * * p < 0 . 0001 , E15 vs P90 * * * * p < 0 . 0001 ; P0 vs P14 $ $ $ p = 0 . 0007 , P0 vs P28 $ $ p = 0 . 001 , P0 vs P90 $ $ p = 0 . 002 ; 4 for each time

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) real time qPCR from brain lysates showing higher PTX3 expression in late embryonic and early postnatal brain, and lower in the adult brain. Data are presented as mean±SEM (B) qPCR fold change normalized on E15, E15=1.037±0.154, P0=0.711±0.094, P7=0.472±0.0524, P14=0.135±0.007, P28=0.168±0.020, P90=0.183±0.029. One-way ANOVA analysis of variance followed by post hoc Tukey test: E15 vs P7 ***p=0.0001, E15 vs P14 **** p<0.0001, E15 vs P2 ****p<0.0001, E15 vs P90 ****p<0.0001; P0 vs P14 $$$p=0.0007, P0 vs P28 $$p=0.001, P0 vs P90 $$p=0.002; 4 animals for each time point


Quantitation of PTX3 in pure astrocyte neuronal cultures . ( C ) ELISA performed on astrocyte neuronal culture mediu Only astrocytes release PTX3 ( ELISA , astro = 3 . 699 ± 1 . 129 , neu = N . D . , not detectable . 3 independent astrocyte cultures evaluated Data are presented as mean ±

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

Quantitation of PTX3 levels in pure astrocyte and neuronal cultures. (C) ELISA assay performed on astrocyte and neuronal culture mediu Only astrocytes release PTX3 (ELISA, astro=3.699±1.129, neu=N.D., not detectable. 3 independent astrocyte cultures evaluated Data are presented as mean±SEM


Quantitation of PTX3 in pure astrocyte neuronal cultures ( D ) rea time qPCR performed on astrocyte neuronal lysates qPCR astro = 1 . 167 ± 0 . 288 ; neu = 0 . 148 ± 0 . 057 ; Mann - Whitney * * p = 0 . 0095 . 2 independent neuronal cultures evaluated . Data are presented as mean ±

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

Quantitation of PTX3 levels in pure astrocyte and neuronal cultures (D) rea time qPCR performed on astrocyte and neuronal lysates qPCR astro=1.167±0.288; neu=0.148±0.057; Mann-Whitney test **p=0.0095. 2 independent neuronal cultures evaluated. Data are presented as mean±SEM


A ) Representative mEPSC traces recorded from control PTX3 - treated ( 1µg / ml ; 48hrs ) neurons . B ) mEPSC frequency quantitation ( Hz , Ctr = 0 . 618 ± 0 . 069 ; PTX3 = 1 . 991 ± 0 . 313 ; Number of neurons : Ctr = 22 , PTX3 = 16 ; 3 independent experiments , Mann - Whitney . Data are presented as a distribution plus mean ± ) . C ) mEPSC amplitude quantitation cumulative probability distribution of mEPSC amplitudes ( pA , Ctr = 12 . 76 ± 0 . 813 ; PTX3 = 16 . 07 ± 0 . 709 . Number of neurons : Ctr = 22 , PTX3 = 16 ; 3 independent experiments , Mann - Whitney . Data are presented as a distribution plus mean ± . Cumulative probability distributions are analyzed by Kolmogorov - Smirnov

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

A) Representative mEPSC traces recorded from control and PTX3-treated (1µg/ml; 48hrs) neurons. B) mEPSC frequency quantitation (Hz, Ctr=0.618±0.069; PTX3=1.991±0.313; Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM). C) mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr=12.76±0.813; PTX3=16.07±0.709. Number of neurons: Ctr=22, PTX3=16; 3 independent experiments, Mann-Whitney test. Data are presented as a distribution plus mean±SEM. Cumulative probability distributions are analyzed by Kolmogorov-Smirnov test


D ) Representative showing 14DIV neurons stained for surface AMPAR ( GluA , green ) , presynaptic Bassoon ( blue ) tubulin ( red ) in different tested conditions . Arrowheads point to postsynaptic GluA clusters . Inset : Example of surface synaptic AMPARs cluster ( GluA & Bsn ) . Scale bar : 5 µm . E ) of surface synaptic AMPARs ( GluA & Bsn ) normalized to total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure ( Ctr = 1 ± 0 . 051 , TTX = 1 . 512 ± 0 . 080 , PTX3 = 1 . 294 ± 0 . 081 , PTX3 heat - inactivated = 0 . 959 ± 0 . 044 ; number of fields examined : 27 , 36 , 37 , 22 , 19 respectively ; one - way ANOVA of variance , p < 0 . 0001 , followed by post hoc Tukey for multiple comparison as indicated in figure ; 3 independent experiments , data are presented as normalized mean values ± ) . F ) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content ( Ctr : 1 ± 0 . 065 , PTX3 : 1 . 023 ± 0 . 068 ; number of fields : Ctr = 26 , PTX3 = 26 ; unpaired t - , 3 independent experiments , data are presented as normalized mean values ± )

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

D) Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA&Bsn). Scale bar: 5 µm. E) Quantification of the surface synaptic AMPARs (GluA&Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr=1±0.051, TTX=1.512±0.080, PTX3=1.294±0.081, PTX3 heat-inactivated=0.959±0.044; number of fields examined: 27, 36, 37, 22, 19 respectively; one-way ANOVA analysis of variance, p<0.0001, followed by post hoc Tukey test for multiple comparison as indicated in figure; 3 independent experiments, data are presented as normalized mean values±SEM). F) 1hr of PTX3 administration is not sufficient to elicit an increase in synaptic GluA content (Ctr: 1±0.065, PTX3: 1.023±0.068; number of fields: Ctr=26, PTX3=26; unpaired t-test, 3 independent experiments, data are presented as normalized mean values±SEM)


