Figure 6.
Figure 6. Cdc42 is essential for antigen presentation. (A) Representative confocal microscopy (with a 63× objective) images showing internalized Alexa Fluor 647 anti-IgM antibody in WT (left) or Cdc42 KO (right) splenic B cells. Bars, 3 µm. Quantification (bottom) indicates percentage of cells with polarized antigen per field of view, each image containing at least 10 cells. Data are representative of 3 independent experiments. (B) Antigen presentation assay showing presentation of Eα peptide on MHCII measured by flow cytometry. Splenic WT (left, blue line) and Cdc42 KO (right, red line) B cells were loaded with beads coated with anti-IgM antibody and Eα peptide and cultured for 3 h (top) or 5 h (bottom). Quantification (right) indicates ratio between mean Eα fluorescence and total MHCII fluorescence. Data are representative of 2 independent experiments. (C) Proliferation profiles of B (left column) and T cells (right column) by flow cytometry. OTII T cells were co-cultured for 3 d with purified WT (blue line) or Cdc42 KO (red line) B cells loaded with anti-IgM- and ovalbumin-coated beads (at the concentrations noted). Data are representative of 2 independent experiments. (D) Representative two-photon microscopy image of a whole fixed popliteal LN after adoptive transfer of HEL-WT B cells (green), HEL-Cdc42 KO B cells (cyan), and OTII T cells (red) into WT mice, and subsequent immunization with Hel- and OVA-coated beads. B cell follicles and T cell area were highlighted in magenta and gray for illustrative purposes. The proportion of cells in B cell follicles versus T cell area were measured and quantification is shown in the chart (bottom right). Bars, 150 µm. Data are representative of 2 independent experiments. (E) Representative image of the visualization of homeostatic movement of WT (green) and Cdc42 KO (cyan) cells by two-photon microscopy. Representative tracks are shown on the image. Quantifications are shown below and indicate the mean speed over the span of the video as well as the mean displacement of cells. Data are representative of 2 independent experiments. (F) Representative images of the whole field of view (left) as well as higher magnification images (7×) of the visualization of B–T cell interactions in explanted LNs by two-photon microscopy. SNARF-1–labeled OTII T cells (red), CFSE-labeled HEL-WT (green), and CTV-labeled MD4+ Cdc42 KO (cyan) B cells were adoptively transferred into WT recipients, which were then immunized intra-footpad with Hel- and OVA-coated beads. Popliteal LNs were explanted and imaged 15 h after immunization. Quantifications are shown below and represent the percentage of B cells interacting with T cells over the span of the video, as well as the mean length and the distribution of the contact time of B–T cell interactions. Data are representative of 3 independent experiments. Mean and SEM. Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001.

Cdc42 is essential for antigen presentation. (A) Representative confocal microscopy (with a 63× objective) images showing internalized Alexa Fluor 647 anti-IgM antibody in WT (left) or Cdc42 KO (right) splenic B cells. Bars, 3 µm. Quantification (bottom) indicates percentage of cells with polarized antigen per field of view, each image containing at least 10 cells. Data are representative of 3 independent experiments. (B) Antigen presentation assay showing presentation of Eα peptide on MHCII measured by flow cytometry. Splenic WT (left, blue line) and Cdc42 KO (right, red line) B cells were loaded with beads coated with anti-IgM antibody and Eα peptide and cultured for 3 h (top) or 5 h (bottom). Quantification (right) indicates ratio between mean Eα fluorescence and total MHCII fluorescence. Data are representative of 2 independent experiments. (C) Proliferation profiles of B (left column) and T cells (right column) by flow cytometry. OTII T cells were co-cultured for 3 d with purified WT (blue line) or Cdc42 KO (red line) B cells loaded with anti-IgM- and ovalbumin-coated beads (at the concentrations noted). Data are representative of 2 independent experiments. (D) Representative two-photon microscopy image of a whole fixed popliteal LN after adoptive transfer of HEL-WT B cells (green), HEL-Cdc42 KO B cells (cyan), and OTII T cells (red) into WT mice, and subsequent immunization with Hel- and OVA-coated beads. B cell follicles and T cell area were highlighted in magenta and gray for illustrative purposes. The proportion of cells in B cell follicles versus T cell area were measured and quantification is shown in the chart (bottom right). Bars, 150 µm. Data are representative of 2 independent experiments. (E) Representative image of the visualization of homeostatic movement of WT (green) and Cdc42 KO (cyan) cells by two-photon microscopy. Representative tracks are shown on the image. Quantifications are shown below and indicate the mean speed over the span of the video as well as the mean displacement of cells. Data are representative of 2 independent experiments. (F) Representative images of the whole field of view (left) as well as higher magnification images (7×) of the visualization of B–T cell interactions in explanted LNs by two-photon microscopy. SNARF-1–labeled OTII T cells (red), CFSE-labeled HEL-WT (green), and CTV-labeled MD4+ Cdc42 KO (cyan) B cells were adoptively transferred into WT recipients, which were then immunized intra-footpad with Hel- and OVA-coated beads. Popliteal LNs were explanted and imaged 15 h after immunization. Quantifications are shown below and represent the percentage of B cells interacting with T cells over the span of the video, as well as the mean length and the distribution of the contact time of B–T cell interactions. Data are representative of 3 independent experiments. Mean and SEM. Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal