A ) SEZ6KO WT neurons were biotinylated with Sulfo - NHS - Biotin surface were enriched by streptavidin bead pull - down . GluK2 / 3 antibody cannot discriminate subunits 2 3 ( Lerma & Marques , 2013 ) , therefore band is commonly indicated with labeling GluK2 / 3 . GluK2 / 3 , GluA2 GluN2B surface were quantified , normalized to SEZ6L2 surface ( negative control ) in same sample ( GluK2 / 3 / SEZ6L2 ) divided by WT , with 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 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 B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD 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

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 of GluA1 , GluA2 , GluK2 , GluK3 GluN2B were quantified in SEZ6KO neurons , normalized to GAPDH β - actin mRNA in same sample compared to mRNA 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 - was used ) .

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

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 WT neurons . editing value was calculated as ( of 376 [ edited ] / of [ 376 ( edited ) + 269 ( unedited ) ] ) × 100 normalized to 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 was used ) .

O O O O B-GENEPROD O O O 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 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

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

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

(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 upper one of lower was missing or running even more closely to lower band of main in SEZ6KO neurons ( schematic representation on right ) . When total lysates of SEZ6KO WT neurons were digested with endoglycosidase H ( EndoH ) - which removes immature , but not mature sugars - two closely comigrating bands were converted to three bands of a lower apparent molecular , consistent with full deglycosylation of lowest band a partial deglycosylation of upper two bands ( marked in blue , red green in right panel ) . In SEZ6KO neurons uppermost ( blue ) band was missing a new band of lower apparent molecular was seen that was overlapping with red labeled band was indicated with purple asterisk in SEZ6KO . No difference in 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 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 B-GENEPROD 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 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 B-GENEPROD 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 ) running pattern of GluK2 / 3 in SEZ6KO WT brains was compared upon EndoH digestion . Similar to neurons , GluK2 / 3 displayed an immature glycosylation pattern in SEZ6KO brains .

O O O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE O O O B-GENEPROD O O O O B-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 were quantified 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 was detectable in SEZ6KO WT adult brains ( plot shows mean ± S . E . M . , 6 replicates , Mann - Whitney was used ) .

O O O B-GENEPROD 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 B-GENEPROD 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

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 ) respective controls ( WT TWT - triple WT ) were digested with EndoH . GluK2 / 3 displayed immature glycosylation in SEZ6KO TKO brains , but not in SEZ6L SEZ6L2 brains , demonstrating non - redundant functions for 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 B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-TISSUE O O O O B-GENEPROD I-GENEPROD B-TISSUE 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 WT mice at different ages were digested with EndoH . GluK2 / 3 displayed immature glycosylation at developmental stages of SEZ6KO mice , suggesting that no compensation effect is occurring with aging .

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

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 I-GENEPROD 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 B-GENEPROD 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 revealed reduction of HNK - 1 epitope on GluK2 / 3 in SEZ6KO brains . Left panels : Comparable amounts of GluK2 / 3 were immunoprecipitated ( IP ) in WT 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 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 NCAM 120 ) . Mid panel : To prove specificity of HNK - 1 antibody , GluK2 / 3 was immunoprecipitated 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 molecular of GluK2 / 3 was reduced ( n = 2 ) . Right panels : In total brain homogenates ( input ) , no difference of general HNK - 1 epitope or NCAM - 1 was detected .

