Figure 1 ( A ) BECN1 expression of microglia from Becn1 + / - and wild type mouse pups in full medium without Bafilomycin A1 ( Baf ) was quantified by Western blotting . Microglia from Becn1 + / - mice show a signifcant reduction of BECN1 ( 48 % ) compared to microglia from wild type mice ; mean ± SEM , wild type n = 3 , Becn1 + / - n = 7 ; two - tailed t - test * * : p < 0 . 01 . Blot shows representative amounts of BECN1 after various treatments . ( B ) Microglia from newborn Becn1 + / - and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 ( Baf ) . LC3 and ACTIN levels were determined by Western blot . LC3 - II was significantly reduced in microglia from Becn1 + / - mice after Baf treatment in full medium compared to wild type microglia ; mean ± SEM , wild type n = 3 , Becn1 + / - n = 4 ; two - tailed t - test * : p < 0 . 05 . ( C ) Microglia from newborn Becn1 + / - and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 ( Baf ) and stained for endogenous LC3 . Exemplary images and the quantification of LC3 - positive vesicles / µm2 cell area show reduced presence of LC3 - positive vesicles in microglia from Becn1 + / - and induction of autophagy by HBSS ; mean ± SEM , wild type n = 4 , Becn1 + / - n = 4 , 3 - 5 fields per view / n and condition and 73 - 271 cells / n and condition ; two - tailed t - test * : p < 0 . 05 ; scale bar : 20

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

Figure 1(A) BECN1 expression of microglia from Becn1+/- and wild type mouse pups in full medium without Bafilomycin A1 (Baf) was quantified by Western blotting. Microglia from Becn1+/- mice show a signifcant reduction of BECN1 (48%) compared to microglia from wild type mice; mean ± SEM, wild type n = 3, Becn1+/- n = 7; two-tailed t-test **: p < 0.01. Blot shows representative amounts of BECN1 after various treatments.(B) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with or without Bafilomycin A1 (Baf). LC3 and ACTIN levels were determined by Western blot. LC3-II was significantly reduced in microglia from Becn1+/- mice after Baf treatment in full medium compared to wild type microglia; mean ± SEM, wild type n = 3, Becn1+/- n = 4; two-tailed t-test *: p < 0.05.(C) Microglia from newborn Becn1+/- and wild type mouse pups were kept for 2 h either in full medium or HBSS with Bafilomycin A1 (Baf) and stained for endogenous LC3. Exemplary images and the quantification of LC3-positive vesicles/µm2 cell area show reduced presence of LC3-positive vesicles in microglia from Becn1+/- and induction of autophagy by HBSS; mean ± SEM, wild type n = 4, Becn1+/- n = 4, 3-5 fields per view/n and condition and 73-271 cells/n and condition; two-tailed t-test *: p < 0.05; scale bar: 20 µm


