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Anti-cancer T cell immunity SMC6052–BIM 6027
Mar 12, 2015
Woong-Kyung Suh, Ph.D.
IRCM
Steve Jobs (1955-2011) Ralph Steinman (1943-2011)
What is common between the two?
Died of pancreatic cancer
Outline Part I
• Cancer and immunity
• Antigen presentation: virus vs. tumor
• Specificity: structure of antigen-T cell receptor
• Context: T cell costimulation
• T cell receptor signaling pathways
Part II
• Immunoediting: 3Es
• Cancer immune-evasion mechanisms
• Anti-tumor immunotherapies: promises and challenges
Douglas Hanahan , Robert A. Weinberg (2000) The Hallmarks of Cancer Cell 100: 57-70
The hallmarks of cancer - 2000
Douglas Hanahan , Robert A. Weinberg (2011) Hallmarks of Cancer: The Next Generation Cell 144: 646-674
Hallmarks of cancer - 2011
Autoimmunity Allergy Immunodeficiency cancer
tolerance immunity
To react or not to react?
Specificity (foreign vs. self) Context (innocuous vs. dangerous)
T cell
Antigen presenting cell
(APC: dendritic cell)
MHC restriction:
T cells only recognize antigen on the
surface of another cell, an APC
From Yewdell and Tscharke Nature 2002 418.923
MHC Major
Histocompatibility
complex proteins
present peptides
to T cells
2 kinds of MHC
Proteins
MHC I- CD8 T cells
intracellular
antigens
MHC II- CD4 T cells
extracellular
antigens
Tania Watts, U of Toronro
MHC IIPlasma
membrane
EndoplasmicReticulum (E.R.)
endocytosedproteins
MIIC/CIIV
cytosolicproteins
MHC IMHC II+ Ii
Lysosome
GolgiApparatus
proteasome
peptidetransporter
cis
medial
trans
InvariantChain (Ii)
MHC I
CD4+
T cell TCR
CD8+
T cellTCR
(degradation of Iiand peptide binding)
CD8 CD4
Comparison of
MHC I
and II
peptide
loading
pathways
David Williams U of Toronto
MHC class I assembly and presentation
• Watch a movie in YouTube. http://www.youtube.com/watch?v=VPvCekgPwRI
Class I Assembly in the Endoplasmic Reticulum
sec61 pore
Peptide Loading Complex
David Williams, U of Toronto
Suh WK, Cohen-Doyle MF, Fruh K, Wang K, Peterson PA, Williams DB.
1994. Interaction of MHC class I molecules with the transporter associated with antigen processing. Science 264:1322–26 (Similar result Cresswell
Lab- Nature 368, 864, 1994) .
TAP associates with 2M associated Endo Hs form of class I
IP:
-“Pulse” EL4 cells with 35S-Met, 5 min -“Chase” with “cold” Met media for up to 4 hrs -Immunoprecipitate MHC class I molecules (Kb or Db) or TAP-class I complexes -Digest immunoprecipitates with endoglycosidase H -Resolve in SDS-PAGE -Autoradiogram
Addition of peptide causes dissociation of TAP/MHC I:
Add Kb binding
Peptide- Kb dissociates from
TAP complex and stable peptide
loaded Kb form appears
Add Db binding
Peptide, Db dissociates
Thermostable- detected by MHC I IP
Class I Assembly in the Endoplasmic Reticulum
sec61 pore
Peptide Loading Complex
David Williams, U of Toronto
Structure of human class I HLA antigen HLA-A2 Bjorkman et al. Nature. 329, 506, 1987
MHC I purified after cleavage from surface of human cell line with papain and purification (3-4mg from 200 liters of cells)
Note that although the 3 and 2M domains
have an Ig superfamily fold, there is quite a
different quaternary structure than in an Fab
Bjorkman Nature 1987
2 exons form single domain unidentified “peptide” Bjorkman PJ, Saper MA, Wiley DC. 1987. Structure of human class I histocompatibility antigen, HLA-A2. Nature 329:506–12 Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512–18
Co-purifying pepides-
what are they?
