EMBO/soda-nel huggingface dataset:
Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants (2016)
Figure 8

Figure 8. EM-Tomography reveals enlargement of synaptic vesicles under continuous exocytosis in OtofI515T/I515T IHCs.

A-D, Exemplary virtual sections through EM tomograms for stimulatory condition at 36°C and 39oC. Scale bar 100nm.

E, Illustration depicting the tomogram analysis (not drawn to scale). Ribbon-associated synaptic vesicles (SVs, green) in the first row ≤80 nm from ribbon (R) and membrane-proximal vesicles (yellow) in ≤50 nm membrane-to-membrane distance from the plasma membrane (blue) and ≤100nm from each side of the presynaptic density.

F-I, 3D reconstruction of the membrane-proximal pool (top view) and J-M, the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.

N-Q, Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane-proximal pool (N-O) and ribbon-associated pool (P-Q). N-P, one way ANOVA followed by Tukey’s test; Q, Kruskal-Wallis test. Round vesicles tested in both the pools appeared larger in OtofI515T/I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes (see also Table EV1).
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 1

Figure 1: TCR activation results in mitochondrial fragmentation and cristae remodelling in T cells.

A. AICD was induced in hPB T as described in Materials and methods. Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown. Data represent mean ± SE of six independent experiments.

B. AICD was induced in Jurkat cells as described in Materials and methods. Apoptotic cells were detected as in (A). Data represent mean ± SE of five independent experiments.

C. hPB Ts were transfected with mtYFP and after 24h AICD was induced. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 30’ after AICD induction is shown. Scale bar, 5μm.

D. Morphometric analysis of mitochondrial shape of cells treated as in (B). Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).

E. Jurkat cells were transfected with mtYFP, 24h after AICD induction cells were fixed. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.

F. Morphometric analysis of mitochondrial shape in cells treated as in (E) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).

G. Representative electron micrographs of hPB T treated as indicated. 30min after AICD induction cells were fixed and processed for electron microscopy. Scale bar, 2μm.

H. 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy. Representative electron micrographs of cells treated as indicated are shown. In EM images white arrowheads point to autophagic vesicles (late autophagosomes or most likely autophagolysosomes, Aφ); black arrows point to mitochondria (mito) with remodelled cristae, specifically touching a representative disorganized crista. Scale bar, 2μm.

I. Representative confocal images of hPB T transfected with mtYFP, fixed 30’ after AICD induction and immunostained with an anti-cytochrome c antibody (red).

J. Cytochrome c localization index was calculated from 30 randomly selected cells treated as in (I). Data represent mean ± SE of three independent experiments.

K. Representative confocal images of Jurkat cells transfected with mtYFP, fixed at 32h after AICD induction and immunostained with an anti-cytochrome c antibody.

L. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of five independent experiments.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 2

Figure 2: AICD depends on mitochondrial fragmentation and cristae remodelling which are rescued by DRP1K38A and OPA1.

A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.

B. Jurkat cells were transfected with pE-GFP and the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of five independent experiments.

C. Jurkat cells were transfected as indicated. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.

D. Morphometric analysis of mitochondrial shape of cells transfected as in (C) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).

E. AICD was induced in Jurkat cells over expressing pE-GFP and the indicated plasmids and sorted for GFP positive fluorescence. 24h after AICD induction cells were fixed and processed for electron microscopy. Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown. White arrow points to tight cristae structure, black arrow points to remodelled cristae and to a swollen cristae junction. Scale bar, 0.2μm.

F. Morphometric analysis of cristae width in cells transfected as in (E).

G. Representative confocal images of cells transfected as indicated, fixed 32h after AICD induction and immunostained with an anti-cytochrome c antibody (red).

H. Cytochrome c localization index was calculated from 30 randomly selected cells (per condition) transfected as in (G). Data represent mean ± SE of five independent experiments.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 3

Figure 3: TCR activation results in early inhibition of autophagy.

