Ligation of the Fas (CD95) receptor leads to an apoptotic death signal in T cells, B cells, and macrophages. However, human CD34 ϩ -derived dendritic cells (DCs) and mouse DCs, regardless of their maturation state, are not susceptible to Fas-induced cell death. This resistance correlates with the constitutive expression of the Fas-associated death domain-like IL-1 ␤ -converting enzyme (FLICE)-inhibitory protein (FLIP) ligand. We demonstrate a new role of Fas in DC physiology. Engagement of Fas on immature DCs by Fas ligand (FasL) or by anti-Fas antibodies induces the phenotypical and functional maturation of primary DCs. Fas-activated DCs upregulate the expression of the major histocompatibility complex class II, B7, and DClysosome-associated membrane protein (DC-LAMP) molecules and secrete proinflammatory cytokines, in particular interleukin (IL)-1 ␤ and tumor necrosis factor ␣ . Mature DCs, if exposed to FasL, produce even higher amounts of IL-1 ␤ . Importantly, it is possible to reduce the production of IL-1 ␤ and interferon (IFN)-␥ during DC-T cell interaction by blocking the coupling of Fas-FasL with a Fas competitor. Finally, during cognate DC-T cell recognition, IL-12 (p70) could not be detected at early or late time points, indicating that Fas-induced, IFN-␥ secretion is independent of IL-12.

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-x L , and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Human interleukin-2 (IL-2) is a glycoprotein of relative molecular mass (Mr) 15,000, which is released by T lymphocytes on stimulation with antigen or mitogen and functions as a T-cell growth factor (TCGF) by inducing proliferation of activated T cells. It is generally accepted that resting or activated B cells do not respond directly to IL-2 but require for their proliferation other T-cell-derived lymphokines usually referred to as B-cell growth factors (BCGFs). Recently, however, a monoclonal antibody reacting with the IL-2 receptor molecules expressed by activated T cells (anti-Tac) was shown to react also with certain B tumour cells; in addition, murine B cells proliferate in response to pure human IL-2. We now show that recombinant IL-2, derived from Escherichia coli expressing the human gene, is able to promote strong proliferation of human B cells activated with protein-A-rich Staphylococcus aureus Cowans strain I. Moreover, we demonstrate that the anti-Tac antibody also reacts with S. aureus-activated normal B cells and inhibits sharply the proliferative response of such cells to IL-2. Finally, immunoprecipitation experiments reveal that anti-Tac defines similar molecules on activated T and B cells.

Interleukin-2 (IL-2) is a T-cell-derived polypeptide hormone of 133 amino acids which exerts its growth-promoting activity via a surface receptor. Originally, IL-2 was believed to be a unique growth factor for activated T cells; more recent studies, however, have demonstrated that certain B-cell tumours as well as normal activated B lymphocytes express a surface molecule which is recognized by monoclonal antibodies directed against the IL-2 receptor. Furthermore, we and others have shown recently that activated B cells proliferate in response to either immunoaffinity-purified or recombinant IL-2. These controversial findings prompted us to undertake a detailed quantitative comparison of IL-2 receptor expression on activated B and T cells. We show here, using biosynthetically labelled IL-2(3H-IL-2) and anti-IL-2 receptor antibody (3H-PC61) that activated B and T cells express both high-affinity (apparent dissociation constant, Kd approximately 20 pM) and low-affinity (Kd approximately 1,000 pM) IL-2 receptors. Binding of IL-2 to both classes of receptor is inhibited by the monoclonal anti-IL-2 receptor antibody PC61. B blasts express half as many total IL-2 binding sites or PC61 binding sites as T blasts, and the ratio of the number of low- to high-affinity receptors for each cell type is approximately 10:1. Immunoprecipitation analysis of surface-labelled blasts indicates that B and T cells have IL-2 receptors of similar relative molecular mass. Taken together, these data suggest strongly that IL-2 can act as a growth hormone for both B and T lymphocytes.

Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus–based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1α and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1α promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1α promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3′ long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.

The analysis of human B cell responses at the clonal level (limiting dilution assay) is still technically difficult. In the present study we report on a culture system that leads to activation, proliferation and differentiation into antibody-secreting cells (ASC) of about 90% of B cells from peripheral blood or spleen. In this system, B cells are cultured in the presence of a mutant subclone of the mouse thymoma EL4 for B cell activation and human T cell plus macrophage supernatant as source of proliferation and differentiation factors. ASC precursors generating clonal responses of IgM only, IgM plus IgG, or IgG only occurred at a ratio of about 6:3:1. The mean clone size was 380 cytoplasmic Ig+ cells; the mean amount of Ig secreted per clone was 20 ng. Furthermore, it has been found using this system that a considerable proportion of peripheral blood B cells from individuals with a history of malaria infection could generate clones of anti-malaria (Plasmodium falciparum) ASC (range of 0.1 to 1%, n = 6). In a control group of blood donors the corresponding frequencies were 10 times lower (range of 0.01 to 0.1%, n = 9). These results show that the EL4 culture system can be applied to the investigation of the human B cell specificity repertoire and of priming effects such as result from infectious disease.