H-2Db with 9-mer peptide bound (ASNENMETM) Young et al. (1994) Cell 76:39
X-ray Crystal Structures of MHC Class I Molecules
16
Interaction between the TCR and MHC class I molecule
Garboczi et al. (1996) Nature 384, 134.
TCR bound to HLA-A2 Variable loops of the TCR V and V domains are centered over the peptide and -helices of the class I H chain.
Peptide is buried in TCR-class I interface
Overall orientation of TCR is diagonal to class I groove. TCR fits between 2 high points on class I surface (top left and bottom right below). Ensures extensive contacts with both peptide and class I.
Footprint of TCR on MHC class I surface
Multiple contacts between residues of TCR and both peptide and class I -helices. Of 46 contacts (H bonds and van der Waals contacts): - 18 are to class I conserved residues (confers general binding to class I)
- 9 are to polymorphic residues (confers allele specificity)
- 19 are to peptide
David Williams, University of Toronto
MHC IIPlasma
membrane
EndoplasmicReticulum (E.R.)
endocytosedproteins
MIIC/CIIV
cytosolicproteins
MHC IMHC II+ Ii
Lysosome
GolgiApparatus
proteasome
peptidetransporter
cis
medial
trans
InvariantChain (Ii)
MHC I
CD4+
T cell TCR
CD8+
T cellTCR
(degradation of Iiand peptide binding)
CD8 CD4
How to generate CTLs
against virus that do not
infect APCs?
What about
tumor cells?
David Williams U of Toronto
Presentation of Exogenous Antigens by MHC Class I Molecules: Cross-presentation
Pathway 1: Pfeifer et al. (1993) Nature 361:359. Song and Harding (1996) J. Immunol. 156:4182.
Why is this important?
Priming naive CD8+ T cells against viruses involves costimulatory molecules that are absent on the majority of cells that are typically infected by viruses. The dominant cell that accomplishes such priming is the dendritic cell that acquires viral antigens largely by phagocytosis and then creates class I-viral peptide complexes at the cell surface. The same applies to tumor cells.
Process is: 1. Inhibited by cytochalasin D which blocks phagocytosis and by
chloroquine which inhibits endosome acidification and proteolysis (peptide production)
2. NOT inhibited by either brefeldin A (blocks ER to Golgi export) or
cycloheximide (protein synthesis inhibitor). Both block the conventional pathway.
3. NOT affected by loss of TAP transporter function Apparently, peptides produced in endocytic compartments either
bind to recycling class I in these same compartments or to "empty" cell surface class I.
X
David Williams, University of Toronto
Presentation of Exogenous Antigens by MHC Class I Molecules
Pathway 2: Kovacsovics-Bankowski and Rock (1995) Science 267:243.
Process is: 1. NOT inhibited by chloroquine
2. inhibited by loss of TAP function 3. inhibited by brefeldin A 4. inhibited by MG115 (inhibits peptide production by the proteasome) Thus, all indications are that the exogenous antigen must pass through the cytosol into
the conventional class I processing pathway.
David Williams, University of Toronto
Lipid Raft Lipid Raft
Fyn Lck
CD4
L A
T
PKC
MAPKKK
IkB
NFkB
NFkB
PIP2
DAG IP3
[Ca2+]
Calcineurin
Calmodulin
NF-AT NF-AT
PLCg1
IL-2
GADS SLP-76 VAV
GDP
GDP
RHO
Actin reorganization SLAP130
Nck PAK
RAS
GRB2
RAF
MEK
ERK1/2
SOS
FOS JUN
Ras
ZAP 70
APC
T Cell
Csk
PAG
CD45
Dominik Filipp, IMG, Czech Republic
Summary – Part I
• Dendritic cells process and present viral antigens or tumor antigens to elicit antigen-specific T cell responses.
