Intracellular amorphous carbonates uncover a new biomineralization process in eukaryotes.

Until now, descriptions of intracellular biomineralization of amorphous inclusions involving alkaline-earth metal (AEM) carbonates other than calcium have been confined exclusively to cyanobacteria (Couradeau et al., 2012). Here, we report the first evidence of the presence of intracellular amorphous granules of AEM carbonates (calcium, strontium, and barium) in unicellular eukaryotes. These inclusions, which we have named micropearls, show concentric and oscillatory zoning on a nanometric scale. They are widespread in certain eukaryote phytoplankters of Lake Geneva (Switzerland) and represent a previously unknown type of non-skeletal biomineralization, revealing an unexpected pathway in the geochemical cycle of AEMs. We have identified Tetraselmis cf. cordiformis (Chlorophyta, Prasinophyceae) as being responsible for the formation of one micropearl type containing strontium ([Ca,Sr]CO3 ), which we also found in a cultured strain of Tetraselmis cordiformis. A different flagellated eukaryotic cell forms barium-rich micropearls [(Ca,Ba)CO3 ]. The strontium and barium concentrations of both micropearl types are extremely high compared with the undersaturated water of Lake Geneva (the Ba/Ca ratio of the micropearls is up to 800,000 times higher than in the water). This can only be explained by a high biological pre-concentration of these elements. The particular characteristics of the micropearls, along with the presence of organic sulfur-containing compounds-associated with and surrounding the micropearls-strongly suggest the existence of a yet-unreported intracellular biomineralization pathway in eukaryotic micro-organisms.

Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its 'undruggable' nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be 'undruggable' as previously thought.

Fast formation of supergene Mn oxides/hydroxides under acidic conditions in the oxic/anoxic transition zone of a shallow aquifer.

Extensive uranium mining in the former German Democratic Republic (GDR) in eastern Thuringia and Saxony took place during the period of 1946-1990. During mining activities, pelitic sediments rich in organic carbon and uranium were processed and exposed to oxygen. Subsequent pyrite oxidation and acidic leaching lead to partial contamination of the area with heavy metals and acid mine drainage (AMD) even few years after completion of remediation. One of those areas is the former heap Gessen (Ronneburg, Germany) were the residual contamination can be found 10 m under the base of the former heap containing partly permeable drainage channels. Actually, in such a system, a rapid but locally restricted mineralization of Mn oxides takes place under acidic conditions. This formation can be classified as a natural attenuation process as certain heavy metals, e.g., Cd (up to 6 µg/g), Ni (up to 311 µg/g), Co (up to 133 µg/g), and Zn (up to 104 µg/g) are bound to this phases. The secondary minerals occur as colored layers close to the shallow aquifer in glacial sediments and could be identified as birnessite and todorokite as Mn phase. The thermodynamic model shows that even small changes in the system are sufficient to shift either the pH or the Eh in the direction of stable Mn oxide phases in this acidic system. As a consequence of 9-15-year-long formation process (or even less), the supergene mineralization provides a cost-efficient contribution for remediation (natural attenuation) strategies of residual with heavy metals (e.g., Cd, Co, Ni, Zn) contaminated substrates.

Interrelationships of breed type, USDA quality grade, cooking method, and degree of doneness on consumer evaluations of beef in Dallas and San Antonio, Texas, USA.

The objective of this research was to evaluate the consumer controlled factors of cooking method and degree of doneness on top loin steaks from different USDA quality grades (Low Choice, High Select or Low Select) and breed-types (English, Continental European Cross or Brahman Cross). In addition, cities within the same region were evaluated for differences in consumer controlled factors and palatability responses. The in-home product test was conducted in Dallas and San Antonio, Texas, USA. Consumers (n=173) evaluated steaks for overall like (OSAT), tenderness (TEND), juiciness (JUIC), and flavor (FLAV) using 23-point hedonic scales. Respondents in Dallas cooked their steaks to higher degrees of doneness than did those in San Antonio. Outdoor grilling was the most frequently used method of cookery for steaks in both cities. Generally, consumers in San Antonio gave higher palatability ratings to Choice steaks and Dallas consumers gave higher ratings to Select steaks. The interactions of city×cooking method, breed-type×cooking method, and degree of doneness×cooking method were significant for all palatability attributes. In addition, the interaction of cooking method×quality grade was significant for TEND, JUIC, and FLAV. Warner-Bratzler shear (WBS) force was determined on a steak from each strip loin. Steaks from Continental European Cross cattle and Low Choice carcasses had the lowest WBS values. Differences in consumer preparation of beef top loin steaks present very unique challenges for the beef industry. Consumer information programs may serve a valuable role in connecting consumer perceptions with the preparation techniques needed to consistently achieve satisfaction.

