Figure 7.
Impaired mobilization of CD107a and CD40L in Stx11−/−CD4 T cells. Mouse: (A) d6 pre-activated SMARTA.Stx11−/− (red, n = 11) or SMARTA.WT (blue, n = 10) (n = 8 independent experiments) were re-stimulated with plate-bound anti-CD3 (10 µg/ml) for 2 h in the presence of anti-CD107a antibodies. Surface CD107a was measured by flow cytometry. Representative overlay of histograms (left), delta of stimulated minus medium-only of percentage (middle), and median fluorescence intensity (MFI) (right). (B) Mobilization of CD107a+ vesicles (red) of CD4 T cells (blue) with LCMV-GP61-80-pulsed WT B cells (green) after 35 min incubation (WT n = 2, Stx11−/−n = 2, one experiment). Scale bar 2 µm; arrows indicate immunological synapses. Human: (C) Western blot for STX11 expression in CD4 T cells of two healthy donors (HD) and after CRISPRCas9 KO of STX11 using four RNP guides (KO1+KO2) or six RNP guides (KO2.2) at d10 of restimulation with anti-CD3/CD28/CD2 beads and rhIL-12. Molecular mass is shown in kilodaltons. (D) Re-stimulation of d10 cultured human CD4 T cells for 3.5 h with anti-CD3 and soluble anti-CD28 (both 2 µg/ml) (representative experiment of n = 2). (E) Restimulation of d10 cultured human CD4 T cells for 3.5 h with PMA/Iono (representative experiment of n = 2). Mouse: (F) Representative dot plots of conjugation assay of CFSE-labeled, LPS-activated, GP61-80-pulsed or unpulsed WT B cells with d7 pre-cultured SMARTA.CD4 T cells. (G) Percentage of CD4.SMARTA.Stx11−/− (red, n = 12) or CD4.SMARTA.WT (blue, n = 11) in conjugates with B cells (from F [red/green] ×100%) after 30 min incubation (n = 8 independent experiments). (H) CD40L mobilization: CD40L+ T cells (as % of total T cells) shown as Δ of stimulated with GP61-80-pulsed minus unpulsed WT B cells (green rectangle shown in F) (left) or CD40L MFI on CD4 T cells (green rectangle shown in F) (right). SMARTA.Stx11−/− (red, n = 12) or SMARTA.WT (blue, n = 11) from n = 8 independent experiments. (I) CD40L mobilization: Δ of PMA/Iono stimulated minus medium-only of d6–7 pre-cultured CD4 T cells. Incubation time 30 min, showing percentage of CD40L+ CD4 T cells (left) and MFI of CD40L (right). SMARTA.Stx11−/− (red, n = 12) or SMARTA.WT (blue, n = 11) from n = 8 independent experiments. (J)Stx11−/− (n = 8), CD8 T cell–depleted (anti-CD8 +) Stx11−/− (n = 16), and WT (C57BL/6N) mice (n = 7) were infected with 200 PFU LCMV. Cells were isolated d12 p.i. and re-stimulated with PMA/Iono. Δ = PMA/Iono minus medium-only control. n = 2 independent experiments. (K) Snapshots from live TIRF imaging of SMARTA.WT CD4 T cells co-transfected with Stx11-mNeonGreen and either Rab11-mCherry or Rab7-mCherry constructs were captured. Cells were positioned on anti-CD3–coated coverslips to observe vesicle polarization and fusion at the synapse. Scale bar 1 µm. (L) Fusion profile analysis at 0.1 and 0.2 s of STX11 and RAB11. Scale bar 1 µm. (M) The video timestamps (Video 1 and Video 2) of the captured frames are shown on the bottom left of each image. Fusion events are indicated by white arrows. Timestamp when a fusion event happens is marked by an asterisk. Scale bar 1 µm. (N) SIM images of a representative WT CD4 T cell transfected with Stx11-mNeonGreen construct. Cells were incubated on anti-CD3–coated coverslips for 30 min, allowing the formation of a synapse and vesicle release. Fixed cells were stained with biotinylated anti-CD40L and anti-LAMP-1 antibodies to analyze co-localization using SIM. Images were acquired at the synapse area above the coverslips. All images were subsequently analyzed and presented after post-processing. The footprint of the cell is marked by white stipple line. Solid arrows point to the colocalization of STX11 and CD40L, whereas the opened arrows point to the colocalization of LAMP1 and CD40L. Scale bar 2 µm. (A–J) Mean and ± SEMs of Mann–Whitney U test are shown; *P < 0.05, **P < 0.01, *** P < 0.001, **** < 0.0001, ns indicates not significant. Source data are available for this figure: SourceData F7.

