Ilka Knippertz

NK cell activation in visceral leishmaniasis requires TLR9, myeloid DCs, and IL-12, but is independent of plasmacytoid DCs

Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-α/β and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-α/β. Depletion of pDCs did not impair the activation of NK cells in L. infantum–infected mice. In contrast, L. infantum–induced NK cell cytotoxicity and IFN-γ production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9−/− mice, which lacked IL-12 expression by mDCs, and in IL-12−/− mice, whereas IFN-α/β receptor−/− mice showed only a minor reduction of NK cell IFN-γ expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-γ release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis.

Mild hyperthermia enhances human monocyte-derived dendritic cell functions and offers potential for applications in vaccination strategies

Dendritic cell (DC)-based immunotherapy has been shown to be a promising strategy for anti-cancer therapy. Nevertheless, only a low overall clinical response rate has been observed in vaccinated patients with advanced cancer and therefore methods to improve DC immuno-stimulatory functions are currently under intense investigation. In this respect, we exposed human monocyte-derived DCs to a physiological temperature stress of 40°C for up to 24 h followed by analysis for (i) expression of different heat shock proteins, (ii) survival, (iii) cell surface maturation markers, (iv) cytokine secretion, and (v) migratory capacity. Furthermore, we examined the ability of heat-shocked DCs to prime naïve CD8+ T cells after loading with MelanA peptide, by transfection with MelanA RNA, or by transduction with MelanA by an adenovirus vector. The results clearly indicate that in comparison to control DCs, which remained at 37°C, heat-treated cells revealed no differences concerning the survival rate or their migratory capacity. However, DCs exposed to thermal stress showed a time-dependent enhanced expression of the immune-chaperone heat shock protein 70A and both an up-regulation of co-stimulatory molecules such as CD80, CD83, and CD86 and of the inflammatory cytokine TNF-α. Moreover, these cells had a markedly improved capacity to prime autologous naïve CD8+ T cells in vitro in an antigen-specific manner, independent of the method of antigen-loading. Thus, our strategy of heat treatment of DCs offers a promising means to improve DC functions during immune activation which, as a physical method, facilitates straight-forward applications in clinical DC vaccination protocols.

Insulated hsp70B' promoter: stringent heat-inducible activity in replication-deficient, but not replication-competent adenoviruses.

Key to the realization of gene therapy is the development of efficient and targeted gene transfer vectors. Therapeutic gene transfer by replication‐deficient or more recently by conditionally replication‐competent/oncolytic adenoviruses has shown much promise. For specific applications, however, it will be advantageous to provide vectors that allow for external control of gene expression. The efficient cellular heat shock system in combination with available technology for focused and controlled hyperthermia suggests heat‐regulated transcription control as a promising tool for this purpose.

Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes

The demand for human monocyte-derived dendritic cells (moDCs), as well as for primary human B and T lymphocytes for immunological research purposes has been increased in recent years. Classically, these monocytes are isolated from blood, leukapheresis products or buffy coats of healthy donors by plastic adherence of peripheral blood mononuclear cells (PBMCs), followed by stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, while lymphocytes are usually isolated from the non-adherent fraction (NAF) by magnetic cell sorting. However, donor-blood is a limited resource and not every blood bank offers leukapheresis products or buffy coats for laboratory use. Additionally, a leukapheresis is very expensive and also the generation/isolation of cells is time- and cost-intensive. To overcome some of these obstacles, we evaluated if low-cost leukoreduction system chambers (LRSCs), which arise after routine donor plateletpheresis procedures, and are usually discarded, would be an alternative and appropriate source of PBMCs to generate moDCs and to isolate lymphocytes. By analyzing the number and phenotype of immature and mature dendritic cells (DCs), as well as of B and T lymphocytes derived from LRSCs, we found all cells to be of high quantity and quality. Further investigations on DCs comprising transwell migration assays, allogeneic mixed lymphocyte reactions (MLR), cytokine secretion assays, and cytotoxic T cell induction assays revealed high migratory, as well as stimulatory capacity of these cells. In addition, DCs and T cells were efficiently electroporated with mRNA and showed characteristic cytokine production after co-culture, demonstrating LRSCs as an efficient, valid, and economic source for generation of moDCs and lymphocytes for research purposes.