Differential transduction efficiency of SCID-repopulating cells derived from umbilical cord blood and granulocyte colony-stimulating factor-mobilized peripheral blood.

The gene transfer efficiency into nonobese diabetic/severe combined immunodeficient (NOD/SCID)-repopulating cells (SRCs) derived from umbilical cord blood (UCB) (n = 11 NOD/SCID mice) and granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) (n = 64 NOD/SCID mice) was compared using a clinically relevant protocol and a retrovirus vector expressing the enhanced green fluorescent protein (EGFP). At 6-9 weeks after transplantation, the frequency of transduced human cells in the bone marrow (BM) (40.5% +/- 2.4% [mean +/- SE]) and spleen (SPL) (36.4% +/- 3.2%) in recipients of UCB cells was significantly higher (p < 0.001) than that observed in the BM (2.2% +/- 1.8%) and SPL (2.0% +/- 2.6%) in recipients of MPB. In subsequent studies, MPB was cultured for 2-8 days in cytokines prior to transduction to determine if longer prestimulation was required for optimal gene transfer. A significant increase in gene transfer into CD45(+) human cells and clonogenic cells derived from MPB SRCs was observed when cells were prestimulated for 6 days compared to 2 days prior to transduction (p = 0.019). However, even after 6 days of prestimulation, transduction was still significantly less than UCB. A substantial discrepancy exists in the ability to introduce genes effectively via retrovirus vectors into SRCs derived from MPB as compared to UCB.

Interleukin-5 inhibition of biliary cell chloride currents and bile flow.

Recent studies have detected significant elevations of interleukin (IL)-5 mRNA in the liver parenchyma of patients with both primary biliary cirrhosis and acute rejection after liver transplantation. In both of these disorders, intrahepatic biliary epithelial cells (BECs) are the targets of injury. We hypothesized that BECs may themselves express IL-5 receptors that may modulate key biliary functions. RNAs coding for IL-5alpha and -beta receptors were amplified by RT/PCR from a biliary cell line derived from a human cholangiocarcinoma (Mz-ChA-1) and verified by DNA sequencing. IL-5 receptor distribution was detected immunocytochemically on Mz-ChA-1 cells, immortalized murine BEC, bile duct-ligated rat liver, and isolated cholangiocytes. Patch-clamp studies on Mz-ChA-1 cells showed that IL-5 inhibits 5'-N-ethylcarboxamidoadenosine-stimulated chloride currents. Additional functional studies showed that IL-5 inhibits secretin-induced bile flow. We conclude that BECs express IL-5 receptors and that IL-5 modulates BEC chloride currents and fluid secretion. Since IL-5 has previously been associated with cholestatic liver disease, we speculate that IL-5 may contribute to liver injury through its effects on biliary secretion.

Simultaneous ex vivo expansion of cytomegalovirus and Epstein-Barr virus-specific cytotoxic T lymphocytes using B-lymphoblastoid cell lines expressing cytomegalovirus pp65.