Figure 2 ( A , B ) Microglia isolated from newborn Becn1 + / - and wild type mouse pups were either non treated or treated with a pro - inflammatory stimulus ( LPS followed by ATP ) . As additional controls cells treated only with LPS or ATP were used in ( A ) . The concentration of the pro - inflammatory cytokines IL - 1beta ( A ) and IL - 18 ( B ) in the cell supernatant was determined by ELISA . Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS / ATP ; mean ± SEM , wild type n ( A / B ) = 9 / 2 , wild type LPS n = 3 / 0 , wild type ATP n = 3 / 0 , wild type LPS / ATP n = 9 / 9 , Becn1 + / - n = 23 / 5 , Becn1 + / - LPS / ATP n = 23 / 23 , two - tailed t - test * : p < 0 . 05 . ( C ) Microglia isolated from wild type mouse pups were pre - incubated in full medium or starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . The autophagy blocker 3 - MA was added for the final 1 h 45 min of the experiment . The concentration of the pro - inflammatory cytokine IL - 1beta in the cell supernatant was determined by ELISA . Incubation with HBSS reduced IL - 1beta release significantly while 3 - MA treatment increased it ; mean ± SEM , full medium n = 6 , full medium LPS / ATP n = 6 , full medium LPS / ATP + 3MA n = 3 , HBSS n = 6 , HBSS LPS / ATP n = 6 , HBSS LPS / ATP + 3MA n = 6 ; two - tailed t - test * : p < 0 . 05 ; * * : p < 0 . 01 . ( D ) Microglia isolated from wild type mouse pups were pre - incubated in starvation medium HBSS before addition of a pro - inflammatory stimulus ( LPS followed by ATP ) . The autophagy blocker 3 - MA was added for the final 1 h 45 min of the experiment , microglia were stained for LC3 and images taken by confocal microscopy . Representative images show a reduction of LC3 - positive vesicles in 3 - MA treated cells . Scale bar : 20 µm . ( E ) Proteins were extracted from brains of wild type , Becn1 + / - , APPPS1 and APPPS1 Becn1 + / - mice at the indicated ages . IL - 1beta was measured by electrochemiluminescence ( MesoScale ) and compared within the repective age groups . IL - 1beta was significantly increased in the TBS and TX fraction ; mean ± SEM , wild type : n ( per age group ) = 2 / 6 / 3 , Becn1 + / - : n = 2 / 5 / 2 , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc test with WT as control group ; * : p < 0 . 05 ; * * : p < 0 . 01 . ( F ) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1 + / - mice at the age as indicated . Amyloid beta 1 - 40 and 1 - 42 were measured by electrochemiluminescence ( MesoScale ) and compared within the repective age groups . No difference in the amyloid beta load between the genotypes is apparent ; mean ± SEM , APPPS1 : n = 5 / 5 / 6 , APPPS1 Becn1 + / - : n = 7 / 9 / 6 ; ANOVA with Dunnett ´ s post hoc test with APPPS1 as control group , ns .

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

Figure 2(A, B) Microglia isolated from newborn Becn1+/- and wild type mouse pups were either non treated or treated with a pro-inflammatory stimulus (LPS followed by ATP). As additional controls cells treated only with LPS or ATP were used in (A). The concentration of the pro-inflammatory cytokines IL-1beta (A) and IL-18 (B) in the cell supernatant was determined by ELISA. Reduction of BECN1 significantly enhanced release of both cytokines after stimulation with LPS/ATP; mean ± SEM, wild type n (A/B) = 9/2, wild type LPS n = 3/0, wild type ATP n = 3/0, wild type LPS/ATP n = 9/9, Becn1+/- n = 23/5, Becn1+/- LPS/ATP n = 23/23, two-tailed t-test *: p < 0.05.(C) Microglia isolated from wild type mouse pups were pre-incubated in full medium or starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment. The concentration of the pro-inflammatory cytokine IL-1beta in the cell supernatant was determined by ELISA. Incubation with HBSS reduced IL-1beta release significantly while 3-MA treatment increased it; mean ± SEM, full medium n = 6, full medium LPS/ATP n = 6, full medium LPS/ATP + 3MA n = 3, HBSS n = 6, HBSS LPS/ATP n = 6, HBSS LPS/ATP + 3MA n = 6; two-tailed t-test *: p < 0.05; **: p < 0.01.(D) Microglia isolated from wild type mouse pups were pre-incubated in starvation medium HBSS before addition of a pro-inflammatory stimulus (LPS followed by ATP). The autophagy blocker 3-MA was added for the final 1 h 45 min of the experiment, microglia were stained for LC3 and images taken by confocal microscopy. Representative images show a reduction of LC3-positive vesicles in 3-MA treated cells. Scale bar: 20 µm.(E) Proteins were extracted from brains of wild type, Becn1+/-, APPPS1 and APPPS1 Becn1+/- mice at the indicated ages. IL-1beta was measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. IL-1beta was significantly increased in the TBS and TX fraction; mean ± SEM, wild type: n (per age group) = 2/6/3, Becn1+/-: n = 2/5/2, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with WT as control group; *: p < 0.05; **: p < 0.01.(F) Proteins were extracted from brains of APPPS1 and of APPPS1 Becn1+/- mice at the age as indicated. Amyloid beta 1-40 and 1-42 were measured by electrochemiluminescence (MesoScale) and compared within the repective age groups. No difference in the amyloid beta load between the genotypes is apparent; mean ± SEM, APPPS1: n = 5/5/6, APPPS1 Becn1+/-: n = 7/9/6; ANOVA with Dunnett´s post hoc test with APPPS1 as control group, ns.


