Dendritic cell and cancer immunobiology

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Dendritic cell and cancer immunobiology
Young Tae Kim, M.D., Ph.D
Department of Obstetrics and Gynecology Yonsei
University College of Medicine
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The immune system has the potential to eliminate
neoplastic cells, as evidenced by occasional
spontaneous remissions in renal-cell carcinomas
and melanomas, regression of distant metastases
after surgical removal of the primary tumor, and
an increased incidence of certain type of tumor (
virus induced ) among immunosuppressed patients.
Therapeutic interventions for tumor
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History of cancer immunotherapy

• Frederick Fehleisen - Erysipelas as a cancer
  therapy (streptococcus erysipelatis)
• Willian B. Coley - Not bacteria - Coleys toxin
  against The New York Surgical Society (1895)
• Issacs Lindemann - discovery of interferon
• Halpern et al. - BCG as tumor therapy
• Discovery of dendritic cells
• Discovery of T-cell growth factor
• Kohler Milstein - Monoclonal Ab technique
• Cloning of Interleukin-2
• Clinical trials pioneered by Rosenberg at NCI
• In vitro generation of large number of DCs
• Clinical trial of fused cell vaccines (allogeneic
  autologous renal cell cancer by Kugler et al.)

1883
1891
1957
1959
1973
1976
1978
1983
1985-1994
1992
2000
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Cancer immunotherapy

• 1. Nonspecific immunotherapy
• Coleys toxin
• Corynebacterium
• BCG
• 2. Specific immunotherapy
• Tumor antigen
• Vaccine tirals
• Autologous challenge/allogeneic tumor
  preparation
• Transfection of protective genes
• Tumor antigen
• MHC antigen
• Costimulatory molecules
• Cytokines
• New vehicles for vaccine preparation

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Tumor vaccines

• Classified as preventive or therapeutic
  (i.e.,FDA-approved
  prophylatic vaccine the recombinant hepatitis B
  vaccine VLP vaccine for cervical carcinoma)
• No FDA-approved therapeutic cancer vaccines
  phase II and III clinical trials in melanoma,
  prostate/renal cell cancer, breast cancer,
  ovarian cancer. Leading to optimism that
  clinically beneficial treatments are on the
  horizon.

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Strategies for tumor vaccination

• 1. Cell-based vaccines
• Tumor cells (TCs) -Autologous or allogeneic
• Dendritic cells (DCs) -Autologous or allogeneic
• 2. Antigen-based vaccines
• Administration of recombinant TSAg
• Recombinant Ag fed DCs
• DNA vaccines-recombinant virus carrying TSAg cDNA

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Tumor cell-based vaccines - Autologous or
allogeneic tumor cells

• TCs modified with physical or chemical agents -
  ionizing radiation, ultraviolet radiation,
  dinitrophenyl(DNP), acetic
  acid, keyhole limpet hemocyanin(KLH)
• TCs modified with biologic extracts -
  Bacillus Calmette-Guerin(BCG), Corynebacterium
  parvum
• TCs lysed by virus ( viral oncolysates)
• TCs lysed by genetically modified virus -
  cytokine-mediated vaccines (IL-2, -4, -7, -12,
  IFN-g , GM-CSF), HSV
  thymidine kinase (HSV tk)/ganciclovir(GCV)
• TCs infected by genetically modified salmonella
  typhimurium

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Tumor vaccines

• Composed of antigens and adjuvants
  (i.e.., activators of the immune
  system)
• Adjuvants play a crucial role in determining the
  quantity and quality of the response to the Ag.
• Dendritic cells(DCs) are called natures adjuvant
  representing an essential component of any
  vaccination strategy.

