Impaired tumor antigen processing by immunoproteasome-expressing CD40-activated B cells and dendritic cells

Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart1_26?C34 (M26) and Cyp1B1_239?C247 (Cyp239)] and one derived from the influenza A viral antigen [FluM1_58?C66 (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens.     

Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells

The development of rapid, efficient, and safe methods for generating Ag-specific T cells is necessary for the clinical application of adoptive immunotherapy. We show that B cells stimulated with CD40 ligand and IL-4 (CD40-B cells) can be efficiently transduced with retroviral vectors encoding a model Ag, CMV tegument protein pp65 gene, and maintain high levels of costimulatory molecules after gene transfer. CTL lines specific for pp65 were readily generated in all four healthy CMV-seropositive donors by stimulating autologous CD8(+) T cells with these transduced CD40-B cells, both of which were derived from 10 ml peripheral blood. ELISPOT assays revealed that the CTL lines used multiple HLA alleles as restricting elements. Thus, CD40-B cells transduced retrovirally with Ag-encoding cDNA can be potent APC and facilitate to generate Ag-specific CTL in vitro.     

Human interdigitating dendritic cells induce isotype switching and IL-13-dependent IgM production in CD40-activated naive B cells

Interdigitating dendritic cells (IDC) represent a mature progeny of dendritic cells (DC) in vivo and are exhibiting a strong lymphocyte stimulatory potential. Because of the restricted localization to secondary lymphoid organs where decisive cellular interactions take place in the initial events of immunity, IDC regulatory function was addressed in relation to naive B cells. In this study, we demonstrate that human tonsillar IDC induce a dual response from CD40-activated IgD+/CD38- naive B lymphocytes. IDC direct naive B cells toward either isotype switching or an IL-13-dependent IgM secretion. IDC-dependent proliferation, isotype switching, and Ig production are all strictly mediated by soluble factors, suggesting that such skewing in B cell activation is the result of differential cytokine expression. Moreover, IDC-expressed IL-13 represents a novel source of a cytokine with recently established effects in Th2 induction as well as in immunological disorders resulting in allergic reactions.     

OK432-activated human dendritic cells kill tumor cells via CD40/CD40 ligand interactions

In vivo, dendritic cells (DC) are programmed to orchestrate innate and adaptive immunity in response to pathogen-derived "danger" signals. Under particular circumstances, DC can also be directly cytotoxic against tumor cells, potentially allowing them to release tumor associated Ags from dying cells and then prime antitumor immunity against them. In this study, we describe the innate characteristics of DC (OK-DC) generated in vitro after exposure of immature human myeloid-derived DC to OK432, a penicillin-inactivated and lyophilized preparation of Streptococcus pyrogenes. OK-DC produced proinflammatory cytokines, stimulated autologous T cell proliferation and IFN-gamma secretion, expressed CCR7, and migrated in response to MIP-3beta. Moreover, OK-DC displayed strong, specific cytotoxicity toward tumor cell targets. This cytotoxicity was associated with novel, OK432-induced up-regulation of CD40L on the cell surface of OK-DC, and was absolutely dependent on expression of CD40 on the tumor targets. These data demonstrate that maturation of human DC with OK432, an adjuvant suitable for clinical use, induces direct tumor cell killing by DC, and describes a novel CD40/CD40L-mediated mechanism for specific DC antitumor cytotoxicity.     

Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: role of memory B cells and CD5+ B cells.

NK cells are a subpopulation of lymphocytes characterized primarily by their cytolytic activity. They are recognized as an important component of the immune response against virus infection and tumors. In addition to their cytolytic activity, NK cells also participate either directly or indirectly in the regulation of the ongoing Ab response. More recently, it has been suggested that NK cells have an important role in the outcome of autoimmune diseases. Here, we demonstrate that human NK cells can induce autologous resting B cells to synthesize Ig, including switching to IgG and IgA, reminiscent of a secondary Ab response. B cell activation by the NK cell is contact-dependent and rapid, suggesting an autocrine B cell-regulated process. This NK cell function is T cell-independent, requires an active cytoplasmic membrane, and is blocked by anti-CD40 ligand (anti-CD154) or CD40-mIg fusion protein, indicating a critical role for CD40-CD40 ligand interaction. Depletion studies also demonstrate that CD5+ B cells (autoreactive B-1 cells) and a heterogeneous population of CD27+ memory B cells play a critical role in the Ig response induced by NK cells. The existence of this novel mechanism of B cell activation has important implications in innate immunity, B cell-mediated autoimmunity, and B cell neoplasia.     

NMDA-receptor antagonists block B-cell function but foster IL-10 production in BCR/CD40-activated B cells

Background

B cells are important effectors and regulators of adaptive and innate immune responses, inflammation and autoimmunity, for instance in anti-NMDA-receptor (NMDAR) encephalitis. Thus, pharmacological modulation of B-cell function could be an effective regimen in therapeutic strategies. Since the non-competitive NMDAR antagonist memantine is clinically applied to treat advanced Alzheimer`s disease and ketamine is supposed to improve the course of resistant depression, it is important to know how these drugs affect B-cell function.