Multiple Interferon Regulatory Factor and NF-κB Sites Cooperate in Mediating Cell-Type- and Maturation-Specific Activation of the Human CD83 Promoter in Dendritic Cells

ABSTRACT CD83 is one of the best-known surface markers for fully mature dendritic cells (mature DCs), and its cell-type- and maturation-specific regulation makes the CD83 promoter an interesting tool for the genetic modulation of DCs. To determine the mechanisms regulating this DC- and maturation-specific CD83 expression, chromatin immunoprecipitation (ChIP)-on-chip microarray, biocomputational, reporter, electrophoretic mobility shift assay (EMSA), and ChIP analyses were performed. These studies led to the identification of a ternary transcriptional activation complex composed of an upstream regulatory element, a minimal promoter, and an enhancer, which have not been reported in this arrangement for any other gene so far. Notably, these DNA regions contain a complex framework of interferon regulatory factor (IRF)- and NF-κB transcription factor-binding sites mediating their arrangement. Mutation of any of the IRF-binding sites resulted in a significant loss of promoter activity, whereas overexpression of NF-κB transcription factors clearly enhanced transcription. We identified IRF-1, IRF-2, IRF-5, p50, p65, and cRel to be involved in regulating maturation-specific CD83 expression in DCs. Therefore, the characterization of this promoter complex not only contributes to the knowledge of DC-specific gene regulation but also suggests the involvement of a transcriptional module with binding sites separated into distinct regions in transcriptional activation as well as cell-type- and maturation-specific transcriptional targeting of DCs.

Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo

The CD83 molecule (CD83) is a well-known surface marker present on mature dendritic cells (mDC). In this study, we show that CD83 is also expressed on a subset of T cells which mediate regulatory T cell (Treg)-like suppressor functions in vitro and in vivo. Treg-associated molecules including CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-induced TNFR family-related gene (GITR), Helios and neuropilin-1 (NRP-1) as well as forkhead box protein 3 (FOXP3) were specifically expressed by these CD83(+) T cells. In contrast, CD83(-) T cells showed a naive T cell phenotype with effector T cell properties upon activation. Noteworthy, CD83(-) T cells were not able to upregulate CD83 despite activation. Furthermore, CD83(+) T cells suppressed the proliferation and inflammatory cytokine release of CD83(-) T cells in vitro. Strikingly, stimulated CD83(+) T cells released soluble CD83 (sCD83), which has been reported to possess immunosuppressive properties. In vivo, using the murine transfer colitis model we could show that CD83(+) T cells were able to suppress colitis symptoms while CD83(-) T cells possessed effector functions. In addition, this CD83 expression is also conserved on expanded human Treg. Thus, from these studies we conclude that CD83(+) T cells share important features with regulatory T cells, identifying CD83 as a novel lineage marker to discriminate between different T cell populations.

Selection of adenovirus-specific and Epstein-Barr virus–specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)–compliant conditions

BACKGROUND AIMS Despite antiviral drug therapies, human adenovirus (HAdV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections still contribute substantially to transplant-related death of patients after hematopoietic stem cell transplantation. Earlier clinical studies demonstrated successful adoptive transfer of magnetically selected CMV-specific T cells via removable, and thus Good Manufacturing Practice-compliant, major histocompatibility class I streptamers. Thus, the primary focus of the present study was the selection of HAdV-streptamer+ T cells, although in three experiments, EBV-streptamer+ T cells were also selected. METHODS Cells from leukaphereses of healthy donors were prepared in large (1-6 × 10(9)) and small (25 × 10(6)) cell batches. Whereas the larger batch was directly labeled with streptamers to select HAdV- and/or EBV-specific T cells (large-scale), the smaller batch was used to generate in vitro virus-specific T-cell lines before streptamer labeling for streptamer selection (small-scale). Isolation of HAdV- and/or EBV-specific T cells was performed with the use of the CliniMACS device. RESULTS The purity of HAdV- and EBV-streptamer+ T cells among CD3+ cells, obtained from large-scale selection, was up to 6.7% and 44%, respectively. If HAdV- and EBV-streptamers were applied simultaneously, the purity of antigen-specific T cells reached up to 50.7%. A further increase in purity reaching up to 98% was achieved by small-scale selection of HAdV-specific T cells. All final products fulfilled the microbiological and chemical release criteria. Interferon-γ-response indicating functional activity was seen in 6 of 9 HAdV and 2 of 3 EBV large-scale selections and in 2 of 3 HAdV small-scale selections. CONCLUSIONS HAdV-streptamers were shown to be clinically feasible for few patients after the large-scale approach but for larger patient numbers if combined with EBV-streptamers or after the small-scale approach.

