Figure 6 . Electron micrographs showing autophagosome - mediated autophagy in attached ( A - C ) and detached ( D and E ) cotyledon cells . ( A ) Two autophagosomes containing cytoplasm were observed . Mitochondria and the cytoplasm appeared to be enclosed with an ER - like double membrane . ( B ) An autophagosome containing mitochondria and cytoplasm was fused with LV ( arrowheads ) . ( C ) Autophagic body containing mitochondria was found in LV . ( D ) An autophagosome containing mitochondria and cytoplasm was observed . The PSV was not converted into the LV . ( E ) Autophagic body was observed in the PSV of detached cotyledon cells . AB , autophagic body ; AP , autophagosome ; ER , endoplasmic reticulum ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars , 200 nm .

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

Figure 6.Electron micrographs showing autophagosome-mediated autophagy in attached (A-C) and detached (D and E) cotyledon cells. (A) Two autophagosomes containing cytoplasm were observed. Mitochondria and the cytoplasm appeared to be enclosed with an ER-like double membrane. (B) An autophagosome containing mitochondria and cytoplasm was fused with LV (arrowheads). (C) Autophagic body containing mitochondria was found in LV. (D) An autophagosome containing mitochondria and cytoplasm was observed. The PSV was not converted into the LV. (E) Autophagic body was observed in the PSV of detached cotyledon cells. AB, autophagic body; AP, autophagosome; ER, endoplasmic reticulum; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


Figure 1E The representative images of IF - IHC staining of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) and DAPI ( blue ) . Scale bar , 200 μm . F Quantification of numbers of Iba - 1 + cells on the meningeal regions . Mock n = 5 ; ZIKV n = 7 embryos . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM . G The representative images of IF - IHC staining of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) and DAPI ( blue ) . Scale bar , 50 μm . H Quantification of total number of Iba - 1 + cells per ChP area . I Percentage of Iba - 1 + cells on the surface of ChP . J Percentage of round - shaped Iba - 1 + cells . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM . Mock n = 5 ; ZIKV n = 6 biological replicatesK The representative images of IF - IHC staining of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) , CD68 ( red ) and DAPI ( blue ) . Scale bar , 50 μm . L Percentage of CD68 + ; Iba - 1 + cells out of total Iba - 1 + cells . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM .

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

Figure 1E The representative images of IF-IHC staining of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 200 μm. F Quantification of numbers of Iba-1+ cells on the meningeal regions. Mock n=5; ZIKV n=7 embryos. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.G The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 50 μm. H Quantification of total number of Iba-1+ cells per ChP area. I Percentage of Iba-1+ cells on the surface of ChP. J Percentage of round-shaped Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. Mock n=5; ZIKV n=6 biological replicatesK The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green), CD68 (red) and DAPI (blue). Scale bar, 50 μm. L Percentage of CD68+; Iba-1+ cells out of total Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


Figure 2B Representative animal tracks in the sociability test . C Percentage of time the subject mice spent in each chamber in the sociability session of three - chamber test . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . empty ( E ) chamber ; ZIKV ( n = 16 ) stranger 1 chamber vs . empty chamber ; one - way ANOVA with Bonferroni post hoc test . D Quantification of time spent investigating the stranger mouse and empty cage during sociability session . Mock ( n = 11 ) sniffing time at stranger 1 mouse vs . empty cup , ZIKV ( n = 16 ) sniffing time at stranger 1 mouse vs . empty cup . E Preference indices in sociability . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . F Representative animal tracks in the social memory test . G Percentage of time the subject mice spent in each in the social memory session . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . stranger 2 ( S2 ) chamber , p < 0 . 0001 ; ZIKV ( n = 16 ) stranger 1 chamber vs . stranger 2 chamber ; one - way ANOVA with Bonferroni post hoc test . H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session . Mock ( n = 11 ) sniffing time at stranger 1 vs . stranger 2 mouse ; ZIKV ( n = 16 ) sniffing time at stranger 1 vs . stranger 2 mouse . I Preference indices in the social novelty session of the three - chamber test . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test unless described otherwise ; n represent biological replicates .