Interleukin 2 (IL-2) 1 was discovered through its function as a T cell growth factor (TCGF) (1, 2). Indeed, the availability of T cell lines for the quantitative measurement of TCGF activity has been crucial for the characterization of IL-2 (3, 4) and IL-2 receptors on activated T cells (5-7) in different species. Human IL-2, which acts on murine and human T cells (4,8,9), can now rapidly be purified to apparent homogeneity with monoclonal antibodies (8, 9) and its cDNA has been cloned (10, 11). Human I L-2 is a T ceil-generated 15 kilodalton peptide hormone of 133 amino acids.Although it was observed in several studies (3, 4, 12-14) that IL-2-rich T cell supernatants (SN) enhance the generation of plaque-forming cells (PFC) in certain B cell culture systems, it is widely believed that B cells do not directly respond to IL-2. Thus, a variety of other T cell-derived factors (but not IL-2) were reported to enhance the proliferation of B cells activated by either Tindependent antigens (15), dextran (16), lipopolysaccharide (LPS) (17, 18), antiimmunoglobulin antibodies (anti-Ig) (16,(19)(20)(21), or other means (22). In one study (23), showing an effect of immunoaffinity-purified IL-2 on anti-Iginduced B cell proliferation, the interpretation of the results was complicated because it was also observed that T cells participated in the B cell response. LPSactivated murine B cells were not found to bind radiolabeled IL-2 (5) but, in another study (7), such LPS blasts reacted weakly with an anti-IL-2 receptor monoclonal antibody.However, there still exist controversies with regard to the activation signals that B cells require to respond to growth factors (24). A recent study (25) showed in a limit-dilution culture system for murine B cells that, in fact, either a cell contact-dependent T-B cell interaction or LPS was required in conjunction with anti-Ig for optimal induction of growth factor responsiveness. Whereas anti-Ig

IntroductionEfficient delivery of genes into primary human B lymphocytes would allow the investigation of gene functions in these cells for the purposes of research and the development of gene therapies. One could then test in mature B cells the promoters/genes potentially suitable for stem cell-based therapies for immunodeficiencies. 1 Vectors achieving the efficient transfection of primary B cells would most likely also be suitable for the delivery of genes into freshly collected B tumor cells-for example, for the development of immune-based anti-B-tumor therapies. 2 Various viral vectors are currently being studied for their ability to transduce hematopoietic cells. [3][4][5] Retroviral vectors derived from murine leukemia virus (MLV) 6,7 can transfer genes into immortal human B-cell lines, such as lymphoblastoid cells, 8 and primary B precursors, 9 but they are inefficient for mature human B cells. 10,11 These simple retroviruses can transduce genes only into actively dividing cells, 12 but a potent T-independent mitogen for human B cells in vitro, such as lipopolysaccharide (LPS) for murine B cells, has not been found. 13 In addition, MLV vectors might not be well adapted for human B cells because of the host species difference. HIV-1 and HIV-derived pseudotyped lentiviral vectors efficiently integrate into human cells, irrespective of cell division. 14-22 High transgene expression from such vectors in human T cells or total lymphocytes has been reported. [23][24][25] Generally, productive HIV infection or lentivectormediated transduction of truly quiescent lymphocytes has not been observed; activation, at least from G 0 to G 1 , seems to be required. 23,[25][26][27][28] Efficient transduction of primary acute lymphoblastic leukemia cells with a bicistronic HIV vector, leading to the expression of a cytokine (granulocyte macrophage-colonystimulating factor [GM-CSF]) and an immunostimulatory molecule (CD80), has also been achieved, 29 indicating a potential use of such vectors in novel anti-B-tumor therapies.In this study we investigated the transduction of peripheral blood B cells with multiply attenuated HIV vectors pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein. 21 Efficient transduction of such B cells occurred after their activation in a culture system using murine EL-4 B5 thymoma cells as helper T cells in conjunction with human cytokines. [30][31][32][33] This system leads to proliferation and subsequent plasmocytic differentiation of all naive and memory human B subsets. 33 Nondividing, freshly isolated multiple myeloma cells were also efficiently transduced by HIV vector. By contrast, an MLV vector pseudotyped with VSV G protein was inefficient even in dividing B cells. F.B. and P.S. contributed equally to this work. D.T. has declared a financial interest as consultant to Cell Genesis, a company whose potential product is related to the HIV-1 vectors used in this study.Reprints: R. Zubler, Division of Hematology, University Hospital, 1211 Geneva-14, Switzerland; e-mail: rudolf.zubl...