Transcriptional analysis of the B cell germinal center reaction

Ulf Klein

Yuhai Tu

Gustavo Stolovitzky

Jeffrey Keller

Riccardo Dalla-Favera

^{Institute for Cancer Genetics, and Departments of Pathology and Genetics and Development, Columbia University, New York, NY 10032;}^{IBM T. J. Watson Research Center, Yorktown Heights, New York, NY 10598;}^{Division of Pediatric Otolaryngology, Columbia–Presbyterian Medical Center, Babies and Children's Hospital, New York, NY 10032; and}^{First Genetic Trust, Inc., Lyndhurst, NJ 07071}

^{To whom correspondence should be addressed. E-mail:}ude.aibmuloc@01dr.

Communicated by Stanley J. Korsmeyer, Dana–Farber Cancer Institute, Boston, MA

Received 2002 Oct 3; Accepted 2002 Dec 30.

Abstract

The germinal center (GC) reaction is crucial for T cell-dependent immune responses and is targeted by B cell lymphomagenesis. Here we analyzed the transcriptional changes that occur in B cells during GC transit (naïve B cells → centroblasts → centrocytes → memory B cells) by gene expression profiling. Naïve B cells, characterized by the expression of cell cycle-inhibitory and antiapoptotic genes, become centroblasts by inducing an atypical proliferation program lacking c-Myc expression, switching to a proapoptotic program, and down-regulating cytokine, chemokine, and adhesion receptors. The transition from GC to memory cells is characterized by a return to a phenotype similar to that of naïve cells except for an apoptotic program primed for both death and survival and for changes in the expression of cell surface receptors including IL-2 receptor β. These results provide insights into the dynamics of the GC reaction and represent the basis for the analysis of B cell malignancies.

Abstract

The germinal center (GC) reaction of antigen-activated B lymphocytes is the hallmark of antibody-mediated immune responses to T cell-dependent antigens (1). Individuals with genetic defects impairing GC formation suffer from immunodeficiency (2), and transgenic mice lacking factors that are required for GC formation do not show affinity maturation of the antibody response or humoral memory (summarized in ref. 3). GC B cells are also thought to be involved in the pathogenesis of most types of human B cell malignancies, including diffuse large cell lymphoma, follicular lymphoma, and Burkitt lymphoma (4, 5).

The GC reaction starts when naïve B cells (IgM^IgD^{) are activated by antigen receptor stimulation and receive costimulatory signals from immune helper cells (}6–8). These events induce the B cell to transform into a centroblast (CB) that proliferates within the histologically defined “dark zone” of the GC (1, 9, 10); CBs express the Ki67 nuclear antigen and can be identified by the expression of the CD77 cell surface marker (11). It is generally thought that CBs revise their antigen receptors through somatic hypermutation of IgV region genes, a process that introduces mainly single nucleotide substitutions into the IgV gene to generate antibodies with a higher or lower affinity to the respective antigen (7, 12). CBs then develop into noncycling centrocytes (CCs), which compose the “light zone” of the GC (9) and are distinguished from CBs by their lack of expression of the CD77 and Ki67 markers (11). In the CC stage, newly generated antibody mutants are selected based on their ability to bind their cognate antigen with the help of follicular dendritic cells and T cells. A large fraction of GC B cells undergoes apoptosis as they have acquired deleterious somatic mutations in their IgV regions that abolish antigen binding, whereas CCs expressing high-affinity antibody mutants eventually differentiate into plasma cells or memory B cells. A fraction of CCs also switches from the expression of IgM and IgD to that of other Ig classes by somatic DNA recombination to generate antibodies with different effector functions. The high-affinity memory B cells released from the GC are long-lived and have acquired the potential to rapidly differentiate into Ig-secreting cells during secondary immune responses (13).

Current knowledge of the physiology of the GC reaction is based on: (i) genetic approaches that have identified molecules required for GC development; (ii) the characterization of GC subpopulations based on the expression of immunophenotypic markers; and (iii) in vitro experiments that attempt to recapitulate the regulatory aspects of in vivo GC development. Although these studies have provided fundamental information on the physiology of GCs, they are based on the analysis of individual or small numbers of genes, proteins, or signaling pathways and cannot fully address the complex dynamics of the GC reaction. To obtain a comprehensive view of GC function and generate a data set for comparing normal versus malignant B cells, we have tracked the expression of ≈12,000 genes during the GC reaction.

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Acknowledgments

We thank Ashlyn Celestine for help with the immunohistological stainings and Richard Baer for comments on the manuscript. U.K. is a recipient of a fellowship granted by the Human Frontiers Science Program. G.C. is a recipient of an Esther Aboodi Associate Professor Fellowship.

Acknowledgments

Abbreviations

GC	germinal center
CB	centroblast
CC	centrocyte
MC	mononuclear cell
o.b.	over background
IL-2Rβ	IL-2 receptor β

Abbreviations

Note Added in Proof.

During the review process, a paper was published by Feldhahn et al. (54), who analyzed genomewide gene expression profiles of human B cell subpopulations by using sequential analysis of gene expression (SAGE).

Note Added in Proof.

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