A. Representative confocal images of cells transfected GFP-LC3 and Parkin-Cherry, treated as indicated, fixed 24h after AICD induction and immunostained with anti-TOM20 antibody are shown. Scale bar, 5μm.

B. Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).

C. Cells were treated as in (A). Where indicated, lysosomal proteases inhibitors E64D and PepA were added. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected cells per condition. Data represent mean ± SE of three independent experiments. Scale bar, 5μm

D. hPB T were transfected with LC3-cherry and treated as indicated. 30min after AICD induction cells were fixed and stained with TUNEL and DAPI. Representative reconstructions of confocal z-stacks of the LC3-Cherry fluorescence and single stacks for DAPI are shown. Scale bar, 5μm.

E. Statistical analysis of the autophagy in cells treated as in (D) is shown. Data represent mean ± SE of three independent experiments (n = 20 cells per condition in each experiment).

F. Jurkat cells were transfected with GFP-LC3 and treated as indicated. Representative reconstructions of confocal z-stacks of the GFP-LC3 fluorescence 16h after AICD induction are shown. Scale bar, 5μm.

G. Statistical analysis of the autophagic flux in cells treated as in (F) is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).

H. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated, lysosomal proteases inhibitors E64D and PepA were added. WB are representative of at least three independent experiments.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 4

Figure 4: Autophagy is inhibited upon AICD induction through PKA activation.

A-B. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Wb are representative of at least four independent experiments. Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown. In these panels, Time states for "time, hours, after AICD treatment" and indicates at which time upon AICD induction samples have been collected.

C. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.

D. Protein samples from Jurkat cells over expressing empty vector or AMPKS173A-S485A were immunoblotted for LC3 and LC3-II/Actin ratio was quantified and analyzed as shown. Data represent mean ± SE of three independent experiments.

E-F. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments. Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown.

G. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as described above.

H-I. Cells where treated as in (A) and where indicated 10μM H89 was added. Quantitative analysis of P-AMPK Ser485-491 phosphorylation (H) and of LC3-II/LC3-I ratio (I) during AICD are shown.

J-K. AICD was induced in Jurkat cells (J) and in hPBT (K) in presence of 10μM H89. At indicated time points (hours) after AICD induction, apoptotic cells were detected by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 5

Figure 5: Pharmacological induction of autophagy during AICD results in cell death inhibition independently from DRP1-dependent mitochondrial shape changes.

A. Apoptotic cells were detected at indicated time points after AICD induction in hPB T cells by flow cytometry as AnnexinV/PI double positive cells. Where indicated hPB T cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Data represent mean ± SE of six independent experiments.

B. AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of six independent experiments.

C. Morphometric analysis of mitochondrial shape 24h after AICD induction. Jurkat cells were transfected with mtYFP 24h before AICD induction and, where indicated, 100nM rapamycin was added. Data represent mean ± SE of four independent experiments (n = 30 cells per condition in each experiment).

D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm.

E. Jurkat cells were transfected with mtYFP and treated as indicated. 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of four independent experiments.

F. Jurkat cells were transfected with DRP1S637A-GFP, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.

G-H. Jurkat cells were co-transfected with mt-RFP and the indicated plasmids, 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index (G) and mitochondrial morphology (H) were calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments.

I. Jurkat cells were transfected with DRP1K38A, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV/PI double negative cells. Data represent mean ± SE of three independent experiments.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 6

Figure 6. Pharmacological induction of autophagy during AICD removes dysfunctional mitochondria decorated by LC3 and Parkin.

A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.

B-C. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown. Data represent mean ± SE of three independent experiments.

D. Jurkat cells were co-transfected with GFP-LC3 and Parkin-Cherry, 32h after AICD induction cells were fixed and immunostained with an anti-TOM20 antibody. Representative images for each condition are presented. Scale bar, 5μm.

E. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. Data referring to Ctrl and AICD-stimulated cells without rapamycin correspond to the ones presented in Fig.3C.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Macroautophagy inhibition maintains fragmented mitochondria to foster T cell receptor-dependent apoptosis (2016)
Figure 7

Figure 7. Autophagy limits AICD in vivo

A. Splenocytes were isolated from Ambragt/+ and wt C57/BL6 mice and activated as indicated in materials and methods. At day 7 after activation, AICD was induced with 0.1μg/ml of plate-bound anti-mCD3 (Clone 17A2). Apoptotic cells were detected 12h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.

B. Splenocytes were isolated from ATG7 fl/fl and wt C57/BL6 mice. Activation of splenocytes and AICD were performed as previously described. At day 5 after activation, cells were infected with retroviral constructs expressing CRE recombinase. Where indicated, cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.

C. Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction. AICD was induced as described in materials and methods. Where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of five independent experiments.

D. Mitochondrial depolarization upon AICD induction was analysed in cells treated as indicated. Data represent mean ± SE of five independent experiments.

E. Protein samples (20μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of three independent experiments.

F. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown. Data represent mean ± SE of three independent experiments.

P values: * p < 0.05; ** p < 0.01; *** p < 0.001.
Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination (2021)
Figure 2

Figure 2: Sporozoite antigen exposure elicits superior peripheral blood CD8+ T cell responses.

(A) Schematic overview of experimental design. C57BL/6 mice received 2x106 OT-I cells alone or were additionally immunised with 10,000 γ-radiation attenuated parasites intravenously.

(B) Flow cytometry plots show the gating strategy for identifying Kb-SIINFEKL+ CD11ahi CD8+ T cells.

(C) Temporal analysis of antigen-specific CD8+ T cell responses. Peripheral blood was obtained on days 4, 7, 14, 21, 42 and 88 post-immunisation with WT (triangles; n=3-9), CSPSIINFEKL (orange squares; n=4-8) or UIS4SIINFEKL (blue circles; n=4-10) sporozoites, or no parasites (diamonds; n=2-5) and stained for Kb-SIINFEKL+ CD11ahi CD8+ T cells. Line graph shows mean values (±SEM) from representative experiments. (*, p<0.05; **, p<0.01; ***, p<0.001; Welch's t-test comparing CSPSIINFEKL and UIS4SIINFEKL). See Appendix Table S1 for the number of mice used per timepoint and per group. See Appendix Table S4 for exact p-values.

(D-H) C57BL/6 mice (n=4 per group), which received 2x106 CFSE-labelled OT-I splenocytes, were immunised with 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites intravenously. 5 days later, mice were sacrificed, spleens harvested and splenocytes assessed for (D) CFSE dilution of CD8+ T cells, (E) CFSE dilution of antigen experienced Kb-SIINFEKL+ CD11ahi CD8+ T cells and stained ex vivo (F-H) for effector CD8+ T cell surface markers. Shown are flow cytometry plots of Kb-SIINFEKL co-staining with markers of effector phenotypes: (F) CD11ahi, (G) CD62Llo and (H) CD49dhi.
Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination (2021)
Figure 3

Figure 3: Sporozoite surface antigen induces a higher CD8+ T cell response than EEF vacuolar membrane antigen in the spleen and liver.

(A) Schematic overview of experimental design. C57BL/6 mice received 2x106 OT-I cells alone (n=2-4) or were additionally immunised with 10,000 γ-radiation attenuated WT (n=3-6), CSPSIINFEKL (n=4) or UIS4SIINFEKL (n=4) sporozoites intravenously. See Appendix Table S2 and S3 for the number of mice used for pentamer staining and peptide restimulation. Spleens and livers were harvested either at day 14 or day 42.

