A synthetic 78 - aa Cse4 peptide ( aa 33 - 110 ) binds to Okp1 / Ame1 in vitro . Microscale thermophoresis ( MST ) measurement of binding of fluorescently labeled Cse4 ( 33 - 110 ) to Okp1 / Ame1 is shown . Normalized fluorescence , from which the fluorescence of unbound Okp1 / Ame1 was subtracted ( ∆ F norm ) , is plotted against Cse4 ( 33 - 110 ) concentration ( logarithmic scale ) . Mean values ± SD ( n = 3 ) are shown .

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

A synthetic 78-aa Cse4 peptide (aa 33 - 110) binds to Okp1/ Ame1 in vitro. Microscale thermophoresis (MST) measurement of binding of fluorescently labeled Cse4 (33 - 110) to Okp1/ Ame1 is shown. Normalized fluorescence, from which the fluorescence of unbound Okp1/ Ame1 was subtracted (∆F norm), is plotted against Cse4(33-110) concentration (logarithmic scale). Mean values ± SD (n = 3) are shown.


Plot of MST measuring the competition of fluorescently labelled Cse4 ( 33 - 110 ) bound to Okp1 / Ame1 ( black ) ( or Okp1 - R164C / Ame1 , red ) with unlabelled Cse4 ( 33 - 87 ) that is unmodified or monomethylated on R37 ( R37Me ) . Normalized fluorescence from which the fluorescence of unbound Cse4 ( 33 - 110 ) was subtracted ( ∆ F norm ) is plotted against peptide concentration . Mean values ± standard deviation ( n = 3 ) are given . Control , normalized fluorescence of Cse4 ( 33 - 110 ) alone at increasing concentrations .

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

Plot of MST measuring the competition of fluorescently labelled Cse4 (33 - 110) bound to Okp1/ Ame1 (black) (or Okp1-R164C/ Ame1, red) with unlabelled Cse4 (33 - 87) that is unmodified or monomethylated on R37 (R37Me). Normalized fluorescence from which the fluorescence of unbound Cse4 (33 - 110) was subtracted (∆F norm) is plotted against peptide concentration. Mean values ± standard deviation (n = 3) are given. Control, normalized fluorescence of Cse4 (33 - 110) alone at increasing concentrations.


E . Immunoblot ( IB ) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h . Data information : Data are representative of three ( E independent experiments with similar outcome .

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

E. Immunoblot (IB) analysis of stable Ub replacement cell lines treated or not with DOX for 72 h. Data information: Data are representative of three (E independent experiments with similar outcome.


F , G . HA - Ub conjugates isolated as in ( D ) were immunoblotted with indicated antibodies . Data information : Data are representative of three independent experiments with similar outcome .

O O O O B-GENEPROD O B-GENEPROD 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

F,G. HA-Ub conjugates isolated as in (D) were immunoblotted with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.


B . Representative images ( upper ) and QIBC analysis ( lower ) ( solid lines , median ; dashed lines , quartiles ; > 10000 cells analyzed per condition ) of Ub ( G76V ) - GFP expression in stable U2OS / Ub ( G76V ) - GFP cells treated or not with p97i for 4 h . Data information : Data are representative of three independent experiments with similar outcome .

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-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O B-GENEPROD 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 O

B. Representative images (upper) and QIBC analysis (lower) (solid lines, median; dashed lines, quartiles; >10000 cells analyzed per condition) of Ub(G76V)-GFP expression in stable U2OS/Ub(G76V)-GFP cells treated or not with p97i for 4 h. Data information: Data are representative of three independent experiments with similar outcome.


D . DOX - treated U2OS / shUb / HA - Ub ( WT ) and U2OS / shUb / HA - Ub ( K27R ) cell lines were transfected with shUb - resistant Ub ( G76V ) - GFP construct and treated or not with p97i for 4 h . Cell extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies . Data information : Data are representative of three independent experiments with similar outcome .

O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

D. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines were transfected with shUb-resistant Ub(G76V)-GFP construct and treated or not with p97i for 4 h. Cell extracts were subjected to GFP IP followed by immunoblotting with indicated antibodies. Data information: Data are representative of three independent experiments with similar outcome.


E . Immunoblot analysis of DOX - treated U2OS / shUb / HA - Ub cell lines treated or not with MG132 for 4 h . Data information : Data are representative of three independent experiments with similar outcome .

O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD 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

E. Immunoblot analysis of DOX-treated U2OS/shUb/HA-Ub cell lines treated or not with MG132 for 4 h. Data information: Data are representative of three independent experiments with similar outcome.


G . DOX - treated U2OS / shUb / HA - Ub ( WT ) and U2OS / shUb / HA - Ub ( K27R ) cell lines transfected with indicated Ub ( G76V ) - GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies . Data information : Data are representative of four ( G ) independent experiments with similar outcome .

O O B-SMALL_MOLECULE O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY 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

G. DOX-treated U2OS/shUb/HA-Ub(WT) and U2OS/shUb/HA-Ub(K27R) cell lines transfected with indicated Ub(G76V)-GFP expression constructs were subjected to GFP IP under denaturing conditions followed by immunoblotting with indicated antibodies. Data information: Data are representative of four (G) independent experiments with similar outcome.


A . FLAG IPs from U2OS cells transfected with indicated FLAG - tagged expression constructs were incubated with purified K27 di - Ub and analyzed by immunoblotting . Data information : Data are representative of three ( A independent experiments with similar outcome .

O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

A. FLAG IPs from U2OS cells transfected with indicated FLAG- tagged expression constructs were incubated with purified K27 di-Ub and analyzed by immunoblotting. Data information: Data are representative of three (A independent experiments with similar outcome.


C . Cells transfected as in ( B ) and treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting . Data information : Data are representative of three independent experiments with similar outcome .

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

C. Cells transfected as in (B) and treated or not with p97i for 4 h were subjected to GFP IP followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.


I . U2OS / Ub ( G76V ) - GFP and U2OS / Ub ( G76V ) - 20AA - GFP cell lines were transfected or not with FLAG - UCHL3 C95S expression construct , treated with MG132 for 2 h and subjected to GFP IP followed by immunoblotting . Data information : Data are representative of three independent experiments with similar outcome .

O O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O

I. U2OS/Ub(G76V)-GFP and U2OS/Ub(G76V)-20AA-GFP cell lines were transfected or not with FLAG-UCHL3 C95S expression construct, treated with MG132 for 2 h and subjected to GFP IP followed by immunoblotting. Data information: Data are representative of three independent experiments with similar outcome.


B ) Protein classification of the total proteins ( 3209 , left panel ) and of the glycoproteins ( 571 , right panel ) detected in 3 out of 3 experiments . More than 90 % of the glycoproteins detected are classified as membrane proteins , as expected for the surface labeling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Protein classification of the total proteins (3209, left panel) and of the glycoproteins (571, right panel) detected in 3 out of 3 experiments. More than 90% of the glycoproteins detected are classified as membrane proteins, as expected for the surface labeling.