G ) Representative mEPSC traces recorded from control , C - term PTX3 fragment or N - term PTX3 fragment - treated cultures . H ) mEPSC frequency quantitation ( Hz , Ctr = 0 . 668 ± 0 . 078 ; C - term = 0 . 523 ± 0 . 056 ; N - term = 1 . 228 ± 0 . 121 . Number of neurons : Ctr = 23 , C - term = 10 , N - term = 23 ; 3 independent experiments . Kruskall - Wallis , p < 0 . 0001 followed by Dunn " s as indicated , data are presented as a distribution plus mean ± ) . I ) mEPSC amplitude quantitation ( pA , Ctr = 16 . 81 ± 0 . 78 ; C - term = 15 . 79 ± 1 . 37 ; N - term = 19 . 78 ± 0 . 87 . Number of neurons : Ctr = 23 , C - term = 10 , N - term = 23 ; 3 independent experiments . One - way ANOVA of variance , p = 0 . 013 followed by post hoc Tukey as indicated in figure , data are presented as a distribution plus mean ± ) cumulative probability distribution of mEPSC amplitudes ( Kolmogorov -

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

G) Representative mEPSC traces recorded from control, C-term PTX3 fragment or N-term PTX3 fragment-treated cultures. H) mEPSC frequency quantitation (Hz, Ctr=0.668±0.078; C-term=0.523±0.056; N-term=1.228±0.121. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. Kruskall-Wallis test, p<0.0001 followed by Dunn"s test as indicated, data are presented as a distribution plus mean±SEM). I) mEPSC amplitude quantitation (pA, Ctr=16.81±0.78; C-term=15.79±1.37; N-term=19.78±0.87. Number of neurons: Ctr=23, C-term=10, N-term=23; 3 independent experiments. One-way ANOVA analysis of variance, p=0.013 followed by post hoc Tukey test as indicated in figure, data are presented as a distribution plus mean±SEM) and cumulative probability distribution of mEPSC amplitudes (Kolmogorov-Smirnov


J ) Representative of 14DIV neurons stained for surface AMPARs ( GluA , green ) , presynaptic Bassoon ( blue ) tubulin ( red ) in different tested conditions . Arrowheads point to postsynaptic GluA clusters . Scale bar : 5µm . K ) of surface synaptic AMPARs normalized to total number of Bsn shows an increase upon N - terminal peptide application , but not upon C - terminal peptide exposure . TTX is used as positive control ( Ctr = 1 . 000 ± 0 . 066 . TTX = 1 . 395 ± 0 . 113 , C - term = 0 . 717 ± 0 . 047 , N - term = 1 . 349 ± 0 . 069 . Number of fields examined : 55 , 31 , 18 , 54 , respectively ; one - way ANOVA of variance , p < 0 . 0001 followed by post hoc Tukey as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± ) .

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

J) Representative images of 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue) and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5µm. K) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn shows an increase upon N-terminal peptide application, but not upon C-terminal peptide exposure. TTX is used as positive control (Ctr=1.000±0.066. TTX=1.395±0.113, C-term=0.717±0.047, N-term=1.349±0.069. Number of fields examined: 55, 31, 18, 54, respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM).


A ) Low high magnification of control PTX3 - treated neurons stained for PNN main component , aggrecan ( red ) , synaptic PSD95 ( green ) Bsn ( blue ) . Scale bar :

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

A) Low and high magnification images of control and PTX3-treated neurons stained for the PNN main component, aggrecan (red), the synaptic proteins PSD95 (green) and Bsn (blue). Scale bar: 5µm


B - D ) PTX3 application induces a remodeling of PNN in culture , as assessed by increase mean integrated density value of synapse - co - localizing aggrecan signal whereas no difference in total of aggrecan is evident ( integrated density : Ctr = 1 ± 0 . 103 , PTX3 = 1 . 874 ± 0 . 197 ; mean : Ctr = 1 . 000 ± 0 . 088 , PTX3 = 1 . 556 ± 0 . 107 ; total : Ctr = 1 ± 0 . 179 , PTX3 = 0 . 982 ± 0 . 149 . Number of fields examined : 26 Ctr , 22 PTX3 ; Mann - Whitney ; 3 independent experiments , data are presented as normalized mean values ± )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) PTX3 application induces a remodeling of the PNN in culture, as assessed by the increase mean intensity and integrated density value of the synapse-co-localizing aggrecan signal whereas no difference in the total area of aggrecan is evident (integrated density: Ctr=1±0.103, PTX3=1.874±0.197; mean intensity: Ctr=1.000±0.088, PTX3=1.556±0.107; total area: Ctr=1±0.179, PTX3=0.982±0.149. Number of fields examined: 26 Ctr, 22 PTX3; Mann-Whitney test; 3 independent experiments, data are presented as normalized mean values±SEM)


E ) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan ( red ) , DAPI ( cyan ) βIII - tubulin ( green ) confocal . Scale bar :

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

E) Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan (red), DAPI (cyan) and βIII-tubulin (green) and confocal analysis. Scale bar: 20µm