Phenotypic and Morphological Properties of Germinal Center Dark Zone Cxcl12-Expressing Reticular Cells

Abstract

The germinal center (GC) is divided into a dark zone (DZ) and a light zone (LZ). GC B cells must cycle between these zones to achieve efficient antibody affinity maturation. Follicular dendritic cells (FDCs) are well characterized for their role in supporting B cell antigen encounter in primary follicles and in the GC LZ. However, the properties of stromal cells supporting B cells in the DZ are relatively unexplored. Recent work identified a novel stromal population of Cxcl12-expressing reticular cells (CRCs) in murine GC DZs. Here we report that CRCs have diverse morphologies, appearing in ‘open’ and ‘closed’ networks, with variable distribution in lymphoid tissue GCs. CRCs are also present in splenic and peripheral lymph node primary follicles. Real-time two-photon microscopy of Peyer’s patch GCs demonstrates B cells moving in close association with CRC processes. CRCs are gp38+ with low to undetectable expression of FDC markers, but CRC-like cells in the DZ are lineage marked, along with FDCs and FRCs, by CD21-Cre and Ccl19-Cre directed fluorescent reporters. In contrast to FDCs, CRCs do not demonstrate dependence on lymphotoxin or TNF for chemokine expression or network morphology. CRC distribution in the DZ does require CXCR4 signaling, which is necessary for GC B cells to access the DZ and likely to interact with CRC processes. Our findings establish CRCs as a major stromal cell type in the GC DZ and suggest CRCs support critical activities of GC B cells in the DZ niche through Cxcl12 expression and direct cell-cell interactions.

IL-4/CXCL12 loop is a key regulator of lymphoid stroma function in follicular lymphoma

“…45 Conversely, DZ CRCs do not require LT and TNF for maintenance of CXCL12 expression and network morphology. 31 In agreement, purified FL B cells exhibit a downregulation of LTA and LTB compared with normal GC B cells (supplemental Figure 10), thus providing an argument in favor of a CRC-like origin for FL stromal cells. The origin of CXCL12 hi FL stroma in invaded BM is even more puzzling.…”

“…42 CRCs lack the main FRC and FDC markers and were thus proposed as a distinct cell type, even if they share a lineage precursor with both FRCs and FDCs. 31 Apart from CRCs, mouse FRCs also express CXCL12, and CRC and FRC networks are tightly connected. Moreover, a subset of FRCs localized in follicular regions has recently been shown to contribute to B-cell homeostasis through the production of BAFF.…”

“…Inside tonsil GCs, CXCL12 staining was always scarce and strictly restricted to CD21L -cells, whereas the CD21L 1 mature FDC network was CXCL12-negative, as previously reported in mice. 31 Conversely, immunohistofluorescence analysis revealed that CXCL12 was overexpressed in stromal cells within follicles in 8 of 14 FL patients and CXCL12 messenger RNA (mRNA) was significantly upregulated in FL LNs compared with tonsil samples (Figure 1A-B; supplemental Figure 3). Interestingly, CXCL12 overexpression was associated with a variable loss of the CD21L FDC marker as revealed by RT-qPCR and immunohistofluorescence experiments (supplemental Figure 3B).…”

Stromal Fibroblasts in Tertiary Lymphoid Structures: A Novel Target in Chronic Inflammation

“…This subset is fate mapped by both the Cd21-cre and Ccl19-cre mouse lines with the fate mapping indicating past and/or present expression (171). It lacks most of the classical markers of FDCs [such as CD21/35, VCAM-1, FDC-M2, FDC-M1, M-2, and CD16/32 (FcγRII/III)] or FRCs (such as laminin and type IV collagen association).…”

Generation of Antibody Diversity

“…CXCL12 is a ligand of CXCR4 and expressed on the cell surface of reticular cells in the dark zone. The CXCL12/CXCR4 signaling of B cells in the dark zone is regarded as a homing signal to keep B cells in the dark zone, if CXCR4 expression is high [31,32]. CXCR4 deficiency in germinal B cells restricted the B cells to the light zone, but the deletion is not sufficient alone for functional transition of dark zone B cells (centroblasts) to light zone B cells (centrocytes) [31].…”