O O B-EXP_ASSAY 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 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 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 B-GENEPROD 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 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 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

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

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 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE 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 in SBP - GFP - GluK2 RUSH experiment with SEZ6 ∆ cytoER - HA ( control ) or SEZ6FL - HA co - transfection . Vesicle from 15 to 28 per timepoint from 2 independent experiments with mean number of vesicles error bars ( SD ) are shown . Mann - Whitney was used to compare HA - tagged SEZ6FL 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 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 B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-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 SBP - mCherry - GluK2 RUSH construct , followed by treating neurons with biotin anti - SBP antibodies ( mouse , green ) for 20 min or 40 min to live - label GluK2 transported to neuronal surface . As a control ( no biotin ) , neurons were incubated only with SBP antibody for 40 min . surface / total of SBP - mCherry - GluK2 constructs was estimated by calculating of SBP ( green ) / mCherry ( red ) signals in same chosen for . B ) results for A , indicating that incubation with biotin increases surface / total of SBP - mCherry - GluK2 over time . No Biotin n = 16 , 20 min n = 13 , 40 min n = 15 , biological replicates . Graph shows mean ± , 1 - way ANOVA p < 0 . 0001 Dunnett ' s post hoc , 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-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 B-GENEPROD O B-GENEPROD O B-GENEPROD O O O 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 O O O B-SMALL_MOLECULE 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 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 SBP - mCherry - GluK2 construct treated with biotin 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 of SBP - mCherry - GluK2 ( ctl n = 25 , SEZ6KO n = 23 biological replicates . Graph shows mean ± , unpaired t - , * * 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE 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 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

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 SEZ6Δcyto were detectable as two bands corresponding to mature ( higher ) immature ( lower ) forms of SEZ6 . No difference in apparent molecular was detected because high of gel used ( 12 % ) small C - terminal deletion ( 39 ) . SEZ6ecto was present as immature band in neuronal lysates SEZ6CTF ( with HA Flag - tags ) was detected at expected low molecular . Additionally , C - terminal fragment generated from SEZ6FL by BACE1 cleavage was detected . Due to its lack of an HA - tag , it had a lower molecular than SEZ6CTF . C - terminal fragment generated from SEZ6Δcyto upon BACE1 cleavage was not visible because of its low molecular . GluK2 / 3 glycosylation was rescued when SEZ6FL , SEZ6Δcyto SEZ6ecto were expressed in neurons , but SEZ6CTF was not able to rescue phenotype . Color coding for asterisks is 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 I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD I-GENEPROD 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 B-GENEPROD 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 B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-EXP_ASSAY 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 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 mature , secreted band in conditioned medium in panel C .

O O B-GENEPROD 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 SEZ6Δcyto . SEZ6ΔcytoER presented only band corresponding to immature form of SEZ6 , as expected for a retained in ER . SEZ6Δcyto , but not SEZ6ΔcytoER , was able to rescue GluK2 / 3 glycosylation , showing that SEZ6 localized to ER is not sufficient to rescue GluK2 / 3 glycosylation ( n = 5 ) . Color coding for asterisks is same as in figure 4A .

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 B-GENEPROD 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 B-SUBCELLULAR 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

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 were co - transfected with a GluK2a plasmid SEZ6 constructs . Expression of SEZ6 constructs was analyzed in total lysates ( " Input " ) revealed a similar expression of different . GluK2a was immunoprecipitated with GluK2 / 3 antibody SEZ6 mutants were detected with HA . 11 antibody ( " IP " ) . SEZ6FL , SEZ6Δcyto 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 chain of antibody used for immunoprecipitation .

O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-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 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 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 + after gating on live single CD3 + CD4 + CD49b + Foxp3 - live single CD3 + CD4 + lymphocytes respectively .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 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 + after gating on live single CD3 + CD4 + CD49b + Foxp3 - live single CD3 + CD4 +

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE 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 B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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+


O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-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 O O B-GENEPROD O B-GENEPROD O B-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 IFN - γ production by ELISA on supernatants of spleen 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 ) cultured for 5 days with increasing concentrations of KLH . Bars are mean + ( 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 Tukey post hoc .

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

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

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

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 21 days before induction of EAE . Results are mean + ( 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 Tukey post hoc .

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 B-DISEASE 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

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 in LNs of naïve mice or 3 days after induction of EAE . 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 O B-TISSUE O O B-ORGANISM O O O O O O B-DISEASE 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 in LNs 3 days after induction of EAE . were gated as single live F4 / 80 - CD8 - B220 - CD4 + CD44 + . Bars are mean + ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - t .

