Dendritic cells (DCs) are special subsets of antigen-presenting cells characterized by their highly potent capacity to activate immunologically naive T cells. Here we report that DCs that are transfected with CD95 ligand (CD95L) cDNA, called 'killer' DCs, deliver death signals, instead of activation signals, to T cells after antigen-specific interaction. Injection of antigen-pulsed killer DCs into mice before sensitization induced antigen-specific immunosuppression. When administered after sensitization, killer DCs suppressed immune responses almost completely after subsequent challenge. Thus, killer DCs represent an entirely new immunomodulatory protocol, which may become directly applicable in preventing and even treating T cell-mediated inflammatory diseases.

Rapamycin (RAP), tacrolimus (FK506), cyclosporin A, and glucocorticoids represent modern and classic immunosuppressive agents being used clinically. Although these agents have distinct molecular mechanisms of action and exhibit different immunoregulatory profiles, their direct influences on Ag presentation processes remain relatively unknown. Here we report quantitative and qualitative differences among the above four immunosuppressants in their impact on Ag-specific, bidirectional interaction between dendritic cells (DC) and CD4+ T cells. In the presence of relevant Ag, bone marrow-derived DC delivered activation signals to CD4+ T cells isolated from the DO11.10 TCR transgenic mice, leading to clonal expansion; secretion of IFN-γ, IL-2, and IL-4; and surface expression of CD69. Conversely, DO11.10 T cells delivered maturation signals to DC, leading to IL-6 and IL-12 production and CD40 up-regulation. FK506 (10−10–10−8 M) and cyclosporin A (10−9–10−7 M) each blocked efficiently and uniformly all the changes resulting from intercellular signaling in both DC→T cell and T cell→DC directions. Dexamethasone (10−9–10−6 M) suppressed all changes, except for CD69 up-regulation, rather incompletely. Remarkably, RAP (10−10–10−8 M) efficiently inhibited DC-induced T cell proliferation and T cell-mediated CD40 up-regulation by DC without abrogating other changes. Interestingly, T cell-independent DC maturation triggered by LPS stimulation was inhibited by dexamethasone, but not by other agents. Our results demonstrate contrasting pharmacological effects of RAP vs calcineurin inhibitors on Ag presentation, thus forming a conceptual framework for rationale-based selection (and combination) of immunosuppressive agents for clinical application.

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-γ or TNF-α plus IFN-γ. LPS- plus IFN-γ-induced dye-coupling was mediated by DC-derived TNF-α. In addition, CpG plus IFN-γ induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-α. LPS- plus IFN-γ-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-γ-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.

IntroductionTransplantation of hematopoietic organs (eg, bone marrow) into immunocompetent hosts induces the activation of alloreactive T cells of both recipient and donor origins, thus causing the host 3 graft reactions expressed clinically as graft rejection and the graft 3 host reactions manifested as graft-versus-host disease (GVHD), respectively. It has become evident that dendritic cells (DCs) play crucial roles in the initiation of both types of alloresponses. 1,2 Host 3 graft reactions are mediated primarily by host T cells that recognize either intact donor major histocompatibility complex (MHC) molecules expressed on donor DCs (direct presentation) or donor-derived peptide antigens being presented by host DCs (indirect presentation). Likewise, donor T cells that recognize intact host MHC molecules on host DCs or host-derived peptide antigens presented by donor DCs function as main effector leukocytes for GVHD. 3,4 Both CD4 ϩ and CD8 ϩ T cells are involved in the above bidirectional allogeneic immune responses, thus providing an additional level of complicity. 5,6 Many therapies have been developed to prevent the onset of allogeneic immune responses after organ transplantation. For example, GVHD, a major complication after allogeneic bone marrow transplantation, has been prevented and treated clinically by conventional immunosuppressive agents and, in selected cases, by ex vivo removal of T cells from the donor inoculum before in vivo infusion. 7,8 GVHD in experimental animals has been successfully treated by more innovative immunomodulatory strategies that are designed to: (1) trigger clonal anergy of effector T cells by blocking costimulatory molecules 9-13 ; (2) control the expansion and differentiation of effector T cells by administration of recombinant cytokines or cytokine inhibitors 7,14-17 ; or (3) interfere with effector T-cell trafficking by blocking adhesion molecules. [18][19][20] Our ultimate goal is to develop a new strategy that is designed to selectively kill alloreactive T cells.Recently, we have created "killer" DCs by introducing the CD95L cDNA into a fully mature DC line (XS106) derived from A/J mice. 21 The resulting CD95L-transduced killer DC clone, when pulsed with ovalbumin (OVA), induced rapid apoptosis of OVA-reactive T cells and prevented the induction of delayedtype hypersensitivity (DTH) responses to OVA in syngeneic A/J mice. Likewise, contact hypersensitivity responses to dinitrofluorobenzene (DNFB) were suppressed almost completely by administration of DNFB-pulsed killer DCs. On the other hand, when administered into allogeneic BALB/c hosts, killer DCs inhibited only partially the host immune responses to A/Jassociated MHC molecules. We have interpreted these results to suggest that killer DCs may deliver apoptotic signals only to the host T cells that recognize allo-MHC molecules via direct presentation. Thus, we have hypothesized that complex alloimmune responses may be suppressed more efficiently if one can Figure 1A). Materials and methods Animals and cell linesAll ...