Results

Non-competitive NMDAR antagonists impaired B-cell receptor (BCR)- and lipopolysaccharide (LPS)-induced B-cell proliferation, reduced B-cell migration towards the chemokines SDF-1α and CCL21 and downregulated IgM and IgG secretion. Mechanistically, these effects were mediated through a blockade of K_v1.3 and K_Ca3.1 potassium channels and resulted in an attenuated Ca²⁺-flux and activation of Erk1/2, Akt and NFATc1. Interestingly, NMDAR antagonist treatment increased the frequency of IL-10 producing B cells after BCR/CD40 stimulation.

Conclusions

Non-competitive NMDAR antagonists attenuate BCR and Toll-like receptor 4 (TLR4) B-cell signaling and effector function and can foster IL-10 production. Consequently, NMDAR antagonists may be useful to target B cells in autoimmune diseases or pathological systemic inflammation. The drugs’ additional side effects on B cells should be considered in treatments of neuronal disorders with NMDAR antagonists.

     

Generation of protective immunity against an immunogenic carcinoma requires CD40/CD40L and B7/CD28 interactions but not CD4+ T cells

Interactions between CD40 and CD40L play a central role in the regulation of both humoral and cellular immunity. Recently, interactions between these molecules have also been implicated in the generation of protective cell-mediated tumor immunity. We have generated a tumor model in which a well-understood and clearly immunostimulatory antigen, influenza hemagglutinin has been transfected into the BALB/c-derived, MHC-class-I-positive, B7-deficient murine mammary carcinoma, MT901. In this model, expression of the influenza hemagglutinin antigen does not alter tumorigenicity in naïve but serves as a tumor-rejection target in immunized mice. T-cell-depletion experiments indicate that successful tumor protection can occur following immunization in mice depleted of CD4⁺ but not CD8⁺ T cells, suggesting that tumor protection is largely CD8-mediated and CD4-independent. Interestingly, despite the ability of tumor protection to be generated in the absence of CD4⁺ T cells, effective immunization was clearly dependent on CD40/CD40L as well as CD28/B7 interactions.     

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (>10⁹ cells per patient) from healthy donors as well as cancer patients^1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro⁵. Furthermore they efficiently take up, process and present antigens to T cells^6,7. CD40-B cells were shown to not only prime naíve, but also expand memory T cells^8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)¹¹. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (>90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach¹².Download video file.^{(141M, mp4)}     

Conditional immortalization of human B cells by CD40 ligation

It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function.     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

CD40-mediated activation of NF-kappa B in airway epithelial cells

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.     

Ectopic CD40 ligand expression on B cells triggers intestinal inflammation

Several studies indicate that CD4(+) T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8-15 wk of age. In contrast, CD40L/B TgxCD40(-/-) double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-gamma production. Furthermore, although mice transferred with CD4(+) T cells alone or with both CD4(+) T and B220(+) B cells, but not B220(+) cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220(+) B cells from diseased CD40L/B Tg mice and CD4(+) T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.     

CD40/CD40 homodimers are required for CD40-induced phosphatidylinositol 3-kinase-dependent expression of B7.2 by human B lymphocytes

Preformed CD40/CD40 homodimers were initially observed on human Burkitt lymphoma cell lines, normal B cells, and transitional bladder carcinoma cell lines. However, the nature and the biological relevance of these homodimers have not yet been investigated. In the present study, we demonstrated that Epstein-Barr virus-transformed B cells and CD40-transfected HEK 293 cells constitutively expressed disulfide-linked CD40/CD40 homodimers at low levels. Oligomerization of CD40 leads to a rapid and significant increase in the disulfide-linked CD40/CD40 homodimer formation, a response that could be prevented using a thiol-alkylating agent. Formation of CD40/CD40 homodimers was found to be absolutely required for CD40-mediated activation of phosphatidylinositol 3-kinase, which, in turn regulated B7.2 expression. In contrast, CD40 monomers provided the minimal signal emerging from CD40, activating p38 MAP kinase and inducing homotypic B cell adhesion. CD40/CD40 homodimer formation was totally independent of TRAF1/2/3/5 associations with the threonine at position 254 in the cytoplasmic tail of the CD40 molecules. This finding may be vital to better understanding the molecular mechanisms that govern cell signaling triggered by CD40/CD154 interactions.     

Generation and characterization of IL-2-activated veto cells.

The regulation of in vivo cytolytic response is important in a model of murine graft-vs-host disease induced by the injection of parental splenocytes into unirradiated B6D2F1 recipients. Injection of C57BL/6J spleen cells into B6D2F1 recipients results in an acute form of graft-vs-host disease that is characterized by the presence of CTL and suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells into B6D2F1 recipients results in a chronic form of graft-vs-host disease that is characterized by the lack of in vivo CTL and hyperproduction of Ig and autoantibodies that results in an SLE-like syndrome. One reason for the lack of donor antirecipient CTL after injection of DBA/2J donor cells is that B6D2F1 recipient cells functionally inactivate the donor DBA/2J CTL precursor cells by expressing veto activity. These B6D2F1 veto cells are radiosensitive, inhibited by anti-CD8 antibodies, found primarily in lymph nodes, and were further characterized by testing the response of these inhibitory cells to lymphokines. These studies indicate that IL-2 can potentiate the activity of the veto cells induced in vivo and veto cells with a similar phenotype can be generated by in vitro incubation of naive lymph node cells with IL-2. These cells have been designated as IL-2-activated veto cells or LAV cells. IL-2 did not increase inhibitory activity by increasing the number of CD8+ cells or the number of CD8 molecules on the LAV cell surface but by altering the activation state of the LAV cell. The inhibitory capabilities of antibodies binding various cell surface molecules indicated that CD2 and intercellular adhesion molecule-1 molecules in addition to CD8 molecules played a role in the function of LAV cells.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Soluble forms of CD40 inhibit biologic responses of human B cells.

