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.

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...

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.Key words: Cell activation . Costimulation . Costimulatory molecules . T cells IntroductionEfficient T-cell activation requires two different signals, provided by MHC-TCR interaction (signal 1) and additional receptorligand interactions that give a costimulatory signal (signal 2) to T cells [1]. Interaction of the immunoglobulin superfamily members CD80/86-CD28 and ICOSL-ICOS can efficiently costimulate proliferation, cytokine production and effector cell generation of T cells [2]. Another group of costimulatory molecules is comprised by members of the tumour necrosis factor receptor (TNFR) superfamily, which can costimulate TCR signals upon interaction with their cognate ligands, members of TNF super-family. Costimulation of T-cell activation has been reported for several members of the TNFR/TNF superfamilies namely for 4-1BB-4-1BBL, OX40-OX40L, CD27-CD70, GITR-GITRL, herpes virus entry mediator (HVEM)-LIGHT and 2678CD30-CD30L [3,4]. Interaction of these TNF family ligands with their receptors leads to recruitment of TNFR-associated factors (TRAF), which initiate signalling cascades that result in T-cell activation. All these TNFR can recruit TRAF2 but there are differences between these molecules in the recruitment of other TRAF proteins [5].Although there is extensive literature on costimulatory pathways involving human 4-1BBL, OX40L and CD70, few reports have described costimulatory functions for human LIGHT and CD30L [6,7]. Signals transduced by members of the TNFR family are regarded as especially important for survival, expansion and effector function of T cells that have initially received activating signals via the CD28 receptor [4]. Consequently a number of reports have addressed the role of members of the TNFR/TNF families on antigen-experienced T cells and have demonstrated potent and important costimulatory functions for these molecules on virus-specific human T cells [8][9][10][11]. Thus blocking or ...

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.

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. (Blood.

Tamm-Horsfall protein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in urine. An important role for THP in antibacterial host defense but also in inflammatory disorders of the urogenital tract has been suggested. In line with this, THP has been shown recently to potently activate macrophages and dendritic cells (DC) via the toll-like receptor 4 (TLR4) pathway. We show here that THP interacts specifically with surface structures on DC and provides evidence that they are distinct from TLR4. Using retroviral expression cloning, we have identified one such receptor as the scavenger receptor (SR) expressed by endothelial cells I (SREC-I). In addition, we found that two other receptors for acetylated low-density lipoprotein (AcLDL), namely scavenger receptors AI (SR-AI) and Cla-1 (SR-BI), also serve as receptors for THP. SREC-I/THP interaction is of high affinity (16.8+/-6.8 nM), whereas Cla-1 and SR-AI have lower affinities for THP (396 nM+/-114 nM and 802 nM+/-157 nM, respectively). The interaction of THP with these molecules is fully blocked by AcLDL. However, AcLDL only partially blocks binding of THP to DC, and a series of experiments did not support a role in DC activation for SR interacting with THP and AcLDL. Thus, our data point to the existence of additional receptors for THP, which mediate TLR4-dependent DC activation. Interaction and up-take of THP by SR might play an important role in local host defense and could contribute to inflammatory kidney diseases associated with THP-specific antibody responses.