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 .

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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 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 .

O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD 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-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD 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 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 .

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C Population composition in UT or bulk edited HSPC from (B) (n=8 for each group, except UT cells from NGFR group, n=7). Paired Wilcoxon's test, to account for the same donor, with p-values adjusted with Bonferroni's correction to account for multiple testing. Analysis was performed separately for NR and NGFR groups. Mean ± SEM.


D Human CD45 + cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in ( B ) and with ( n = 4 ) or without ( n = 5 ) GSE56 . Comparison between groups was performed by NLME model with an asymptotic model ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the first time - point ( i . e . 6 weeks ; * p = 0 . 0213 and * * p = 0 . 0069 ) . Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups ( p = 0 . 0034 for NGFR + GSE56 and p = 0 . 0080 for NR + GSE56 ) . Median ± IQR .

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D Human CD45+ cell engraftment in PB after transplantation of HSPC edited with the two donor templates described in (B) and with (n=4) or without (n=5) GSE56. Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the first time-point (i.e. 6 weeks; *p=0.0213 and **p=0.0069). Group transplanted with HSPC treated without GSE56 needs significantly greater time for reaching the final plateau with respect to both GSE56 groups (p=0.0034 for NGFR+GSE56 and p=0.0080 for NR+GSE56). Median ± IQR.


E Percentage of HDR within human cells over time , measured by ddPCR in PB of mice from ( D ) . Longitudinal comparisons were performed with an LME model with time treated as a continuous variable , and followed by an appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the last time - point ( i . e . 20 weeks ; * * * * p < 0 . 0001 in both comparisons ) . NR + GSE56 group shows a constant behavior over time ( p = 0 . 0588 ) . The slope of the linear trajectory over time is not significantly different between GSE56 groups ( p = 0 . 1624 ) , indicating gene editing stability of both groups over time . Mice transplanted with HSPC treated without GSE56 shows significantly lower slope than GSE56 groups ( p < 0 . 0001 in respect of NGFR group , p = 0 . 0054 in respect of NR group ) , thus highlighting decreased gene editing efficiency over time . Median ± IQR .

O O O B-EXP_ASSAY O B-ORGANISM O O O O O O B-EXP_ASSAY O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O 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.


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

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


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

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


H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by MFI on pooled CD4 + T cells retrieved from spleen of mice from ( D ) ( n = 4 NGFR + , 4 NGFR - ) . Mean ± SEM .

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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 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 .

O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O B-GENEPROD O O O O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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D, E Percentage of donor CD4+ and CD8+ cells gated within CD3+CD45.1.2+ cells in PB of CPA treated (D) or NC mice (E) from (B). Mean ± SEM.


F TNP - KLH specific IgG in sera of transplanted mice collected at the times indicated in ( A ) ( n = 7 CPA 300 , 13 NC for first boost and 4 CPA 300 , 9 NC for second boost ) . Sera from vaccinated WT ( n = 5 ) and CD40lg - / - ( n = 5 ) mice were used as positive and negative controls , respectively . Two independent experiments . Comparisons of first boost data were performed with an LME model , accounting for multiple experiments , followed by appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups ( * * * * p < 0 . 0001 in all comparisons ) . Median ± IQR .

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F TNP-KLH specific IgG in sera of transplanted mice collected at the times indicated in (A) (n=7 CPA 300, 13 NC for first boost and 4 CPA 300, 9 NC for second boost). Sera from vaccinated WT (n=5) and CD40lg-/- (n=5) mice were used as positive and negative controls, respectively. Two independent experiments. Comparisons of first boost data were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.


G Average area of PNA + foci in splenic sections of experimental mice from ( B ) , calculated as ratio between total PNA + area and number of PNA + foci / cells . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test ( n = 9 CD40LG , 6 WT , 18 NC , 8 CPA300 ) . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0149 , * * p = 0 . 0065 and * * * * p < 0 . 0001 ) . Mean ± SEM .

