Otto Majdic

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 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 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 predominately in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion.

B7-H1 (Programmed Death-1 Ligand) on Dendritic Cells Is Involved in the Induction and Maintenance of T Cell Anergy

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.

1,25-Dihydroxyvitamin D3 inhibits dendritic cell differentiation and maturation in vitro

OBJECTIVE Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.

Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains

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.

CD20 antibody (C2B8)-induced apoptosis of lymphoma cells promotes phagocytosis by dendritic cells and cross-priming of CD8+ cytotoxic T cells.

C2B8 (Rituximab, MabThera) is a chimeric mouse/human monoclonal antibody (mAb) directed against the human B cell-restricted cell surface antigen CD20 which is used as an alternative medication in the treatment of B cell non-Hodgkin lymphomas (NHL). Treatment of CD20+ B cells with C2B8 triggers different cell damaging effects including complement-dependent lysis of tumor cells, antibody-dependent cellular cytotoxicity and induction of apoptosis. Dendritic cells (DC) have recently been shown to ingest cell debris and to present associated antigens even on MHC class I molecules, a mechanism called cross-presentation. In this study, we investigated whether C2B8 treatment of lymphoma promotes the induction of CD8+ T cell responses against lymphoma cell-associated antigens via cross-presentation. We used Daudi lymphoma cells as a model system in our studies and could demonstrate, that C2B8-treated Daudi cells undergo apoptosis, are phagocytosed by DC and induce in DC typical features of maturation; among them, the induction of CD83 expression as well as the up-regulation of prominent accessory molecules (CD40, CD86) and MHC molecules. Importantly, upon co-culture of such lymphoma cell-pulsed DC with autologous T cells, we could induce efficient cytotoxic T cell (CTL) responses against Daudi cell-associated antigens. These findings suggest that antibody treatment of tumor cells can, in addition to its direct cell damaging effects, under certain conditions, contribute to an induction of potentially protective cytotoxic T cell responses.

VIL-A1, a monoclonal antibody reactive with common acute lymphatic leukemia cells.

The VIL-A1 monoclonal antibody raised against Reh cells reacts with common acute lymphatic leukemia (CALL) cells but not with normal or malignant B or T lymphocytes. It also shows no binding to normal or malignant myeloid, monocytic or erythroid cells, nor does it react with thrombocytes. The antibody is of IgM class and lyses CALL cells very efficiently in the presence of rabbit but not human complement. Immunoprecipitation experiments followed by SDS-polyacrylamide gel electrophoresis under reducing conditions revealed that VIL-A1 defines a 95,000 mol. wt membrane protein. Approximately 40% of it binds to lens culinaris lectin. Capping experiments showed that the membrane component defined by VIL-A1 co-caps with the one recognized by another recently described monoclonal antibody to CALL cells (J5).

Cell lineage heterogeneity in blast crisis of chronic myeloid leukaemia

Summary Blast cells from 45 patients with chronic myeloid leukaemia in blast crisis (CML‐BC) were immunologically phenotyped with a panel of 26 monoclonal antibodies and studied for terminal deoxynucleotidyl transferase (TdT) content. Out of 45 blast‐populations, 28 showed a myeloid, 14 a lymphoid, two a mixed and one an unclassifiable marker profile.

Cross-Priming of Cytotoxic T Cells Promoted by Apoptosis-Inducing Tumor Cell Reactive Antibodies?

Humanizing xenogenic monoclonal antibodies (MAbs) by genetic engineering has greatly improved their therapeutic utility and efficacy. The chimeric CD20 MAb C2B8 (Rituximab) is a prominent representative of this new generation of therapeutic MAbs and has been proposed as a treatment of choice for recurrent follicular non-Hodgkins lymphomas. Treatment of CD20+ B cells with MAb C2B8 triggers several cell-damaging actions including complement-mediated lysis (CDL), antibody-dependent cellular cytotoxicity (ADCC), and MAb-induced induction of apoptosis. We provide an overview of the most prominent mechanisms underlying the efficacy of antibody treatment. We introduce our concept of cross-priming of cytotoxic T-cell responses promoted by apoptosis incucing antibodies. Treatment of tumor cells with antibodies that are capable of inducing a proapoptotic signal via their cell surface target structure may not only contribute to their direct killing but also may induce cellular responses against the tumor, which may have a long-lasting protective effect. We report, using the example of C2B8 anti-CD20 treatment of lymphoma cells, that MAb C2B8-induced apoptosis of lymphoma cells not only kills these cells but also promotes uptake and cross-presentation of lymphoma cell-derived peptides by antigen-presenting dendritic cells (DC), induces maturation of DC, and allows the generation of specific CTL.

Antigenic analysis of human haemopoietic progenitor cells expressing the growth factor receptor c-kit.

Summary. The cell surface molecule encoded by the proto‐oncogene c‐kit has recently been identified as the receptor for a growth factor variously termed stem cell factor (SCF), mast cell growth factor or steel factor. Using the c‐kit antibody 17F11 we analysed, in triple staining experiments, the surface molecule profile and scatter characteristics of c‐kit+CD34+ human haemopoietic progenitor cells. In 10 normal bone marrow samples we found 19–51% of CD34+ bone marrow progenitor cells to coexpress c‐kit. These c‐kit+CD34+ bone marrow cells turned out to represent a phenotypically heterogeneous population. A considerable proportion coexpressed CD33 (52±23%), and/or CD71 (62 ±26) antigens, marker molecules previously shown to be expressed by committed in vitro colony forming cells but not by their precursors. In line with a relatively differentiated phenotype c‐kit+CD34+ cells also gave rise to on average higher forward and right‐angle light scattering signals. The proportions of CD38 and/or HLA‐D expressing cells were similar in the c‐kit+ and in the c‐kit− subsets of CD34+ progenitor cells. Coexpression of CD19 was found to be less frequent in the c‐kit+ (4 ± 5%) as compared to the c‐kit− (17 ± 14%) fraction of CD34+ cells. CD7+CD34+ bone marrow cells were hardly detectable and their numbers too low to allow further subdivision in c‐kit+ and c‐kit− subsets.

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes.

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.