Deletion of 2D2 CD4⁺ T cells in the thymus is specifically induced by B cells.

<p>LN of 2D2 and 2D2/JHT (left row) as well as B^MOG/2D2 and B^MOG/2D2/JHT (right row) mice were analyzed by FACS analysis for presence of CD4⁺ Vα3.2⁺ T cells. Genotypes and antibodies used are as indicated. Cell surface markers are shown as coordinates. Cells were gated on live lymphocytes. Numbers besides gates or in quadrants indicate percent positive cells in each.</p

FACS analysis of Foxp3-expressing T cells in B^MOG/2D2 mice.

<p>(A) Thymus and LN of WT 2D2 and B^MOG/2D2 mice were analyzed for expression of Foxp3 by intracellular FACS staining. Genotypes and antibodies used are as indicated. Cells were gated on CD4⁺ cells. Numbers in quadrants indicate percent positive cells. (B) Total numbers of CD4⁺ Foxp3⁺ T cells from thymus and LN were calculated. Data represent mean values ± SEM of several individual experiments. ns, not significant. (C) LN cells from B^MOG/2D2 and WT 2D2 mice were gated on CD4⁺ Va3.2⁺ or CD4+ Va3.2⁻ T cells, respectively, and percentage of Foxp3-expressing cells among these populations is shown by histograms. The blue line in the histograms refers to the blue gate of CD4⁺ Va3.2⁻ cells, the red line refers to CD4⁺ Va3.2⁺ cells (red gate). Colored numbers above the marker line indicate percent positive cells for the respective populations.</p

Residual MOG-specific T cells in B^MOG/2D2 mice downregulate Vβ11.

<p>LN of WT 2D2 and B^MOG/2D2 mice were analyzed by FACS analysis for presence of CD4, Vα3.2 and Vβ11. Cells were analyzed for expression of Vα3.2 vs CD4 (dot blots). Cells were then gated on CD4⁺ Vα3.2⁺ T cells and the percentage of Vβ11-expressing cells among this population is shown by histogram overlay. The red line in the histograms refers to the red gate of CD4⁺ Va3.2⁺ cells in 2D2 mice, the blue line refers to CD4⁺ Va3.2⁺ cells (blue gate) of B^MOG/2D2 mice. Colored numbers above the marker line indicate MFI (mean fluorescence intensity) of anti-Vβ11-PE for the respective populations.</p

Deletion of 2D2 CD4⁺ T cells upon MOG encounter by B cells in the thymus.

<p>Thymus and LN from WT 2D2 (upper row) and B^MOG/2D2 (lower row) mice were analyzed by FACS analysis for presence of CD8⁺ and CD4⁺ Va3.2⁺ T cells, respectively (A). Genotypes and antibodies used are as indicated. Cell surface markers are shown as coordinates. Cells were gated on live lymphocytes. Numbers besides gates or in quadrants indicate percent positive cells in each. Total and CD4 single positive thymocyte numbers (B) and LN total CD4⁺ as well as CD4⁺ Va3.2⁺ and CD4⁺ Va3.2⁻ T cell numbers (C) of 2D2 and B^MOG/2D2 mice were calculated. Values represent mean ± SEM. ns, not significant.</p

Figure 5

<p>(<b>A</b>) <b>2D2 CD4⁺ T cells from B^MOG/2D2 mice do not proliferate upon </b><b><i>in vivo</i></b><b> MOG stimulation.</b> CD4⁺ Thy1.1⁺ T cells from WT 2D2 and B^MOG/2D2 mice were MACS-purified, CFSE-labeled, and transferred to APC^MOG mice (∼10×10⁶/mouse). Prior to transfer, APC^MOG mice were injected with anti-CD40. Five days after transfer, cells were monitored for proliferation by FACS analysis and loss of CFSE is depicted by representative dot blots of LN cells. Genotypes and antibodies used are as indicated. Cell surface markers are shown as coordinates. Cells were gated on live lymphocytes. (<b>B</b>)<b> B^MOG/2D2 mice are resistant to EAE.</b> EAE was actively induced by immunization of 2D2 (squares) and B^MOG/2D2 (circles) mice with MOGp35-55. B^MOG/2D2 mice are protected from EAE compared to 2D2 WT mice (p<0,05: days 12–27). Values represent mean (± SEM) clinical scores. Shown is one representative of two individual experiments (n≥4 mice/group).</p

Total serum immunoglobulin levels after depletion of B cells.

<p>B-DTR mice and control mice were injected daily four times with 25 ng/g body weight DT. Mice were bled just before DT injection and in weekly intervals thereafter. Shown are the total immunoglobulin levels at the indicated time points. The values are shown as average of groups of at least five mice.</p

Efficiency of B cell depletion.

<p>A. Number of marginal zone, follicular, immature and mature B cells in spleen. B. Depletion of B cells in bone marrow, gated for B220⁺. C. Depletion of transitional B cells in spleen, gated for CD19⁺ cells. D. Depletion of mature and immature B cells in spleen, gated for CD19⁺ cells. E. Depletion of marginal zone and follicular B cells gated for CD19⁺ cells. F. Depletion of B1a and B2 B cells in peritoneal cavity, gated for CD19⁺. G. Depletion of resting B cells in peritoneal cavity gated for CD19⁺ the percentages of cells in the different shown B cell subpopulations are indicated.</p

Plasma cells identified with Syndecan-1 in cytospins.

<p>Bone marrow cells from NP-CG immunized and boosted cells were analyzed for plasma cells. Plasma cells are shown in red with a surface staining of syndecan-1, the remaining cell population was counter stained with Hoechst (blue). The bar chart represents plasma cells found in the regarding groups (n = 5). The pictures show representative sections of the cytospins.</p

Diphtheria toxin mediated depletion of B cells in B-DTR mice.

<p>The mice were treated once daily for 4 days with a dose of 25 ng/g body weight of diphtheria toxin. The decrease of B cells in comparison to T cells is shown in spleen, lymph nodes, bone marrow and peritoneal cavity; dot blots in the left panel and the corresponding bar charts in the right panel.</p

eYFP expression in CD19-Cre/eYFP mice.

<p>A. CD19-Cre/eYFP mice were analyzed for eYFP expression in the B cell compartment of spleen, lymph nodes, Peyer's patches, peritoneal cavity and bone marrow. The shown histograms are gated on live lymphocytes and CD19. The filled histogram curve represents CD19-Cre/eYFP mice and the unfilled control mice. Percentages of eYFP⁺ CD19⁺ cells are shown in the histograms, upper number/percentage B-DTR mice, lower panel shows cells of control mice. B. To identify the CD19⁺ eYFP⁻ B-cell fraction in bone marrow of CD19-Cre/eYFP mice, bone marrow cells were stained for AA4.1 and CD23.</p