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Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.

Epstein-Barr virus (EBV) latently infects and immortalizes B lymphocytes and causes lymphoproliferative malignancies. We show here that the EBV nuclear antigen EBNA2 induces expression of the 2 chains of the interleukin-18 receptor (IL-18R) in Burkitt lymphoma (BL) cell lines and in nontransformed B cells. Activation of IL-18R expression by EBNA2 is independent of its interaction with the transcriptional repressor RBPJκ. It occurs in the absence of any other viral protein but requires de novo synthesis of cellular proteins. IL-18R induction is a highly specific function of EBNA2, because neither other EBV latent proteins nor the cellular proteins c-myc or Notch can exert this effect. Using cDNA microarray expression profiling, we find that the IL-18 receptor expressed in EBV-infected BL cells has signaling capacity, because IL-18 significantly modified gene expression. We report that EBNA2 expression is associated with IL-18R expression in vivo in EBV-positive B-lymphomas from AIDS patients. (Blood. 2005;105:1632-1639)

Abstract 3004 Clinical studies exploiting the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation (HSCT) have provided promising results. It is known that NK cells are a heterogeneous population and can be divided into functionally distinct NK cell subpopulations. Murine NK cells can be separated along their expression of CD27 and CD11b and CD117 (c-kit). However, the functional relevance of distinct NK cell subsets in graft-versus-host-disease (GVHD) has not been investigated in detail so far. We have established different protocols for ex vivo isolation and expansion of murine NK cell subpopulations. These NK subsets were further analyzed in vitro and in vivo in an allogeneic murine GVHD model. Here we report on different genomic, phenotypic and functional properties of 4 NK cell subsets. Our data clearly demonstrate that CD27+ NK cells revealed the highest IFN-g production upon coculture with tumor cells and/or IL-2. Interestingly, the CD11b+ NK cells express multiple genes of cytotoxic pathways and develop the highest cytotoxic capacity towards tumor cells. We observed up to 60% tumor lysis by CD27- CD11b+ NK cells compared to 40–45% by CD27+ CD11b+, about 25% by CD27+ CD11b- and 10% by c-kit+ CD11b- NK cells at an effector-target ratio of 5:1, respectively. Furthermore, the CD11b+ NK cell subset significantly reduced T cell proliferation induced by allogeneic dendritic cells in mixed lymphocytes reactions. Next, we analyzed the migratory capacity and tissue-specific homing of FACS-sorted NK cell subsets by adoptive transfer of congeneic CD45.1+ and Luc+ NK cell subpopulations in autologous and allogeneic bone marrow transplantation. Of interest, FACS analysis and in vivo imaging showed that CD11b+ NK cells migrated to peripheral GVHD target organs, whereas CD27+ NK cells preferentially homed to the bone marrow. Finally, this study addressed for the first time the role of distinct NK cell subpopulations in the development of GVHD in a fully MHC mismatched HSCT mouse model. Importantly, we identified the CD11b+ NK cell population as the NK cell subset that significantly diminished GVHD. In vivo imaging of Luc+CD11b+ NK cells revealed that this subset migrates to the colonic tissue to prevent development of GVHD colitis as shown by colonoscopy. In summary, our comparative study outlines that only CD11b+ NK cells, migrating to the peripheral GVHD target organs and providing the most efficient cytolytic capacity directed against allogeneic dendritic cells, protect against GVHD. These new insights are highly relevant for the selection of optimal NK cell subsets in the field of cellular immunotherapy. Disclosures: No relevant conflicts of interest to declare.