Human adenovirus-specific γ/δ and CD8+ T cells generated by T-cell receptor transfection to treat adenovirus infection after allogeneic stem cell transplantation.

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/β T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/β T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/β T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/β T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.

Triggering of NF-κB in cytokine-matured human DCs generates superior DCs for T-cell priming in cancer immunotherapy

Understanding the signaling that governs the immunogenicity of human dendritic cells (DCs) is a prerequisite for improving DC‐based therapeutic vaccination strategies, in which the ability of DCs to induce robust and lasting Ag‐specific CTL responses is of critical importance. Cytokine‐matured DCs are regularly used, but to induce memory‐type CTLs, they require additional activation stimuli, such as CD4+ T‐cell help or TLR activation. One common denominator of these stimuli is the activation of NF‐κB. Here, we show that human monocyte‐derived, cytokine cocktail‐matured DCs transfected with constitutively active mutants of IκB kinases (caIKKs) by mRNA electroporation, further upregulated maturation markers, and secreted enhanced amounts of cytokines, including IL‐12p70, which was produced for more than 48 h after transfection. Most importantly, cytotoxic T cells induced by caIKK‐transfected DCs combined high CD27 expression, indicating a more memory‐like phenotype, and a markedly enhanced secondary expandability with a high lytic capacity. In contrast, CTLs primed and expanded with unmodified cytokine cocktail‐matured DCs did not maintain their proliferative capacity upon repetitive stimulations. We hypothesize that “designer” DCs expressing constitutively active IκB kinases will prove highly immunogenic also in vivo and possibly emerge as a new strategy to improve the clinical efficacy of therapeutic vaccinations against cancer and other chronic diseases.

Human dendritic cells efficiently phagocytose adenoviral oncolysate but require additional stimulation to mature

Oncolytic adenoviruses are emerging agents for treatment of cancer by tumor‐restricted virus infection and cell lysis. Clinical trials have shown that oncolytic adenoviruses are well tolerated in patients but also that their antitumor activity needs improvement. A promising strategy toward this end is to trigger systemic and prolonged antitumor immunity by adenoviral oncolysis. Antitumor immune activation depends in large part on antigen presentation and T cell activation by dendritic cells (DCs). Thus, it is likely that the interaction of lysed tumor cells with DCs is a key determinant of such “oncolytic vaccination.” Our study reveals that human DCs effectively phagocytose melanoma cells at late stages of oncolytic adenovirus infection, when the cells die showing preferentially features of necrotic cell death. Maturation, migration toward CCL19 and T cell stimulatory capacity of DCs, crucial steps for immune induction, were, however, not induced by phagocytosis of oncolysate, but could be triggered by a cytokine maturation cocktail. Therefore, oncolytic adenoviruses and adenoviral oncolysate did not block DC maturation, which is in contrast to reports for other oncolytic viruses. These results represent a rationale for inserting immunostimulatory genes into oncolytic adenovirus genomes to assure critical DC maturation. Indeed, we report here that adenoviral transduction of melanoma cells with CD40L during oncolysis triggers the maturation of human DCs with T cell stimulatory capacity similar to DCs matured by cytokines. We conclude that triggering and shaping DC‐induced antitumor immunity by oncolytic adenoviruses “armed” with immunostimulatory genes holds promise for improving the therapeutic outcome of viral oncolysis in patients.