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G Average area of PNA+ foci in splenic sections of experimental mice from (B), calculated as ratio between total PNA+ area and number of PNA+ foci/cells. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test (n=9 CD40LG, 6 WT, 18 NC, 8 CPA300). P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0149, **p=0.0065 and ****p<0.0001). Mean ± SEM.


H Detection of splenic TNP - KLH specific IgM ( left ) and IgG ( right ) - secreting cells by ELISPOT assay ( n = 7 NC , 4 CPA300 , 3 WT , 2 CD40LG ) . Spots were counted by an ELISPOT Reader using a size range of 0 . 005 - 1 mm . Median ± IQR .

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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 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 .

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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 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 .

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C Total counts of engrafted donor CD45.1+ cells in PB of mice from (B). Three independent experiment. Comparisons at day 18\24 were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods; ***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. Mean ± SEM.


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

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O 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-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE 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 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 .

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E TNP-KLH specific IgG concentration in sera of mice from (B) collected before (day 35; pre) and after (day 49; post) the first boost (n=13 NC, 19 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4; three independent experiments) and at day 81 (pre) and 89 (post) for the second boost (n=4 NC, 4 CPA 300, 4 CPA 200, 5 ALS, 6 ANTI-CD4). Sera from vaccinated WT (n=5) and Cd40lg-/- (n=5) mice were used as positive and negative controls, respectively. For early challenge data, comparisons were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (***p=0.0002 and ****p<0.0001 in all comparisons). CPA200 was not included in the analysis because n=4. WT group, not indicated in the figure, is significantly different from all groups (p<0.0001 in all comparisons). Of note, regarding the comparisons between time-points, the groups CPA 300 and WT show a significant increase between pre and post values (p<0.0001 for both), while all other groups show a significant decrease between pre and post values (p<0.0001 for all except p=0.0022 for ANTI-CD4). Median ± IQR.


F Total counts of recipient CD3 + cells gated within CD45 . 2 + cells after transfer of primed donor T cells in PB of mice treated with 300 mg / kg CPA ( n = 11 ) , 200 mg / kg CPA ( n = 11 ) or NC ( n = 6 ) . Longitudinal comparisons were performed by LME model followed by an appropriate post - hoc analysis ( Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to day 4 ( * * * * p < 0 . 0001 in all comparisons ) . Mean ± SEM .

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F Total counts of recipient CD3+ cells gated within CD45.2+ cells after transfer of primed donor T cells in PB of mice treated with 300 mg/kg CPA (n=11), 200 mg/kg CPA (n=11) or NC (n=6). Longitudinal comparisons were performed by LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 4 (****p<0.0001 in all comparisons). Mean ± SEM.


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

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G Total counts of engrafted donor CD45.1+ cells in PB of mice from (F). Longitudinal comparisons were performed with an LME model followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 25. At day 215 a significant difference was observed between CPA 300 vs NC and CPA 300 vs CPA 200 (****p<0.0001 for both). Mean ± SEM.


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

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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 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 .

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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 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 .

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C TNP-KLH specific IgG concentration in sera of mice from (A) collected seven days before (pre) and after (post) TNP-KLH vaccination (n=13 100% WT, 13 25% WT, 14 10% WT, 6 1% WT, 14 0% WT). Three independent experiments. Comparisons were performed with an LME model, accounting for multiple experiments, followed by an appropriate post-hoc analysis (Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.


D Percentage of PNA + GL7 + GC B cells within the spleen of mice from ( A ) ( n = 16 100 % WT , 6 25 % WT , 16 10 % WT , 11 1 % WT , 18 0 % WT ) . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons ( * p = 0 . 0385 for 25 % WT vs 1 % WT , * p = 0 . 0129 for 100 % WT vs 1 % WT , * * * p = 0 . 0001 and * * * * p < 0 . 0001 in both comparisons ) . Median ± IQR .

