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Accumulating evidence indicates that the cellular microenvironment plays a key role in follicular lymphoma (FL) pathogenesis, both within tumor lymph nodes (LNs) and in infiltrated bone marrow where ectopic LN-like reticular cells are integrated within malignant B-cell nodular aggregates. In normal secondary lymphoid organs, specific stromal cell subsets provide a highly specialized microenvironment that supports immune response. In particular, fibroblastic reticular cells (FRCs) mediate immune cell migration, adhesion, and reciprocal interactions. The role of FRCs and their postulated progenitors, that is, bone marrow mesenchymal stem cells (MSCs), in FL remains unexplored. In this study, we investigated the relationships between FRCs and MSCs and their capacity to sustain malignant B-cell growth. Our findings strongly suggest that secondary lymphoid organs contain MSCs able to give rise to adipocytes, chondrocytes, osteoblasts, as well as fully functional B-cell supportive FRCs. In vitro, bone marrow–derived MSCs acquire a complete FRC phenotype in response to a combination of tumor necrosis factor-α and lymphotoxin-α1β2. Moreover, MSCs recruit primary FL cells that, in turn, trigger their differentiation into FRCs, making them able to support malignant B-cell survival. Altogether, these new insights into the cross talk between lymphoma cells and their microenvironment could offer original therapeutic strategies.

There is accumulating evidence that cellular microenvironment plays a key role in follicular lymphoma (FL) pathogenesis, both within tumor lymph nodes (LN) and in infiltrated bone marrow (BM) where ectopic LN-like reticular cells are integrated within malignant B-cell nodular aggregates. In normal secondary lymphoid organs, specific stromal cell subsets provide a highly specialized microenvironment that supports immune response. In particular, fibroblastic reticular cells (FRC) mediate immune cell migration, adhesion, and reciprocal interactions. The role of FRC and their postulated progenitors, i.e. bone marrow mesenchymal stem cells (MSC), in FL remains unexplored. In this study, we have investigated the relationships between FRC and MSC and their capacity to sustain malignant B-cell growth. Our findings strongly suggest that secondary lymphoid organs contain bona-fide MSC able to give rise at single-cell level to adipocytes, chondrocytes, and osteoblasts. These LN-derived MSC could also differentiate, in response to a combination of tumor necrosis factor-α (TNF) and lymphotoxin-α1β2 (LT), into fully functional FRC, able to construct a dense extracellular reticular meshwork positive for transglutaminase and fibronectin staining, to produce inflammatory (CXCL9, CXCL10, CCL5, CCL2) and LN-specific (CCL19) chemokines, and to favour lymphoma B-cell growth. Bone marrow-derived MSC (BM-MSC) acquire in vitro a complete FRC phenotype in the same culture conditions. As an exemple, BM-MSC had a strong, although not complete, protective effect on serum deprivation-induced apoptosis of BL2 cell line (mean percentage of CD20posCaspase-3pos cells: 24.8 +/− 17.5% in coculture with BM-MSC versus 80.7 +/- 10.4% in medium alone; P < .05; n =5) and pretreatment with TNF/LT fully restored BL2 viability (mean percentage of CD20posCaspase-3pos cells: 7.4 +/− 4.7%; P < .05; n = 5). Moreover, stimulation of stromal cells by TNF/LT before coculture enhanced the number of viable CD19pos primary FL B cells by 2.4-fold for BM-MSC and 2.3 fold for LN-MSC compared with the culture without stromal cells (P < .05; n = 6). Interestingly, cell contact with lymphoma B-cell lines or purified FL B cells trigger the differentiation of BM-MSC into FRC that, in turn, support malignant B-cell migration, adhesion and survival. Altogether, these new insights into the interactions between lymphoma cells and their microenvironment could offer original therapeutic strategies.

Several subsets of stromal cells are found among secondary lymphoid organs where they play a key role in the initiation and maintenance of immune response. In particular, fibroblastic reticular cells (FRC) of the paracortex secrete extracellular matrix (ECM) components that constitute a dense network of conduits allowing antigens carried within the subcapsular afferent lymph to reach the lumen of the medullary high endothelial venules. FRC produce also several chemokines that recruit T, B, and dendritic cells from blood and favour their reciprocal interactions. In addition, follicular dendritic cells (FDC) are located exclusively into germinal centers and allow normal B-cell selection through a complex set of survival signals, including BCR-mediated signal, chemokines and adhesion molecules. FRC and FDC networks are phenotypically and probably functionally altered during development of follicular lymphomas and diffuse large B cell lymphomas, the two most frequent Non-Hodgkin Lymphomas. FRC and FDC are supposed to be of mesenchymal origin even if no conclusive work has been conducted to date in human. We have obtained 15 tonsil-derived stromal cell lines, that displayed all the morphologic, phenotypic, and functional characteristics of FRC, including synthesis of inflammatory (CXCL10, CXCL9, CCL5) and lymph-node specific (CCL19, CCL21) chemokines, and secretion of ECM organized in a reticular meshwork after long-term culture in the presence of TNF-α and lymphotoxin-α1β2 (LT). These cells induced tonsil leukocyte migration and adhesion in vitro. Tonsil-derived stromal cells expressed LTβR, TNFR, and CD40 but were negative for FDC specific markers, such as CD21 or CXCL13, even following in vitro stimulation by TNF-α, LT, and trimeric CD40L. Interestingly, such TNF and LT-dependent FRC differentiation could also be induced in adult bone marrow-derived mesenchymal stem cells (MSC). In addition, MSC-like cells able to differentiate along osteogenic, adipogenic, and chondrogenic lineages at the clonal level were found in normal tonsils. These data shed new lights on our current understanding of lymph node stromal cell origin and strongly suggest that MSC are the precursors of FRC in secondary lymphoid organs, and perhaps in bone marrow in case of FL involvement where ectopic lymph node-like stromal cells are detected in close association with tumor cells. In conclusion, MSC and their progeny trigger differential immune effects, depending on cytokine context, localization and cell contact with immune cells. These properties are probably modified during lymphomas where the contact between malignant B cells and stromal cells is crucial for tumor development.