A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein

Abstract

Dendritic cells (DCs) are known to activate naive T cells to become effective helper cells. In addition, recent evidence suggests that DCs may influence naive B cells during the initial priming of antibody responses. In this study, using three-color confocal microscopy and three-dimensional immunohistograms, we have observed that in the first few days after a primary immunization, cells with dendritic morphology progressively localize within primary B cell follicles. These cells were identified by their ability to bind a fusion protein consisting of the terminal cysteine-rich portion of the mouse mannose receptor and the Fc portion of human immunoglobulin (Ig)G1 (CR-Fc). In situ, these CR-Fc binding cells express major histocompatibility complex class II, sialoadhesin, and CD11c and are negative for other markers identifying the myeloid DC lineage, such as (CD11b), macrophages (F4/80), follicular DCs (FDC-M2), B cells (B220), and T cells (CD4). Using CR-Fc binding capacity and flow cytometry, the cells were purified from the draining lymph nodes of mice 24 h after immunization. When injected into naive mice, these cells were able to prime T cells as well as induce production of antigen-specific IgM and IgG1. Furthermore, they produced significantly more of the lymphocyte chemoattractant, macrophage inflammatory protein (MIP)-1alpha, than isolated interdigitating cells. Taken together, these results provide evidence that a subset of DCs enters primary follicles, armed with the capacity to attract and provide antigenic stimulation for T and B lymphocytes.

Figure 1

Localization of CR-Fc binding cells in lymph nodes with time after a primary immunization. Three-color confocal analysis was used in order to determine when and where CR-Fc binding cells appeared in the tissue. Cryostat sections prepared from murine lymph nodes obtained at days 0–4 (A–E) of a primary response were processed for IgM expression (red), N418 labeling (green), and CR-Fc binding (blue). The images were then digitally transformed using a computer program in order to map the MFI (in arbitrary units) for either single (CR-Fc⁺; N418⁺) or double (CR-Fc⁺/ N418⁺) positive cells. The resulting 3D immunohistograms revealed that CR-Fc binding cells accumulated with time in the outer part of the B cell follicles. Many of these cells were also N418⁺ (see the CR-Fc⁺/N418⁺ column). The scale 0–250 represents the MFI, with the colors yellow, blue, green, and red representing values in the 100–150, 150–200, 200–250, and >250 range, respectively.

Figure 3

Profile of CR-Fc binding cells by flow cytometry. 24 h after a primary immunization, low buoyant density cells were labeled for CR-Fc binding and N418 expression. Setting a gate (R1) using the scatter parameters demonstrated that all N418⁺ cells (gray) produced an elevated side scatter pattern. The N418 single positive cells represented 50% of the total low density population while 10% were N418/CR-Fc double positive.

Figure 2

B cells, T cells, and CR-Fc binding cells colocalize in the outer B cell follicles. Cryostat sections prepared from murine lymph nodes obtained at day 3 (A, C, and E) and day 4 (B, D, and F) of a primary response were processed for IgM expression (red), CD4 labeling (green), and CR-Fc binding (blue). Especially at the higher magnification (E and F) of the areas outlined in C and D, CR-Fc binding cells (blue) and T cells (green) can be observed within the B cell follicles (red).

Figure 5

OVA-specific antibody titers in B cell supernatants from the lymph nodes of mice receiving CR-Fc binding versus nonbinding or no cells. CR-Fc binding and nonbinding cells were isolated from OVA-immune mice, then injected subcutaneously into naive mice. 7 d later, the B cells were isolated from the lymph nodes on the side receiving CR-Fc binding (black bars) or nonbinding (stippled bars) cells. In addition, B cells from lymph nodes draining sites where no cells were injected were also evaluated (white bars). The B cells were cultured for 7 d, and then the supernatants were subjected to ELISA to determine the level of isotype-specific anti-OVA titers. The data are presented as the maximum dilution at which a titer was still observed. These data are representative of five experiments.

Figure 4

T cell priming by CR-Fc binding cells. (A) CR-Fc binding cells were isolated from OVA-immune mice, then injected subcutaneously into naive mice. 7 d later, the T cells were isolated from the lymph nodes on the side receiving the injection and compared with T cells isolated from nondraining lymph nodes for in vitro restimulation. In the presence of splenic antigen-presenting cells, both T cell populations were incubated without (−) or with (+) OVA. At 24 h, [3H]thymidine was added, and 24 h later, incorporation was assessed. (B) CR-Fc binding cells were isolated from immune mice and then incubated at varying concentrations with 10⁵ OVA-specific transgenic T cells. At 24 h, [3H]thymidine was added, and 24 h later, incorporation was assessed. Both sets of data are representative of three experiments.

Figure 4

T cell priming by CR-Fc binding cells. (A) CR-Fc binding cells were isolated from OVA-immune mice, then injected subcutaneously into naive mice. 7 d later, the T cells were isolated from the lymph nodes on the side receiving the injection and compared with T cells isolated from nondraining lymph nodes for in vitro restimulation. In the presence of splenic antigen-presenting cells, both T cell populations were incubated without (−) or with (+) OVA. At 24 h, [3H]thymidine was added, and 24 h later, incorporation was assessed. (B) CR-Fc binding cells were isolated from immune mice and then incubated at varying concentrations with 10⁵ OVA-specific transgenic T cells. At 24 h, [3H]thymidine was added, and 24 h later, incorporation was assessed. Both sets of data are representative of three experiments.

Figure 7

Cell surface molecules expressed by isolated CR-Fc binding cells. CR-Fc binding cells were isolated 24 h after OVA injection. The cells were then double labeled for CR-Fc plus one of the markers indicated. The fine line represents the level of fluorescence of CR-Fc binding cells with a control antibody, and the bold line represents the level of expression on CR-Fc binding cells of the specific marker.

Figure 6

OVA-specific serum titers of mice receiving CR-Fc binding cells. CR-Fc binding cells were isolated from OVA-immune mice, then adoptively transferred subcutaneously into naive mice (10⁵ per mouse). 14 d later, the same mice were injected subcutaneously a second time with a freshly isolated preparation of CR-Fc binding cells from OVA-immune mice (5 × 10⁴ per mouse). Serum was obtained before the primary (D0) and secondary (D14) injections. Isotype-specific anti-OVA titers were assessed by ELISA. Each data point represents the maximum dilution at which a titer was still observed for an individual mouse.

Figure 8

Chemokine production by the different subsets of DC. Day 6 BM-DCs, CR-Fc binding cells (CR-Fc⁺/N418⁺; isolated 24 h after OVA injection), and interdigitating cells (CR-Fc⁻/N418⁺; isolated 24 h after OVA injection) were isolated and cultured for 24 h in medium. Supernatants were then harvested, and the levels of MIP-1α and RANTES were determined by ELISA.