Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of human B cells from patients with chronic lymphocytic leukemia after co-culture with nurse-like cells

ABSTRACT: In the marrow and lymphatic tissues, chronic lymphocytic leukemia (CLL) cells interact with accessory cells that constitute the leukemia microenvironment. In lymphatic tissues, CLL cells are interspersed with CD68+ nurselike cells (NLC) and T cells. However, the mechanism regulating co-localization of CLL cells and these accessory cells are largely unknown. To dissect the molecular cross-talk between CLL and NLC, we profiled the gene expression of CD19-purified CLL cells before and after co-culture with NLC. NLC co-culture induced high-level expression of B cell maturation antigen (BCMA) and two chemoattractants (CCL3, CCL4) by CLL cells. Supernatants from CLL-NLC co-cultures revealed high CCL3/CCL4 protein levels. B cell receptor triggering also induced a robust induction of CCL3 and CCL4 expression by CLL cells, which was almost completely abrogated by a specific Syc inhibitor, R406. High CCL3 and CCL4 plasma levels in CLL patients suggest that activation of this pathway plays a role in vivo. These studies reveal a novel mechanism of cross-talk between CLL cells and their microenvironment, namely the secretion of two T cell chemokines by CLL-NLC interaction and in response to BCR stimulation. Through these chemokines, CLL cells can recruit accessory cells, and thereby actively create a microenvironment that favors their growth and survival. Experiment Overall Design: The microarray part of this study included samples of CLL cells from 9 different patients, analyzed directly after purification and after 14 days of co-culture with Nurse like cells . Experiment Overall Design: In detail, RNA was isolated from CD19-purified CLL cells from 9 different patientsâ peripheral blood mononuclear cells (PBMC) after Ficoll separation and subsequent purification with CD19 MicroBeads and the MACSÂ® technology according to the manufacturerâs instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). For comparison, the same CLL cell samples were co-cultured for 14 days with NLC (""14d NLC"").""). For co-culture with NLC, PBMC from patients with CLL were suspended in complete RPMI medium (RPMI1640 with 10% FCS, penicillin-streptomycin-glutamine, Gibco-BRL, Grand Island, NY) to a concentration of 1 x 107/ml (total 20 ml) and incubated for 14 days in 75 cm2 tissue culture flasks (Techno Plastic Products AG) as described previously3. Nonadherent lymphoid cells then were removed and the NLC layer was washed two times with phosphate-buffered saline (PBS). The complete removal of lymphocytes was verified by phase-contrast microscopy. The nonadherent cells together with the wash-fractions were then used for RNA preparation. In order to purify the CLL B cells prior to RNA isolation, CLL PBMC were passed through a 30 Âµm nylon mesh to obtain a single-cell suspension. Then CLL B cells were purified with CD19 MicroBeads. Subsequently RNA was extracted.

ORGANISM(S): Homo sapiens

SUBMITTER: Andreas Rosenwald

PROVIDER: E-GEOD-13811 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation.

Burger Jan A JA Quiroga Maite P MP Hartmann Elena E Bürkle Andrea A Wierda William G WG Keating Michael J MJ Rosenwald Andreas A

Blood 20081212 13

In lymphatic tissues, chronic lymphocytic leukemia (CLL) cells are interspersed with CD68(+) nurselike cells (NLCs), T cells, and other stromal cells that constitute the leukemia microenvironment. However, the mechanism regulating colocalization of CLL and these accessory cells are largely unknown. To dissect the molecular cross talk between CLL and NLCs, we profiled the gene expression of CD19-purified CLL cells before and after coculture with NLCs. NLC coculture induced high-level expression o ...[more]

PMID: 19074730

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Project description:Chronic lymphocytic leukemic B cells (CLL) reside in close contact with activated endothelial cells (EC) in infiltrated tissues. Here, we investigated the interactions between EC and CLL cells, highlighting molecular networks involved in this cellular crosstalk. We co-cultured purified CLL cells on HUVEC monolayer (HC) or in medium alone. We found that EC protected CLL from spontaneous apoptosis. A 2.2-fold increase in relative viability in IGHV mutated CLL and a 6.1-fold increase in IGHV unmutated CLL was detected in co-culture. Moreover, the endothelial cell layer decreased the in vitro sensitivity of CLL cells to Fludarabine-induced apoptosis. Physical contact with EC is essential for protection to apoptosis. The insertion of a microporous membrane or blocking adhesion with anti-CD106 and anti-CD18 antibodies determined the complete abrogation of apoptosis protection. On the other hand, a reduction of apoptosis was measured in CLL cells cultured with conditioned medium collected from HC, implying that survival is partially mediated by soluble factors. Overall 1944 genes were modulated in CLL by co-culture. The EC contact seem to determine on CLL a kind of microenvironmental-driven angiogenic switch, improve the secretion of cytokines regulating tissue elements such as stromal cells and macrophages and also increase anti-apoptotic molecules. Our study support the line of evidence indicating endothelial cells as a major player in the CLL-infiltrated microenvironments are able to create a vicious cycle of cooperation that strongly sustains leukemic cell survival, protects CLL from drug-induced apoptosis and widely modifies CLL phenotype. Large-scale gene expression profiling (GEP) was performed on total RNA extracted from purified CD19+ cells isolated from 9 individual CLL patients which were separated in 3 experimental subsets: (i) freshly isolated cells (CLL baseline), (ii) CLL cells cultured in medium alone for 48 hours (CLL only) and (iii) CLL cells co-cultured 48h on HUVEC layer (CLL HC).

Dataset Information

Transcription profiling by array of human B cells from patients with chronic lymphocytic leukemia after co-culture with nurse-like cells

Publications

High-level expression of the T-cell chemokines CCL3 and CCL4 by chronic lymphocytic leukemia B cells in nurselike cell cocultures and after BCR stimulation.

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