Germinal center (GC) dark and light zones segregate cells undergoing somatic hypermutation and antigen-driven selection, respectively, yet the factors guiding this organization are unknown. We report here that GC organization was absent from mice deficient in the chemokine receptor CXCR4. Centroblasts had high expression of CXCR4 and GC B cells migrated toward the CXCR4 ligand SDF-1 (CXCL12), which was more abundant in the dark zone than in the light zone. CXCR4-deficient cells were excluded from the dark zone in the context of a wild-type GC. These findings establish that GC organization depends on sorting of centroblasts by CXCR4 into the dark zone. In contrast, CXCR5 helped direct cells to the light zone and deficiency in CXCL13 was associated with aberrant light zone localization.

A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.

Homo-and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo-and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo-and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.In recent years, the concept of GPCR 1 dimerization has raised questions about the molecular details and functional role of such oligomeric assembly (for a recent review, see Ref.1). Given the clinical interest in GPCRs, insights into the structural and functional organization of the receptor complexes have the potential to facilitate the design of new drug candidates with increased efficacy and selectivity. Resonance energy transfer (RET) techniques have emerged as methods of choice to study receptor dimerization in living cells. Although most RET studies indicate that many if not all GPCRs exist as dimers or higher oligomers under basal conditions, apparent contradictions exist concerning their potential dynamic regulation upon ligand binding. Although numerous authors did not find any effects of ligands on constitutive RET signals in their systems (2-8), others observed ligand-promoted increases or decreases that were interpreted as either the formation (9 -11) or the dissociation (12-15) of GPCR dimers in response to receptor activation. Conformational changes within pre-existing constitutive dimers have also been proposed as alternative explanations for agonist or antagonist-induced changes in .Chemokine receptors such as CCR2 and CXCR4 have been reported to form homo-and heterodimers (3, 4, 19 -24). In early co-immunoprecipitation studies, proposed that the dimerization of CXCR4 is induced upon activation by its chemokine ligand SDF-1. In contrast, data obtained with RET techniques revealed that CXCR4 homo-dimers form spontaneously in the absence of ligand (3,4,24). In one study, no significant effect of SDF-1 was observed on the constitutive energy transfer (4), whereas a small but reproducible increase was detected by others (24). As for CXCR4, agonist stimulation of CCR2 was found to promote the formation of dimers as revealed by chemical cross-linking followed by immunoprecipitati...

IntroductionB-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of a monoclonal population of CD5 ϩ neoplastic B cells in secondary lymphoid organs, marrow, and blood. Because most of the circulating leukemia cells are arrested in the G 0 /G 1 phase of the cell cycle, the primary defect may be one of resistance to programmed cell death rather than accelerated cell division. 1,2 However, CLL cells can rapidly undergo spontaneous apoptosis under culture conditions that support the growth of human B-cell lines. This implies that such ex vivo conditions lack factors necessary for leukemia cell survival or that the resistance to apoptosis is not intrinsic to the leukemia B cell.The leukemia cell microenvironment in the marrow or in secondary lymphoid tissues may contribute to the noted resistance of CLL cells to apoptosis in vivo. 3,4 Normal B-cell development depends on complex interactions with accessory cells that define the so-called specialized microenvironments. T cells and a variety of different types of adherent cells, generally defined as stromal cells, are the main elements of the microenvironment. 3 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the pattern and extent of marrow involvement correlates with clinical stage and prognosis. 5,6 As such, interactions with stromal cells in the marrow microenvironment appear to play a role in disease progression and resistance to therapy. [7][8][9] In addition, we found that a small proportion of the mononuclear cells from the blood of patients with CLL can differentiate into large, round, adherent cells that attract CLL cells and protect them from undergoing spontaneous or drug-induced cell death. 10,11 Because these cells share features with thymic nurse cells that nurture developing thymocytes, we designated them nurselike cells (NLCs). Although NLCs differentiate from blood mononuclear cells after several days in vitro, fully differentiated NLCs can be found in the spleen and secondary lymphoid tissue of patients with CLL, 11 where they might play a role in protecting CLL cells from apoptosis in vivo. This model implies that CLL cells depend on specific extrinsic factors from NLCs and other stromal elements for their survival. Conceivably, CLL cells recirculate from the blood through secondary lymphoid tissues and back into the systemic circulation in response to certain chemokines.One such chemokine is stromal cell-derived factor-1/pre-B cell growth-stimulating factor (SDF-1/PBSF), which recently has been designated CXCL12. CXCL12 is a member of a family of chemotactic cytokines (chemokines) that initially were characterized as growth-stimulating factors for B-cell precursors. 12 CXCR4 is a primary physiologic receptor for CXCL12 and functions as a coreceptor for entry of T-tropic strains of HIV-1. Mutant mice with targeted gene disruption of CXCL12 or CXCR4 have defects in the We previously demonstrated that stromal cells can attract CLL cells through the production of CXCL12. 13 In addition, NLCs e...