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Mechanisms of action of adjuvants
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Dendritic cell-based vaccines - Autologous or
allogeneic DCs

• DCs incubated with whole tumor cells modified by
  the following - ionizing radiation, ultraviolet
  radiation, cell fragmentation
• DCs incubated with the followings - apoptotic
  bodies, tumor lysates, known
  recombinant tumor antigen, RNA encoding known
  tumor antigen, transduction with specific
  Antigen cDNA
• DCs transduced with specific cDNA -
• IL-12, -18, GM-CSF
• Fusion cell tumor-DC heteroconjugates

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Active and passive immunity
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The multipotential hematopoietic stem cell and
its progeny
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Properties and functions of antigen-presenting
cells
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The immunoglobulin superfamily
(a) Two domain structure of an immunoglobulin
(b) Some immunoglobulin-related molecule showing
C-like and V-like domains. TCR, T cell receptor
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Antigen processing presentation

• Antigen processing
• the partial digestion of a polymeric structure
  (mainly protein, but also glycolipids) into
  oligomeric fragment (8-33 aminoacid peptides or
  small lipids or lipo-oligosaccharides)
• 2 type
• 1) cytosolic pathway
• 2) endocytic pathway

• Antigen presentation
• Surface expression of that oligomeric antigen
  bound with special antigen binding grooves of the
  MHC class I proteins, MHC class II antigen, or
  CD1 molecules
• MHC I II for peptide
• CD1 for lipids and lipo-oligosaccharides

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Pathways of antigen processing and presentation
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Adhesion molecules interaction
between Dendritic cell and T lymphocyte
Cell membrane
Cell membrane
ICAM-3
SIGN
T lymphocyte
Dendritic cell
SIGN specific ICAM-3 grabbing nonintegration
molecule, ICAM intercellular adhesion molecule,
LFA leukocyte function-associated antigen
( Cell 2000100575 )
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Role of co-stimulatory pathway in T-cell
activation
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Discovery of dendritic cells(DCs)

• In 1973 Steinman and Cohn the 1st to describe
  DCs in the murine system as highly efficient Ag
  presenting cells
• They represent a minor cell population in all
  tissues.
• In 1992 Culture system In vitro generation of
  large numbers of mouse and human DCs
• Mouse Flt3 ligand(Flt3-L) GM-CSF
• Human from bone marrow progenitors (CD34
  cells)
• cultured in GM-CSF TNF
• from PBMC (CD14 cells)
• cultured with GM-CSF IL-4
• GM-CSF IL-13

