Human tonsillar B cells were separated into naive IgD+CD38-CD23- (Bm1) and IgD+CD38-CD23 (Bm2), germinal center IgD-CD38+CD23- centroblasts (Bm3) and IgD-CD38+CD77- centrocytes (Bm4) and memory IgD-CD38- (Bm5) subsets. Previous IgVH sequence analysis concluded that the triggering of somatic mutations occurs during the transition from Bm2 subset into the Bm3 subset. To determine the initiation of isotype switching, sterile transcript expression was analyzed by amplification, cloning, and sequencing. A selective sterile I gamma, I alpha, and I epsilon expression was observed at centrocyte (Bm4) stage, suggesting that isotype switch is triggered within germinal centers, after somatic mutation is initiated with centroblasts (Bm3). Finally, the high level of 5'S gamma-S mu 3' DNA switching circles observed in germinal center B cells indicates that within human tonsils, germinal center is a major location for isotype switching.

Somatic hypermutation in immunoglobulin variable region genes occurs within germinal centers. Here, we describe a subset of germinal center dark zone centroblasts that express only sIgD and have accumulated up to 80 mutations per heavy chain variable region (IgVH delta gene). Over half of the hypermutated IgVH delta sequences were found to be clonally related. This level of mutation is not observed in either IgVH gamma transcripts from the same sample or IgVH delta transcripts from peripheral blood, suggesting that these cells neither undergo isotype switch nor mature into circulating memory B cells. Optimal growth of these cells in vitro depends on CD40 ligand, T cell cytokines, and a fibroblast stroma, a combination possibly mimicking the dark zone microenvironment. Our hypothesis is that these cells may be sequestered within germinal centers, where their somatic mutation machinery is triggered. The isolation of these hypermutated B cells may represent a critical step for studying both the biology and biochemistry of somatic hypermutation.

SummaryDuring T cell-dependent antibody responses, B cells within germinal centers (GC) alter the affinity of their antigen receptor by introducing somatic mutations into variable region of immunoglobulin (IgV) genes. During this process, GC B cells are destined to die unless positively selected by antigens and CD40-1igand. To understand survival/death control of germinal center B cell, the expression of four apoptosis-inducing genes, Fas, c-myc, Bax, and p53, together with the survival gene bcl-2, has been analyzed herein among purified tonsillar naive, GC, and memory B cells. IgD+CD38 -naive B cells were separated into CD23-(mature B cell IBm) 1) subset and CD23 + (Bm2), IgD-CD38 + GC B cells were separated into subsets ofCD77 + centroblasts (Bm3) and CD77-centrocytes (Bin4), whereas IgD-CD38-cells represented the Bm5 memory B cell subset. Sequence analysis of IgV region genes indicated that somatic hypermutation was triggered in the Bm3 centroblast subset. Here we show that bcl-2 is only detectable with naive (Bin1 and 2) and memory B cell (Bin5) subsets, whereas all four apoptosisinducing genes were most significantly expressed within GC B cells. Fas was equally expressed in Bin3 centroblasts and Bin4 centrocytes, whereas Bax was most significantly expressed in Bin4 centrocytes, c-myc, a positive regulator of cell cycle, was most significantly expressed in proliferating Bin3 centroblasts, whereas p53 a negative regulator of cell cycle, was most significantly expressed in nonproliferating Bm4 centrocytes. The present results indicate that the survival/death of GC B cells are regulated by the up-and downregulation of multiple genes, among which the expression of c-myc and p53 in the absence of bcl-2 may prime the proliferating Bin3 centroblasts and nonproliferating Bin4 centrocytes to apoptosis.

Purpose: Heat shock protein 90 (HSP90) is a chaperone for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSP90 is abundantly expressed by a variety of tumor types and has been recently targeted for cancer therapy. The objective of this study was to determine the role of HSP90 in promoting growth and survival of Hodgkin's lymphoma and to determine the molecular consequences of inhibiting HSP90 function by the small-molecule 17-allylamino-17-demethoxy-geldanamycin (17-AAG) in Hodgkin's lymphoma. Experimental Design: HSP90 expression in Hodgkin's lymphoma cell lines was determined by Western blot and in primary lymph node sections from patients with Hodgkin's lymphoma by immunohistochemistry. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Apoptosis and cell cycle fractions were determined by flow cytometry. Expression of intracellular proteins was determined by Western blot. Results: HSP90 is overexpressed in primary and cultured Hodgkin's lymphoma cells. Inhibition of HSP90 function by 17-AAG showed a time-and dose-dependent growth inhibition of Hodgkin's lymphoma cell lines.17-AAG induced cell cycle arrest and apoptosis, which were associated with a decrease in cyclin-dependent kinase (CDK) 4, CDK 6, and polo-like kinase1 (PLK1), and induced apoptosis by caspase-dependent and caspase-independent mechanisms. Furthermore, 17-AAG depleted cellular contents of Akt, decreased extracellular signal^regulated kinase (ERK) phosphorylation, and reduced cellular FLICE-like inhibitory protein levels (FLIP), and thus enhanced the cytotoxic effect of doxorubicin and agonistic anti^tumor necrosis factor^related apoptosisinducing ligand (TRAIL) death receptor antibodies. Conclusion: Inhibition of HSP90 function induces cell death and enhances the activity of chemotherapy and anti^tumor necrosis factor^related apoptosis-inducing ligand death receptor antibodies, suggesting that targeting HSP90 function might be of therapeutic value in Hodgkin's lymphoma.

SummaryUpon activation, B lymphocytes can change the isotype of the antibody they express by immunoglobulin (Ig) isotype switch recombination. In previous studies on the regulation of human IgG expression, we demonstrated that interleukin 10 (IL-10) could stimulate IgG1 and IgG3 secretion by human CD40-activated naive (slgD +) tonsillar B cells. To assess whether IL-10 actually promotes the DNA recombination underlying switching to these isotypes, we examined the effect of IL-10 on the generation of reciprocal products that form DNA circles as byproducts of switch recombination. The content of reciprocal products characteristic of p.-~/recombination was elevated after culture of CD40-activated tonsillar slgD + B cells with either IL-4 or IL-10, although high levels of IgG secretion were observed only with IL-10. Unlike IL-4, IL-10 did not induce reciprocal products of p~-e and ~/-e switch recombination. These results demonstrate that IL-10 promotes both switching to ",/and IgG secretion.