Plasma cell purification from murine bone marrow using a two-step isolation approach.

Interest in plasma cells has increased greatly in the past decade. While several studies have examined the longevity and transcriptional control of antibody secreting cells in vivo, few studies have examined freshly isolated plasma cells ex vivo. Studies of primary plasma cells have been limited primarily due to the difficulty of isolating the large numbers of plasma cells necessary for experiments. In this protocol, plasma cells are purified from murine bone marrow in approximately 5 hours, following a two-step isolation method utilizing column enrichment and FACS-sorting based on CD138 (Syndecan-1) surface expression.

The effects of microenvironment and internal programming on plasma cell survival.

Two populations of plasma cells (PCs) are formed after immunization. A short-lived population in the spleen and lymph nodes provides rapid protection. A long-lived population, mainly in the bone marrow, provides lasting immunity. The mechanisms responsible for the differences in PCs life span remain largely unknown. The goal of the current study was to compare the intrinsic survival capacity of isolated short-lived (spleen) versus long-lived (bone marrow) PCs. We approached this question by using a previously established in vitro model that measures PC survival in a supportive stromal environment. Regardless of the tissue source or isolation time point after immunization, the two PC populations showed similar intrinsic ability to survive in vitro. To test differences in the stromal microenvironments, stromal cells from marrow, spleen or lymph nodes were evaluated for ability to support PCs survival. Survival of isolated PC was always greater when co-cultured with marrow stromal cells compared with those from spleen (or lymph node) despite the finding that IL-6, necessary for PC survival in culture, was secreted by all three stromal cell sources. Additionally, low expression of B-cell-activating factor belonging to the tumor necrosis factor-family was detected in all three stromal isolates. In contrast, marrow stromal cells were distinguished by cell-surface phenotype and CXC chemokine ligand (CXCL)12, IL-7 and stem cell factor expression. Although CXCL12 has been suggested as a possible survival factor for PC, addition or neutralization of CXCL12 had minimal effect on PC survival. We conclude the mechanisms regulating PC longevity appear extrinsically driven and marrow favored, but the factors that give marrow stromal cells a unique advantage remain unknown.

The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity.

Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors.