Fig 1.
Expression of CD5 on dendritic cells.
DC from various lymphoid and non-lymphoid organs are analyzed. In histograms, CD5 staining (solid line) is compared to fluorescence minus one (FMO) control (dashed line). (A) Gated SP DC (Lin-/CD11c+/Ia-IE+) express CD5. Both CD8α+ and CD8α- subsets express a similar level of CD5. (B) Resident and migratory DC from LN are gated and analyzed. CD8α+ and CD8α- resident DC express a higher level of CD5 than migratory DC which include four subpopulations (CD11b+, CD11b-, CD103+ and Langerhans CD207+/CD103-). (C) Gated thymus DC (Lin-/CD11c+/Ia-IE+) express CD5. (D) Dermal CD11c+ DC express CD5. Only few CD11c+ cells from the epidermal cells can be detected. (E) DC from the lung (Lin-/Gr1-/F4/80-/CD11c+/Ia/IE+) express CD5. The data are representative of 2–3 independent experiments.
Fig 2.
Effects of CD5 deficiency on DC.
(A) DC from wild-type (WT) (upper panels) and CD5-/- (bottom panels) mice are gated as described in the Fig 1 and resident and migratory DC populations are analyzed. The numbers in the graphs show the percentage of the indicated DC subsets. (B) CD5 deficiency alters cytokine production by BMDC. Levels of cytokine mRNA from WT and CD5-/- BMDC are quantified by real time RT-PCR (n = 4, * P<0.05). (C) CD5 deficiency increases IL-12 production by DC. SP DC from WT and CD5-/- were purified by CD11c coupled MACS beads and stimulated with LPS for 24 hours. Cytokine concentrations in culture supernatants were measured by ELISA (n = 3, **P<0.01). The data are representative of 2–3 independent experiments (two-tailed Student’s t-test).
Fig 3.
CD5 deficiency enhances the capacity of DC to activate CD4+ and CD8+ T cells.
OT-II (A) or OT-I (B) cells were cultured with OVA peptides pulsed BMDC. The cultures without OVA peptides served as controls (T Ctrl) (n = 5). (C) CD4+ T cells from 2D2 mice were stimulated with CD11c+ SP DC from WT or CD5-/- mice in the presence of MOG35-55 peptide (n = 5). (D) Naïve WT mice were injected subcutaneously with OVA peptides pulsed BMDC into rear footpads followed by intravenous injection of CFSE labeled OT-II or OT-I cells. Control mice received CFSE labeled T cells but no DC (Ctrl). The draining popliteal LN were analyzed (n = 6). The data are representative of 3–5 independent experiments (two-tailed Student’s t-test, *P<0.05, **P<0.01).
Fig 4.
CD5 deficiency enhances the ability of DC to stimulate the production of Th1 cytokines by T cells.
(A) BMDC were cultured with OT-II cells for 4 days in the presence of the OVA peptides. Control T cells (Ctrl) were cultured with BMDC which were not pulsed with OVA. Supernatants from T cell/DC cultures were harvested and cytokine concentrations were measured by ELISA (n = 4, *P<0.05). UD: undetected. (B) Purified CD11c+ SP DC or BMDC were pulsed with OVA peptides and cultured with OT-II cells for 4 days. The percent of cytokine producing cells were detected by intracellular cytokine staining and analyzed by flow cytometry. The data are representative of 2–3 independent experiments (two-tailed Student’s t-test).
Fig 5.
CD5-/- DC are more potent than WT DC to induce anti-tumor immunity in animals.
WT mice were immunized subcutaneously with OVA pulsed BMDC generated from WT or CD5-/- mice. Control mice were not immunized. The mice were then inoculated subcutaneously with mouse EG.7 tumor cells. Tumor size was measured every 3 days (n = 5, *P<0.05). The data are presented as mean ± SEM and are representative of 2 independent experiments (two-tailed Student’s t-test).
Fig 6.
CD5-/- DC enhance contact hypersensitivity responses (CHS).
SP DC or BMDC from WT (WT-DC) or CD5-/- (CD5-/-DC) mice were pulsed with DNBS and injected subcutaneously in naïve mice. (A). Sensitized mice were challenged with DNFB and ear swelling was measured. Naïve mice which were not injected with DC but challenged with DNFB served as controls (n = 5, * P<0.05). (B) T cells from the draining LN of the immunized mice were stimulated with DNBS-pulsed WT BMDC. Cytokine concentrations were measured by ELISA. T cells from naïve mice served as controls (n = 4, * P<0.05). The data presented are mean ± SEM and are representative of 2–3 independent experiments (two-tailed Student’s t-test).
Fig 7.
Re-expression of CD5 restores regulatory activity to CD5-/- DC.
(A). Restoration of CD5 in CD5-/- BMDC transfected with Ad-GFP (CD5-/- /AdGFP) or Ad-CD5 (CD5-/- /AdCD5). Ctrl: Isotype-matched IgG control. (B). The restoration of CD5 diminishes the activity of CD5-/- DC to stimulate proliferation of CD4+ and CD8+ T cells. T cell control (T Ctrl) was cultured without DC (n = 4). (C) The re-expression of CD5 expression inhibits cytokine production by CD5-/- DC (n = 4). Note that IL-12 was below the detectable level (UD) in the CD5-/-/AdCD5 group. (D) The re-expression of CD5 expression attenuates the ability of CD5-/- DC to induce CHS responses (n = 6). The data are presented as mean ± SEM and are representative of 2–3 independent experiments (two-tailed Student’s t-test, *P<0.05, ** P<0.01).