C ) Changes of the 3209 proteins detected on the cell surface of SEZ6KO neurons compared to WT . The average log2 fold change of SEZ6KO neurons over WT ( log2 SEZ6KO / WT ) is plotted against the negative log10 of each protein ' s p - value according to a one - sample t test . Blue labeled dots are considered as hits ( including GluK2 and GluK3 ) and are proteins with a p - value less than 0 . 05 ( neg . log10 ( 0 . 05 ) = 1 . 3 ; horizontal dotted line ) and a fold - change to less than - 0 . 5 or more than 0 . 5 ( vertical dotted lines ) . GluK2 and GluK3 are labeled in pink , GluA1 and GluA2 are indicated with green lines and dots and GluN1 and GluN2B are marked with red lines and dots .

O O O O O O O O O O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O B-GENEPROD B-CELL_TYPE 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 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 B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

C) Changes of the 3209 proteins detected on the cell surface of SEZ6KO neurons compared to WT. The average log2 fold change of SEZ6KO neurons over WT (log2 SEZ6KO/WT) is plotted against the negative log10 of each protein's p-value according to a one-sample t test. Blue labeled dots are considered as hits (including GluK2 and GluK3) and are proteins with a p-value less than 0.05 (neg. log10(0.05)=1.3; horizontal dotted line) and a fold-change to less than -0.5 or more than 0.5 (vertical dotted lines). GluK2 and GluK3 are labeled in pink, GluA1 and GluA2 are indicated with green lines and dots and GluN1 and GluN2B are marked with red lines and dots.


A ) SEZ6KO and WT neurons were biotinylated with Sulfo - NHS - Biotin and surface proteins were enriched by streptavidin bead pull - down . The GluK2 / 3 antibody cannot discriminate the subunits 2 and 3 ( Lerma & Marques , 2013 ) , therefore the band is commonly indicated with the labeling GluK2 / 3 . GluK2 / 3 , GluA2 and GluN2B surface levels were quantified , normalized to SEZ6L2 surface levels ( negative control ) in the same sample ( GluK2 / 3 / SEZ6L2 ) and divided by the WT levels , with the ratio for WT being set to 1 . 0 ( plot shows mean ± S . E . M . , at least 10 replicates in 4 independent biological experiments , Mann - Whitney * * * * p - value < 0 . 005 ) .

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

A) SEZ6KO and WT neurons were biotinylated with Sulfo-NHS-Biotin and surface proteins were enriched by streptavidin bead pull-down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma & Marques, 2013), therefore the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2 and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3 / SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± S.E.M., at least 10 replicates in 4 independent biological experiments, Mann-Whitney ****p-value<0.005).


B ) mRNA levels of GluA1 , GluA2 , GluK2 , GluK3 and GluN2B were quantified in SEZ6KO neurons , normalized to GAPDH and β - actin mRNA in the same sample and compared to the mRNA levels in WT neurons . No difference in GluK2 / 3 mRNA was detected in SEZ6KO neurons compared to WT ( plot shows mean ± SD , 6 replicates in 2 independent biological experiments , multiple t - test was used ) .

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

B) mRNA levels of GluA1, GluA2, GluK2, GluK3 and GluN2B were quantified in SEZ6KO neurons, normalized to GAPDH and β-actin mRNA in the same sample and compared to the mRNA levels in WT neurons. No difference in GluK2/3 mRNA was detected in SEZ6KO neurons compared to WT (plot shows mean ± SD, 6 replicates in 2 independent biological experiments, multiple t-test was used).


C ) Editing of GluK2 mRNA was tested in SEZ6KO and WT neurons . The editing value was calculated as ( intensity of 376 [ edited ] / intensity of [ 376 ( edited ) + 269 ( unedited ) ] ) × 100 and normalized to the band at 76 bp . No difference was detected in SEZ6KO neurons compared to WT ( plot shows mean ± S . E . M . , 6 replicates in 2 independent biological experiments , Mann - Whitney test was used ) .

O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O 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 B-GENEPROD 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

C) Editing of GluK2 mRNA was tested in SEZ6KO and WT neurons. The editing value was calculated as (intensity of 376 [edited] / intensity of [376 (edited) + 269 (unedited)]) × 100 and normalized to the band at 76 bp. No difference was detected in SEZ6KO neurons compared to WT (plot shows mean ± S.E.M., 6 replicates in 2 independent biological experiments, Mann-Whitney test was used).


( A ) Left : Current trace resulting from whole - cell voltage - clamp recordings at a holding potential of - 70 mV in a CA1 pyramidal neuron in a WT mouse hippocampal slice ( P15 ) . The current was recorded in the presence of 10 µM bicuculline , 20 µM GYKI53655 and 30 µM APV in the ACSF . Perfusion of the recording chamber with 10 µM kainate and 100 µM 2 , 3 - dioxo - 6 - nitro - 1 , 2 , 3 , 4 - tetrahydrobenzo [ f ] quinoxaline - 7 - sulfonamide ( NBQX ) are indicated with green and blue bars , respectively . Right : Analogous experiment in a SEZ6KO mouse ( P15 ) . ( B ) Summary of the experiments in A . Bar graph illustrates mean charge with standard error of the mean carried by the inward currents ( measured as area under the curve , AUC ) for the two genotypes at P15 ( WT 46 . 6 ± 0 . 5 nC ( n = 10 cells ) , SEZ6KO 30 . 8 ± 0 . 5 nC ( n = 9 cells ) , * p = 0 . 0375 Mann - Whitney - U - test ) .

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

(A) Left: Current trace resulting from whole-cell voltage-clamp recordings at a holding potential of -70 mV in a CA1 pyramidal neuron in a WT mouse hippocampal slice (P15). The current was recorded in the presence of 10 µM bicuculline, 20 µM GYKI53655 and 30 µM APV in the ACSF. Perfusion of the recording chamber with 10 µM kainate and 100 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) are indicated with green and blue bars, respectively. Right: Analogous experiment in a SEZ6KO mouse (P15). (B) Summary of the experiments in A. Bar graph illustrates mean charge with standard error of the mean carried by the inward currents (measured as area under the curve, AUC) for the two genotypes at P15 (WT 46.6 ± 0.5 nC (n=10 cells), SEZ6KO 30.8 ± 0.5 nC (n=9 cells), *p=0.0375 Mann-Whitney-U-test).


A ) In WT neurons , GluK2 / 3 was seen as two closely comigrating bands , whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons ( schematic representation on the right ) . When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H ( EndoH ) - which removes immature , but not mature sugars - the two closely comigrating bands were converted to three bands of a lower apparent molecular weight , consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands ( marked in light blue , red and green in the right panel ) . In SEZ6KO neurons the uppermost ( light blue ) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO . No difference in the glycosylation of GluA2 was detectable , pointing to a specific effect of SEZ6 on GluK2 / 3 .

