Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

Abstract

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4+ and CD8+ populations and assessed for the ability to process and present particulate antigen to CD4+ and CD8+ T cells. CD4+ and CD8+ DCs both processed exogenous particulate antigen, but CD8+ DCs were much more efficient than CD4+ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8+ DCs, our studies also revealed an important contribution of CD24. CD8+ DCs were also more efficient than CD4+ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8+ T cells and interferon (IFN)-gamma-producing CD4+ T cells. In summary, CD8+ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.

Figure 1

Phenotypically distinct splenic dendritic cell (DC) populations. Freshly isolated CD4⁺ or CD8⁺ DCs were stained for expression of CD11c, CD11b, DEC-205 and 33D1 to distinguish between the two populations. CD4⁺ DCs expressed higher levels of CD11b and lower levels of DEC-205 when compared with CD8⁺ DCs. 33D1 was effective in differentiating CD4⁺ DCs from CD8⁺ DCs.

Figure 2

CD8⁺ dendritic cells (DCs) are more efficient than CD4⁺ DCs for processing and presentation of latex-ovalbumin (L-OVA) to T hybridoma cells. CD4⁺ or CD8⁺ DCs (2 × 10⁴) were cultured with L-OVA for 1 hr prior to addition of 10⁵ CD8OVA1·3 [a, major histocompatibility complex class I (MHC-I)-restricted] or DOBW (b, MHC-II-restricted) T hybridoma cells. Cultures were then incubated at 37° for 24 hr, and supernatants were collected for a CTLL assay to assess interleukin (IL)-2 production. (a) CD8OVA1·3 responses. (b) DOBW responses. Data points are mean ± standard deviation (*, P < 0·01 by Student's t-test). When not visible, error bars are smaller than the symbol. Results are representative of five independent experiments.

Figure 3

CD8⁺ dendritic cells (DCs) are more efficient than CD4⁺ DCs for induction of naïve CD4⁺ and CD8⁺ T cell proliferative responses. CD4⁺ or CD8⁺ DCs (4 × 10⁴) were cultured with latex-ovalbumin (L-OVA) for 1 hr prior to addition of 10⁵ T cells. Cultures were then incubated at 37° for 96 hr with 1 µCi [³H]thymidine added for the last 8 hr. Cells were harvested and [³H]thymidine incorporation was assessed. (a) Major histocompatibility complex class I (MHC-I)-restricted OT-I T-cell responses. (b) MHC-II-restricted OT-II T-cell responses. Data points represent mean ± standard deviation (*, P < 0·01 by Student's t-test). When not visible, error bars are smaller than the symbol. Results are representative of six independent experiments. CPM, counts per minute.

Figure 4

CD24 expression distinguishes CD4⁺ and CD8⁺ dendritic cells (DCs). Splenic CD4⁺ and CD8⁺ DCs were stained for expression of costimulatory molecules CD24, CD80 and CD86 and assessed by flow cytometry. Bold line, CD8⁺ DCs; thin line, CD4⁺ DCs. Shaded histogram, staining of CD8⁺ DCs with isotype-matched negative control antibody (CD4⁺ DCs gave similar staining with control antibody). Results are representative of five independent experiments.

Figure 5

CD24 functions as a costimulatory molecule for CD4⁺ and CD8⁺ T cells. CD8⁺ dendtritic cells (DCs) (4 × 10⁴) were cultured for 1 hr with latex-ovalbumin (L-OVA) (1 µg/ml) with or without blocking antibodies (5 µg/ml) to CD24 (clone M1/69), CD80 (clone 16·10A1), and/or CD86 (clone GL1) for 1 hr. The isotype control is represented by rat immunoglobulin G2a (IgG2a) (clone R35-95). There was no change in T-cell proliferation when other isotype controls were used (rat IgG2b or Armenian hamster IgG). T cells (10⁵) were then added and cultures were incubated for 96 hr, with 1·0 µCi [³H]thymidine added for the last 8 hr. Cells were then harvested and [³H]thymidine incorporation was assessed. (a) Major histocompatibility complex class I (MHC-I)-restricted OT-I responses. (b) MHC-II-restricted OT-II responses. Data points represent mean ± standard deviation (*, P < 0·01 by Student's t-test). The results are representative of three independent experiments. CPM, counts per minute.

Figure 6

CD8⁺ dendritic cells (DCs) are more efficient at generating CD8⁺ cytotoxic T lymphocytes (CTLs). CD4⁺ or CD8⁺ DCs (2 × 10⁵) were cultured with 2 × 10⁶ OT-I CD8⁺ T cells in 24-well plates for 5 days. EL4 cells were incubated with [³H]thymidine and ovalbumin (OVA_323-339), and labelled EL4 target cells (10⁴ cells/well) were plated in a 96-well plate with the indicated ratio of cultured OT-I CD8⁺ T cells for 5 hr at 37°. The percentage cytotoxicity was determined as described in ‘Materials and methods’. Data points represent mean ± standard deviation (*, P < 0·01 by Student's t-test). When not visible, error bars are smaller than the symbol Results are representative of six independent experiments.

Figure 7

CD4⁺ and CD8⁺ dendritic cells (DCs) both generate T helper type 1 (Th1)-like responses by CD4⁺ T cells. CD4⁺ or CD8⁺ DCs (4 × 10⁴) were cultured for 1 hr with latex-ovalbumin (L-OVA) (1 µg/ml). OT-II cells were then added for 96 hr. Cells were then washed and added to 96-well plates coated with anti-CD3 in the presence of 1 µg/ml brefeldin A for 5–6 hr. Cells were washed, stained with anti-CD3, fixed with formaldehyde, labelled with anticytokine antibodies in the presence of saponin, and assessed by flow cytometry. Results are representative of four independent experiments. IFN, interferon; IL, interleukin.

Figure 8

CD4⁺ and CD8⁺ dendritic cells (DCs) support production of interferon (IFN)-γ and interleukin (IL)-2 by ovalbumin (OVA)-specific CD4⁺ T cells. Latex-ovalbumin (L-OVA)-pulsed CD4⁺ or CD8⁺ T cells were cultured with OT-II T cells for 96 hr. (a) Cell supernatant was collected and production of cytokines was determined using a cytokine antibody array. (b) The relative levels of each cytokine were compared to those for T cells cultured in media alone, as described in ‘Material and methods’. CD8⁺ DCs promoted increased production of IL-2 and IFN-γ when compared with T cells cultured with CD4⁺ DCs (*, P < 0·01 by Student's t-test). Results are representative of three independent experiments.