Ulrich Grigoleit

Aspergillus fumigatus antigens activate innate immune cells via toll-like receptors 2 and 4

Invasive aspergillosis (IA) is a leading cause of mortality in haematological patients. Appropriate activation of the innate immune system is crucial for the successful clearance of IA. Therefore, we studied the Aspergillus fumigatus‐mediated activation of human granulocytes and monocyte‐derived immature dendritic cells (DCs), as well as murine bone marrow‐derived DCs (BMDCs) from wild type, toll‐like receptor (TLR)4‐deficient, TLR2 knockout, and TLR2/TLR4 double deficient mice. Aspergillus fumigatus antigens induced the activation and maturation of immature DCs as characterized by CD83 expression, upregulation of major histocompatibility complex and co‐stimulatory molecules. Moreover, fungal antigens enhanced the phagocytosis and production of interleukin (IL)‐8 in granulocytes. The release of IL‐12 by BMDCs in response to A. fumigatus antigens was dependent on the expression of TLR2, whereas the release of IL‐6 was dependent on the expression of functional TLR4 molecules. The protein precipitate of A. fumigatus supernatant provided strong stimulation of DCs and granulocytes, indicating that a factor secreted by A. fumigatus might activate innate immune cells. In conclusion, A. fumigatus antigens induced the activation of DCs and granulocytes. Our results indicated that this activation was mediated via TLR2 and TLR4. Future studies are needed to assess the clinical impact of these findings in patients at high risk for IA.

Ex vivo generation of human cytomegalovirus‐specific cytotoxic T cells by peptide‐pulsed dendritic cells

Adoptive transfer of donor‐derived human cytomegalovirus (HCMV)‐specific T‐cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV‐specific T cells using HCMV‐infected fibroblasts as stimulator cells confines this approach to HCMV‐seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV‐specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV‐specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide‐pulsed dendritic cells as antigen‐presenting cells. HCMV‐specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV‐seropositive and 2 out of 11 HCMV‐seronegative donors. The CTLs recognized HCMV‐infected autologous fibroblasts. No lysis was observed with either non‐infected autologous or HLA‐mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide‐specific T cells of up to 28% and > 90% of CD8+ T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 × 109 HCMV‐specific T cells was possible after 6–7 weeks when cultures were initiated with 1–5 × 106 responder cells. Thus, the approach with peptide‐pulsed DCs to generate HCMV‐specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.

Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells

Summary. The hypothesis that productive infection of monocyte‐derived immature dendritic cells (DCs) by the human cytomegalovirus (HCMV) is associated with decreased immunostimulatory capacity was tested in this study. DCs were infected with 60–80% efficiency by HCMV strain TB40/E. Infected versus uninfected cells were analysed by fluorescence‐activated cell sorting and by immunocytochemistry for surface expression of major histocompatibility complex (MHC) and co‐stimulatory molecules as well as cytokine secretion during the 3 d after infection. The immunostimulatory capacity of these cells was measured by mixed leucocyte reaction. In spite of the fact that HCMV infection of DCs induced an increased release of tumour necrosis factor‐α (TNF‐α) and a decreased interleukin 10 (IL‐10) production, expression of MHC class I and II, as well as CD40 and CD80 molecules, were downregulated on infected DCs. The mixed leucocyte reaction showed significantly reduced immunostimulatory capacity of infected DC cultures. Simultaneous detection of MHC antigens and virus antigens by double immunofluorescence revealed that downregulation occurred only on infected cells, but not on uninfected bystander cells. These findings demonstrate on a single cell level, together with the marked downregulation of MHC and co‐stimulatory molecules in the presence of high TNF‐α and low IL‐10 levels, a direct inhibitory effect of HCMV on antigen presentation by immature DCs independent of soluble mediators.

Induction of CMV-specific T-cell lines using Ag-presenting cells pulsed with CMV protein or peptide

BACKGROUND CMV disease is still associated with a high morbidity and mortality in recipients of a solid organ or stem cell graft, especially in patients undergoing allogenic stem cell transplantation. Reconstitution of CMV-specific CD4(+) and CD8(+) cytotoxic T cell responses are essential to control CMV infection following allogenic stem cell transplantation. The transfer of unselected populations of lymphocytes from the peripheral blood of a CMV-scropositive donor to a transplant recipient can be used to control CMV infection. However, such transfer of unselected donor lymphocytes is limited by potentially fatal complications that arise from alloreactive T cells, also present in the unselected donor lymphocytes. Thus to make infusion of donor T cells safe and also more effective in controlling CMV infection in the recipient of the T cell infusion, T cells are manipulated in vitro to deplete alloreactive T cells and to enrich for CMV-specific T cells. METHODS Using various antigen-presenting cells (monocytes/PBMNCs/dendritic cells) and different modes of antigen presentation (infected APCs, pulsing of protein or peptide antigen) different CMV-specific T cell populations can be generated and expanded. RESULTS Using protein-/or peptide-pulsed DCs CMV-specific CD8(+) cytoxic T cell lines (can be generated and expanded) in addition CMV-specific CD4(+) T cell lines can be generated when CMV-protein-pulsed DCs are used as antigen-presenting cells. When peripheral blood mononuclear cells were stimulated with CMV lysates predominantly CMV-specific CD4(+) T cells are generated and expanded ex vivo. DISCUSSION Depending on the APC used (monocytes versus DC) and the mode of antigen presentation (protein versus peptide pulsing) different CMV-specific T cell populations of varying purity can be generated which show preserved function when tested for specific proliferation, cytokine production and cytotoxicity.