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D Percentage of PNA+GL7+ GC B cells within the spleen of mice from (A) (n=16 100% WT, 6 25% WT, 16 10% WT, 11 1% WT, 18 0% WT). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0385 for 25% WT vs 1% WT, *p=0.0129 for 100% WT vs 1% WT, ***p=0.0001 and ****p<0.0001 in both comparisons). Median ± IQR.


E Quantitation of P . murina rRNA in lung homogenate of mice from ( A ) transplanted with different ratios of WT HSPC cells ( n = 8 100 % WT , 7 25 % WT , 7 10 % WT , 8 0 % WT ) and infected with the pathogen . Results are expressed in P . murina / HPRT RNA copies . Kruskal - Wallis test followed by post - hoc analysis with Dunn ' s test . P - values were adjusted with Bonferroni ' s correction to account for multiple comparisons . Only the groups 100 % WT and 0 % WT resulted to be significantly different ( * * p = 0 . 0028 ) . Median ± IQR .

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E Quantitation of P. murina rRNA in lung homogenate of mice from (A) transplanted with different ratios of WT HSPC cells (n=8 100% WT, 7 25% WT, 7 10% WT, 8 0% WT) and infected with the pathogen. Results are expressed in P.murina/HPRT RNA copies. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (**p=0.0028). Median ± IQR.


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

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F Lung weight of mice from (E). Kruskal-Wallis test followed by post-hoc analysis with Dunn's test. P-values were adjusted with Bonferroni's correction to account for multiple comparisons. Only the groups 100% WT and 0% WT resulted to be significantly different (*p=0.0254). Median ± IQR.


G Left : Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody ( upper left : negative sample ; lower left : positive sample ) . Brown areas represent intra - alveolar aggregates of P . murina organisms . Right : Representative pictures of P . murina organisms detected by immunofluorescence in lungs homogenate ( upper right : negative sample ; lower right : positive sample ) .

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G Left: Representative pictures of lung tissue sections stained immunohistochemically with a rabbit primary antibody (upper left: negative sample; lower left: positive sample). Brown areas represent intra-alveolar aggregates of P. murina organisms. Right: Representative pictures of P. murina organisms detected by immunofluorescence in lungs homogenate (upper right: negative sample; lower right: positive sample).


H Quantitation of P . Murina rRNA in lung homogenate of mice adoptively transferred ( n = 9 CD40LG + T cells ) or not ( n = 8 CD40LG ) with in vivo primed CD4 + T cells in absence of conditioning and infected with the pathogen . Results are expressed in P . Murina / HPRT RNA copies . Mann - Whitney test . * * * P - value of the comparison = 0 . 0003 . Median ± IQR .

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H Quantitation of P. Murina rRNA in lung homogenate of mice adoptively transferred (n=9 CD40LG + T cells) or not (n=8 CD40LG) with in vivo primed CD4+ T cells in absence of conditioning and infected with the pathogen. Results are expressed in P.Murina/HPRT RNA copies. Mann-Whitney test. ***P-value of the comparison = 0.0003. Median ± IQR.


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

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


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

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


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

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


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

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


( 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O 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-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O 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-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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-ORGANISM O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O 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-ORGANISM O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM 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 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 .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O B-CELL_TYPE 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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O B-CELL_TYPE 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O B-CELL_TYPE 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 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 .

O O O O O B-ORGANISM O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-ORGANISM O O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O B-CELL_TYPE O B-CELL_TYPE O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O O O O O 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-CELL_TYPE O O O O O O B-ORGANISM O O B-CELL_TYPE O O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM 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 - 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 .

O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O 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-CELL_TYPE O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM O O B-ORGANISM 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 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

O O O B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ±

O O B-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O B-CELL_TYPE O O O B-CELL_TYPE O B-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE 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 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 ±

O O B-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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 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

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O 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-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O 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 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 )

O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD 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 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 -

O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 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 ) .