Figure 3 ( A ) The expression of NLRP3 protein in non treated and LPS / ATP treated wild type and Becn1 + / - microglia was determined by Western blot and normalised to ACTIN . Reduction of BECN1 significantly enhanced expression of NLRP3 ; mean ± SEM , wild type n = 4 , wild type LPS / ATP n = 9 , Becn1 + / - n = 4 , Becn1 + / - LPS / ATP n = 23 ; ANOVA with Tukey ´ s post hoc test * : p < 0 . 05 . ( B ) The expression of Nlrp3 mRNA in non treated and LPS / ATP treated wild type and Becn1 + / - microglia was determined by qPCR . No significant differences can be detected between wild type and Becn1 + / - microglia ; mean ± SEM , wild type : n = 7 , Becn1 + / - : n = 3 ; ANOVA with Tukey ´ s post hoc test , ns for LPS / ATP treated wild type versus Becn1 + / - . ( C ) LPS / ATP treated and non treated wild type and Becn1 + / - microglia were immunolabeled for ASC ( red ) and imaged by confocal microscopy to investigate the assembly of inflammasomes . In non stimulated cells ASC is distributed diffusely in the cytoplasm ( upper panels ) . However , after stimulation formation of inflammasomes ( arrowheads ) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis / ejection ( arrow ) . Quantification showed a significantly increased percentage of inflammasomes in or around Becn1 + / - microglia ; mean ± SEM , wild type LPS / ATP n = 4 , Becn1 + / - LPS / ATP n = 7 ; two - tailed t - test * : p < 0 . 05 ; scale bar : 50 µm , insert scale bar : 10 µm . ( D ) The presence of cleaved CASP1 ( p10 ) protein in the supernatant of LPS / ATP treated wild type and Becn1 + / - microglia was determined by Western blot and normalised to ACTIN . Reduction of BECN1 significantly enhanced release of CASP1 ; mean ± SEM , wild type LPS / ATP n = 8 , Becn1 + / - LPS / ATP n = 18 ; two - tailed t - test * : p < 0 . 05 .

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

Figure 3(A) The expression of NLRP3 protein in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild type n = 4, wild type LPS/ATP n = 9, Becn1+/- n = 4, Becn1+/- LPS/ATP n = 23; ANOVA with Tukey´s post hoc test *: p < 0.05.(B) The expression of Nlrp3 mRNA in non treated and LPS/ATP treated wild type and Becn1+/- microglia was determined by qPCR. No significant differences can be detected between wild type and Becn1+/- microglia; mean ± SEM, wild type: n = 7, Becn1+/- : n = 3; ANOVA with Tukey ´s post hoc test, ns for LPS/ATP treated wild type versus Becn1+/-.(C) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non stimulated cells ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1+/- microglia; mean ± SEM, wild type LPS/ATP n = 4, Becn1+/- LPS/ATP n = 7; two-tailed t-test *: p < 0.05; scale bar: 50 µm, insert scale bar: 10 µm.(D) The presence of cleaved CASP1 (p10) protein in the supernatant of LPS/ATP treated wild type and Becn1+/- microglia was determined by Western blot and normalised to ACTIN. Reduction of BECN1 significantly enhanced release of CASP1; mean ± SEM, wild type LPS/ATP n = 8, Becn1+/- LPS/ATP n = 18; two-tailed t-test *: p < 0.05.


Figure 4 ( A ) LPS / ATP treated and non treated wild type and Becn1 + / - microglia were immunolabeled for NLRP3 ( green ) and LC3 ( red ) and imaged by confocal microscopy . Stimulation resulted in the appearance of many NLRP3 - containing aggregates of different sizes ( arrows ) . In non treated cells , only a few overlapping signals with LC3 - stained autophagosomes ( arrowheads ) are visible . Stimulated microglia however , showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates ; scale bar : 7 . 5 µm . ( B ) LPS / ATP treated wild type microglia were immunolabeled for NLRP3 ( green ) and LC3 ( red ) and analysed by super resolution microscopy ( SIM ) . NLRP3 aggregates of different sizes coclustering with LC3 - positive autophagosomes are clearly visible . 3D volume rendering of large ( I ) and small ( II ) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes ; scale bar : 10 µm ; magnified ROIs : 1 µm . ( C ) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia , centered on the maxima of NLRP3 clusters . The radial profiles confirm that both proteins colocalize to the same organelle ; mean ± SEM , n = 4 .