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

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

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

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

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

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

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

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 in spleen on day 7 of EAE in mice immunized with MOG , RA IL - 2 or vehicle ( Veh ) only . were gated as single live CD45 + CD3 + CD4 + .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE O O O O B-DISEASE O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD 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 in spinal cords of mice on day 12 of EAE . were gated as single live CD45 + . D Mean absolute numbers of infiltrating CD45 + in spinal cords of mice on day 12 of EAE . Bars are mean + ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - t .

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

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 in spinal cords of mice on day 12 of EAE . were gated as single live CD45 + .

O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE I-TISSUE O B-ORGANISM O O O O B-DISEASE 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 + IFN - γ + CD4 T in spinal cords on day 12 of EAE . were gated as single live CD45 + CD3 + CD4 + . F Mean absolute numbers of infiltrating IL - 17 + IFN - γ + CD4 T in spinal cords on day 12 of EAE . Bars are mean + ( n = 5 or 6 ) . * p < 0 . 05 by unpaired two - t .

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

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 + IFN - γ + CD4 T in spinal cords on day 12 of EAE . were gated as single live CD45 + CD3 + CD4 + .

O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE I-TISSUE O O O O B-DISEASE 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 in spinal cords on day 12 of EAE . 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 O B-TISSUE I-TISSUE O O O O B-DISEASE 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 IL - 17 IFN - γ expression on CD4 T in spinal cords on day 12 of EAE . * p < 0 . 05 by unpaired two - t .

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

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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 amplified in vitro with MOG , RA IL - 2 . Results are mean + ( n = 4 or 6 ) . * p < 0 . 05 * * p < 0 . 01 by two - way ANOVA with Bonferroni post hoc .

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 B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 in spinal cords on day 20 of EAE . were gated as live single CD45 . 2 + CD3 + CD4 + . D Mean absolute numbers of IL - 17 + CD4 T in spinal cords on day 20 of EAE . Results are mean + ( n = 6 ) . * p < 0 . 05 by unpaired two - t .

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

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 in spinal cords on day 20 of EAE . were gated as live single CD45 . 2 + CD3 + CD4 + .

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD 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 in spleens of mice with EAE . were gated as live single CD45 . 1 + CD3 + CD4 + . F Mean percentages of CTLA - 4 + CD4 T in spleens of mice with EAE . Results are mean + ( n = 6 ) . * * p < 0 . 01 by unpaired two - t .

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

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 in spleens of mice with EAE . 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 O B-TISSUE O B-ORGANISM O B-DISEASE 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 IL - 10 - / - mice immunized s . c . with MOG + RA + IL - 2 , or PBS + DMSO ( Veh ) , 7 21 days before induction of EAE . Results are mean + ( 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 Tukey post hoc .

O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY 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 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 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

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 7 days before induction of EAU by s . c . injection of IRBP emulsified in CFA . Bars are mean + ( n = 6 ) , * p < 0 . 05 * * * 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 .

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 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 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

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 of each group of mice 35 days after induction of EAU . Arrowheads indicate with retinal vasculitis asterisk indicates a region of vitreous haze , where 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 25 ( 2x ) . Results are mean + ( n = 3 , 4 or 5 ) . * p < 0 . 05 * * * 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 .

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

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 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 in retina on day 36 of HEL - TCR transgenic mice . were gated on live single CD3 + CD4 + CD3 + CD4 + LAG - 3 + Foxp3 - respectively .

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

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

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

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 infected with 105 AF CEA10 conidia intranasally . Mortality was assessed using a 20 % 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 , 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 inflammation quantified by pixel threshold in ImageJ . FK506 - treated had increased inflammation at day 2 p . i . compared to control . P = 0 . 0126 ( using Student ' s t - ) , n = 3 - 5 per group . ( C ) Close - up of an infectious focus showing increased inflammation fungal growth in FK506 - treated group . Scale bar : 400 μm .

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

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 , effects of FK506 on shuttling of glucocorticoid receptor were assessed by confocal immunofluorescence microscopy . nuclei are indicated in red , GR is indicated in green . Scale bar : 50 μm . FK506 did not cause increased translocation of glucocorticoid receptor . Data represent mean + 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 B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR 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 B-GENEPROD I-GENEPROD 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 was monitored daily for 7 days p . i . Immunosuppressed mice showed increased loss through days 1 - 3 p . i . Data points represent mean ± .

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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 . fungal killing assessed by enumerating CFUs . Fungal killing was significantly impaired in immunosuppressed mice . Error bars indicate .

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 B-EXP_ASSAY O O O O O O O O B-ORGANISM 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 of CD45 + leucocyte populations in BAL . Representative dot plots for CD45 + ( C ) percentages of Ly - 6G - positive neutrophils infiltrating infected lungs over a 48 - h p . i . time course ( D ) . bars indicate mean ( ± ) 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 B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE 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 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 FK506 - treated at 6 h p . i . Scale bar : 50 μm . IC showed inflammatory infiltrates around conidia ( black arrows ) , whereas in FK506 - treated , only uptake of conidia by 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 B-SMALL_MOLECULE 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 B-ORGANISM 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 using ImageJ . Bars represent mean percentages ( ± ) , n = 3 .

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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 of BAL SNs at 6 h p . i . Bars represent mean ± .

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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 hindbrain mortality , fungal burden neutrophil recruitment were assessed . A , B Absolute neutrophil numbers in treated untreated mpx : GFPlarvae were quantified by direct counting of neutrophils in non - infected whole fish ( n = 11 - 13 per group ) . There were no significant differences .

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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 hindbrain mortality , fungal burden neutrophil recruitment were assessed . C For survival , lyz : dsRedlarvae were microinjected with ˜ 10 conidia effects of FK506 on outcome from invasive aspergillosis was determined by Kaplan - Meier for survival over 6 days post - infection ( n = 16 per group ) . Immunosuppression led to a significant increase in mortality .

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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 hindbrain mortality , fungal burden neutrophil recruitment were assessed . D TNF - α mRNA were measured in whole non - infected or infected larvae 48 h p . i . Immunosuppression led to significant almost complete inhibition of TNF - α expression during infection .

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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 hindbrain mortality , fungal burden neutrophil recruitment were assessed . E - G Lyz : dsRedlarvae were infected with ˜ 50 eGFP - expressing conidia of A . fumigatus were imaged daily from 24 h p . i . ( E ) effects of FK506 on neutrophil recruitment were assessed by integrated density of red neutrophils at sites of infection in ImageJ . A representative image showing impaired neutrophil recruitment to site of infection in immunosuppressed larvae is shown ( 60 h p . i . ) . arrows indicate infectious foci . ( F ) Neutrophil recruitment was minimal throughout first 60 h p . i . in immunosuppressed larvae ( n = 8 per group ) . ( G ) Fungal growth was determined by measurement of of hyphal elements ( n = 8 per group ) .

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

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

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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 ) stimulated with either AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in culture supernatant by ELISA . CnA siRNA led to significant reductions in both AF - zymosan - dependent TNF - α , but had no significant effect on LPS responses .

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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 ) , macrophage extracts were probed by Western blot for NFATc2 . NFATc2 shuttling to nucleus was observed was maximal at 2 h p . i .

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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 ) , NFAT translocation was assessed by confocal microscopy . NFAT is indicated in green , nuclei are indicated in red , AF conidia can be seen in pink . Live conidia were observed to stimulate sustained translocation compared to dead conidia or zymosan . Data represent mean ± . Scale bar : 50 μm . Arrows indicate A . fumigatus or zymosan - positive .

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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 , macrophage extracts were probed by Western blot for NFATc2 NF - κB translocation . NFATc2 NF - κB 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 .