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

A) In WT neurons, GluK2/3 was seen as two closely comigrating bands, whereas the upper one of lower intensity was missing or running even more closely to the lower band of main intensity in SEZ6KO neurons (schematic representation on the right). When the total lysates of SEZ6KO and WT neurons were digested with endoglycosidase H (EndoH) - which removes immature, but not mature sugars - the two closely comigrating bands were converted to three bands of a lower apparent molecular weight, consistent with full deglycosylation of the lowest band and a partial deglycosylation of the upper two bands (marked in light blue, red and green in the right panel). In SEZ6KO neurons the uppermost (light blue) band was missing and a new band of lower apparent molecular weight was seen that was overlapping with the red labeled band and was indicated with the purple asterisk in the SEZ6KO. No difference in the glycosylation of GluA2 was detectable, pointing to a specific effect of SEZ6 on GluK2/3.


B ) The running pattern of GluK2 / 3 in SEZ6KO and WT brains was compared upon EndoH digestion . Similar to primary neurons , GluK2 / 3 displayed an immature glycosylation pattern in SEZ6KO brains .

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

B) The running pattern of GluK2/3 in SEZ6KO and WT brains was compared upon EndoH digestion. Similar to primary neurons, GluK2/3 displayed an immature glycosylation pattern in SEZ6KO brains.


C ) Total GluK2 / 3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates ( plot shows mean ± S . E . M . , 9 replicates in 3 independent biological experiments , Mann - Whitney * * p - value = 0 . 0012 ) . D ) No significant change in GluK2 / 3 total levels was detectable in SEZ6KO and WT adult brains ( plot shows mean ± S . E . M . , 6 replicates , Mann - Whitney test was used ) .

O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD 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 B-GENEPROD O O O O O O O B-GENEPROD O O O B-TISSUE 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 GluK2/3 levels were quantified and showed a significant but moderate reduction in SEZ6KO compared to WT neuronal lysates (plot shows mean ± S.E.M., 9 replicates in 3 independent biological experiments, Mann-Whitney **p-value=0.0012). D) No significant change in GluK2/3 total levels was detectable in SEZ6KO and WT adult brains (plot shows mean ± S.E.M., 6 replicates, Mann-Whitney test was used).


E ) Synaptosomes from knock - out ( KO ) brains of single SEZ6 family members ( SEZ6 , SEZ6L , SEZ6L2 ) , triple knock - out ( TKO ) and respective controls ( WT and TWT - triple WT ) were digested with EndoH . GluK2 / 3 displayed the immature glycosylation in SEZ6KO and TKO brains , but not in SEZ6L and SEZ6L2 brains , demonstrating non - redundant functions for the SEZ6 family members . ( n = 4 )

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

E) Synaptosomes from knock-out (KO) brains of single SEZ6 family members (SEZ6, SEZ6L, SEZ6L2), triple knock-out (TKO) and respective controls (WT and TWT - triple WT) were digested with EndoH. GluK2/3 displayed the immature glycosylation in SEZ6KO and TKO brains, but not in SEZ6L and SEZ6L2 brains, demonstrating non-redundant functions for the SEZ6 family members. (n=4)


F ) Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH . GluK2 / 3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice , suggesting that no compensation effect is occurring with aging .

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

F) Brain homogenates from SEZ6KO and WT mice at different ages were digested with EndoH. GluK2/3 displayed the immature glycosylation at all developmental stages of SEZ6KO mice, suggesting that no compensation effect is occurring with aging.


A ) The glycome fingerprint of GluK2 / 3 was analyzed by lectin chip microarray ( LecChip ) . Lectin PHAL and PHAE ( indicated with asterisks ) detected reduced amounts of the oligosaccharide Galβ1 - 4GlcNAcβ1 on GluK2 / 3 in SEZ6KO neurons ( plot shows mean ± S . E . M . , 3 WT replicates and 4 SEZ6KO replicates were used , discoveries were determined using the FDR method of Benjamini and Hochberg , with Q = 2 % ) .

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

A) The glycome fingerprint of GluK2/3 was analyzed by lectin chip microarray (LecChip). Lectin PHAL and PHAE (indicated with asterisks) detected reduced amounts of the oligosaccharide Galβ1-4GlcNAcβ1 on GluK2/3 in SEZ6KO neurons (plot shows mean ± S.E.M., 3 WT replicates and 4 SEZ6KO replicates were used, discoveries were determined using the FDR method of Benjamini and Hochberg, with Q = 2%).


B ) Immunoblot analysis revealed reduction of HNK - 1 epitope on GluK2 / 3 in SEZ6KO brains . Left panels : Comparable amounts of GluK2 / 3 were immunoprecipitated ( IP ) in WT and SEZ6KO brains using excess of brain homogenates compared to beads . Anti - HNK - 1 antibody was used for detection . HNK - 1 band on GluK2 / 3 was detected only in WT brains but not in SEZ6KO brains ( n = 6 ) . As a control , HNK - 1 modification of NCAM - 1 was unaltered , as revealed after IP of NCAM - 1 from WT and SEZ6KO brains ( n = 3 ) , followed by detection with anti - HNK - 1 antibody . Different isoforms of NCAM - 1 are annotated with arrowheads ( NCAM 180 , NCAM 140 and NCAM 120 ) . Mid panel : To prove the specificity of the HNK - 1 antibody , GluK2 / 3 was immunoprecipitated and digested with Peptide - N - Glycosidase F ( PNGase F ) , which removes N - linked oligosaccharides . Upon PNGase F digestion , HNK - 1 was not detectable in WT brains and the molecular weight of GluK2 / 3 was reduced ( n = 2 ) . Right panels : In total brain homogenates ( input ) , no difference of general HNK - 1 epitope or NCAM - 1 levels was detected .

O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD B-TISSUE O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD B-TISSUE O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-TISSUE O O O B-GENEPROD B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

B) Immunoblot analysis revealed reduction of HNK-1 epitope on GluK2/3 in SEZ6KO brains. Left panels: Comparable amounts of GluK2/3 were immunoprecipitated (IP) in WT and SEZ6KO brains using excess of brain homogenates compared to beads. Anti-HNK-1 antibody was used for detection. HNK-1 band on GluK2/3 was detected only in WT brains but not in SEZ6KO brains (n=6). As a control, HNK-1 modification of NCAM-1 was unaltered, as revealed after IP of NCAM-1 from WT and SEZ6KO brains (n = 3), followed by detection with anti-HNK-1 antibody. Different isoforms of NCAM-1 are annotated with arrowheads (NCAM 180, NCAM 140 and NCAM 120). Mid panel: To prove the specificity of the HNK-1 antibody, GluK2/3 was immunoprecipitated and digested with Peptide-N-Glycosidase F (PNGase F), which removes N-linked oligosaccharides. Upon PNGase F digestion, HNK-1 was not detectable in WT brains and the molecular weight of GluK2/3 was reduced (n=2). Right panels: In total brain homogenates (input), no difference of general HNK-1 epitope or NCAM-1 levels was detected.


B , C ) The constitutively ER - retained SEZ6 mutant SEZ6 ∆ cytoER - HA ( B ) and full length wild - type SEZ6FL - HA ( C ) were co - transfected with SBP - GFP - GluK2 . Biotin was added to elicit release of SBP - GFP - GluK2 from the ER ( 0 min ) and cells were fixed at different time points ( 0 , 20 and 40 min ) . HA - tagged SEZ6FL and SEZ6 ∆ cytoER transgenes were labeled with anti - HA ( mouse ) and anti - mouse - Alexa594 antibodies . Size bars represent 5 µm .

O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR 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 B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

B,C) The constitutively ER-retained SEZ6 mutant SEZ6∆cytoER-HA (B) and full length wild-type SEZ6FL-HA (C) were co-transfected with SBP-GFP-GluK2. Biotin was added to elicit release of SBP-GFP-GluK2 from the ER (0 min) and cells were fixed at different time points (0, 20 and 40 min). HA-tagged SEZ6FL and SEZ6∆cytoER transgenes were labeled with anti-HA (mouse) and anti-mouse-Alexa594 antibodies. Size bars represent 5 µm.


D ) Scatter dot plot represents number of vesicles per cell in SBP - GFP - GluK2 RUSH experiment with SEZ6 ∆ cytoER - HA ( control ) or SEZ6FL - HA co - transfection . Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars ( SD ) are shown . Mann - Whitney test was used to compare HA - tagged SEZ6FL and SEZ6 ∆ cytoER at each time point . At 20 min * * * p - value = 0 . 0007 , at 40 min * * * * p - value < 0 . 0001 .

O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD 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 O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Scatter dot plot represents number of vesicles per cell in SBP-GFP-GluK2 RUSH experiment with SEZ6∆cytoER-HA (control) or SEZ6FL-HA co-transfection. Vesicle counts from 15 to 28 cells per timepoint from 2 independent experiments with mean number of vesicles and error bars (SD) are shown. Mann-Whitney test was used to compare HA-tagged SEZ6FL and SEZ6∆cytoER at each time point. At 20 min ***p-value=0.0007, at 40 min ****p-value<0.0001.


A ) Representative pictures of WT hippocampal neurons transfected with the SBP - mCherry - GluK2 RUSH construct , followed by treating neurons with biotin and anti - SBP antibodies ( mouse , green ) for 20 min or 40 min to live - label the GluK2 transported to the neuronal cell surface . As a control ( no biotin ) , neurons were incubated only with SBP antibody for 40 min . The surface / total ratio of SBP - mCherry - GluK2 constructs was estimated by calculating the ratio of SBP ( green ) / mCherry ( red ) fluorescence signals in the same area chosen for quantification . B ) Quantification results for A , indicating that incubation with biotin increases the surface / total ratio of SBP - mCherry - GluK2 over time . No Biotin n = 16 , 20 min n = 13 , 40 min n = 15 , all biological replicates . Graph shows mean ± SEM , 1 - way ANOVA p < 0 . 0001 and Dunnett ' s post hoc test , No Biotin vs 20 min * p = 0 . 0030 ; No Biotin vs 40 min * * * p < 0 . 0001 .

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

A) Representative pictures of WT hippocampal neurons transfected with the SBP-mCherry-GluK2 RUSH construct, followed by treating neurons with biotin and anti-SBP antibodies (mouse, green) for 20 min or 40 min to live-label the GluK2 transported to the neuronal cell surface. As a control (no biotin), neurons were incubated only with SBP antibody for 40 min. The surface/total ratio of SBP-mCherry-GluK2 constructs was estimated by calculating the ratio of SBP (green)/ mCherry (red) fluorescence signals in the same area chosen for quantification. B) Quantification results for A, indicating that incubation with biotin increases the surface/total ratio of SBP-mCherry-GluK2 over time. No Biotin n=16, 20 min n=13, 40 min n=15, all biological replicates. Graph shows mean±SEM, 1-way ANOVA p<0.0001 and Dunnett's post hoc test, No Biotin vs 20 min *p=0.0030; No Biotin vs 40 min ***p<0.0001.


C - D ) SEZ6 flox / flox neurons were lentivirally infected with inactivated Cre ( Y324F , Ctrl ) or Cre recombinase ( SEZ6KO ) , followed by transfection with the SBP - mCherry - GluK2 construct and treated with biotin and anti - SBP antibodies ( mouse , green ) for 20 min . Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti - Cre antibody ( rabbit , blue ) . SEZ6KO neurons showed a significantly reduced surface / total ratio of SBP - mCherry - GluK2 ( ctl n = 25 , SEZ6KO n = 23 biological replicates . Graph shows mean ± SEM , unpaired t - test , * * p = 0 . 0068 ) .

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

C-D) SEZ6 flox/flox neurons were lentivirally infected with inactivated Cre (Y324F, Ctrl) or Cre recombinase (SEZ6KO), followed by transfection with the SBP-mCherry-GluK2 construct and treated with biotin and anti-SBP antibodies (mouse, green) for 20 min. Expression of inactivated Cre or Cre recombinase were confirmed by immunostaining with an anti-Cre antibody (rabbit, blue). SEZ6KO neurons showed a significantly reduced surface/total ratio of SBP-mCherry-GluK2 (ctl n=25, SEZ6KO n=23 biological replicates. Graph shows mean ± SEM, unpaired t-test, **p=0.0068).


B , SEZ6 constitutive KO neurons were transduced with SEZ6 constructs . SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature ( higher ) and immature ( lower ) forms of SEZ6 . No difference in the apparent molecular weight was detected because the high percentage of the gel used ( 12 % ) and the small C - terminal deletion ( 39 amino acids ) . SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF ( with HA and Flag - tags ) was detected at the expected low molecular weight . Additionally , the C - terminal fragment generated from SEZ6FL by BACE1 cleavage was detected . Due to its lack of an HA - tag , it had a lower molecular weight than SEZ6CTF . The C - terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight . GluK2 / 3 glycosylation was rescued when SEZ6FL , SEZ6Δcyto and SEZ6ecto were expressed in the neurons , but SEZ6CTF was not able to rescue the phenotype . Color coding for asterisks is the same as in figure 4A . Representative blot of 3 independent experiments .