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

Figure 4(A) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for NLRP3 (green) and LC3 (red) and imaged by confocal microscopy. Stimulation resulted in the appearance of many NLRP3-containing aggregates of different sizes (arrows). In non treated cells, only a few overlapping signals with LC3-stained autophagosomes (arrowheads) are visible. Stimulated microglia however, showed multiple colocalizations of LC3 positive vesicles and NLRP3 aggregates; scale bar: 7.5 µm.(B) LPS/ATP treated wild type microglia were immunolabeled for NLRP3 (green) and LC3 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates of different sizes coclustering with LC3-positive autophagosomes are clearly visible. 3D volume rendering of large (I) and small (II) NLRP3 aggregates from the magnified ROIs showed engulfment of NLRP3 by autophagosomes; scale bar: 10 µm; magnified ROIs: 1 µm.(C) 3D Radial intensity profiles of NLRP3 and LC3 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize to the same organelle; mean ± SEM, n = 4.


Figure 5 ( A ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) and CALCOCO2 ( red ) and analysed by confocal microscopy . Colocalizations of NLRP3 aggregates ( arrows ) and CALCOCO2 ( arrowheads ) are apparent ; scale bar : 10 µm . ( B ) LPS / ATP treated wild type microglia were stained for NLRP3 ( green ) and CALCOCO2 ( red ) and analysed by super resolution microscopy ( SIM ) . NLRP3 aggregates coclustering with CALCOCO2 - positive signals are visible . 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2 ; scale bar : 10 µm ; magnified ROIs : 1 µm . ( C ) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia , centered on the maxima of NLRP3 clusters . The radial profiles confirm that both proteins colocalize ; mean ± SEM , n = 3 . ( D ) In SIM images from wild type microglia the sub - autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2 . LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster . While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity , the relative LC3 intensity there was significantly lower ; mean ± SEM , LC3 : n = 4 , CALCOCO2 : n = 3 ; two - tailed t - test * * : p < 0 . 01 . ( E ) Microglia from Becn1 + / - mice were transfected with scrambled siRNA or with siRNA for CALCOCO2 . 6 d after transfection cells were stimulated with LPS / ATP . The expression of CALCOCO2 protein was determined by Western blot . Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2 ; mean ± SEM , n = 3 ; two - tailed t - test * : p < 0 . 05 . ( F ) The amount of IL - 1beta in the supernatant released from the cells described in ( E ) was determined by ELISA . Microglia with a CALCOCO2 knockdown released significantly more IL - 1beta ; mean ± SEM , n = 3 ; two - tailed t - test * : p < 0 . 05 .

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

Figure 5(A) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by confocal microscopy. Colocalizations of NLRP3 aggregates (arrows) and CALCOCO2 (arrowheads) are apparent; scale bar: 10 µm.(B) LPS/ATP treated wild type microglia were stained for NLRP3 (green) and CALCOCO2 (red) and analysed by super resolution microscopy (SIM). NLRP3 aggregates coclustering with CALCOCO2-positive signals are visible. 3D volume rendering of NLRP3 aggregates from the magnified ROIs showed close contact of NLRP3 with the autophagic receptor CALCOCO2; scale bar: 10 µm; magnified ROIs: 1 µm.(C) 3D Radial intensity profiles of NLRP3 and CALCOCO2 signals derived from SIM images in wild type microglia, centered on the maxima of NLRP3 clusters. The radial profiles confirm that both proteins colocalize; mean ± SEM, n = 3.(D) In SIM images from wild type microglia the sub-autophagosomal localization of NLRP3 was analysed in relation to LC3 and CALCOCO2. LC3 and CALCOCO2 intensities from the center of mass of NLRP3 aggregates were normalised to their maximum within the cluster. While the center of NLRP3 aggregates shows near maximal CALCOCO2 intensity, the relative LC3 intensity there was significantly lower; mean ± SEM, LC3: n = 4, CALCOCO2: n = 3; two-tailed t-test **: p < 0.01.(E) Microglia from Becn1+/- mice were transfected with scrambled siRNA or with siRNA for CALCOCO2. 6 d after transfection cells were stimulated with LPS/ATP. The expression of CALCOCO2 protein was determined by Western blot. Application of CALCOCO2 siRNA resulted in significantly reduced expression of CALCOCO2; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.(F) The amount of IL-1beta in the supernatant released from the cells described in (E) was determined by ELISA. Microglia with a CALCOCO2 knockdown released significantly more IL-1beta; mean ± SEM, n = 3; two-tailed t-test *: p < 0.05.