O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 :

O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE B-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD 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 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 )

O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD 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 ) and βIII - tubulin ( green ) and confocal analysis . Scale bar :

O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY 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


F ) Representative images showing 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 :

O O O O O O B-CELL_TYPE B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O

F) Representative images showing 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


G ) HAse treatment blocks PTX3 - induced synaptic surface AMPA receptors clustering ( Ctr = 1 . 000 ± 0 . 075 , PTX3 = 1 . 425 ± 0 . 088 , PTX3 + HAse = 0 . 961 ± 0 . 053 , HAse = 1 . 080 ± 0 . 087 . Number of fields examined : 37 , 23 , 53 , 28 respectively ; Kruskall - Wallis test , p = 0 . 0004 followed by post hoc Tukey test as indicated in figure ; 3 independent experiments , data are presented as normalized mean value ± SEM )

O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HAse treatment blocks PTX3-induced synaptic surface AMPA receptors clustering (Ctr=1.000±0.075, PTX3=1.425±0.088, PTX3+HAse=0.961±0.053, HAse=1.080±0.087. Number of fields examined: 37, 23, 53, 28 respectively; Kruskall-Wallis test, p=0.0004 followed by post hoc Tukey test as indicated in figure; 3 independent experiments, data are presented as normalized mean value±SEM)


H ) Representative images showing 14DIV WT and TSG6 KO 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 :

O O O O O O O O B-GENEPROD O B-CELL_TYPE B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O

H) Representative images showing 14DIV WT and TSG6 KO 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


I ) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures . On the contrary WT cultures ( from littermates ) display increased surface GluA & Bsn / Bsn upon PTX3 treatment . A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures ( WT = 1 ± 0 . 06 ; WT + PTX3 = 1 . 389 ± 0 . 113 ; WT + TTX = 1 . 698 ± 0 . 109 ; Number of fields examined : 40 , 39 , 32 respectively ; Kruskall - Wallis test p < 0 . 0001 followed by post hoc Dunn " s test . TSG6 KO = 1 . 000 ± 0 . 047 ; TSG6 KO + PTX3 = 0 . 979 ± 0 . 048 ; TSG6 KO + TTX = 1 . 363 ± 0 . 077 . Number of fields examined : 54 , 57 , 29 respectively ; one - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure . n = 3 independent experiments , data are presented as normalized mean

O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures. On the contrary WT cultures (from littermates) display increased surface GluA&Bsn/Bsn upon PTX3 treatment. A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures (WT=1±0.06; WT+PTX3=1.389±0.113; WT+TTX=1.698±0.109; Number of fields examined: 40, 39, 32 respectively; Kruskall-Wallis test p<0.0001 followed by post hoc Dunn"s test. TSG6 KO=1.000±0.047; TSG6 KO+PTX3=0.979±0.048; TSG6 KO+TTX=1.363±0.077. Number of fields examined: 54, 57, 29 respectively; one-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. n=3 independent experiments, data are presented as normalized mean values


J ) Representative mEPSC traces recorded from WT and TSG6 KO littermates cultures treated or not with PTX3 ( 1μg / ml for 48hrs ) . K ) On the contrary of WT cultures , mEPSC frequency quantitation shows no increase in frequency in TSG6 KO cultures when treated with PTX3 ( Normalized Frequency , WT Ctr : 1 . 000 ± 0 . 074 ; Wt + PTX3 = 1 . 912 ± 0 . 289 ; TSG6 KO Ctr = 1 ± 0 . 079 ; TSG6 KO + PTX3 = 1 . 226 ± 0 . 096 ; 22 , 20 , 30 , 24 cells respectively , 6 TSG6 KO mice and 4 WT littermates . One way ANOVA test p = 0 . 002 followed by post hoc Dunn ' s test , data are presented as normalized mean ± SEM plus the distribution ) . L ) Cumulative probability plot of mEPSC amplitude and average mEPSC amplitude quantitation ( inset ) showing no difference in amplitude ( WT Ctr : 1 . 000 ± 0 . 034 , Wt + PTX3 = 1 . 069 ± 0 . 046 , TSG6 KO Ctr = 1 . 000 ± 0 . 028 ; TSG6 KO + PTX3 = 1 . 074 ± 0 . 67 ; 22 , 20 , 30 , 24 cells respectively , 6 TSG6 KO mice and 4 WT littermates . One way ANOVA test p = 0 . 817 followed by post hoc Dunn ' s test , data are presented as normalized mean ± SEM plus the distribution . Cumulative distribution is analyzed by Kolmogorov - Smirnov test ) .