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

B, SEZ6 constitutive KO neurons were transduced with SEZ6 constructs. SEZ6FL and SEZ6Δcyto were detectable as two bands corresponding to mature (higher) and immature (lower) forms of SEZ6. No difference in the apparent molecular weight was detected because the high percentage of the gel used (12%) and the small C-terminal deletion (39 amino acids). SEZ6ecto was present as the immature band in the neuronal lysates SEZ6CTF (with HA and Flag-tags) was detected at the expected low molecular weight. Additionally, the C-terminal fragment generated from SEZ6FL by BACE1 cleavage was detected. Due to its lack of an HA-tag, it had a lower molecular weight than SEZ6CTF. The C-terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular weight. GluK2/3 glycosylation was rescued when SEZ6FL, SEZ6Δcyto and SEZ6ecto were expressed in the neurons, but SEZ6CTF was not able to rescue the phenotype. Color coding for asterisks is the same as in figure 4A. Representative blot of 3 independent experiments.


C ) SEZ6ecto was present as the mature , secreted band in the conditioned medium in panel C .

O O B-GENEPROD O O O O O O O O O O O O O O O O

C) SEZ6ecto was present as the mature, secreted band in the conditioned medium in panel C.


D ) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto . SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6 , as expected for a protein retained in the ER . SEZ6Δcyto , but not SEZ6ΔcytoER , was able to rescue GluK2 / 3 glycosylation , showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2 / 3 glycosylation ( n = 5 ) . Color coding for asterisks is the same as in figure 4A .

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

D) Sez6 constitutive KO neurons were transduced with SEZ6ΔcytoER and SEZ6Δcyto. SEZ6ΔcytoER presented only the band corresponding to the immature form of SEZ6, as expected for a protein retained in the ER. SEZ6Δcyto, but not SEZ6ΔcytoER, was able to rescue GluK2/3 glycosylation, showing that SEZ6 localized to the ER is not sufficient to rescue the GluK2/3 glycosylation (n=5). Color coding for asterisks is the same as in figure 4A.


E ) HEK293T cells were co - transfected with a GluK2a plasmid and the SEZ6 constructs . Expression of SEZ6 constructs was analyzed in the total lysates ( " Input " ) and revealed a similar expression of the different proteins . GluK2a was immunoprecipitated with GluK2 / 3 antibody and the SEZ6 mutants were detected with the HA . 11 antibody ( " IP " ) . SEZ6FL , SEZ6Δcyto and SEZ6ecto , but not SEZ6CTF were co - immunoprecipitated with GluK2a , suggesting that SEZ6 extracellular domain is necessary for GluK2a binding . Representative blot of 2 independent experiments . * indicates the light chain of the antibody used for immunoprecipitation .

O O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O

E) HEK293T cells were co-transfected with a GluK2a plasmid and the SEZ6 constructs. Expression of SEZ6 constructs was analyzed in the total lysates ("Input") and revealed a similar expression of the different proteins. GluK2a was immunoprecipitated with GluK2/3 antibody and the SEZ6 mutants were detected with the HA.11 antibody ("IP"). SEZ6FL, SEZ6Δcyto and SEZ6ecto, but not SEZ6CTF were co-immunoprecipitated with GluK2a, suggesting that SEZ6 extracellular domain is necessary for GluK2a binding. Representative blot of 2 independent experiments. * indicates the light chain of the antibody used for immunoprecipitation.


A Ex vivo flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + IL - 2 , KLH + RA , KLH + RA + IL - 2 . Results are representative FACS plots of LAG3 + CD4 + or CD25 + Foxp3 + cells after gating on live single CD3 + CD4 + CD49b + Foxp3 - and live single CD3 + CD4 + lymphocytes respectively .

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

A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+ lymphocytes respectively.


A Ex vivo flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + IL - 2 , KLH + RA , KLH + RA + IL - 2 . Results are representative FACS plots of LAG3 + CD4 + or CD25 + Foxp3 + cells after gating on live single CD3 + CD4 + CD49b + Foxp3 - and live single CD3 + CD4 +

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

A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+


C Expression of Tr1 markers PD - 1 , CD226 , ICOS , CD25 and CTLA - 4 on CD49b + LAG - 3 + Foxp3 - CD4 + T cells or on CD49b - CD4 + T cells .

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

C Expression of Tr1 markers PD-1, CD226, ICOS, CD25 and CTLA-4 on CD49b+ LAG-3+ Foxp3-CD4+ T cells or on CD49b-CD4+ T cells.


D IL - 10 , IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO ( Veh ) , KLH + DMSO ( KLH ) , KLH + RA , KLH + IL - 2 , KLH + RA + IL - 2 or KLH + LP - GMP ( LP1569 + c - di - GMP ) and cultured for 5 days with increasing concentrations of KLH . Bars are mean + SEM ( n = 4 or 5 ) . * * p < 0 . 01 , * * * p < 0 . 001 , KLH + IL - 2 + RA vs KLH , # # p < 0 . 01 , # # # p < 0 . 001 KLH + IL - 2 + RA vs KLH + RA , † † † p < 0 . 001 KLH + IL - 2 + RA vs KLH + IL - 2 by two - way ANOVA and Tukey post hoc test .

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

D IL-10, IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + RA, KLH + IL-2, KLH + RA + IL-2 or KLH + LP-GMP (LP1569 + c-di-GMP) and cultured for 5 days with increasing concentrations of KLH. Bars are mean + SEM (n=4 or 5). **p<0.01, ***p<0.001, KLH + IL-2 + RA vs KLH, ##p<0.01, ###p <0.001 KLH + IL-2 + RA vs KLH + RA, †††p<0.001 KLH + IL-2 + RA vs KLH + IL-2 by two-way ANOVA and Tukey post hoc test.


A Flow cytometry on LN cells of mice 7 days after two s . c . immunizations ( 14 days apart ) with PBS + DMSO ( Veh ) , MOG + DMSO ( MOG ) , MOG + RA + IL - 2 . Cells were gated on live single CD3 + lymphocytes . FMO : Fluorescence Minus One . B Mean percentages of IL - 10 + , LAG - 3 + , PD1 + or Foxp3 + CD25 + CD4 T cells as described in A . Cells were gated on live single CD3 + lymphocytes ( top left panel ) or on live single CD3 + CD4 + lymphocytes . Bars are mean + SEM ( n = 5 ) . * p < 0 . 05 , * * * p < 0 . 01 by one - way ANOVA and Tukey post hoc test .

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

A Flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), MOG + DMSO (MOG), MOG + RA + IL-2. Cells were gated on live single CD3+ lymphocytes. FMO: Fluorescence Minus One. B Mean percentages of IL-10+, LAG-3+, PD1+ or Foxp3+ CD25+ CD4 T cells as described in A. Cells were gated on live single CD3+ lymphocytes (top left panel) or on live single CD3+ CD4+ lymphocytes. Bars are mean + SEM (n=5). *p <0.05, ***p <0.01 by one-way ANOVA and Tukey post hoc test.