Figure 2okp1 - R164C suppressed the temperature - sensitive growth defect of cbf1 ∆ cse4 - R37A . Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC . The original okp1 - R164C isolate from the suppressor screen is indicated as " UV mutagenesis " . Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC . The original okp1 - R164C isolate from the suppressor screen is indicated as " UV mutagenesis " . okp1 - R164C suppressed the growth defect of cse4 - R37A with chl4 ∆ . okp1 - R164C was unable to suppress the lethality of cse4 - R37A with ame1 - 4 . Tetrad dissection of a genetic cross between a cse4 - R37A okp1 - R164C and an ame1 - 4 strain is shown . The four spores from individual asci are aligned in vertical rows . okp1 - R164C suppressed the maintenance defect of cse4 - R37A for plasmids lacking the CDEI sequence of CEN6 ( CEN ∆ CDEI , at 37ºC ) . Error bars give SD of at least three independent transformants . * p = 0 . 03 . The okp1 mutations I45T , S94T and E208V suppressed the temperature - sensitive growth defect of cbf1 ∆ cse4 - R37A . Strains with the indicated okp1 alleles on a plasmid ( derivatives of AEY5584 )

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

Figure 2okp1-R164C suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis".Serial dilutions of strains with the indicated genotypes were spotted on full medium and grown for three days at 30ºC or 37ºC. The original okp1-R164C isolate from the suppressor screen is indicated as "UV mutagenesis". okp1-R164C suppressed the growth defect of cse4-R37A with chl4∆.okp1-R164C was unable to suppress the lethality of cse4-R37A with ame1-4. Tetrad dissection of a genetic cross between a cse4-R37A okp1-R164C and an ame1-4 strain is shown. The four spores from individual asci are aligned in vertical rows.okp1-R164C suppressed the maintenance defect of cse4-R37A for plasmids lacking the CDEI sequence of CEN6 (CEN ∆CDEI, at 37ºC). Error bars give SD of at least three independent transformants. * p = 0.03.The okp1 mutations I45T, S94T and E208V suppressed the temperature-sensitive growth defect of cbf1∆ cse4-R37A. Strains with the indicated okp1 alleles on a plasmid (derivatives of AEY5584)


Figure 3okp1 - R164C restored binding of Okp1 to centromeric sequences . ChIP of 9xmyc - tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP . Enrichment of CEN4 and POL1 ( as a control ) relative to input is given . Below , Western blot analysis of the amounts of 9xmyc - Okp1 and histone H2B ( loading control ) in whole cell extracts . okp1 - R164C restored binding of Ame1 to centromeric sequences . ChIP of 9xmyc - tagged Ame1 was performed as in ( A ) . Below , Western blot analysis of the amounts of 9xmyc - Ame1 and H2B as in ( A ) . okp1 - R164C restored binding of Mtw1 to centromeric sequences . ChIP of 9xmyc - Mtw1 and Western blots ( right ) okp1 - R164C improved in vivo association of Okp1 with Cse4 - R37A . Co - immunoprecipitation of Cse4 - R37A and Okp1 was carried out in cells carrying cse4 - R37A with ( + ) or without ( - ) 3xHA - tag and OKP1 or okp1 - R164C with ( + ) or without ( - ) 9xmyc - tag ( AEY5040 , AEY5972 , AEY5973 , AEY6589 , AEY6592 ) . Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting . Cse4 - R37A was immunoprecipitated using an α - HA antibody , and the presence of Cse4 - R37A and Okp1 or Okp1 - R164C in the immunoprecipitate was tested by Western blotting with α - HA and α - myc antibody , respectively . Left , inputs ( the two bands indicate Okp1 and a shorter degradation product ) ; right , α - HA immunoprecipitates .

