Establishment of Lymphotoxin β Receptor Signaling-Dependent Cell Lines with Follicular Dendritic Cell Phenotypes from Mouse Lymph Nodes

Abstract

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin β receptor mAb and TNF-α. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, FcγRIIB, lymphotoxin β receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.

Splenic CD19−CD35+B220+ cells function as an inducer of follicular dendritic cell network formation

“…The strong association of classic FDC/FDC-like cells with stromal cells has not been described elsewhere. 27,[38][39][40] Moreover, they possess stromallike fibroblastic or dendritic appearance, in contrast to CD35 ϩ B220 ϩ cells ( Figure 1C; Figure S2). However, as shown in Figure 3 and Figure S3, CD35 ϩ B220 ϩ cells and stromal cells efficiently interact with each other, resulting in the appearance of double-nuclear cells of stromal origin (ie, GFP ϩ ) that acquire the surface phenotype of CD35 ϩ B220 ϩ cells.…”

IL-21–Dependent B Cell Death Driven by Prostaglandin E2, a Product Secreted from Follicular Dendritic Cells

“…An FDC line, pFL, was established from the primary culture of immunized BALB/c lymph node (LN) cells in the presence of lymphotoxin (LT)-expressing feeder cells as described previously (20). FL-Y, a phenotypically stable and more rapidly growing FDC line, was isolated from the long-term culture of pFL cells (20).…”

In silico subtraction approach reveals a close lineage relationship between follicular dendritic cells and BP3hi stromal cells isolated from SCID mice

“…To overcome these problems, FDC lines have been established, however, as these cells are maintained over several weeks in culture, their phenotype no longer reflects the in vivo situation [8][9][10][11][12][13]. A number of different approaches for the enrichment and gene expression analysis of FDC have been shown to be more representative of the in vivo situation [6,8,11].…”

“…To overcome these problems, FDC lines have been established, however, as these cells are maintained over several weeks in culture, their phenotype no longer reflects the in vivo situation [8][9][10][11][12][13]. A number of different approaches for the enrichment and gene expression analysis of FDC have been shown to be more representative of the in vivo situation [6,8,11].From a number of experiments, it is apparent that FDC are a highly specialized subset of reticular cells [14][15][16][17][18]. Using BP3, an ectoenzyme of the NAD glycohydrolase family, as a marker antigen for the splenic reticular network of stromal cells, one can distinguish the reticulum cells in the B-cell area from those in the T-cell zone.…”