Cytokine-Like 1 Is a Novel Proangiogenic Factor Secreted by and Mediating Functions of Endothelial Progenitor Cells.

RATIONALE: Endothelial colony forming cells (ECFCs) or late blood outgrowth endothelial cells can be isolated from human cord or peripheral blood, display properties of endothelial progenitors, home into ischemic tissues and support neovascularization in ischemic disease models. OBJECTIVE: To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation. METHODS AND RESULTS: We show by transcriptomic analysis that ECFCs are distinguished from endothelial cells of the vessel wall by production of high amounts of CYTL1. Modulation of expression demonstrates that the factor confers increased angiogenic sprouting capabilities to ECFCs and can also trigger sprouting of mature endothelial cells. The data further display that CYTL1 can be induced by hypoxia and that it functions largely independent of VEGF-A (vascular endothelial growth factor-A). By recombinant production of CYTL1 we confirm that the peptide is indeed a strong proangiogenic factor and induces sprouting in cellular assays and functional vessel formation in animal models comparable to VEGF-A. Mass spectroscopy corroborates that CYTL1 is specifically O-glycosylated on 2 neighboring threonines in the C-terminal part and this modification is important for its proangiogenic bioactivity. Further analyses show that the factor does not upregulate proinflammatory genes and strongly induces several metallothionein genes encoding anti-inflammatory and antiapoptotic proteins. CONCLUSIONS: We conclude that CYTL1 can mediate proangiogenic functions ascribed to endothelial progenitors such as ECFCs in vivo and may be a candidate to support vessel formation and tissue regeneration in ischemic pathologies.

Lactoferrin is a natural inhibitor of plasminogen activation.

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

Quantification of human diamine oxidase.

OBJECTIVES: Diamine oxidase (DAO) is essential for extracellular degradation of histamine. For decades activity assays with inherent limitations were used to quantify the relative amounts of DAO. No reference DAO standard is available. Absolute DAO amounts cannot be determined. Controversy exists, whether DAO is circulating or not in non-pregnant individuals. The role of DAO as biomarker in various diseases is ambiguous. It is not clear, whether precise quantification of human DAO antigen using commercially available enzyme-linked immunosorbent assays (ELISAs) is possible. The objective was to develop a precise and robust ELISA to quantify DAO in various biological fluids. DESIGN AND METHODS: A research prototype ELISA was established using a mouse monoclonal antibody for capturing and a polyclonal rabbit serum IgG fraction for the detection of human DAO. The limit of blank (LoB), limit of detection (LoD) and estimated limit of quantification (eLoQ) and normal DAO concentrations in serum and plasma were determined. RESULTS: The LoB, LoD and eLoQ derived from 42 standard curves are 0.27, 0.48 and 0.7ng/mL respectively. The detection range using the LoD as the lower and the highest DAO standard as the upper boundary is 0.5 to 450ng/mL. Serum and plasma mean/median concentrations are between 0.5 and 1.5ng/mL in healthy volunteers (n=58) and mastocytosis patients (n=19) and plateau at approximately 145ng/mL (n=16) during pregnancy. Accurate quantification was not influenced by heparin (DAO is a heparin-binding protein), lipaemic or hemolytic serum. The measured DAO antigen concentrations are in close agreement with published enzymatic activity data using radioactive putrescine as substrate. CONCLUSIONS: This research prototype ELISA is able to reliably and accurately quantify human DAO in different biological fluids. The potential of DAO as biomarker in various diseases can be evaluated.

Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses.

The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.

Human Langerhans cells control Th cells via programmed death-ligand 1 in response to bacterial stimuli and nickel-induced contact allergy.

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4(+)T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6(+)/CCR4(+) T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6(+) cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6(+)/CCR4(+) cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.

The effects of Cyclosporine A and azathioprine on human T cells activated by different costimulatory signals.

Immunosuppression is an important treatment modality in transplantation and human diseases that are associated with aberrant T cell activation. There are considerable differences regarding the cellular processes targeted by the immunosuppressive drugs that are in clinical use. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like cyclosporine A (CsA) specifically target signaling pathways in T cells. Since the outcome of T cell responses critically depends on the quality and strength of costimulatory signals, this study has addressed the interplay between costimulation and the immunosuppressive agents CsA and Aza during the in vitro activation of human T cells. We used an experimental system that allows analyzing T cells activated in the presence of selected costimulatory ligands to study T cells stimulated via CD28, CD2, LFA-1, ICOS or 4-1BB. The mean inhibitory concentrations (IC(50)) for Aza and CsA were determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC(50) for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation.

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1.

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.

Costimulatory signals potently modulate the T cell inhibitory capacity of the therapeutic CD11a antibody Efalizumab.