C EAE clinical scores in mice immunized s . c . with MOG + RA , MOG + IL - 2 , MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 12 , except for MOG + IL - 2 , where n = 6 ; combined from two experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , MOG + IL - 2 + RA vs Veh , # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 , MOG + IL - 2 + RA vs MOG + RA , † p < 0 . 05 , † † p < 0 . 01 , † † † p < 0 . 001 , MOG + IL - 2 + RA vs MOG + IL - 2 by two - way ANOVA and Tukey post hoc test .

O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O B-SMALL_MOLECULE 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 O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

C EAE clinical scores in mice immunized s.c. with MOG + RA, MOG + IL-2, MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=12, except for MOG + IL-2, where n=6; combined from two experiments). *p<0.05, **p<0.01, ***p <0.001, MOG + IL-2 + RA vs Veh, #p<0.05, ##p<0.01, ###p<0.001, MOG + IL-2 + RA vs MOG + RA, †p<0.05, ††p<0.01, †††p <0.001, MOG + IL-2 + RA vs MOG + IL-2 by two-way ANOVA and Tukey post hoc test.


D Representative FACS plots of MOG dextramer + CD4 T cells in LNs of naïve mice or 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + .

O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O B-ORGANISM O O O O O O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

D Representative FACS plots of MOG dextramer+ CD4 T cells in LNs of naïve mice or 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+.


E Mean percentages of MOG dextramer + CD4 T cells in LNs 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O O O B-SMALL_MOLECULE O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD 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 O O O O O O

E Mean percentages of MOG dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+. Bars are mean + SEM (n=5 or 6). *p <0.05 by unpaired two-tailed t test.


F Mean fluorescence intensity ( MFI ) of CD49b , ICOS and PD - 1 by MOG - dextramer + CD4 T cells in LNs 3 days after induction of EAE . Cells were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + dextramer + . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 , * * p < 0 . 01 by unpaired two - tailed t test .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD 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 O O O O O O O O O O O O O O O O

F Mean fluorescence intensity (MFI) of CD49b, ICOS and PD-1 by MOG-dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+ dextramer+. Bars are mean + SEM (n=5 or 6). *p <0.05, **p <0.01 by unpaired two-tailed t test.


G MOG - specific IL - 17 and IFN - γ production by spleen cells from day 7 of EAE , quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG . Bars are mean + SEM ( n = 3 or 4 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - ANOVA and Tukey post hoc test .

O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O B-DISEASE 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 O O O O O O O O O O O O O O O O O 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 MOG-specific IL-17 and IFN-γ production by spleen cells from day 7 of EAE, quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG. Bars are mean + SEM (n=3 or 4). *p<0.05, **p<0.01 and ***p<0.001 by two-ANOVA and Tukey post hoc test.


A Representative FACS plots of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 or vehicle ( Veh ) only . Cells were gated as single live CD45 + CD3 + CD4 + . B Mean absolute numbers of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 . Bars are mean + SEM ( n = 4 or 5 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-DISEASE O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-DISEASE O B-ORGANISM O O B-SMALL_MOLECULE 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

A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+. B Mean absolute numbers of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2. Bars are mean + SEM (n=4 or 5). *p<0.05 by unpaired two-tailed t test.


A Representative FACS plots of CD11a + CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG , RA and IL - 2 or vehicle ( Veh ) only . Cells were gated as single live CD45 + CD3 + CD4 + .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-DISEASE O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+.


C Representative FACS plots of CD45 + CD4 T cells in the spinal cords of mice on day 12 of EAE . Cells were gated as single live CD45 + . D Mean absolute numbers of infiltrating CD45 + cells in the spinal cords of mice on day 12 of EAE . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O B-ORGANISM O O O O B-DISEASE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O B-ORGANISM O O O O B-DISEASE O O O O 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 FACS plots of CD45+ CD4 T cells in the spinal cords of mice on day 12 of EAE. Cells were gated as single live CD45+. D Mean absolute numbers of infiltrating CD45+ cells in the spinal cords of mice on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.


C Representative FACS plots of CD45 + CD4 T cells in the spinal cords of mice on day 12 of EAE . Cells were gated as single live CD45 + .

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

C Representative FACS plots of CD45+ CD4 T cells in the spinal cords of mice on day 12 of EAE. Cells were gated as single live CD45+.


E Representative FACS plots of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + . F Mean absolute numbers of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Bars are mean + SEM ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O 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 Representative FACS plots of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+. F Mean absolute numbers of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.


E Representative FACS plots of infiltrating IL - 17 + and IFN - γ + CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + .

O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

E Representative FACS plots of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.


G Representative histogram of LAG - 3 expression on CD4 T cells in the spinal cords on day 12 of EAE . Cells were gated as single live CD45 + CD3 + CD4 + .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O

G Representative histogram of LAG-3 expression on CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.


H Inverse correlation between LAG - 3 and IL - 17 and IFN - γ expression on CD4 T cells in the spinal cords on day 12 of EAE . * p < 0 . 05 by unpaired two - tailed t test .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O O O O O O O O O

H Inverse correlation between LAG-3 and IL-17 and IFN-γ expression on CD4 T cells in the spinal cords on day 12 of EAE. *p<0.05 by unpaired two-tailed t test.


A IL - 17 and IFN - γ production by ELISA on supernatants of spleen cells from mice with EAE ( EAE ) co - cultured for 3 days with MOG - specific Tr1 cells ( from mice immunized twice with MOG , RA and IL - 2 and amplified in vitro with MOG , RA and IL - 2 for 8 days ) or control T cells from LNs of mice injected with PBS + DMSO ( Veh ) . Bars are mean + SD of one experiment . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by unpaired two - tailed t test .

O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O B-TISSUE O O B-ORGANISM O B-DISEASE O B-DISEASE O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-ORGANISM O O B-SMALL_MOLECULE 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

A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test.


B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG - specific Tr1 cells amplified in vitro with MOG , RA and IL - 2 . Results are mean + SEM ( n = 4 or 6 ) . * p < 0 . 05 and * * p < 0 . 01 by two - way ANOVA with Bonferroni post hoc test .

O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-DISEASE O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O

B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test.


C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells . D Mean absolute numbers of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Results are mean + SEM ( n = 6 ) . * p < 0 . 05 by unpaired two - tailed t test .

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O 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 FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells. D Mean absolute numbers of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Results are mean + SEM (n=6). *p<0.05 by unpaired two-tailed t test.


C Representative FACS plots of IL - 17 + CD4 T cells in the spinal cords on day 20 of EAE . Cells were gated as live single CD45 . 2 + CD3 + CD4 + cells .

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O

C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells.


E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + . F Mean percentages of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Results are mean + SEM ( n = 6 ) . * * p < 0 . 01 by unpaired two - tailed t test .

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-ORGANISM O B-DISEASE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-ORGANISM O B-DISEASE O O O 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 Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+. F Mean percentages of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Results are mean + SEM (n=6). **p<0.01 by unpaired two-tailed t test.


E Representative FACS plots of CTLA - 4 + CD4 T cells in the spleens of mice with EAE . Cells were gated as live single CD45 . 1 + CD3 + CD4 + .

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

E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+.


G EAE clinical scores of WT and IL - 10 - / - mice immunized s . c . with MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 and 21 days before induction of EAE . Results are mean + SEM ( n = 6 or 7 ) . * * * p < 0 . 001 , WT MOG + IL - 2 + RA vs WT Veh , # # p < 0 . 01 , # # # p < 0 . 001 , IL - 10 - / - MOG + IL - 2 + RA vs IL - 10 - / - Veh by two - way ANOVA and Tukey post hoc test .

O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-DISEASE 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-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test.


Retinal inflammatory scores of mice immunized s . c . with PBS + DMSO ( Veh ) , IRBP + DMSO ( IRBP ) or IRBP + RA + IL - 2 21 and 7 days before induction of EAU by s . c . injection of IRBP emulsified in CFA . Bars are mean + SEM ( n = 6 ) , * p < 0 . 05 and * * * p < 0 . 001 , IRBP + IL - 2 + RA vs Veh , # # # p < 0 . 001 IRBP + IL - 2 + RA vs IRBP by two - way ANOVA with Tukey post hoc test .

B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-DISEASE 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O

Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test.


Representative fundus images of each group of mice 35 days after induction of EAU . Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze , where the image appears blurred .

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Representative fundus images of each group of mice 35 days after induction of EAU. Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze, where the image appears blurred.


Retinal inflammation scores in HEL - TCR transgenic mice were s . c . injected with PBS + DMSO ( Veh ) or HEL + RA + IL - 2 on pn 18 ( 1x ) or on pn 18 and 25 ( 2x ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * p < 0 . 05 and * * * p < 0 . 001 , HEL + IL - 2 + RA 1x vs Veh , # # p < 0 . 01 HEL + IL - 2 + RA 2x vs Veh by two - way ANOVA with Tukey post hoc test .

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Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test.


Representative photographs of each group of retinal fundus of HEL - TCR transgenic mice at pn 35 . Arrowheads indicate representative areas with retinal vasculitis .

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Representative photographs of each group of retinal fundus of HEL-TCR transgenic mice at pn 35. Arrowheads indicate representative areas with retinal vasculitis.


E Representative FACS plots of IL - 17 + CD4 or IL - 10 + CD4 T cells in the retina on day 36 of HEL - TCR transgenic mice . Cells were gated on live single CD3 + CD4 + and CD3 + CD4 + LAG - 3 + Foxp3 - respectively .

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E Representative FACS plots of IL-17+ CD4 or IL-10+ CD4 T cells in the retina on day 36 of HEL-TCR transgenic mice. Cells were gated on live single CD3+CD4+ and CD3+CD4+LAG-3+Foxp3- respectively.


F Mean absolute number of IL - 17 + CD4 T cells in the retina ( upper panels ) or mean percentages of IL - 10 + LAG - 3 + Foxp3 - CD4 T cells in the spleens of of HEL - TCR transgenic mice with vasculitis ( lower panels ) . Results are mean + SEM ( n = 3 , 4 or 5 ) . * * p < 0 . 01 and * * * p < 0 . 001 by one way ANOVA with Bonferroni post hoc test .

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F Mean absolute number of IL-17+ CD4 T cells in the retina (upper panels) or mean percentages of IL-10+ LAG-3+ Foxp3- CD4 T cells in the spleens of of HEL-TCR transgenic mice with vasculitis (lower panels). Results are mean + SEM (n=3, 4 or 5). **p <0.01 and ***p <0.001 by one way ANOVA with Bonferroni post hoc test.


A C57BL / 6 WT or Rag2 − / − mice were immunosuppressed with hydrocortisone + / − FK506 and infected with 105 AF CEA10 conidia intranasally . Mortality was assessed using a 20 % weight loss endpoint . Mortality was significantly increased in FK506 immunosuppressed mice reaching 100 % by day 10 p . i . ( P = 0 . 003 for HC versus HC + FK506 in Rag2 − / − using log - rank test , n = 10 per group ) .

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A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group).


B , C PAS - stained lung sections of Rag2 − / − mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ . FK506 - treated animals had increased inflammation at day 2 p . i . compared to control . P = 0 . 0126 ( using Student ' s t - test ) , n = 3 - 5 per group . ( C ) Close - up of an infectious focus showing increased inflammation and fungal growth in the FK506 - treated group . Scale bar : 400 μm .

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B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm.


D , E Murine macrophages ( J774A . 1 ) were pre - treated with hydrocortisone , and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy . The nuclei are indicated in red , and the GR is indicated in green . Scale bar : 50 μm . FK506 did not cause increased translocation of the glucocorticoid receptor . Data represent mean + SEM calculated from 10 fields of vision . One out of 2 experiments is shown .

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

D, E Murine macrophages (J774A.1) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . A Weight was monitored daily for 7 days p . i . Immunosuppressed mice showed increased weight loss through days 1 - 3 p . i . Data points represent mean ± SEM .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O B-ORGANISM O O O O O B-ORGANISM O B-ORGANISM 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 O O O O O O O O O O O O O O O

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1-3 p.i. Data points represent mean ± SEM.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . B BALs were performed over a 3 - day period p . i . and fungal killing assessed by enumerating CFUs . Fungal killing was significantly impaired in immunosuppressed mice . Error bars indicate SEM .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O B-ORGANISM 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 B-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O O

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . C , D Flow cytometry analysis of the CD45 + leucocyte populations in BAL . Representative dot plots for CD45 + cells ( C ) and percentages of Ly - 6G - positive neutrophils infiltrating the infected lungs over a 48 - h p . i . time course ( D ) . The bars indicate the mean percentage ( ± SEM ) of neutrophils of total CD45 + leucocytes ( n = 5 mice per group ) . Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p . i .

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

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n. C, D Flow cytometry analysis of the CD45+leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . E PAS - stained lung sections of immunocompetent and FK506 - treated animals at 6 h p . i . Scale bar : 50 μm . IC animals showed inflammatory cell infiltrates around conidia ( black arrows ) , whereas in FK506 - treated animals , only uptake of conidia by the resident macrophage population could be seen .

