After 24 hours, cocultivated cells were collected, and Ramos (PINCO) cells, following labeling with CD19 MicroBeads (Miltenyi Biotec, Auburn, CA), were positively selected by magnetic cell separation using the MidiMACS system (Miltenyi Biotec). The plates were centrifuged at 290 rcf (centrifuge 5810R Eppendorf, Hamburg, Germany) for 5 minutes. BCR signaling was induced by adding 10 μg/mL anti-IgM antibodies (Southern Biotechnology Associates, Birmingham, AL) to Ramos (PINCO) cocultivated with NIH3T3 (empty vector). Ramos (PINCO) cells were cocultivated with NIH3T3 (empty vector) or NIH3T3 (mCD40L) in a 2:1 ratio in 6-well plates at a cell density of 0.75 × 10 6/mL. Ramos (PINCO) cells were maintained in Iscove modified Dulbecco medium supplemented with 10% fetal bovine serum and 100 IU/mL penicillin and 100 μg/mL streptomycin (Mediatech).

22 NIH3T3 (murine CD40L ) and NIH3T3 (empty vector), kindly provided by Dr Hilda Ye (Albert Einstein College of Medicine, Bronx, NY), were cultivated in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and 100 IU/mL penicillin and 100 μg/mL streptomycin (Mediatech, Herndon, VA). The Epstein-Barr virus-negative (EBV -) Burkitt lymphoma cell line Ramos was transduced with a retroviral expression vector (PINCO) carrying cDNA coding for the enhanced green fluorescence protein (EGFP). The data indicate the absence of CD40 signaling in centroblasts (CBs) and in most centrocytes (CCs), while they suggest that it may be active in pre- and post-GC B cells. The results were validated by examining NF-κB activation as well as the induction of CD40-induced genes as a readout of CD40 signaling in normal human lymphoid tissues by immunohistochemical analysis. This signature was used to investigate the presence of CD40 signaling in purified human B cells corresponding to defined developmental stages of the GC. To address this question, we first identified, using an in vitro system, the CD40 gene expression signature represented by genes that change expression after CD40 activation. In particular, neither in vivo genetic ablation nor in vitro B-cell stimulation experiments could determine whether CD40 signaling is active at the beginning of, throughout, or at the end of the GC reaction. Nonetheless, it is not yet understood at which stages of B-cell development signaling through CD40 occurs. Thus, the CD40-CD40L interaction has been extensively studied in the past years, and its critical role in B-cell responses has been established. Together, these results show that GC expansion occurs in the absence of CD40 signaling, which may act only in the initial and final stages of the GC reaction. Nevertheless, a subset of centrocytes and B cells in the subepithelium showed nuclear staining of multiple NF-κB subunits, suggesting that a fraction of naive and memory B cells may be subject to CD40 signaling or to other signals that activate NF-κB. This finding was validated in lymphoid tissues by showing that the nuclear factor-κB (NF-κB) transcription factors, which translocate to the nucleus upon CD40 stimulation, are retained in the cytoplasm in most GC B cells, indicating the absence of CD40 signaling. This signature was then investigated in the gene expression profiles of normal B cells and found in pre- and post-GC B cells (naive and memory) but, surprisingly, not in GC B cells. To address this question, we induced CD40 signaling in human transformed GC B cells in vitro and identified a CD40 gene expression signature by DNA microarray analysis. However, it is not fully understood at which stages of B-cell development the CD40 pathway is activated in vivo.

Substantial evidence indicates that signaling through the CD40 receptor (CD40) is required for germinal center (GC) and memory B-cell formation.

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