Cytomegalovirus (CMV) infection and Epstein-Barr virus (EBV)-induced lymphoproliferative disease are serious complications associated with allogeneic stem cell transplantation. Immunotherapy using ex vivo expanded, virus-specific cytotoxic T lymphocytes (CTL) has been explored and proven to be effective in therapeutic or prophylactic regimens for CMV and EBV infections. To generate CTL specific for both CMV and EBV, we engineered EBV-transformed B-lymphoblastoid cell lines (BLCL) to express CMV pp65 for use as antigen-presenting cells (APC). BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. Western blot analysis and immunocytochemistry confirmed the expression of pp65 in the transduced cells. Peripheral blood mononuclear cells (PBMC) from healthy CMV seropositive donors were stimulated with autologous pp65-expressing BLCL weekly for 3 weeks. Chromium release assays showed that the resulting CTL cultures possessed specific cytotoxicity against EBV and CMV. Recombinant vaccinia viruses encoding individual CMV peptides were used to demonstrate that this CMV-specific cytotoxicity was specific for pp65. Assays on CD4- and CD8-depleted CTL fractions indicated that CD8(+) CTL mediated the pp65-specific cytotoxicity. These CMV/EBV-specific CTL recognized CMV- and EBV-infected targets sharing HLA class I antigens, but not HLA mismatched targets. Our results demonstrate that BLCL can be used as APC to stimulate expansion of EBV- and CMV-specific CTL simultaneously. These findings have potential implications for posttransplant CMV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus.

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.

CD4(+) Epstein-Barr virus-specific cytotoxic T-lymphocytes from human umbilical cord blood.

Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants.

Simultaneous infection with retroviruses pseudotyped with different envelope proteins bypasses viral receptor interference associated with colocalization of gp70 and target cells on fibronectin CH-296.

Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34(+) hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34(+) hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.

Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T lymphocytes.

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.

Facilitation of retrovirus-mediated gene transfer into hematopoietic stem and progenitor cells and peripheral blood T-lymphocytes utilizing recombinant fibronectin fragments.

A transduction strategy has been developed, using fibronectin (FN)-assisted retroviral-mediated gene transfer, based on the observation that hematopoietic stem and progenitor cells bind to specific adhesion domains of fibronectin, via the integrins, very late antigen-4 (VLA-4)alpha 4 beta 1 and very late antigen-5 (VLA-5)alpha 5 beta 1. Retrovirus-mediated transduction on a recombinant FN fragment, FN CH-296, containing binding sites for VLA-4 and VLA-5, separated by type III repeats 12 to 14, makes it possible to efficiently target hematopoietic stem and progenitor cells and T-lymphocytes due to colocalization of target cells and retrovirus particles. These gene therapy strategies are applicable to the potential treatment of a variety of acquired and inherited immune disorders.

High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments.

Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876-882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3+ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.

Treatment of posttransplant lymphoproliferative disease in the central nervous system of a lung transplant recipient using allogeneic leukocytes.

Posttransplant Epstein-Barr virus-related lymphoproliferative disease (PT-LPD) is a common and often fatal complication following solid organ and hematopoietic stem cell transplantation. PT-LPD following solid organ transplantation generally occurs in B cells of recipient origin in contrast to PT-LPD in marrow transplant recipients, which is exclusively of donor origin. The efficacy of adoptive immunotherapy using donor leukocytes to treat PT-LPD in bone marrow transplant recipients has recently been reported. Because PT-LPD in solid organ transplant recipients is generally of recipient origin, the potential application of adoptive immunotherapy of PT-LPD in solid organ recipients obligates the use of either autologous or allogeneic HLA identical leukocytes, with the attendant risk of organ rejection if cells mismatched with the transplanted organ are used. Nonirradiated allogeneic mononuclear cells from an Epstein-Barr virus (EBV)-seropositive, HLA-identical normal sibling were used to treat a monoclonal EBV lymphoma of recipient origin in the central nervous system of a child who had undergone an HLA-mismatched cadaveric lung transplant. The patient received three separate mononuclear cell infusions over a 9-month period, each containing 1 x 10(6) CD3+ mononuclear cells per kilogram. Complete clinical, radiological, and pathological remission was achieved with this treatment regimen. The response correlated with in vivo reconstitution of normal EBV-specific cytotoxic activity and cytotoxic T lymphocyte precursor frequency. Use of allogeneic HLA-compatible mononuclear cells may thus offer an additional mode of therapy for EBV-related lymphoproliferative disease in selected solid organ transplant recipients refractory to conventional therapies.

Post-transplant EBV induced lymphoproliferative disorders.