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

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ . Bars represent mean percentages ( ± SEM ) , n = 3 .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . G Luminex multiplex cytokine analysis of BAL SNs at 6 h p . i . Bars represent mean ± SEM .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-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 O

Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . A , B Absolute neutrophil numbers in treated and untreated mpx : GFPlarvae were quantified by direct counting of fluorescent neutrophils in non - infected whole fish ( n = 11 - 13 per group ) . There were no significant differences .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O B-TISSUE 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 B-GENEPROD O B-GENEPROD 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

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.A, B Absolute neutrophil numbers in treated and untreated mpx:GFPlarvae were quantified by direct counting of fluorescent neutrophils in non-infected whole fish (n = 11-13 per group). There were no significant differences.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . C For survival analysis , lyz : dsRedlarvae were microinjected with ˜ 10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan - Meier analysis for survival over 6 days post - infection ( n = 16 per group ) . Immunosuppression led to a significant increase in mortality .

O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O 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 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

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.C For survival analysis, lyz:dsRedlarvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan-Meier analysis for survival over 6 days post-infection (n = 16 per group). Immunosuppression led to a significant increase in mortality.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . D TNF - α mRNA levels were measured in whole non - infected or infected larvae 48 h p . i . Immunosuppression led to significant and almost complete inhibition of TNF - α expression during infection .

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

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. D TNF-α mRNA levels were measured in whole non-infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF-α expression during infection.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFP larvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . E - G Lyz : dsRedlarvae were infected with ˜ 50 eGFP - expressing conidia of A . fumigatus and were imaged daily from 24 h p . i . ( E ) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ . A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown ( 60 h p . i . ) . The arrows indicate infectious foci . ( F ) Neutrophil recruitment was minimal throughout the first 60 h p . i . in immunosuppressed larvae ( n = 8 per group ) . ( G ) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements ( n = 8 per group ) .

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

At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. E-G Lyz:dsRedlarvae were infected with ˜50 eGFP-expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group).


A J774A . 1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . FK506 significantly reduced TNF - α levels at 5 and 10 ng / ml for AF . A significant reduction in TNF - α was also seen for zymosan , but not LPS .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS.


B J774A . 1 cells were treated with FK506 ( 10 ng / ml ) and either left non - stimulated ( NS ) or stimulated with AF SC ( MOI = 1 ) for 6 h . TNF - α mRNA levels were quantified from whole - cell RNA extracts by RT - PCR . FK506 significantly reduced TNF - α expression .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-ORGANISM O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O

B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT-PCR. FK506 significantly reduced TNF-α expression.


C J774A . 1 macrophages were treated with control ( Ctrl . ) or calcineurin A ( CnA ) - targeting siRNA ( 50 nM ) and stimulated with either AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . CnA siRNA led to significant reductions in both AF - and zymosan - dependent TNF - α levels , but had no significant effect on LPS responses .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O

C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.


A J774A . 1macrophages were stimulated with live AF SC ( MOI = 5 ) , and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2 . NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p . i .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O

A J774A.1macrophages were stimulated with live AF SC (MOI=5), and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2. NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p.i.


B J774A . 1 macrophages were stimulated with live or fixed swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFAT translocation was assessed by confocal microscopy . NFAT is indicated in green , the nuclei are indicated in red , and AF conidia can be seen in pink . Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan . Data represent mean ± SEM . Scale bar : 50 μm . Arrows indicate A . fumigatus or zymosan - positive cells .

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

B J774A.1 macrophages were stimulated with live or fixed swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFAT translocation was assessed by confocal microscopy. NFAT is indicated in green, the nuclei are indicated in red, and AF conidia can be seen in pink. Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan. Data represent mean ± SEM. Scale bar: 50 μm. Arrows indicate A. fumigatus or zymosan-positive cells.


C J774A . 1 macrophages were stimulated with zymosan after FK506 ( 10 ng / ml ) or control treatment , and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF - κB translocation . NFATc2 and NF - κB nuclear translocation was observed , which was maximal at 1 h p . i . , in stimulated macrophages . FK506 specifically inhibited NFATc2 translocation , but had no effect on NF - κB .

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

C J774A.1 macrophages were stimulated with zymosan after FK506 (10 ng/ml) or control treatment, and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF-κB translocation. NFATc2 and NF-κB nuclear translocation was observed, which was maximal at 1 h p.i., in stimulated macrophages. FK506 specifically inhibited NFATc2 translocation, but had no effect on NF-κB.


D J774A . 1 macrophages were pre - treated with FK506 ( 10 ng / ml ) or vehicle and stimulated with live swollen conidia or zymosan . NFATc2 translocation was evaluated 30 min after stimulation . Box and whisker plot shows median ± maximum / minimum . Scale bar : 50 μm . FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan . Statistical analysis was carried out by Student ' s t - test . NS , non - stimulated . Arrows indicate A . fumigatus or zymosan - positive cells .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-GENEPROD O O O B-ORGANISM 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 B-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O O O O

D J774A.1 macrophages were pre-treated with FK506 (10 ng/ml) or vehicle and stimulated with live swollen conidia or zymosan. NFATc2 translocation was evaluated 30 min after stimulation. Box and whisker plot shows median ± maximum/minimum. Scale bar: 50 μm. FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan. Statistical analysis was carried out by Student's t-test. NS, non-stimulated. Arrows indicate A. fumigatus or zymosan-positive cells.


A BMDMs from WT , Dectin - 1 − / − or MyD88 − / − mice were infected with swollen conidia ( MOI = 1 ) , and TNF - α was measured in the SN after overnight incubation . TNF - α levels were significantly reduced in Dectin1 − / − and Myd88 − / − BMDMs .

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

A BMDMs from WT, Dectin-1−/− or MyD88−/− mice were infected with swollen conidia (MOI = 1), and TNF-α was measured in the SN after overnight incubation. TNF-α levels were significantly reduced in Dectin1−/− and Myd88−/− BMDMs.


B BMDMs were stimulated with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation . Whilst almost complete abrogation of NFAT translocation was seen in Dectin1 − / − macrophages stimulated with zymosan , NFAT responses to AF were unaffected .

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

B BMDMs were stimulated with swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation. Whilst almost complete abrogation of NFAT translocation was seen in Dectin1−/−macrophages stimulated with zymosan, NFAT responses to AF were unaffected.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( C ) TNF - α was measured in the SN after overnight incubation . Syk inhibition significantly reduced swollen conidia - dependent or zymosan - dependent TNF - α levels .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O

C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (C) TNF-α was measured in the SN after overnight incubation. Syk inhibition significantly reduced swollen conidia-dependent or zymosan-dependent TNF-α levels.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( D ) NFATc2 translocation was quantified by confocal microscopy . Syk inhibition impaired zymosan - dependent NFAT translocation , but did not affect AF - dependent NFAT translocation .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-ORGANISM O O B-GENEPROD O O

C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (D) NFATc2 translocation was quantified by confocal microscopy. Syk inhibition impaired zymosan-dependent NFAT translocation, but did not affect AF-dependent NFAT translocation.