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

CD80CD86

CD40

T-Cell activated T-cellNK-Cell

IFN-

IL-12IFN-

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-12IL- 15IFN- T-Cell activated 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

CD123BDCA-2/-4

PlasmacytoidDendritic Cell

CD11c

MyeloidDendritic Cell

PDC and MDC in Blood and Tumor of HNSCC-Patients

PBMC

Lineage-negativeCD4-positive

PDC MDC

Production of Typ I Interferon (IFN-)

Priming of T- CellsCytokine production

UK-SH - HNO Lübeck

TLR1

TLR2TLR4 TLR5 TLR6

TLR7 TLR8

TLR9TLR3

Long dsRNA

CpG-DNACG

LPS FlagellinPeptidoglycan

Lipoprotein

Lipoprotein

ssRNAisRNA

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 2216 5’-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

PDCCpG+ +

CD123+, BDCA2/4+

B-Zelle

MDC

IFN-

CD11c

-T-Zelle NK-Zelle

IFN-

CpG and the immune system

T-Zelle

TH1 CTL

CD4 CD8

IL8IL12TNF

IL12

UK-SH - HNO Lübeck

Monocyte

NK cell

CD4T cell

CDT cell

Innate immunity

Granulocyte

Acquired immunity

B cell

Recognition of conserved

molecular patterns

Recognition of newly acquired protein antigens

UK-SH - HNO Lübeck

MDC PDCHNSCC

„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

100

50

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

100

50

IL

1 6 10

Cytokine production of MDC under HNSCC and CpG

UK-SH - HNO Lübeck

Induction of high levels of IL1 and IL10 by MDC through HNSCCcan be limited by CpG-DNA

2h 4h 8h 12h

HNSCC

70

50

30

10

PPD inducedIFNy-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

MDCHNSCC

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 toTLR-downregulation after incubation in HNSCC supernatant

UK-SH - HNO Lübeck

TLR1

TLR mRNA[copy numberper 1000 copies ofhouse keeping gene]

0

100

200

300

400

500

600

TLR6

TLR7 TLR9

TLR10

Tumorsupernatant - + - + - + - + - +

700

MFI 22.5

Medium 40 hTumor single cell suspension

MFI 3,1CD86

CD123

CpG- DNA activates PDC in HNSCC

MFI 323

CpG-2216 40 h

CD86

UK-SH - HNO Lübeck

050

100150200250300350

p=0.225

p=0.049

Medium ODN 2216

CD86[MFI]

PDC in tumor-draining lymph nodesA

MediumDay 0 Day 2 Day 2

0100200300400500600700800 p=0.109

p=0.033

Medium ODN 2216MediumDay 0 Day 2 Day 2

CD86[MFI]

MDC in tumor-draining lymph nodesB

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 h CpG-2216 40 h

Lymph-node

UK-SH - HNO Lübeck

Tumorcell

immature pDCimmature pDCdysregulated mDCdysregulated mDC

Tumor-infiltrating, non activated DCs:

T-cell-anergy CD4/CD8T-cell

TH2:Tolerance, Tolerance,

Tumorgrowth promotionTumorgrowth promotion

UK-SH - HNO Lübeck

immature pDCimmature pDCdysregulated mDCdysregulated mDC

Activation of the innate immunity by PAMP´s

Activated DC´sActivated DC´s

PAMP´s (CpG, RNA...)

Activated TcellActivated Tcell

Tumorcell

UK-SH - HNO Lübeck

IL12, IFN alpha

CD4/CD8T-Cell

-T-Cell NK-Cell

IFN-

Thanks a lot to …Thanks a lot to …

Nicole BohnertNicole Bohnert

Carsten BrocksCarsten Brocks

Evelyn HartmannEvelyn Hartmann

Gunther HartmannGunther Hartmann

Stephan LangStephan Lang

Ray XieRay Xie

Brigitte WollmannBrigitte Wollmann

Ralph PriesRalph Pries