Figure 1.
T cell activation mobilizes monocytes into LNs and promotes formation of CD209+ DC.
A) Mice were injected in the footpad with 10µg anti-CD3 or an isotype control antibody. 18 hours later cells from the draining LNs were stained for the early T cell activation marker CD69. Cells shown were gated on DAPI-, Thy1.2+ cells. N=2 per group. Both duplicates are shown. B) Mice were injected in the footpad with 10µg anti-CD3 or PBS. 18 hours later, bone marrow, cardiac blood or draining LNs were stained for DAPI-, side scatter -/int, CD11b+, CD11c-, Ly6c+ monocytes. Shown are mean cell percentage and SEM, N=2 mice per group. C) Mice were injected in the footpad with 10µg anti-CD3 or PBS. 18 hours later, draining LNs were evaluated for the frequency of CD11b+ CD11c+ DC or CD11b+ CD11c- monocytes. Shown is one example representative of more than 5 individual experiments. D) Mice were injected in the footpad with 10µg anti-CD3, isotype control, anti-CD28 or PBS. The absolute number of CD209+, CD11b+, CD11c+, MHC II+, CD40+, Ly6c-, DAPI- DC from 3 draining LNs is shown. Shown are mean cell number and SEM, N=4, representative of more than 5 individual experiments. E) Shown is one example of CD209 staining compared to isotype control staining of LN CD11c+, CD11b+ DC 18 hours after anti-CD3 antibody injection. The results shown are representative of 2 independent experiments. F) Mice were injected in the footpad with the indicated amounts of anti-CD3 antibody. 18 hours later the absolute number of LN CD209+ DC was determined with flow cytometry. G) Mice were injected in the footpad with 10µg anti-CD3 or an isotype control. 18 hours later draining LNs were removed, fixed and frozen. The sections were then stained for the presence of CD209 and B220. The scale bar is 50µM. H) Mice were injected IV with 25µg TSST-1 or PBS. 16 hours later, both inguinal and brachial LNs were harvested and the absolute number of CD209+, CD11b+, CD11c+, MHC II+, CD40+, Ly6c-, DAPI- DC are shown as mean and SEM, N=4 mice per group.
Figure 2.
Activated T cell driven CD209+ Mo-DC stimulate T cell proliferation, but do not induce T cell polarization.
A) Mice were injected in the footpad with 10µg anti-CD3. 18 hours later, LN monocytes gated as Ly6c+, CD11c-, side scatterlo/inT cells, Mo-DC gated as Ly6c-, CD11c+, CD209+, CD205- cells and cDC gated as Ly6c-, CD11c+, CD209-, CD205+ cells were evaluated for expression of various surface proteins. Shown is a representative example from one mouse. B) The gating scheme for sorting CD209+ Mo-DC and CD209- cDC is shown. C) DC sorted as in B) were plated on cover slip bottom chamber slides in medium and immediately imaged. The scale bar is 20µM. D) Cells sorted as in B) along with monocytes and B-cells were pulsed with 2.5µg/ml MHC class II restricted OVA peptide ISQ for 90 minutes at 37°C. They were then washed twice and plated at various ratios with OT-II CD4+ T cells from RAG KO mice. After 72 hours the cells were pulsed with H3-thymidine and harvested 18 hours later. Results of triplicate cultures are shown as mean and SEM, and are representative of 3 independent experiments. E) Cultures were set up as in D) except in some conditions 1µg/ml LPS was added during pulsing with peptide. After 72 hours, the cell-free supernatant was harvested and cytokines were measured by ELISA. Results are shown as mean and SEM, performed in triplicate from the 1 DC : 20 T cells condition, and are representative of 3 independent experiments. ND = not detected.
Figure 3.
CD40L but not GM-CSF is needed for CD209+ Mo-DC formation.
A) Serum from cardiac blood was assayed for GM-CSF at various times after footpad injection of 10µg anti-CD3 antibody. N=2 mice per time point. Results are shown as mean and SEM. B) CD4+ and CD8+ T cells were positively selected from the LNs of wild-type mice using biotinylated antibodies and anti-biotin microbeads. CD4- CD8- negative LN cells were also collected via negative selection. 1 x 106 cells were cultured in triplicate in plates that were previously coated with 1µg/ml anti-CD3 antibody. Cell free supernatant was harvested after 24 and 48 hours for ELISA. C) Mice were injected IP with 100µg anti-GM-CSF blocking antibody or isotype control antibody. 4 hours later, the mice were injected in the footpad with 10µg/ml anti-CD3. 18 hours later the frequency of CD209+, CD11b+, CD11c+, MHC II+, CD40+, Ly6c-, DAPI- Mo-DC was determined. Cells shown were first gated on Thy1.2-, CD19-, DX5- cells. N=2 mice per group. The data shown are representative of 2 independent experiments. D) Bone marrow monocytes from wild-type or GM-CSFR KO mice were cultured with 50µg/ml GM-CSF and 20µg/ml IL-4 for 5 days. The cells were then analyzed by flow cytometry. Cells shown were gated on DAPI-, CD1lb+ cells. The data shown are representative of 2 independent experiments, 1 mouse per group. E) GM-CSFR heterozygous and GM-CSFR KO mice were injected in the footpad with 10µg anti-CD3. 18 hours later the frequency of CD209+, CD11b+, CD11c+, Mo-DC in the draining LNs was determined and the results are shown as the percentage of live LN cells. N=2 mice per group. F) Wild-type and CD40L KO mice were injected in the footpad with 10µg anti-CD3. 18 hours later the absolute number of CD209+, CD11b+, CD11c+ Mo-DC in the draining LNs was determined with flow cytometry. N=4 mice per group.
Figure 4.
Upregulated PDL-2 expression inhibits CD209+ Mo-DC from polarizing naïve T cells.
A) Mice were injected in the footpad with 10µg anti-CD3 or PBS. 18 hours later, the draining LNs were evaluated for the expression of alternative costimulatory molecules on CD11c+, CD11b+, Ly6c- DC subsets. Results shown are representative of 2 independent experiments. Red (Mo-DC [CD209+] from anti-CD3 treated mice), black (cDC [CD209-] from anti-CD3 treated mice) and gray (total DC from PBS treated control mice). B) The DC subsets shown in boxes in A) were evaluated for PDL-2 expression (MFI = median fluorescence intensity). C) CD209+ and CD209- DC were sorted and cultured at a 1:10 ratio with naïve CD4+ T cells from OT-II RAG KO mice in the presence of high (2.50µg/ml) or low (0.25µg/ml) ISQ peptide and 10µg/ml anti-PDL-2 antibody or an isotype control antibody. After 72 hours, the cells were pulsed with H3-thymidine and harvested 20 hours later. The results of triplicate cultures are shown as mean and SEM, done in triplicate. D) Aliquots of the supernatants from the cultures in C) were removed after 72 hours of culture and tested for IFNγ by ELISA. The results of triplicate cultures are shown as mean and SEM. Iso refers to isotype control antibody.