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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 stimulated with live swollen conidia or zymosan . NFATc2 translocation was evaluated 30 min after stimulation . Box whisker plot shows median ± maximum / minimum . Scale bar : 50 μm . FK506 completely inhibited translocation of NFATc2 in response to AF or zymosan . Statistical was carried out by Student ' s t - . NS , non - stimulated . Arrows indicate A . fumigatus or zymosan - positive .

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 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 B-SMALL_MOLECULE O O 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 B-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE 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 ) , TNF - α was measured in SN after overnight incubation . TNF - α were significantly reduced in Dectin1 − / − Myd88 − / − BMDMs .

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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 ) , 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 .

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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 ) 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 SN after overnight incubation . Syk inhibition significantly reduced swollen conidia - dependent or zymosan - dependent TNF - α .

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


C - E J774A . 1 macrophages were pre - treated with ( C , D ) 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 ) . ( E ) Whole - lysates of Syk or Ctrl . siRNA - treated were separated by SDS - PAGE , followed by Western blotting , membranes were probed with anti - Syk anti - β - actin antibodies .

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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). (E) Whole-cell lysates of Syk or Ctrl. siRNA-treated cells were separated by SDS-PAGE, followed by Western blotting, and membranes were probed with anti-Syk and anti-β-actin antibodies.


F J774A . 1 macrophages were pre - treated with piceatannol infected with swollen conidia ( MOI = 5 ) . Phagocytosis was quantified by FACS 30 min after stimulation . Syk inhibition led to impaired phagocytosis of zymosan , but had no effect on phagocytosis of SC .

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F J774A.1 macrophages were pre-treated with piceatannol and infected with swollen conidia (MOI = 5). Phagocytosis was quantified by FACS 30 min after stimulation. Syk inhibition led to impaired phagocytosis of zymosan, but had no effect on phagocytosis of SC.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( G ) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (G) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( H ) For of NF - κB translocation , extracts were separated by SDS - PAGE , followed by Western blotting . Membranes were probed with anti - NF - κB p65 anti - HDAC1 antibodies .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (H) For quantification of NF-κB translocation, nuclear extracts were separated by SDS-PAGE, followed by Western blotting. Membranes were probed with anti-NF-κB p65 and anti-HDAC1 antibodies.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( I ) After overnight stimulation , TNF - α was measured in SN by ELISA . Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF , but not ionomycin ( G ) . In addition , inhibition of phagocytosis lead to increased translocation of NF - κB ( H ) increased of TNF - α in response to either zymosan or AF .

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

G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (I) After overnight stimulation, TNF-α was measured in the SN by ELISA. Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF, but not ionomycin (G). In addition, inhibition of phagocytosis lead to increased translocation of NF-κB (H) and increased levels of TNF-α in response to either zymosan or AF.


A J774A . 1 macrophages were pre - treated with bafilomycin A1 ( 100 nM ) stimulated with swollen conidia ( MOI = 3 ) for 30 min . NFATc2 translocation was assessed by confocal microscopy . Acidification was required for NFATc2 translocation .

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A J774A.1 macrophages were pre-treated with bafilomycin A1 (100 nM) and stimulated with swollen conidia (MOI = 3) for 30 min. NFATc2 translocation was assessed by confocal microscopy. Acidification was required for NFATc2 translocation.


B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP - expressing swollen conidia by confocal microscopy . Scale bar : 10 μm . arrow indicates a TLR9 - positive AF - containing phagosome .

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B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP-expressing swollen conidia by confocal microscopy. Scale bar: 10 μm. The arrow indicates a TLR9-positive AF-containing phagosome.


C BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 1 ) , after overnight stimulation , TNF - α was measured in SN by ELISA . TLR9 − / − BMDMs had significantly lower TNF - α .

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C BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 1), and after overnight stimulation, TNF-α was measured in the SN by ELISA. TLR9−/− BMDMs had significantly lower TNF-α levels.


D , E BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 5 ) for 30 min , NFATc2 translocation was quantified by ( D ) confocal microscopy . TLR9 − / − BMDMs exhibited reduced NFATc2 translocation by both approaches .

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D, E BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 5) for 30 min, and NFATc2 translocation was quantified by (D) confocal microscopy. TLR9−/− BMDMs exhibited reduced NFATc2 translocation by both approaches.


G BMDMs from WT or MyD88 − / − mice were pre - treated with ODN2088 ( 10 μM ) infected with swollen conidia ( MOI = 5 ) for 30 min . NFATc2 translocation was quantified by confocal microscopy . In both , WT MyD88 − / − BMDMs , blocking TLR9 significantly impaired NFATc2 translocation .

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G BMDMs from WT or MyD88−/−mice were pre-treated with ODN2088 (10 μM) and infected with swollen conidia (MOI = 5) for 30 min. NFATc2 translocation was quantified by confocal microscopy. In both, WT and MyD88−/− BMDMs, blocking TLR9 significantly impaired NFATc2 translocation.


A , B J774A . 1 macrophages were pre - treated with BTK inhibitor LFM - A13 ( 12 . 5 μM ) infected with swollen conidia . ( A ) After overnight stimulation , TNF - α was measured in SN by ELISA .

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A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA.


A , B J774A . 1 macrophages were pre - treated with BTK inhibitor LFM - A13 ( 12 . 5 μM ) infected with swollen conidia . ( B ) After 30 min of stimulation , NFATc2 translocation was quantified by confocal microscopy . Both TNF - α NFATc2 translocation were significantly reduced by BTK blockade .

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A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade.


C J774A . 1 macrophages were treated with a control or BTK - targeting siRNA ( 25 nM ) , NFAT translocation was quantified by confocal microscopy . BTK knock - down led to a significant reduction in NFATc2 translocation in response to AF .

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C J774A.1 macrophages were treated with a control or BTK-targeting siRNA (25 nM), and NFAT translocation was quantified by confocal microscopy. BTK knock-down led to a significant reduction in NFATc2 translocation in response to AF.


D , E J774A . 1 macrophages were treated with a control , BTK ( 25 nM ) - or TLR9 ( 75 nM ) - targeting siRNA additionally pre - treated with either ODN2088 ( 10 μM ) or BTK inhibitor LFM - A13 ( 12 . 5 μM ) . were infected with swollen conidia ( MOI = 5 ) , NFATc2 translocation was measured by confocal microscopy . Blocking both TLR9 BTK signalling had no additive effect on NFAT translocation blockade .

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D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.


Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . were pre - treated for 1 h with FK506 ( 10 ng / ml ) , NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in culture supernatant by ELISA . A Representative FACS - phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three samples .

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

Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples.


Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . were pre - treated for 1 h with FK506 ( 10 ng / ml ) , NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in culture supernatant by ELISA . B Inhibition of alveolar macrophage responses to AF with FK506 , MCV1 or SC514 led to a significant reduction in TNF - α .

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

Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.


B . Survival curves of Bcs1l homozygotes without ( GRAC ) with ( GROX ) alternative oxidase ( AOX ) expression ( n = 18 - 21 / group ) . median survival of GRAC mice was 210 days of GROX mice 589 days with no gender difference Statistics : survival data was analyzed using Log - rank ( Mantel - Cox ) ( p < 0 . 0001 )

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B. Survival curves of Bcs1l homozygotes without (GRAC) and with (GROX) alternative oxidase (AOX) expression (n=18-21/group). The median survival of GRAC mice was 210 days and of GROX mice 589 days with no gender difference Statistics: The survival data was analyzed using Log-rank (Mantel-Cox) test (p<0.0001)


C . of mice from P50 to P200 ( n > 10 / group ) at P600 ( n = 4 - 6 / group ) .

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C. Weight of mice from P50 to P200 (n>10/group) and at P600 (n=4-6/group).