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

Figure 2B Representative animal tracks in the sociability test. C Percentage of time the subject mice spent in each chamber in the sociability session of three-chamber test. Mock (n=11) stranger 1 (S1) chamber vs. empty (E) chamber; ZIKV (n=16) stranger 1 chamber vs. empty chamber; one-way ANOVA with Bonferroni post hoc test. D Quantification of time spent investigating the stranger mouse and empty cage during sociability session. Mock (n=11) sniffing time at stranger 1 mouse vs. empty cup, ZIKV (n=16) sniffing time at stranger 1 mouse vs. empty cup. E Preference indices in sociability. Mock (n=11) vs. ZIKV (n=16). F Representative animal tracks in the social memory test. G Percentage of time the subject mice spent in each in the social memory session. Mock (n=11) stranger 1 (S1) chamber vs. stranger 2 (S2) chamber, p< 0.0001; ZIKV (n=16) stranger 1 chamber vs. stranger 2 chamber; one-way ANOVA with Bonferroni post hoc test. H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session. Mock (n=11) sniffing time at stranger 1 vs. stranger 2 mouse; ZIKV (n=16) sniffing time at stranger 1 vs. stranger 2 mouse. I Preference indices in the social novelty session of the three-chamber test. Mock (n=11) vs. ZIKV (n=16). Data information: All data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t-test unless described otherwise; n represent biological replicates.


Figure 3B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC . C Quantitation of sEPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 4 mice ; ZIKV , n = 15 neurons from 5 mice in B and C . Data information : Data were collected from PL and IL regions of mPFC , and were presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . Two - tailed unpaired t - test unless described otherwise . E Quantifications of sIPSC frequency from pyramidal neurons in mPFC . F Quantifications of sIPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 5 mice ; ZIKV , n = 18 neurons from 6 mice in E and F . Data information : Data were collected from PL and IL regions of mPFC , and were presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

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

Figure 3B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC. C Quantitation of sEPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 4 mice; ZIKV, n= 15 neurons from 5 mice in B and C. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.E Quantifications of sIPSC frequency from pyramidal neurons in mPFC. F Quantifications of sIPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 5 mice; ZIKV, n= 18 neurons from 6 mice in E and F. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


Figure 4A Representative images of PV + , SST + and CAL + cells in the prelimbic ( PL , upper panel ) and infralimbic ( IL , lower panel ) cortex . Scale bars : 200 μm . H IHC staining of mPFC stained with antibodies against vGLUT1 . Scale bars , 5 μm . I Quantification of vGLUT1 + puncta numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . Two - tailed unpaired t - test unless described otherwise . J IHC staining of mPFC stained with antibodies against vGAT . Scale bars , 5 μm . K Quantification of vGAT + puncta numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

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

Figure 4A Representative images of PV+, SST+ and CAL+ cells in the prelimbic (PL, upper panel) and infralimbic (IL, lower panel) cortex. Scale bars: 200 μm.H IHC staining of mPFC stained with antibodies against vGLUT1. Scale bars, 5 μm. I Quantification of vGLUT1+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.J IHC staining of mPFC stained with antibodies against vGAT. Scale bars, 5 μm. K Quantification of vGAT+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


Figure 5B Representative confocal images of the vHIP stained with mCherry ( red ) and c - Fos ( green ) . C Quantification of mCherry and c - Fos double positive cells out of total mCherry - positive cells in the vHIP CA1 regions . Scale bar , 50 μm . Mock ( n = 5 mice ) vs . ZIKV ( n = 5 mice ) , p = 0 . 0013 . Data information : Data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise . F Representative EPSC traces of the pyramidal neurons in the mPFC . G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC ( PL and IL ) . Mock ( n = 17 neurons from 5 mice ) vs . ZIKV ( n = 19 neurons from 5 mice ) , p = 0 . 0122 . Data information : Data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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

Figure 5B Representative confocal images of the vHIP stained with mCherry (red) and c-Fos (green). C Quantification of mCherry and c-Fos double positive cells out of total mCherry-positive cells in the vHIP CA1 regions. Scale bar, 50 μm. Mock (n= 5 mice) vs. ZIKV (n= 5 mice), p=0.0013. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.F Representative EPSC traces of the pyramidal neurons in the mPFC. G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC (PL and IL). Mock (n=17 neurons from 5 mice) vs. ZIKV (n=19 neurons from 5 mice), p=0.0122. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