O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-EXP_ASSAY 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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 mEPSC traces recorded from WT and TSG6 KO littermates cultures treated or not with PTX3 (1μg/ml for 48hrs). K) On the contrary of WT cultures, mEPSC frequency quantitation shows no increase in frequency in TSG6 KO cultures when treated with PTX3(Normalized Frequency, WT Ctr: 1.000±0.074; Wt+PTX3=1.912±0.289; TSG6 KO Ctr=1±0.079; TSG6 KO+PTX3=1.226±0.096; 22, 20, 30, 24 cells respectively, 6 TSG6 KO mice and 4 WT littermates. One way ANOVA test p=0.002 followed by post hoc Dunn's test, data are presented as normalized mean±SEM plus the distribution). L) Cumulative probability plot of mEPSC amplitude and average mEPSC amplitude quantitation (inset) showing no difference in amplitude (WT Ctr: 1.000±0.034, Wt+PTX3=1.069±0.046, TSG6 KO Ctr =1.000±0.028; TSG6 KO+PTX3=1.074±0.67; 22, 20, 30, 24 cells respectively, 6 TSG6 KO mice and 4 WT littermates. One way ANOVA test p=0.817 followed by post hoc Dunn's test, data are presented as normalized mean±SEM plus the distribution. Cumulative distribution is analyzed by Kolmogorov-Smirnov test).


A ) Representative images showing 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 :

A) Representative images showing 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


B ) Blocking β1 integrins activity by using the specific anti - β1 Integrin monoclonal antibody prevents the PTX3 - induced postsynaptic AMPARs recruitment ( Ctr = 1 . 000 ± 0 . 074 , PTX3 = 1 . 504 ± 0 . 098 , αCD29 + PTX3 = 0 . 766 ± 0 . 097 , αCD29 = 0 . 810 ± 0 . 108 . Number of fields examined : 37 , 33 , 23 , 24 respectively ; one way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test ; 3 independent experiments , data are presented as normalized mean values ± SEM )

O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Blocking β1 integrins activity by using the specific anti-β1 Integrin monoclonal antibody prevents the PTX3-induced postsynaptic AMPARs recruitment (Ctr=1.000±0.074, PTX3=1.504±0.098, αCD29+PTX3=0.766±0.097, αCD29=0.810±0.108. Number of fields examined: 37, 33, 23, 24 respectively; one way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test; 3 independent experiments, data are presented as normalized mean values±SEM)


C ) Western blotting analysis of p - ERK levels on lysates from control and PTX3 - treated neurons upon 30 minutes stimulation ( Ctr = 1 ± 0 ; PTX3 = 1 . 453 ± 0 . 195 , 6 independent experiments , unpaired t - test , data are presented normalized on control and as mean ± SEM )

O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O 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) Western blotting analysis of p-ERK levels on lysates from control and PTX3-treated neurons upon 30 minutes stimulation (Ctr=1±0; PTX3=1.453±0.195, 6 independent experiments, unpaired t-test, data are presented normalized on control and as mean±SEM)