The therapeutic CD11a antibody Efalizumab interferes with psoriasis pathogenesis by blocking T cell activation and migration. We have performed a detailed analysis on its effects during the activation of human T cells and found that the capability of Efalizumab to inhibit proliferation and cytokine production of T cells critically depends on the quality and quantity of costimulatory signals. Efalizumab potently inhibited the proliferation and cytokine production of human T cells costimulated via ICOS, OX40, CD27 or 4-1BB, but did not significantly inhibit T cells that received stimuli via CD2 or CD28. The capacity of CD2 and CD28 signals to interfere with the T cell inhibitory effects of Efalizumab was also observed upon stimulation of T cells with allogeneic DC. Furthermore, studies with T cells from psoriasis patients indicated that Efalizumab therapy induces inhibition of T cell responses that can be reverted by CD2 or CD28 signals.

T cell stimulator cells, an efficient and versatile cellular system to assess the role of costimulatory ligands in the activation of human T cells.

It is well established that full activation of T cells requires the interaction of the TCR complex with the peptide-MHC complex (Signal 1) and additional signals (Signal 2). These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with activating receptors on T cells. In addition, T cell responses are negatively regulated by inhibitory costimulatory pathways. Since professional antigen presenting cells (APC) harbour a plethora of stimulating and inhibitory surface molecules, the contribution of individual costimulatory molecules is difficult to assess on these cells. We have developed a system of stimulator cells that can give signal 1 to human T cells via a membrane bound anti-CD3 antibody fragment. By expressing human costimulatory ligands on these cells, their role in T cell activation processes can readily be analyzed. We demonstrate that T cell stimulator cells are excellent tools to study various aspects of human T cell costimulation, including the effects of immunomodulatory drugs or how costimulatory signals contribute to the in vitro expansion of T cells. T cell stimulator cells are especially suited for the functional evaluation of ligands that are implicated in costimulatory processes. In this study we have evaluated the role of the CD2 family member CD150 (SLAM) and the TNF family member TL1A (TNFSF15) in the activation of human T cells. Whereas our results do not point to a significant role of CD150 in T cell activation we found TL1A to potently costimulate human T cells. Taken together our results demonstrate that T cell stimulator cells are excellent tools to study various aspects of costimulatory processes.

Allogeneic disparities in immunoglobulin-like transcript 5 induce potent antibody responses in hematopoietic stem cell transplant recipients.

In hematopoietic stem cell transplant (HSCT) recipients, the recognition of polymorphic antigens by the donor-derived immune system is an important mechanism underlying both graft-versus-host disease and graft-versus-leukemia (GVL) effect. Here we show that a subset of HSCT recipients (13.9%, n = 108) have antibodies directed to surface molecules of dendritic cells. We have used one such serum in conjunction with retroviral expression cloning to identify the highly polymorphic surface molecule immunoglobulin-like transcript 5 (ILT5) as one of the targets of dendritic cell-reactive antibodies. ILT5 reactive antibodies were found in 5.4% of HSCT patients but not in solid organ transplantation recipients, patients with collagen diseases, multiparous women, or polytransfused or healthy persons. We show that ILT5-specific antibodies can mediate killing of ILT5-bearing cells and furthermore demonstrate ILT5 expression in some leukemic cells, indicating that it might be a target for GVL effects. Thus, our results represent the first description of potent allogeneic antibody responses to a non-major histocompatibility complex cell surface molecule in hematopoietic stem cell transplanted patients and warrant further studies to elucidate the role of antibodies to polymorphic cell surface molecules in GVL and graft-versus-host responses.

B7-H3 is a potent inhibitor of human T-cell activation: No evidence for B7-H3 and TREML2 interaction.

B7-H3 belongs to the B7 superfamily, a group of molecules that costimulate or down-modulate 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.

The capacity of the TNF family members 4-1BBL, OX40L, CD70, GITRL, CD30L and LIGHT to costimulate human T cells.

Activating signals generated by members of the tumour necrosis factor receptor superfamily upon interaction with their cognate ligands play important roles in T-cell responses. Members of the tumour necrosis factor family namely 4-1BBL, OX40L, CD70, GITRL, LIGHT and CD30L have been described to function as costimulatory molecules by binding such receptors on T cells. Using our recently described system of T-cell stimulator cells we have performed the first study where all these molecules have been assessed and compared regarding their capacity to costimulate proliferation and cytokine production of human T cells. 4-1BBL, which we found to be the most potent molecule in this group, was able to mediate sustained activation and proliferation of human T cells. OX40L and CD70 were also strong inducers of T-cell proliferation, whereas the costimulatory capacity of human GITRL was significantly lower. Importantly CD30L and LIGHT consistently failed to act costimulatory on human T cells, and we therefore suggest that these molecules might be functionally distinct from the costimulatory members of this family.

The cytoplasmic tail of CD45 is released from activated phagocytes and can act as an inhibitory messenger for T cells.

