The architecture of neoplastic follicles in follicular lymphoma; analysis of the relationship between the tumor and follicular helper T cells.

CD4+ T-follicular helper cells are essential for the survival, proliferation, and differentiation of germinal center B cells and have been implicated in the pathogenesis of follicular lymphoma (FL). To further define the role of these cells in FL, we used multiparameter confocal microscopy to compare the architecture of normal and neoplastic follicles and next generation sequencing to analyze the T-cell receptor repertoire in FL lymph nodes (LN). Multiparameter analysis of LN showed that the proportion of T-follic-ular helper cells (TFH) in normal and neoplastic follicles is the same and that the previously reported increase in TFH numbers in FL is thus due to an increase in the number and not content of follicles. As in normal germinal centers, TFH were shown to have a close spatial correlation with proliferating B cells in neoplastic follicles, where features of immunological synapse formation were observed. The number of TFH in FL correlate with the rate of B-cell proliferation and TFH co-localized to activation induced cytidine deaminase expressing proliferating B cells. T-cell receptor repertoire analysis of FL LN revealed that follicular areas are significantly more clonal when compared to the rest of the LN. These novel findings show that neoplastic follicles and germinal centers share important structural features and provide further evidence that TFH may play a role in driving B-cell proliferation and genomic evolution in TFH Our results also suggest that targeting this interaction would be an attractive therapeutic option.

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration.

Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4(dim)CD5(bright) phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of α4ß1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4(dim)CD5(bright) with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d(hi) CLL cells showed an enhanced capacity to activate T cells compared with CD49d(lo) subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4(+) T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells.

CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis.

Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase- and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved.