The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand

Abstract

A subset of CD4+CD11c-CD3- blood cells was recently shown to develop into dendritic cells when cultured with monocyte conditioned medium. Here, we demonstrate that CD4+ CD11c-CD3- cells, isolated from tonsils, correspond to the so-called plasmacytoid T cells, an obscure cell type that has long been observed by pathologists within secondary lymphoid tissues. They express CD45RA, but not markers specific for known lymphoid- or myeloid-derived cell types. They undergo rapid apoptosis in culture, unless rescued by IL-3. Further addition of CD40-ligand results in their differentiation into dendritic cells that express low levels of myeloid antigens CD13 and CD33.

Figure 1

Anatomical localization of CD4⁺CD11c⁻CD3⁻ plasmacytoid cells. To distinguish CD4⁺CD11c⁻CD3⁻ plasmacytoid cells from CD4⁺CD3⁺ T cells and CD4⁺CD11c⁺ germinal center dendritic cells, double staining with anti-CD3 (blue), anti-CD11c (blue), and anti-CD4 (red) on tonsillar sections was performed. Accordingly, whereas CD4⁺CD3⁺ T cells and CD4⁺CD11c⁺ germinal center dendritic cells should be double stained purple, CD4⁺CD11c⁻CD3⁻ plasmacytoid cells should be single stained red. (A) shows a germinal center and its adjacent T cell–rich extrafollicular areas containing HEV. Red CD4⁺CD11c⁻CD3⁻ plasmacytoid cells can only be found in T cell area but not in germinal center. (B) shows that the same germinal center displayed in A does not contain red CD4⁺CD11c⁻CD3⁻ plasmacytoid cells. (C) shows that the same T cell area displayed in A contains many red CD4⁺CD11c⁻CD3⁻ plasmacytoid cells, located within or around HEV. (D) shows a red CD4⁺ CD11c⁻CD3⁻ plasmacytoid cell within the lumen of HEV. (E) shows many red CD4⁺CD11c⁻CD3⁻ plasmacytoid cells, one in particular is within the endothelial wall of HEV. Original magnification: (A) ×100; (B, C) ×200; (D, E) ×400.

Figure 2

Cell sorting of CD4⁺CD11c⁻CD3⁻ plasmacytoid cells. After preparation of tonsillar cells that are negative for CD3, CD14, CD19, CD20, and CD56 (detailed in Materials and Methods), cells were stained with mouse anti–CD4-PE-Cy5 (Immunotech), anti–CD11c-PE (Becton Dickinson), and a cocktail of FITC-labeled mAbs anti-CD3 and CD34 (Immunotech), anti-CD20, anti-CD57, anti-CD7 and anti-CD16 (Becton Dickinson), and anti-CD1a (Ortho). As shown in the left panel, cells were first selected by their cocktail FITC⁻ (Lin⁻). This selected population contains CD4⁺CD11c⁺ germinal center dendritic cells (14) and CD4⁺ CD11c⁻ plasmacytoid cells, as shown in the right panel.

Figure 3

Morphology of freshly isolated CD4⁺CD11c⁻CD3⁻ plasmacytoid cells. (A) Giemsa staining; (B) SEM; (C) TEM; (D) TEM. (E) negative antiIg κ+λ light chain staining of CD4⁺CD11c⁻Lin⁻ plasmacytoid cells; (F) positive anti-Ig κ+λ light chain staining of CD38⁺⁺CD20⁻ Ig–secreting plasma cells. Original magnification: (A, F) ×1,000; (B) ×3,500; (C) ×2,000; (D) ×8,000.

Figure 4

Surface phenotype of isolated CD4⁺CD11c⁻CD3⁻ plasmacytoid cells analyzed by flow cytometry. Filled histograms represent isotype matched controls, open histograms represent staining with specific antibodies. Data shown are one representative of six experiments.

Figure 5

CD4⁺CD11c⁻CD3⁻ plasmacytoid cells undergo rapid apoptosis when cultured in medium, unless rescued by IL-3. (A) Many cell clusters at 4 h of culture with IL-3; (B) Dispersed cells at 4 h of culture in medium alone; (C) Few apoptotic cells within clusters at 4 h of culture with IL-3; (D) Many apoptotic cells at 4 h of culture in medium alone; (E) Cells display pseudopods at 16 h of culture with IL-3; (F). All cells are dead at 16 h of culture in medium; (G) Mitotic figures at 3 d of culture with IL-3; (H) cells with pseudopods at 6 d of culture with IL-3. Data shown are one representative of six experiments. Original magnification: (B) ×200.

Figure 6

IL-3 maintains the viability of CD4⁺CD11c⁻CD3⁻ plasmacytoid cells for up to 6 d. (A) Total numbers of viable cells at day 1, day 3, and day 6 of culture with medium (open circles), IL-3 (open squares), CD40ligand transfected L cells (open triangles), CD40-ligand negative L cells = ORIENT (solid squares) and CD40-ligand transfected L cells plus IL-3 (solid triangles). (B) IL-3 induces [³H]thymidine incorporation by plasmacytoid cells at day 3 of culture. Data shown are one representative of four experiments.

Figure 7

CD4⁺CD11c⁻CD3⁻ plasmacytoid cells acquire the morphology of mature interdigitating dendritic cells after 6 d of culture with IL-3 and CD40-ligand. (A) Culture morphology; (B) Giemsa staining; (C) SEM; (D) TEM. Original magnification: (A) ×100; (B) ×400; (C) ×3,000; (D) ×6,000.

Figure 8

CD4⁺CD11c⁻CD3⁻ plasmacytoid cells express high levels of costimulatory molecules, but few myeloid antigens, after 6 d of culture with IL-3 or IL-3+CD40-ligand. Filled histograms represent isotype matched controls, open histograms represent staining with specific antibodies. Data shown are one representative of six experiments.

Figure 9

Dendritic cells derived from CD4⁺CD11c⁻CD3⁻ plasmacytoid cells after 6 d of culture with IL-3+CD40-ligand did not uptake FITC-dextran. Methods are as described by Sallusto et al. (11). Time 0 represents cells that were incubated with FITC–dextran for 30 min at 4°C. After three washes, the FITC–dextran uptake by plasmacytoid cells derived DC cultured with IL-3 (open squares) or with IL-3 plus CD40ligand (solid circles) and monocyte-derived DC (solid triangles) at 37°C were analyzed with a FACScan^® flow cytometer. MFI, mean fluorescence intensity. Data shown are one representative of three experiments.

Figure 10

DC derived from CD4⁺CD11c⁻CD3⁻ plasmacytoid cells induces strong proliferation of CD4⁺CD45RA⁺ naive T cells. Purified allogeneic blood CD4⁺CD45RA⁺ naive T cells were cocultured with different numbers of DC generated from: CD14⁺ monocyte–derived DCs (solid circle), CD34+ progenitor–derived DCs (solid square), plasmacytoid cells after 6 d of culture with IL-3 (open square) and plasmacytoid cells after 6 d of culture with IL-3 and CD40L (open circle). After 5 d of DC-T cell cocultures, cells were pulsed with 1 μCi [³H]thymidine for 8 h before harvesting and counting. Data shown are one representative of six experiments.