• Endogenous antigens are presented by MHC class I proteins to prime CD8 killer T cells whereas exogenous antigens are presented by MHC class II proteins to elicit CD4 helper T cell responses.
• However, exogenous antigens (viral particles, dead tumor cells) can be presented by dendritic cells through MHC class I proteins (cross-presentation).
• T cell receptor-peptide/MHC interaction allows discrimination between self and foreign antigens.
• Strength of costimulatory signals provide the context – dangerous or innocuous.
Outline Part I
• Cancer and immunity
• Antigen presentation: virus vs. tumor
• Specificity: structure of antigen-T cell receptor
• Context: T cell costimulation
• T cell receptor signaling pathways
Part II
• Immunoediting: 3Es
• Cancer immune-evasion mechanisms
• Anti-tumor immunotherapies: promises and challenges
Dunn et al. Nature
Reviews Immunology 6,
836–848 (November
2006) |
doi:10.1038/nri1961
3Es in Immunoediting
Experimental test of “elimination” in mice
_Create mouse models with altered immune cells or
target cellls
_KO
_immune cells: RAG,
_immune cell weapons: perforin, trail, IFN-g
_target cell responsiveness: STAT1, IFN-gR, proteasome subunits
_mAb-mediated depletion: anti-Thy1.1, NK1.1, asialo-GM1
_activation of immune cells: -GalCer, IL-12
_Score incidence, latency, and spectrum of
_spontaneous tumors in aging mice
_tumors induced by chemical carcinogens: MCA, DMBA, TPA
_spontaneous tumors in p53 ko mice
Evidence for tumor cells in equilibrium (dormant tumors)
Low dose MCA
>eliminate mice (20%) that
develop progressively growing sarcomas
at d200
treat the mice having no tumors or
stable tumor masses with antibodies that block
components of innate or adaptive immunity
measure tumor sizes
Koebel C.M. et al. (2007) Nature 450: 903-907
Evidence for tumor cells in equilibrium (dormant tumors)
tumors outgrew if T cells or IFN-γ
pathways were blocked but unaffected
when innate immunity was blocked
Thus, adaptive immunity prevents late
MCA-induced sarcoma outgrowth
Koebel C.M. et al. (2007) Nature 450: 903-907
Check anti-CD4 alone or anti-Cd8 alone works
Evidence for tumor immune escape
• Many established murine cancer cells induce anti-tumor immune responses but grow well when injected in immune competent hosts
• These escapes often down regulate MHC class I or secrete immunosuppressive factors
Questions
1) Do cancer cells provoke T cell attack? If so, how?
2) How do cancer cells avoid or subvert T cell attack?
3) How can we harness T cells to fight cancer?
Do cancer cells provoke T cell attack? If so, how?
• Carcinogens, or UV induces mutations. Some
of the mutations can give rise to tumor-specific antigens
• Rapid proliferation in the absence of tumor suppressors lead to DNA damage and mutations. Many mutations can be immunogenic .
Tumor specific T cell antigen (Rejection antigen)
Questions:
1) New tumor specific rejection antigens arise in nascent tumor cells?
2) Rejection antigens are sufficient to induce protective anti-tumor immunity?
Approaches:
Take tumor cells grown in immunodeficient (RAG2 KO) mice
1) Clone single cells, analyze their growth patterns in WT mice
(progressor vs regressor), and identify “neoantigens” uniquely expressed
by regressors
2) Express identified rejection antigens in progressor clones and see if this can
convert progressors into regressors and induce anti-tumor T cells
Mastushita et al. (2012) Nature 482:400-404
Progressor (20%)
Regressor (80%)
Single cell cloning
Mastushita et al. (2012) Nature 482:400-404
Are the escapers pre-existing?