(B-G) Proportions and numbers of (B-D) Kb-SIINFEKL+ CD8+ T cells were enumerated, or (E-G) IFN-γ-secreting CD8+ T cells following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with CD11a and (B) Kb-SIINFEKL or (E) IFN-γ. The upper panel of bar charts (C, F) show the percentage of co-stained CD8+ T cells and the lower panel (D, G) the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (*, p<0.05; **, p<0.01; ***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.
Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination (2021)
Figure 4

Figure 4: Higher magnitude of endogenous SIINFEKL-specific CD8+ T cell responses induced by a sporozoite antigen.

(A) Schematic overview of experimental design.

(B-G) C57BL/6 mice received 10,000 γ-radiation attenuated WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites, either (B-D) intravenously (n=6-8 mice per group) or (E-G) intradermally (n=4 mice per group). Additional control mice did not receive sporozoites (n=6-8 intravenously, n=4 intradermally). Spleens and livers were harvested either at day 14 (n=5 livers and n=6 spleens for intravenous immunisation) or day 42 (n=8 for intravenous immunisation), and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified. Flow cytometry plots show representative percentages of CD8+ T cells co-stained with IFN-γ and CD11a (B, E). The upper panel of bar charts (C, F) show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel (D, G) the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (*, p<0.05; **, p<0.01; ***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.
Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination (2021)
Figure 5

Figure 5: Increasing antigen dose does not improve antigen-specific CD8+ T cell responses to an EEF vacuolar membrane protein.

(A) Schematic overview of experimental design. C57BL/6 mice (n=4 per group) received an intravenous dose of 8,000 γ-radiation attenuated CSPSIINFEKL or UIS4SIINFEKL sporozoites or 64,000 γ-radiation attenuated UIS4SIINFEKL sporozoites. Spleens and livers were harvested at day 12 and IFN-γ-secreting lymphocytes following restimulation ex vivo with SIINFEKL peptide were quantified.

(B) Flow cytometry plots show representative CD8+ T cells co-stained with IFN-γ and CD11a.

(C) The upper panel of bar charts show the percentage of CD11ahi IFN-γ+ CD8+ T cells and the lower panel the absolute cell counts. Bar charts show mean values (±SEM) from representative experiments (***, p<0.001; one-way ANOVA with Tukey's multiple comparison test). See Appendix Table S4 for exact p-values.
Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination (2021)
Figure 6

Figure 6: Both sporozoite surface and EEF vacuolar membrane antigens are susceptible to vaccine-induced CD8+ T cells for killing, resulting in sterile protection.

(A) Schematic overview of experimental design. Mice received 2x106 OT-I splenocytes, and one day later they were either vaccinated with 1x108 ifu recombinant AdHu5 expressing whole ovalbumin (AdOVA) (n=5 per challenge group) or left untreated (n=4 per challenge group).

(B, C) Flow cytometry (B) and scatter plots (C) represent CD8+ T cells derived from peripheral blood co-stained with IFN-γ and CD11a, following ex vivo restimulation with SIINFEKL.

(D) Protective efficacy as measured by quantitative real-time PCR. Groups of mice were vaccinated as described and challenged 19 days later with 10,000 WT (n=7 non-vaccinated, n=9 vaccinated), CSPSIINFEKL (n=6 non-vaccinated, n=4 vaccinated) or UIS4SIINFEKL sporozoites (n=3 non-vaccinated, n=11 vaccinated). 42 hours later livers were removed, and parasite load was assessed by qPCR. Plots show the relative parasite load of mice in each condition (**, p<0.01; Mann-Whitney U test). See Appendix Table S4 for exact p-values.

(E) Proportion of sterile protection after immunisation. Mice (vaccinated group, n=8; control group, n=4) were vaccinated as described and were challenged with 800 WT, CSPSIINFEKL or UIS4SIINFEKL sporozoites. Daily blood smears were taken from day 3-14 post challenge to check for parasitaemia.

Data information: Data for B-E are representative of two experiments performed with scatter plots showing mean values (±SEM).

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