Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs

Abstract

Background: There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naive cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments.

Results: We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known.

Conclusions: Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors.

Figure 1

Three different B-cell compartments isolated by LCM. Frozen sections of reactive tonsils or spleens were fixed, and a consecutive section was immunostained for CD3 to guide the dissection. The three B-cell compartments (GC, MNZ and MGZ) were isolated using LCM with the Arcturus PixCell II system (Arcturus Engineering, Mountain View, CA). Cells were captured at the 15-μm with laser set to pulse at 60 mW for 200 ms. The GC and MNZ were clearly recognizable, and the MGZ was obtained from a spleen with a morphologically clearly defined MGZ. Only well-defined GC, MNZ and MGZ were dissected to avoid contamination.

Figure 2

FACS-sorting of naïve and memory B-cells from splenocytes. A single B-cell suspension prepared from a fresh spleen was isolated using the Human B-cell Isolation Kit (See methods) and subjected to 3-color cell sorting. Memory B-cells from the splenic B-cells were gated on the IgM^highIgD^lowCD27⁺ fraction, while naïve B-cells were gated at IgM^lowIgD^highCD27^-. FACS-sorted populations were over 90 % pure, judging from post sort immunophenotyping.

Figure 3

Correlation Coefficient Mapping. Reproducibility of the different hybridization experiments was checked through correlation coefficient mapping programmed in MATLAB. High correlation is seen among samples from same compartment or FACS-sorted population.

Figure 4

Confirmation of the Microarray analysis by semi quantitative RT-PCR. aRNA amplified from GC, MNZ and MGZ -cells was reversely transcribed and amplified by PCR. The human HPRT gene was used as the comparative standard. Five fold serially dilutions (4 dilutions) of each cDNA amplified with gene specific primers and analyzed by electrophoresis in 2% agarose gel. The transcripts had either an almost exclusive expression in one compartment (ARK2 in GC, CCL20 in MNZ and CMRF-35H in MGZ), or they were expressed in all compartments with a significant differential expression in one – for example, SET and FAIM in GC, Cyclin G2 in MNZ and NM23-H1, CARD11 and GAS2 in MGZ.

Figure 5

Cell proliferation and quiescence. Differential gene expression among B-cell compartments. Genes identified in SAM analyses were merged according to functional or operational categories and visualized in Tree View. Color changes within a row indicate expression levels relative to the median of the sample population. Only transcripts differing 2-fold or more in their magnitude than the median or mean of the other two compartments are shown.

Figure 6

DNA repair, replication and protein synthesis.

Figure 7

(A) Apoptosis and cell survival and (B) Cytokines and chemokines and their receptors.

Figure 8

Stromal signature.

Figure 9

Other unique compartment markers.