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Figure 3okp1-R164C restored binding of Okp1 to centromeric sequences. ChIP of 9xmyc-tagged Okp1 was performed in strains with the indicated genotypes that were grown at 37ºC for 4 hours before ChIP. Enrichment of CEN4 and POL1 (as a control) relative to input is given. Below, Western blot analysis of the amounts of 9xmyc-Okp1 and histone H2B (loading control) in whole cell extracts.okp1-R164C restored binding of Ame1 to centromeric sequences. ChIP of 9xmyc-tagged Ame1 was performed as in (A). Below, Western blot analysis of the amounts of 9xmyc-Ame1 and H2B as in (A).okp1-R164C restored binding of Mtw1 to centromeric sequences. ChIP of 9xmyc-Mtw1 and Western blots (right)okp1-R164C improved in vivo association of Okp1 with Cse4-R37A. Co-immunoprecipitation of Cse4-R37A and Okp1 was carried out in cells carrying cse4-R37A with (+) or without (-) 3xHA-tag and OKP1 or okp1-R164C with (+) or without (-) 9xmyc-tag (AEY5040, AEY5972, AEY5973, AEY6589, AEY6592). Cells were grown at 23ºC or were shifted to 37ºC for 5 hours prior to harvesting. Cse4-R37A was immunoprecipitated using an α-HA antibody, and the presence of Cse4-R37A and Okp1 or Okp1-R164C in the immunoprecipitate was tested by Western blotting with α-HA and α-myc antibody, respectively. Left, inputs (the two bands indicate Okp1 and a shorter degradation product); right, α-HA immunoprecipitates.


Figure 4Cse4 ( 21 - 129 , Cse4N ) and Okp1 / Ame1 were expressed and purified separately from bacteria and co - incubated before separation by analytical gel filtration . Top , representative size - exclusion chromatography ( SEC ) chromatogram with absorbance measurements at 280 nanometers ( nm ) is shown . Below , image of Coomassie Blue - stained SDS - PAGE gels with fractions from principal SEC peaks of runs with Okp1 / Ame1 alone ( top ) , Cse4N alone ( middle ) and Okp1 / Ame1 pre - incubated with Cse4N before SEC ( bottom ) . Molecular weight standards are shown in kilodalton ( kDa ) . The ame1 mutations S59P , N159H , S275N and a C - terminal truncation after aa 273 ( ame1 - 273 * ) suppressed the growth defect of cbf1 ∆ cse4 - R37A . cbf1 ∆ cse4 - R37A ame1 ∆ strains carrying the indicated plasmid - borne ame1

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Figure 4Cse4(21-129, Cse4N) and Okp1/ Ame1 were expressed and purified separately from bacteria and co-incubated before separation by analytical gel filtration. Top, representative size-exclusion chromatography (SEC) chromatogram with absorbance measurements at 280 nanometers (nm) is shown. Below, image of Coomassie Blue-stained SDS-PAGE gels with fractions from principal SEC peaks of runs with Okp1/ Ame1 alone (top), Cse4N alone (middle) and Okp1/ Ame1 pre-incubated with Cse4N before SEC (bottom). Molecular weight standards are shown in kilodalton (kDa).The ame1 mutations S59P, N159H, S275N and a C-terminal truncation after aa 273 (ame1-273*) suppressed the growth defect of cbf1∆ cse4-R37A. cbf1∆ cse4-R37A ame1∆ strains carrying the indicated plasmid-borne ame1 alleles