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Research output on dendritic cells(DCs) and
natural killer (NK) cells
Number of articles in MEDLINE
Year
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Dendritic cell morphology demonstrates
characteristic dendrites after 7 days culture in
vitro.
Mature DC Presence of
membrane processes - dendrites, pseudopods, veils
Intracellular structures related Ag processing
endosomes,lysosomes, Birbeck granules of
Langerhans cell(LC)
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Dendritic cells within the skin are evident on
silver stain.
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Langerhans cells in the epidermis of human skin
staining with an anti-class II MHC antibody
Mature DC cell surface phenotype Characterized
by large amounts of class II MHC molecules and
absence of lineage markers including
CD14(monocyte), CD3(T cell), CD19,20,24(B cell),
CD56(NK cell) CD66b(granulocyte) Adhesion
molecules CD11a (LFA-1), CD11c, CD50(ICAM-2),
CD54(ICAM-1), CD58(LFA-3), CD102(ICAM3) Costimulat
ory molecules CD80(B7.1), CD86(B7.2), CD40
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DC surface phenotype
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Development of DC-changes of cell surface
molecules
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Scanning electron microscopy of human dendritic
cells
Dendritic cells generated from peripheral blood
monocytic cells(PBMC) by cultivation in the
presence of GM-CSF and IL-4 show remarkable
morphological variations.
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Scanning electron microscopy of human dendritic
cells
Within minutes nonadherent suspension cells can
transform into fully adherent flat cells.
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Scanning electron microscopy of human dendritic
cells
Mature dendritic cells preferentially show a
nonadherent phenotype with numerous membrane
folds.
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4 Different maturation stages of DC
(1)Bone marrow progenitors (2)Patrolling DC
through blood lymphatics (3)Tissue-residing
immature DC (4)Mature DC within 2ndary lymphoid
organs
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Dendritic cells
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Dendritic cell(DC) generation
Start
6 7 days
10 days
TNF
GM-CSF IL- 4
Immature DC
Plastic adherent cell
Blood collection
Mature DC
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Effect of coculture with melanoma cell lines on
differentiation of DC from PBMC
mb-GM-CSF
mb-GM-CSF
IL-4 alone IL-4 GM-CSF No cytokine
mb-GM-CSF
DC generation ?
mb-GM-CSF
mb-GM-CSF
Conley BF8 cell line
IL-4 alone IL-4 GM-CSF No cytokine
DC generation?
Conley cell line(wild type)
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Schematic representation of culture conditions in
Conley Conley B-F8
Conley (106 cells)
Conley B-F8 (106 cells)
Conley B-F8 (105 cells)
IL-4
IL-4
IL-4
0.11 ratio
11 ratio
Conley B-F8 (106 cells)
Conley (106 cells)
Monocyte only (106 cells)
IL-4 (1000IU/ml)
IL-4
IL-4
GM-CSF (1000IU/ml)
GM-CSF
GM-CSF
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Schematic representation of culture conditions in
Jorp Jorp C-E6
Jorp C-E6 (106 cells)
Jorp C-E6 (2x106 cells)
Jorp C-E6 (105 cells)
Jorp (106 cells)
IL-4
IL-4
IL-4
IL-4
0.11 ratio
11 ratio
21 ratio
Jorp C-E6 (106 cells)
Jorp (106 cells)
Monocyte only (106 cells)
IL-4
IL-4
IL-4 (1000IU/ml)
GM-CSF
GM-CSF
GM-CSF (1000IU/ml)
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Fig. 1 Soluble GM-CSF detected in the
supernatant of melanoma cell lines by ELISA
GM-CSF (ng/106cells/24h)
ley B-F8
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Cell interaction in growth regulation
Endocrine
Autocrine
Paracrine
Juxtacrine
Direct cell-cell contact
Diffusible factors
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Table 1 Percentage of cells positive for marker
molecules in the presence(pre) or absence(abs) of
GM-CSF
Cells cocultured with Conley B-F8 Conley Jorp
C-E6a Jorpa Monocytesa
HLA DR pre abs 83 76 91
72 6613 6013 6711 5111 806
CD1a pre abs 60 31 81
2 62 2 21 9 50 1 4421 64 4
CD86 pre abs 62 3 61
2 26 9 2219 3715 35 1 4417
aResult are expressed as mean SE of four
experiments.
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Table 2 Percentage of cells positive for marker
molecules in the presence(pre) or absence(abs) of
GM-CSF
Cells cocultured with Conley B-F8 Conley Jorp
C-E6a Jorpa Monocytesa
CD14 pre abs 2 48 3
63 21 6314 21 21 3 33
mbGM-CSF pre abs 3 2 lt1
lt1 61 4 2 87 7 3 43
CD83 pre abs 14 3 13
2 156 10.3 30.9 181 2520
aResult are expressed as mean SE of four
experiments.
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HLA DR
mbGM-CSF
CD 83
CD 1a
CD 14
CD 86
Fig. 3 Flow cytometric phenotyping of DC grown in
the presence of GM-CSF/IL-4 and cocultured with
Conley B-F8 melanoma cell line. Cells were
stained with control isotype (open histogram) or
with the indicated monoclonal antibody(mAb) cell
surface markers (solid histogram).
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IL-4 GM-CSF (11)
IL-4 alone (11)
IL-4 alone (0.11)
Percentage of cells ()
Fig. 4 Phenotypic expression of cells cocultured
with Conley B-F8
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Clinical trials of DC-based vaccine for cancer