D ) Representative mEPSC traces recorded from the indicated experimental conditions . E ) mEPSC frequency quantitation showing that pre - incubation with PD98059 ( 30μM ) completely prevents the PTX3 - dependent increase of mEPSC frequency ( Hz , Ctr = 0 . 911 ± 0 . 104 ; PTX3 = 1 . 748 ± 0 . 273 ; PTX3 + PD = 0 . 518 ± 0 . 055 ; PD = 0 . 491 ± 0 . 104 . Number of neurons : Ctr = 14 , PTX3 = 14 , PTX3 + PD = 13 ; PD = 9 ; 3 independent experiments . One - way ANOVA analysis of variance , p < 0 . 0001 followed by post hoc Tukey test as indicated in figure . Data are presented as a distribution plus mean ± SEM ) . F ) inset : average mEPSC amplitude quantitation ( pA ) , Ctr = 14 . 19 ± 0 . 70 ; PTX3 = 19 . 09 ± 1 . 71 ; PTX3 + PD = 14 . 31 ± 0 . 70 , PD = 16 . 51 ± 1 . 17 . Number of neurons : Ctr = 14 , PTX3 = 14 , PTX3 + PD = 13 ; PD = 9 ; 3 independent experiments . Kruskall - Wallis test , p = 0 . 015 followed by Dunn " s test as indicated in figure , data are presented as a distribution plus mean ± SEM . Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov - Smirnov test : Ctr vs PTX3 : p < 0 . 0001 , D = 0 . 183 , PTX3 vs PTX3 + PD : p < 0 . 0001 , D = 0 . 251 , PTX3 vs PD : p < 0 . 0001 , D =

O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O

D) Representative mEPSC traces recorded from the indicated experimental conditions. E) mEPSC frequency quantitation showing that pre-incubation with PD98059 (30μM) completely prevents the PTX3-dependent increase of mEPSC frequency (Hz, Ctr=0.911±0.104; PTX3=1.748±0.273; PTX3+PD=0.518±0.055; PD=0.491±0.104. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. One-way ANOVA analysis of variance, p<0.0001 followed by post hoc Tukey test as indicated in figure. Data are presented as a distribution plus mean±SEM). F) inset: average mEPSC amplitude quantitation (pA), Ctr=14.19±0.70; PTX3=19.09±1.71; PTX3+PD=14.31±0.70, PD=16.51±1.17. Number of neurons: Ctr=14, PTX3=14, PTX3+PD=13; PD=9; 3 independent experiments. Kruskall-Wallis test, p=0.015 followed by Dunn"s test as indicated in figure, data are presented as a distribution plus mean±SEM. Cumulative probability plot of mEPSC amplitudes are analyzed with Kolmogorov-Smirnov test: Ctr vs PTX3: p<0.0001, D=0.183, PTX3 vs PTX3+PD: p<0.0001, D=0.251, PTX3 vs PD: p<0.0001, D=0


G ) Representative images showing 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 :

G) Representative images showing 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


H ) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [ ( GluA & Bsn ) / Bsn ] shows a statistically significant increase after PTX3 exposure , while pre - incubation with PD98059 prevents the PTX3 - dependent increase ( Ctr = 1 ± 0 . 042 , PTX3 = 1 . 499 ± 0 . 121 , PTX3 + PD = 1 . 093 ± 0 . 060 , PD = 1 . 017 ± 0 . 117 ; number of fields examined : 49 , 15 , 15 , 20 respectively ; one - way ANOVA analysis of variance , p = 0 . 0002 followed by post hoc Tukey test as indicated in figure ; at least 3 independent experiments , data are presented as normalized mean values ± SEM )

O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the surface synaptic AMPARs normalized to the total number of Bsn [(GluA&Bsn)/Bsn] shows a statistically significant increase after PTX3 exposure, while pre-incubation with PD98059 prevents the PTX3-dependent increase (Ctr=1±0.042, PTX3=1.499±0.121, PTX3+PD=1.093±0.060, PD=1.017±0.117; number of fields examined: 49, 15, 15, 20 respectively; one-way ANOVA analysis of variance, p=0.0002 followed by post hoc Tukey test as indicated in figure; at least 3 independent experiments, data are presented as normalized mean values±SEM)