We have expressed the CD40 surface Ag as both a soluble 28-kDa molecule and a 57-kDa Fc fusion protein containing the human IgG1 Fc region. Soluble CD40 and the Fc fusion protein inhibited the proliferative response of anti-IgM-activated human B cells to the CD40 mAb G28-5. Similarly, G28-5- and IL-4-induced IgE secretion from PBMC depleted of T cells was effectively blocked by both forms of soluble CD40. Although the soluble constructs of CD40 had only a minimal inhibitory effect on IL-4-mediated proliferation of anti-IgM-activated B cells, IL-4-induced soluble CD23 shedding from both PBMC and T cells depleted of PBMC, and IgE secretion from PBMC, were significantly reduced in a concentration-dependent manner when soluble CD40 was present in the culture. The data presented demonstrate that both soluble forms of the CD40 molecule are biologically active, and suggest that the ligand for CD40 is inducible in IL-4-stimulated cultures and that it mediates both shedding of sCD23 and IgE secretion.     

Generation of nondividing high rate Ig-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells.

The capacity of human B cells to differentiate into high rate nondividing antibody-secreting plasma cells was investigated. Highly purified human peripheral blood B cells were stimulated with polyclonal B cell activators in the presence of a variety of recombinant cytokines (IL-2, IL-4, IL-6). Maximal production of Ig of all isotypes was observed when B cells were stimulated with intact T cells that were activated with mAb to the CD3 molecular complex. In these cultures, Ig production continued for more than 16 days. Moreover, differentiation to nondividing high rate Ig-producing cells was induced, as evidenced by a ninefold increase in the amount of Ig produced per Ig-secreting cell and the acquisition of resistance of ongoing Ig secretion to the inhibitor of DNA synthesis, hydroxyurea. To determine whether intact T cells were required for the entire culture period to achieve maximal Ig production, B cells were cultured with activated T cells for various lengths of time, reisolated and cultured with fresh activated T cells or various cytokines, then analyzed for Ig secretion. B cells preactivated for 6 days with anti-CD3-stimulated T cells required contact with intact T cells for continued Ig secretion. However, after 9 days of preactivation, dividing B cells responded maximally to anti-CD3-stimulated T cells, whereas cytokines were able to drive continued IgG secretion by nondividing B cells in the absence of intact T cells. IL-6 alone, or in combination with either IL-2 or IL-4, was the major cytokine driving ongoing Ig secreting by nondividing preactivated B cells. These results suggest that continued clonal expansion of Ig-secreting B cell blasts requires intact anti-CD3-activated T cells, whereas terminal differentiation of B cells into plasma cells after extensive clonal expansion is driven by cytokines, most notably IL-6.     

CD40-activated B cell cancer vaccine improves second clinical remission and survival in privately owned dogs with non-Hodgkin's lymphoma

Cell-based active immunotherapy for cancer is a promising novel strategy, with the first dendritic cell (DC) vaccine achieving regulatory approval for clinical use last year. Manufacturing remains arduous, especially for DC vaccines, and the prospect of using cell-based immunotherapy in the adjuvant setting or in combination with chemotherapy remains largely untested. Here, we used a comparative oncology approach to test the safety and potential efficacy of tumor RNA-loaded, CD40-activated B cells in privately owned dogs presenting with non-Hodgkin's lymphoma (NHL), a clinical scenario that represents not only a major problem in veterinary medicine but also a bona fide spontaneous animal model for the human condition. When administered to NHL dogs in remission after induction chemotherapy, CD40-B cells electroporated ex vivo with autologous tumor RNA safely stimulated immunity in vivo. Although chemotherapy plus CD40-B vaccination did not improve time-to-progression or lymphoma-specific survival compared to dogs treated with chemotherapy alone, vaccination potentiated the effects of salvage therapy and improved the rate of durable second remissions as well as subsequent lymphoma-specific survival following salvage therapy. Several of these relapsed dogs are now long-term survivors and free of disease for more than a year. Overall, these clinical and immunological results suggest that cell-based CD40 cancer vaccination is safe and synergizes with chemotherapy to improve clinical outcome in canine NHL. More broadly, our findings underscore the unique value of clinical investigations in tumor-bearing companion animals.     

Triggering of murine NK cells by CD40 and CD86 (B7-2)

NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab')2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)-/- or CD28-/- mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L-/- and CD28-/- mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.