Epstein Barr virus induced lymphoproliferative disease (EBV-LPD) is a heterogeneous disorder, ranging from polyclonal lymphoproliferations to malignant lymphoma, typically seen in individuals with inadequate cellular immunity to EBV. The diagnosis of EBV-LPD following transplant requires a high index of suspicion in those patients at risk. Unlike organ transplant patients, in whom lymphomas are generally of host origin and respond to decreases in immunosuppression, bone marrow transplant recipients have donor origin tumors that are not as responsive to conservative treatment modalities. For the latter group, adoptive immunotherapy with donor lymphocytes is the treatment of choice and generally results in complete eradication of these tumors. Whether adoptive immunotherapy can be used for EBV-LPD in patients following organ transplantation is currently being investigated.

Cord blood mononuclear cell transformation assay for screening for the presence of Epstein-Barr virus.

EBV-induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication following stem cell transplantation. Strategies have been developed for the cultivation of donor-derived, EBV-specific cytotoxic T lymphocytes (CTL) for stem cell transplant (SCT) patients affected with these disorders, using donor-derived, EBV-transformed B lymphoblastoid cell lines (BLCL) as stimulators. Although cultivation of EBV-transformed BLCL is possible without using an exogenous source of EBV, transformation of autologous B cells with endogenous virus may be slow and inconsistent. Therefore, if exogenous strains of EBV are used to generate BLCL, it may be beneficial to patients to ensure that these cell lines are not producing virus that potentially could be conveyed at the time of CTL infusion. A reliable method of screening for EBV using a cord blood transformation assay has been developed and is described.

Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28.

The expression of the murine T cell Ag 4-1BB, a member of the TNF-R family, is induced by T cell activation. Previously, we and others had shown that signaling through 4-1BB enhanced proliferative T cell responses. To investigate a potential role for the interaction of 4-1BB with its ligand (4-1BBL) in T cell activation, we studied the ability of a soluble chimera of 4-1BB (4-1BBFc) to interfere with proliferative responses and cytokine production in models of activation dependent in intercellular interactions. The potential blocking effect of 4-1BBFc was compared with that of the chimeric molecule CTLA-4Ig, a reagent known to interfere with the interaction of CD28 (and/or CTLA-4) with B7 costimulatory receptors. In this study, we report that 4-1BBFc partially blocked both the activation of unfractionated splenocytes triggered by soluble anti-CD3 (anti-CD3s), and the more physiologically relevant responses to alloantigen. In addition, we show that both chimeric molecules partially blocked proliferative responses and IL-2 secretion by highly purified resting T cells activated with anti-CD3s in the presence of fixed accessory cells that express B7 receptors and 4-1BBL. Furthermore, in this model system, the blocking capacity of 4-1BBFc and CTLA-4Ig appears to correlate with the relative expression of their respective cognate receptors (4-1BBL and B7) on the accessory cell. Simultaneous addition of both blocking reagents produced an additive effect in the model systems studied.

Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP.

K46J B lymphomas express a T cell costimulatory activity that is not inhibited by CTLA-4Ig, anti-B7-1, anti-B7-2, anti-intercellular adhesion molecule 1 or antibodies to heat stable antigen. In this paper we report that this costimulatory activity is mediated at least in part by 4-1BB ligand, a member of the tumor necrosis factor (TNF) gene family that binds to 4-1BB, a T cell activation antigen with homology to the TNF/nerve growth factor receptor family. A fusion protein between 4-1BB and alkaline phosphatase (4-1BB-AP) blocks T cell activation by K46J lymphomas in both an antigen-specific system and with polyclonally (anti-CD3) activated T cells. 4-1BB-AP also blocks antigen presentation by normal spleen cells. When the antigen-presenting cells express B7 molecules as well as 4-1BB ligand, we find that B7 molecules and 4-1BB-AP both contribute to T cell activation. These data suggest that 4-1BB ligand plays an important role in costimulation of IL-2 production and proliferation by T cells. The B lymphoma M12 expresses low levels of 4-1BB-L but can be induced to express higher levels by treatment of the B cells with cAMP, which also induces B7-1 and B7-2 in these cells. Thus cAMP appears to coordinately induce several costimulatory molecules on B cells.