Figure 6B Representative examples of GFP - labeled presynaptic boutons . Scale bars : 2 . 5 μm . C Quantification of presynaptic bouton density and weighted size in the mPFC ( PL and IL ) . Mock : n = 57 axons from 6 mice ; ZIKV : n = 63 axons from 7 mice . D Quantification of presynaptic bouton weighted size in the mPFC ( PL and IL ) . Mock : n = 2974 boutons from 6 mice ; ZIKV : n = 3486 boutons from 7 mice . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise . H Comparisons of percentages of vHIP - innervated mPFC ( PL and IL ) neuron subtypes . Mock , n = 4 mice ; ZIKV , n = 7 mice . P = 0 . 5343 in CaMKIIa , 0 . 0117 in PV , 0 . 7491 in CAL , 0 . 4484 in SST , 0 . 1519 in other uncharacterized neuron subtypes . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise . J Representative confocal images of CTB488 - labeled tdTomato - positive neurons in the vHIP . White arrowheads indicate CTB488 single positive neurons . Scale bars : 200 μm . K Quantification of the percentage of tdTomato ; CTB488 double - positive cells out of total CTB488 - positive cells . Mock , n = 7 mice ; ZIKV , n = 7 mice . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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

Figure 6B Representative examples of GFP-labeled presynaptic boutons. Scale bars: 2.5 μm. C Quantification of presynaptic bouton density and weighted size in the mPFC (PL and IL). Mock: n= 57 axons from 6 mice; ZIKV: n= 63 axons from 7 mice. D Quantification of presynaptic bouton weighted size in the mPFC (PL and IL). Mock: n= 2974 boutons from 6 mice; ZIKV: n= 3486 boutons from 7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.H Comparisons of percentages of vHIP-innervated mPFC (PL and IL) neuron subtypes. Mock, n=4 mice; ZIKV, n=7 mice. P= 0.5343 in CaMKIIa, 0.0117 in PV, 0.7491 in CAL, 0.4484 in SST, 0.1519 in other uncharacterized neuron subtypes. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.J Representative confocal images of CTB488-labeled tdTomato-positive neurons in the vHIP. White arrowheads indicate CTB488 single positive neurons. Scale bars: 200 μm. K Quantification of the percentage of tdTomato;CTB488 double-positive cells out of total CTB488-positive cells. Mock, n= 7 mice; ZIKV, n=7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


Figure 7C Representative animal tracks in three - chamber test . D Discrimination indices of the social memory session of three - chamber test . mCherry mock , n = 8 mice ; mCherry ZIKV , n = 10 mice ; hM4Di ZIKV n = 10 mice , mCherry mock vs . mCherry ZIKV p = 0 . 0041 ; mCherry ZIKV vs . hM4Di ZIKV p = 0 . 0115 ; and mCherry mock vs . hM4Di ZIKV p = 0 . 7076 . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test . E Representative confocal images of mCherry ( red ) and c - Fos ( green ) double - labeled cells in the vHIP CA1 regions . Scale bars , 50 μm . F . Quantification of the percentage of c - fos ; mCherry double positive cells out of total mCherry cells . mCherry mock , n = 6 mice ; mCherry ZIKV , n = 4 mice ; hM4Di ZIKV n = 6 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test .

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

Figure 7C Representative animal tracks in three-chamber test. D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.E Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm. F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


Figure 3 Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia . Select compounds were injected 24 hours before and on the day of LPS administration , using routes and doses specified in the methods . C57bl / 6 female mice were injected with a high - lethality dose of E . coli LPS ( 38 - 40 μg / g ) followed by a subcutaneous injection of sterile saline . Significant differences in concentration between drug and vehicle - treated pre - and post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . Percent survival was compared using a log rank Mantel Cox test

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

Figure 3 Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test


Figure 1D ) In the 6 months old p32cKO heart , p62 and multi - ubiquitin staining patterns were co - localized . Scale bar , 5 µm . E ) In the 6 months old p32cKO heart , the staining patterns of p62 and the mitochondrial marker protein , pyruvate dehydrogenase ( PDH ) , were co - localized . Scale bar , 5 µm . ( 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 .

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

Figure 1D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.(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.


Figure 2 ( 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 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 .

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

Figure 2(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 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.


Figure 3 ( 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 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 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 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 .

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

Figure 3(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 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 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 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.


Figure 4 ( 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 ± SEM of 4 independent experiments . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 001 .

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

Figure 4(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 ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.


Figure 5 ( 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 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 .

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

Figure 5(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 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.


Figure 6 ( 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 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 .

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

Figure 6(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 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.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


Figure 1 ( 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 . ( 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 .

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

Figure 1(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.(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.


Figure 2 ( 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 . ( 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 .

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

Figure 2(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.(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.