Tumor specific T cell antigen (Rejection antigen)
-Identify tumor-specific mutations by exome sequencing -Choose potential class I binding epitopes -Predict affinity values -Choose mutant epitopes uniquely expressed in regressors R913L mutation in spectrin β2 is one of them (750 fold increase in binding affinity)
Mastushita et al. (2012) Nature 482:400-404
Tumor specific T cell antigen (Rejection antigen)
Estimation of tumor specific T cell antigens in human cancers
Neil, H. et al. (2008) Cancer Res. 68:889-892
• Analyzed 1,152 peptides with missense mutations in breast and colorectal cancer using epitope prediction algorithms
• Identified average ~10 novel and ~7 unique epitopes that can bind to human MHC class I molecule HLA-A0201
• Suggest that tumor cell destruction in situ (e.g. by chemotherapy) in combination with adjuvants may induce polyvalent anti-tumor T cell immunity
How do cancer cells avoid or subvert T cell attack?
• Decrease surface MHC class I molecules
• Become insensitive to IFN-γ: no upregulation of MHC molecules, TAP, and proteasome subunits.
• Suppress anti-tumor immune cells: TGF-β, VEGF, Indoleamine 2,3-dioxygenase (IDO) > recruit pro-tumor immune cells such as tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg)
How can we harness T cells to fight cancer?
• Engineer tumor-specific T cells in vitro and infuse back into patients: chimeric antigen receptor (CAR)
• Elicit endogenous T cell responses by boosting antigen presentation: ex vivo or in vivo dendritic cell therapies
• T cell immune checkpoint blockade
Stanley R. Riddell , Michael C. Jensen , Carl H. June (2012) Biology of Blood and Marrow Transplantation Volume 19, Issue 1, Supplement 2013 S2 - S5
Chimeric antigen receptor (CAR)
Extracelular: Single chain Fv to a tumor antigen Intracellular: Combination of TCR and costimulatory signaling motifs
CD19 CAR: a success story
Porter, D.L. et al. (2011) N Engl J Med 365:725-33
-CD19 is highly expressed in chronic lymphocytic B cell leukemia as well as normal B cells. -T cells were taken from CLL patients and modified by lentiviral transduction to express anti-CD19 CAR. -Reinfusion of modified T cells led to complete remission in one out of three patients.
DC-mediated cancer immunotherapies
Palucka, K and Banchereau, J (2012) Nature Reviews Cancer 12: 265-277
Chemotherapy inducing immunogenic cell death (ICD)
Kroemer, G. et al. (2012) Annu. Rev. Immunol. 31: 51-72
Immunogenic cell death induces anti-tumor T cell response
Kroemer, G. et al. (2012) Annu. Rev. Immunol. 31: 51-72
T cell immune checkpoint blockade
• Ipilimumab (anti-CTLA-4): first FDA approved immunotherapeutic drug (2011), humanized mAb that blocks CTLA-4.
• 15-25% of melanoma patients show complete remission.
• Being tested for lung cancer, prostate cancer in combination of other treatments. Need to improve efficacy.
• PD-1, PD-L1, and B7-H4 blockade under clinical trial
Steve Jobs (1955-2011) Ralph Steinman (1943-2011)
Pancreatic cancer: How Nobelist Ralph Steinman beat the odds, but Steve Jobs didn’t By Janet Fang | October 10, 2011, 9:53 PM PDT
Jobs: Diagnosed pancreatic neuroendocrine tumor (curable by surgery) in 2003. But he insisted on alternative medicine 9 months before agreeing to get surgery. In 2009, cancer spread to liver and he had to get a liver transplant. To prevent graft rejection, he had to take immunosuppressant drug. Steinman: Diagnosed pancreatic adenocarcinoma (fatal) 1997. Chemotherapy combined with eight DC-based experimental immunotherapies. 4.5 yr survival with adenocarcimoma was extremely rare.
Summary
• Cancer cells do produce tumor-specific antigens and elicit anti-tumor T cell reaction.
• The immune system can function as an extrinsic tumor suppressor and influence the repertoire of outgrowing tumor cells (3E hypothesis).
• Immune system can be harnessed to fight cancer: dendritic cells and T cells are most promising.