Dendritic Cells – Target of HNSCC Immune Escape can be supported with PAMP´s Barbara Wollenberg Universitätsklinikum Schleswig-Holstein, Campus Lübeck

The Dendritic Cell: Key Function in the Immune System Bacteria Virus MHC I MHC II CD80 CD86 CD40 T-Cellactivated T-cellNK-Cell IFN-  IL-12 IFN-  UK-SH - HNO Lübeck

Current DC Vaccination Trials Adherent Fraction of PBMC´s (Monocytes) IL4 / GM-CSF Tumor - Fragments Peptide –Pulsing RNA – Transfection Hybridoma + IL-12 IL- 15 IFN-  T-Cellactivated T-cell NK-Cell IFN-  i.v. s.c. i.n. UK-SH - HNO Lübeck

Current DC Vaccination Trials – Lessons learned Ineffective stimulation of T-Cells is due to : Quick DC Suppression by the tumor milieu Inflammatory mediators are insufficient for full DC activation and promote expansion of CD4 T cell populations lacking helper functions !!! (Spörri et al. Nature Immunology 2005) Insufficient response due antigenic shift (– even in mixtures) Duration of therapy less than 6 months Artificial generation of DC´s limits biological effectiveness UK-SH - HNO Lübeck

Current DC Vaccination Trials – New Needs „Natural cells“ – new isolation techniques Activation of DC´s In vivo to target all possible TA antigens present - not only selected artificial epitopes New „natural molecular activation mechanisms of DC´s“ : PAMP`s - Pathogen associated molecular patterns: isRNA, ssRNA, dsRNA, DNA-Fragments UK-SH - HNO Lübeck

CD123 BDCA-2/-4 Plasmacytoid Dendritic Cell CD11c Myeloid Dendritic Cell PDC and MDC in Blood and Tumor of HNSCC-Patients PBMC Lineage-negative CD4-positive PDC MDC Production of Typ I Interferon (IFN-  ) Priming of T- Cells Cytokine production UK-SH - HNO Lübeck

TLR1 TLR2 TLR4TLR5 TLR6 TLR7 TLR8 TLR9 TLR3 Long dsRNA CpG-DNA CG LPSFlagellin Peptidoglycan Lipoprotein ssRNA isRNA Toll-like-Receptors and PAMP´s Cell surface Endosome UK-SH - HNO Lübeck PAMP´S- Pathogen associated molecular patterns

CpG-Oligonucleotides (DNA) mimick viral infections ODN ’-GGG GGA CGA TCG TCG GGG GG-3’ - GT CG TT - CpG Sequences in bacterial DNA 1:16, in human DNA 1:60 -> DANGER SIGNAL UK-SH - HNO Lübeck

PDC CpG ++ CD123+, BDCA2/4+ B-Zelle MDC IFN-  CD11c  -T-Zelle NK-Zelle IFN-  CpG and the immune system T-Zelle TH1CTL CD4CD8 IL8 IL12 TNF  IL12 UK-SH - HNO Lübeck

Monocyte NK cell CD4 T cell CD  T cell Innate immunity Granulocyte Acquired immunity B cell Recognition of conserved molecular patterns Recognition of newly acquired protein antigens UK-SH - HNO Lübeck MDCPDC HNSCC

„The MDC Story“ MDC 1. Pick up and process the antigen in the periphery 2. Migrate to the locoregional lymph node 3. Present the antigen to immune effector cells T-CellNK-Cell IFN-  UK-SH - HNO Lübeck

Medium 12h HNSCC 2h CpG 12h CpG Migration of MDC under the influence of HNSCC and CpG UK-SH - HNO Lübeck HNSCC accelerates the migration of MDC, CpG slows the migration of MDC

Medium 12h HNSCC 2h HNSCC 4h HNSCC 8h HNSCC 12h HNSCC 2h CpG 2h CpG 2h CpG 2h CpG 2h CpG IL Cytokine production of MDC under HNSCC and CpG UK-SH - HNO Lübeck Induction of high levels of IL1 and IL10 by MDC through HNSCC can be limited by CpG-DNA

2h 4h 8h 12h HNSCC PPD induced IFNy-spots HNSCC limit the antigen presentation of MDC and CD8 T cell function (Ellispot) PPD: Purified Protein Derivate- (Mycobacterium tuberculosis) UK-SH - HNO Lübeck

HNSCC accelerates the migration of immunologically defect, tolerance- inducing myeloid dendritic cells MDC HNSCC CpG IL-10 Migration IL-6 IL-1 T-cell activation Model of MDC Dysregulation by HNSCC UK-SH - HNO Lübeck

PDC in HNSCC: the majority is not activated PDC* CD86 Fresh HNSCC-Suspension CD123 UK-SH - HNO Lübeck PDC are forced into a TH2 Milieu by HNSCC -> reduced activation -> reduced secretion of IFN- 

Tumormilieu regulates TLR´s on pDC reduced secretion of IFN-  is due to TLR-downregulation after incubation in HNSCC supernatant UK-SH - HNO Lübeck TLR1 TLR mRNA [copy number per 1000 copies of house keeping gene] TLR6 TLR7 TLR9 TLR10 Tumor supernatant

MFI 22.5 Medium 40 h Tumor single cell suspension MFI 3,1 CD86 CD123 CpG- DNA activates PDC in HNSCC MFI 323 CpG h CD86 UK-SH - HNO Lübeck

p=0.225 p=0.049 MediumODN 2216 CD86 [MFI] PDC in tumor-draining lymph nodes A Medium Day 0 Day p=0.109 p=0.033 MediumODN 2216Medium Day 0 Day 2 CD86 [MFI] MDC in tumor-draining lymph nodes B UK-SH - HNO Lübeck CpG activates PDC in suspensions of tumor draining lymph node

CD69 CD8 Tumor Activation of CD4-and CD8-TIL Tumor cellsuspension Medium 40 hCpG h Lymph- node UK-SH - HNO Lübeck

Tumorcell immature pDC dysregulated mDC Tumor-infiltrating, non activated DCs: T-cell-anergyCD4/CD8 T-cell TH2:Tolerance, Tumorgrowth promotion UK-SH - HNO Lübeck

immature pDC dysregulated mDC Activation of the innate immunity by PAMP´s Activated DC´s PAMP´s (CpG, RNA...) Activated Tcell Tumorcell UK-SH - HNO Lübeck IL12, IFN alpha CD4/CD8 T-Cell  -T-Cell NK-Cell IFN- 

Thanks a lot to … Nicole Bohnert Carsten Brocks Evelyn Hartmann Gunther Hartmann Stephan Lang Ray Xie Brigitte Wollmann Ralph Pries