Figure 5Cse4 - K49Ac and - R37Me are increased in S - phase of the cell cycle . Cells carrying 3xHA - tagged Cse4 were used to immunoprecipitate Cse4 with an α - Cse4 - K49Ac ( left , top ) and an α - HA antibody ( left , bottom ) . To detect Cse4 - R37 methylation , 3xHA - tagged Cse4 was immunoprecipitated with an α - Cse4 - R37Me2a ( right , top ) ( Samel et al . , 2012 ) and an α - HA antibody ( right , bottom ) . Western blots were probed for the presence of Cse4 with the α - HA antibody . G1 , treatment with α - factor ; S , arrest with hydroxyurea ; G2 / M , arrest in nocodazole ; asynch , asynchronous culture . Mimicking the unacetylated state of Cse4 - K49 ( cse4 - K49R ) partially suppressed the temperature - sensitivity of cbf1 ∆ cse4 - R37A . cbf1 ∆ cse4 ∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and growncse4 - K49R suppressed the lethality of cse4 - R37A with ctf19 ∆ . Tetrad dissection of a ctf19 ∆ strain crossed with cse4 - R37A ( left ) and cse4 - R37A K49R ( right ) . RepresentationAcetylation of Cse4 - K49 was reduced in the absence of Gcn5 and the SAGA complex , but not in the absence of the HAT Sas2 . Cse4 was precipitated as in ( A ) from strains wild - type ( wt ) or gcn5 ∆ ( top ) or sas2 ∆ strains ( bottom ) carrying HA - tagged Cse4 . w / o , control immunoprecipitation without antibody . Cse4 - K49Ac depended on the SAGA components Ada2 and Spt7 . Cse4 - K49Ac was determined as in ( A , left ) with cells carrying ada2 ∆ or spt7 ∆ .

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Figure 5Cse4-K49Ac and -R37Me are increased in S-phase of the cell cycle. Cells carrying 3xHA-tagged Cse4 were used to immunoprecipitate Cse4 with an α-Cse4-K49Ac (left, top) and an α-HA antibody (left, bottom). To detect Cse4-R37 methylation, 3xHA-tagged Cse4 was immunoprecipitated with an α-Cse4-R37Me2a (right, top) (Samel et al., 2012) and an α-HA antibody (right, bottom). Western blots were probed for the presence of Cse4 with the α-HA antibody. G1, treatment with α-factor; S, arrest with hydroxyurea; G2/ M, arrest in nocodazole; asynch, asynchronous culture.Mimicking the unacetylated state of Cse4-K49 (cse4-K49R) partially suppressed the temperature-sensitivity of cbf1∆ cse4-R37A. cbf1∆ cse4∆ strains carrying the indicated cse4 alleles on a plasmid were spotted on full medium and growncse4-K49R suppressed the lethality of cse4-R37A with ctf19∆. Tetrad dissection of a ctf19∆ strain crossed with cse4-R37A (left) and cse4-R37A K49R (right). RepresentationAcetylation of Cse4-K49 was reduced in the absence of Gcn5 and the SAGA complex, but not in the absence of the HAT Sas2. Cse4 was precipitated as in (A) from strains wild-type (wt) or gcn5∆ (top) or sas2∆ strains (bottom) carrying HA-tagged Cse4. w/o, control immunoprecipitation without antibody.Cse4-K49Ac depended on the SAGA components Ada2 and Spt7. Cse4-K49Ac was determined as in (A, left) with cells carrying ada2∆ or spt7∆.


Figure 6A 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 .

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Figure 6A 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.


Figure 1E . 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 .

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Figure 1E. 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.


Figure 3F , 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 .

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Figure 3F,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.


Figure 4B . 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 . 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 .

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Figure 4B. 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.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.


Figure 5A . 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 . 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 .

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Figure 5A. 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.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.


Figure 1B ) 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 .

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Figure 1B) 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.


Figure 2A ) 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 ) . 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 ) .

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Figure 2A) 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).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).


Figure 3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).


Figure 4A ) 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 . 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 ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) 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.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)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.


Figure 5A ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) 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.


Figure 6B , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B,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.


Figure 7A ) 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 ) .

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Figure 7A) 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).


Figure 8B , 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8B, 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.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.


Figure 1 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 A 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.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.


Figure 2 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 . 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 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.  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. 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.


Figure 3 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 . 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 . 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A 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.  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.  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.  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.


Figure 4 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 . 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 . 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 A 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.  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.  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.  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.


Figure 5 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 . 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 . 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 .

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

Figure 5 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.  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.  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.


Figure 1A 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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.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.