Impaired mobilization of CD107a and CD40L in Stx11 −/− CD4 T cells. Mouse: (A) d6 pre-activated SMARTA.Stx11−/− (red, n = 11) or SMARTA.WT (blue, n = 10) (n = 8 independent experiments) were re-stimulated with plate-bound anti-CD3 (10 µg/ml) for 2 h in the presence of anti-CD107a antibodies. Surface CD107a was measured by flow cytometry. Representative overlay of histograms (left), delta of stimulated minus medium-only of percentage (middle), and median fluorescence intensity (MFI) (right). (B) Mobilization of CD107a+ vesicles (red) of CD4 T cells (blue) with LCMV-GP61-80-pulsed WT B cells (green) after 35 min incubation (WT n = 2, Stx11−/−n = 2, one experiment). Scale bar 2 µm; arrows indicate immunological synapses. Human: (C) Western blot for STX11 expression in CD4 T cells of two healthy donors (HD) and after CRISPRCas9 KO of STX11 using four RNP guides (KO1+KO2) or six RNP guides (KO2.2) at d10 of restimulation with anti-CD3/CD28/CD2 beads and rhIL-12. Molecular mass is shown in kilodaltons. (D) Re-stimulation of d10 cultured human CD4 T cells for 3.5 h with anti-CD3 and soluble anti-CD28 (both 2 µg/ml) (representative experiment of n = 2). (E) Restimulation of d10 cultured human CD4 T cells for 3.5 h with PMA/Iono (representative experiment of n = 2). Mouse: (F) Representative dot plots of conjugation assay of CFSE-labeled, LPS-activated, GP61-80-pulsed or unpulsed WT B cells with d7 pre-cultured SMARTA.CD4 T cells. (G) Percentage of CD4.SMARTA.Stx11−/− (red, n = 12) or CD4.SMARTA.WT (blue, n = 11) in conjugates with B cells (from F [red/green] ×100%) after 30 min incubation (n = 8 independent experiments). (H) CD40L mobilization: CD40L+ T cells (as % of total T cells) shown as Δ of stimulated with GP61-80-pulsed minus unpulsed WT B cells (green rectangle shown in F) (left) or CD40L MFI on CD4 T cells (green rectangle shown in F) (right). SMARTA.Stx11−/− (red, n = 12) or SMARTA.WT (blue, n = 11) from n = 8 independent experiments. (I) CD40L mobilization: Δ of PMA/Iono stimulated minus medium-only of d6–7 pre-cultured CD4 T cells. Incubation time 30 min, showing percentage of CD40L+ CD4 T cells (left) and MFI of CD40L (right). SMARTA.Stx11−/− (red, n = 12) or SMARTA.WT (blue, n = 11) from n = 8 independent experiments. (J)Stx11−/− (n = 8), CD8 T cell–depleted (anti-CD8 +) Stx11−/− (n = 16), and WT (C57BL/6N) mice (n = 7) were infected with 200 PFU LCMV. Cells were isolated d12 p.i. and re-stimulated with PMA/Iono. Δ = PMA/Iono minus medium-only control. n = 2 independent experiments. (K) Snapshots from live TIRF imaging of SMARTA.WT CD4 T cells co-transfected with Stx11-mNeonGreen and either Rab11-mCherry or Rab7-mCherry constructs were captured. Cells were positioned on anti-CD3–coated coverslips to observe vesicle polarization and fusion at the synapse. Scale bar 1 µm. (L) Fusion profile analysis at 0.1 and 0.2 s of STX11 and RAB11. Scale bar 1 µm. (M) The video timestamps (Video 1 and Video 2) of the captured frames are shown on the bottom left of each image. Fusion events are indicated by white arrows. Timestamp when a fusion event happens is marked by an asterisk. Scale bar 1 µm. (N) SIM images of a representative WT CD4 T cell transfected with Stx11-mNeonGreen construct. Cells were incubated on anti-CD3–coated coverslips for 30 min, allowing the formation of a synapse and vesicle release. Fixed cells were stained with biotinylated anti-CD40L and anti-LAMP-1 antibodies to analyze co-localization using SIM. Images were acquired at the synapse area above the coverslips. All images were subsequently analyzed and presented after post-processing. The footprint of the cell is marked by white stipple line. Solid arrows point to the colocalization of STX11 and CD40L, whereas the opened arrows point to the colocalization of LAMP1 and CD40L. Scale bar 2 µm. (A–J) Mean and ± SEMs of Mann–Whitney U test are shown; *P < 0.05, **P < 0.01, *** P < 0.001, **** < 0.0001, ns indicates not significant. Source data are available for this figure: SourceData F7.

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