In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-γ production.

SummaryThe glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells, uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix, uPA-R is also involved in induction of ceU adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cdlular events. By size ffactionation of monocyte lysate and af~nity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fY ", p53/56 lyn, p58/64 hck, and p59f~ as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning 32-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as weU as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for call migration.

Human tumors frequently escape immune destruction, despite the presence of cyototoxic T cells (CTL) recognizing tumor-associated antigens (TAA). We have previously shown that programmed death ligand-1 (PD-L1), a recently identified ligand of the B7 superfamily, is expressed on murine tumors and can inhibit antitumor immune responses. To evaluate the clinical relevance of our animal model findings, we examined human tumors and tumorspecific T cells. We found PD-L1 to be constitutively expressed on human renal cell carcinoma (RCC) cell lines and upregulated on human melanoma cell lines upon exposure to interferon-gamma. Similarly, we found binding of anti-PD-L1 monoclonal antibody (mAb) on frozen sections from RCC and melanomas, but not on normal tissues. The corresponding inhibitory receptor of PD-L1, PD-1, revealed a higher expression on tumor-infiltrating lymphocytes than on peripheral blood lymphocytes (PBL) from melanoma patients upon specific antigen stimulation. Stimulation of PBL from healthy donors with peptide-loaded dendritic cells in the presence of anti-PD-L1 mAb altered neither the total T cell numbers after expansion, nor the percentage of peptide-specific CTL, when providing a T cell help by addition of cytokines. However, when stimulating TAA-specific CTL and T helper cells with Ag-pulsed dendritic cells in the absence of exogenous cytokines, PD-L1 blockade increased the cytokine production. Similar to the data achieved in the murine system, the blockade of PD-L1 on human tumors resulted in enhanced cytolytic activity of TAA-specific CTLs and cytokine production of TAA-specific T helper cells when interacting directly with the tumor. In summary, our data suggest that PD-L1/PD-1 interactions negatively regulate T cell effector functions predominately in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion. ' 2006 Wiley-Liss, Inc.Key words: RCC; PD-L1; PD-1; B7-H1; melanoma Although the prognosis for patients with advanced solid tumors still remains poor, 1 a number of promising approaches, such as cancer immunotherapy, have been developed over the past decade. However, it is still an unsolved question why existing tumor-specific T cells in cancer patients fail to effectively prevent tumor progression. Inefficient T cell stimulation, either due to the absence of efficient antigen-presentation and costimulation or due to the presence of coinhibitory molecules, may contribute to this phenomenon.Data from animal models showing that CD8 1 T cells can lead to regression and rejection of solid tumors, and metastases 2 were reproduced with human cancer patients in clinical studies only partially. [3][4][5][6] Despite the fact that tumor-specific T cells can be expanded and transferred effectively and thus survive and localize into tumors, 7,8 tumor growth is not always controlled.It appears that the microenvironment of cancers protects tumor cells from immune destruction. 9,10 Increasing appreciation of costimulation (via CD28 or ICOS) and coinhibition (...

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B7-H3 belongs to the B7 superfamily, a group of molecules that costimulate or downmodulate T-cell responses. Although it was shown that B7-H3 could inhibit T-cell responses, several studies -most of them performed in murine systems -found B7-H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7-H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T-cell subsets. We show that B7-H3 does not costimulate human T cells. In the presence of strong activating signals, B7-H3 potently and consistently down-modulated human T-cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre-activated T cells. Furthermore, we demonstrate that B7-H3-T-cell interaction is characterised by an early suppression of IL-2 and that T-cell inhibition can be reverted by exogenous IL-2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT-2) has been recently described as costimulatory receptor of murine B7-H3 we have extensively analysed interaction of human B7-H3 with TREML2/TLT-2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7-H3. Key words: Costimulatory molecules . Immune regulation . T cells Supporting Information available online IntroductionFor fine-tuning the immune response, several costimulatory and coinhibitory signals are needed, in addition to signal 1 provided via the peptide-MHC/TCR-complex interaction. CD80 (B7-1) and CD86 (B7-2) serve as primary costimulatory ligands. Recently, additional members of the B7 family -the so-called B7 homologshave been identified [1]. The functional role of several of these B7 homologs is still controversially discussed. One of these molecules is B7-H3, which was originally described as a potent costimulatory molecule and inducer of IFN-g in human T cells [2]. In contrast, Ling et al. found human B7-H3 to strongly down-regulate T-cell proliferation and cytokine production [3]. It was suggested that the presence of two B7-H3 receptors with different functions could explain these divergent results [3]. Recent data that showed opposing effects of B7-H3 on resting and cytokine-activated T cells as well as contradicting results on the function of murine B7-H3 would also be in support for such a constellation [4][5][6][7]. Such receptor molecules could either be differentially regulated on T cells or be expressed on different T-cell subsets. Depending on the experimental system used the effects of the costimulatory or the inhibitory receptor could prevail and explain the discrepancies in different studies.Here, we have specifically addressed a potential functional dualism of B7-H3 by studying B7-H3 effects under varying experimental conditions as well as on different subsets of human T cells. Our results point to a potent and consistent inhibitory role of h...

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-α, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.

Pure populations of human basophilic granulocytes were obtained from chronic myeloid leukemia (CML) blood by negative selection using a mixture of monoclonal antibodies and complement. '25Iradiolabeled recombinant human interleukin 3 (rhIL-3) bound to purified basophils in a specific manner. Quantitative binding studies and Scatchard plot analyses performed on samples from two donors revealed the presence of a single class of high-affinity IL-3 binding sites (500 and 2100 sites per cell; dissociation constant at equilibrium, 230 and 160 pmol/liter, respectively). Purified CML basophils maintained in suspension in the presence of rhIL-3 (100 units/ml) incorporated up to 12 times more [Hlthymidine than basophils in control cultures. Furthermore, after preincubation in vitro with rhIL-3 (100 units/mi) for 30 min, normal blood basophils released 2-to 3-fold more histamine than basophils pretreated with control medium when exposed to various concentrations ofan anti-IgE antibody. Together, these results show that rhIL-3 binds to a specific receptor on blood basophils and is a regulator of basophil function.

Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.