CD45 is the prototypic transmembrane protein tyrosine phosphatase (PTP), which is expressed on all nucleated hematopoietic cells and plays a central role in the integration of environmental signals into immune cell responses. Here we report an alternative function for the intracellular domain of CD45. We dis-covered that CD45 is sequentially cleaved by serine/metalloproteinases and gamma-secretases during activation of human monocytes and granulocytes by fungal stimuli or phorbol 12-myristate 13-acetate but not by other microbial stimuli. Proteolytic processing of CD45 occurred upon activation of monocytes or granulocytes but not of T cells, B cells, or dendritic cells and resulted in a 95-kDa fragment of the cytoplasmic tail of CD45 (ct-CD45). ct-CD45 was released from monocytes and granulocytes upon activation-induced cell death. Binding studies with ct-CD45 revealed a counter-receptor on preactivated T cells. Moreover, T-cell proliferation induced by dendritic cells or CD3 antibodies was inhibited in the presence of ct-CD45. Taken together, the results of our study demonstrate that fragments of the intracellular domain of CD45 from human phagocytes can function as intercellular regulators of T-cell activation.

Oxidized phospholipids induce anergy in human peripheral blood T cells.

Lipids are key regulators of immune responses. In this study we investigated the direct impact of oxidized phospholipids (ox-PL) on T cell activation and function. We could demonstrate that ox-PL strongly inhibit proliferation of purified human T cells induced with anti-CD3/CD28 or anti-CD3/CD63 mAb, whereas proliferation of naive T cells from human cord blood was not affected by ox-PL. Unoxidized phospholipids showed no such effect. Inhibition of T cell proliferation by ox-PL was not due to cell death. Moreover, T cell proliferation triggered by PMA/ionomycin activation was not diminished by ox-PL. T cells activated in the presence of ox-PL produced and released low amounts of IFN-gamma and IL-2, whereas IL-4 was only slightly diminished. Ox-PL prevented the expression of de novo synthesized activation markers (CD25, MHC class II) but not expression of CD63 or CD69. We further observed that T cells stimulated in the presence of ox-PL are poorly cytotoxic T cells. Most importantly, T cells activated in the presence of ox-PL failed to proliferate in response to restimulation. This hypo-proliferative state was accompanied with an up-regulation of early growth response gene 3 and Casitas B-lineage lymphoma protein B. Taken together, our results demonstrate that ox-PL are potent and specific regulators of T cell activation and function.

No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation.

The B7 family member programmed-death-1-ligand 2 (PD-L2/B7-DC) is a ligand for programmed-death-receptor 1 (PD-1), a receptor involved in negative regulation of T cell activation. Several independent studies have reported that PD-L2, however, can also potently costimulate murine T cells via an additional yet unidentified receptor. In this study, we evaluated the contribution of PD-L2 to the activation of human T cells using a novel system of engineered T cell stimulators that expresses membrane-bound anti-CD3 antibodies. Analyzing early activation markers, cytokine production and proliferation, we found PD-L2 to consistently inhibit T cell activation. PD-L2 inhibition affected CD4+ and CD8+ T cells and was not abrogated by costimulation via CD28. Blocking PD-1 reverted the inhibitory effect of PD-L2, demonstrating involvement of this pathway. In human T cells, we found no evidence for any of the costimulatory effects described for PD-L2 in murine systems. In line with our functional data that do not point to stimulatory PD-L2-ligands, we show that binding of PD-L2-immunoglobulin to activated human T cells is abrogated by PD-1 antibodies. Our results demonstrate that PD-L2 negatively regulates human T cell activation and thus might be a candidate molecule for immunotherapeutic approaches aimed to attenuate pathological immune responses.

Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro.

Human tumors frequently escape immune destruction, despite the presence of cytotoxic 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 tumor-specific 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 predominantly in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion.

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression.

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.

Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40.

Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cytokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation.

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells.

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN-gamma for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN-gamma for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.

CD63 as an activation-linked T cell costimulatory element.

Dendritic cells (DC) are unique in their capacity to either stimulate or regulate T cells, and receptor/ligand pairs on DC and T cells are critically involved in this process. In this study we present such a molecule, which was discovered by us when analyzing the functional effects of an anti-DC mAb. This mAb, 11C9, reacted strongly with DC, but only minimally with lymphocytes. In MLR it constantly reduced DC-induced T cell activation. Therefore, we assumed that mAb 11C9 primarily exerts its functions by binding to a DC-structure. This does not seem to be the case, however. Preincubation of DC with mAb 11C9 before adding T cells had no inhibitory effect on T cell responses. Retroviral expression cloning identified the 11C9 Ag as CD63. This lysosomal-associated membrane protein (LAMP-3), is only minimally expressed on resting T cells but can, as we show, quickly shift to the surface upon stimulation. Cross-linkage of that structure together with TCR-triggering induces strong T cell activation. CD63 on T cells thus represents an alternative target for mAb 11C9 with its binding to activated T cells rather than DC being responsible for the observed functional effects. This efficient CD63-mediated costimulation of T cells is characterized by pronounced induction of proliferation, strong IL-2 production and compared with CD28 enhanced T cell responsiveness to restimulation. Particularly in this latter quality CD63 clearly surpasses several other CD28-independent costimulatory pathways previously described. CD63 thus represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion.