Mechanisms of Immune Evasion by Tumors

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Mechanisms of Immune Evasion by Tumors

• W.H. Chambers, Ph.D.
• Associate Director for Basic Research
• University of Pittsburgh Cancer Institute
• Associate Professor of Pathology
• University of Pittsburgh School of Medicine

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DCs migrate to Secondary Lymphoid Organs To
Stimulate Immunity
Tumor specific T cells migrate into tumors And
mediate specific anti-tumor functions
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Innate capacity of lysis
LGL
CD3-, CD16, CD56, CD158, CD161
NK- more HSV, more tumors
NK IT better prognosis
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NK Infiltration at Tumor SiteCorrelates with
Improved Prognosis

• Hepatocellular carcinoma (Taketomi 1998) plt.0001
• Adenocarcinoma Lung (Takanami 2001) plt.0002
• Laryngeal carcinoma (Gonzalez 1998)
• ? Cirrhosis (Kawarabayashi 2000)
• Leukemia (Lowdell 2002)
• Gastric carcinoma (Ishigami 2000) plt.01
• Colorectal carcinoma (Coca 1997) plt.001
• Gastric carcinoma (Takeuchi 2001) plt.05
• Squamous Cell Lung (Villegas 2002) p.03
• Uterine Cervix (Vaquer 1990)

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Stellate Morphology
Most Efficient APCs
CD80, CD86, CD83, Class I/II, CD205, CD209
pDC, lyDCs, myDCs
Promote Th1 DC1 or Th2 DC2
Produce IL12, IL2
DC IT better prognosis
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Figure 8. Day 9 bone marrow
derived cultures are enriched for mature DCs.
FACS analyses for the indicated
cell surface markers was performed on day 8 of
cultures generated with GM-CSF,
IL4 and FL (top panels), and on day 9 after 18-24
hrs of subculture in the
absence of FL (bottom panels). Shaded area,
specific Ab staining. Light area, isotype control
Ab staining.
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DC Infiltration at Tumor SitesCorrelates with
Improved Prognosis

• Basal Cell (Bergefelt 1992,1994)
• Melanoma (Toriyama 1993)
• Cervical (Nakono 1992,1993)
• Esophogeal (Furihata 1992)
• Gastric (Tsujitani 1992,1993,1995)
• Hodgkins (Alavaikko 1994)
• Lung (Zeid 1993)
• Tongue (Goldman and Lotze 1998)
• Pancreas (Dallal and Lotze 2002)
• HN (Whiteside and Storkus 2002)

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Gliomas Incidence and Prognosis

• Most commonly diagnosed primary brain tumor
• Include astrocytomas (gt90 of gliomas),
  oligodendrogliomas, and ependymomas
• 15-17,000 new cases/yr in the US
• 15-17,000 deaths/yr in the US
• Brain and CNS tumors 3rd leading cause of
  cancer-related deaths in US males (15-34 yr) 4th
  leading cause of cancer-related deaths in US
  females (15-34 yr)
• Median survival (conventional therapy) for high
  grade gliomas is 10-12 months

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The Nature and Anatomic Location of Gliomas
Limits Conventional Approaches to Therapy

• Surgery complete resection cannot be
  accomplished because of diffuse infiltratation
  into surrounding tissue and because of the need
  to preserve neurologic function
• Chemotherapy affected to some extent by
  blood-brain-barrier, drug resistance, and by
  neurotoxic effects of drugs
• Radiotherapy targeting difficult because of the
  infiltrative nature of gliomas, their relative
  radiation insensitivity, and the delayed
  radiation necrotic effects of doses sufficient to
  kill tumor cells

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The location and biology of gliomas limits the
efficacy of conventional therapies
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Gliomas Have a Number of Mechanisms for
Suppression or Evasion of the Immune System

• Production of soluble suppressive factors - TGFb,
  IL10, sFHL
• Disruption of antigen presentation TAP
  deficiency, b2-M deficiency, Class I deficiency
  (some or all)
• Elimination of effector cells CD95L
• Protection from lytic mechanisms sCD95, CD36,
  CD46, CD55, CD59

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9L Tumors Are Infiltrated by NK Cells, But There
Are Some Surprises!
NK Cells
NK/T Cells
T Cells
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Confocal Analyses Confirming Relative
Infiltration of 9L Glioma by CD161bright and
CD161dim Lymphocytes
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Table 1. Comparison of Lytic Activity of CD161
Cells Isolated from Normal Splenocytes vs
Established (Day 14) 9L Gliosarcoma
CD161bright CD161dim Normal
Spleen 9L-Derived Normal Spleen 9L-Derived Exp.
1 501 697.4 414.1 60.8
13.0 251 603.6 ND
50.6 10.5 12.51 502.8
ND 32.9 31.3 6.251
335.4 ND 50.7
31.5 Exp. 2 501 92 9.8 ND
303.8 226.9 251 8112.4
344.5 212.4 172.8 12.51 72
6.8 295.2 140.6 110.5 6.251
3310.7 243.0 116.5 113.5
LU30/106 206 32 12
8 ET ratio Percent specific
cytotoxicity in 51Cr release assay
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Elimination or reversal of the effects of TGFb
has been embraced as a means of enhancing
immunity to gliomas

• Constitutive expression of TGFb anti-sense
  results in reduced tumorigenicity
• Constitutive expression of TGFb anti-sense
  results in enhanced immunogenicity
• Treatment with TGFb anti-sense oligos results in
  reduced tumorigenicity
• Unfortunately, clinical application has not
  proven effective

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Receptors for TGFb

• Heteromeric complex
• Type I - 53 kDa (2 isoforms)
• Type II - 75 kDa (2 isoforms)
• Type III - 300 kDa (beta-glycan)
• Type II has serine-threonine kinase activity
• Receptors are expressed ubiquitously

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NK
Tu
NK
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9L-TGFbsr is more susceptible to NK cell-mediated
lysis than 9L-neo
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9L-TGFbsr is less tumorigenic than 9L
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9L-neo NK depletion
9L-TGFßsr NK depletion
9L-neo
9LTGFbsr
Tumor size cm3




Days After Implantation








b
sr Mediated
Figure 7. Depletion of CD161
Cells Results in Reversal of the TGF
Loss of Tumorigenicity.
Rats were given either 9L-neo or 9L-TGF
b
sr coupled with
either mAb 3.2.3 or an isotype control mAb.
Tumor size was determined at various
intervals using calipers. P lt0.05.
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Enhanced Survival of Rats with IC 9L-TGFbsr vs
9L-neo
120
100
80
9L-NEO
Percent Survival
60
9L-TGFBsr
40
20
0
0
5
10
15
20
25
30
35
Days After Tumor Implantation
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What can we conclude from these experiments?

• Local expression of TGFbsr reduces tumorigenicity
• CD161 cells are important in the anti-tumor
  effects in vivo, and their activity is regulated
  by TGFb
• Some increase 22-40 in survival is associated
  with local expression of TGFbsr

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DCs migrate to Secondary Lymphoid Organs To
Stimulate Immunity
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Intra-tumoral Delivery of iDCs into Intracranial
9L Does Not Enhance Survival
Yang et al., Cancer Res. 622583, 2002
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MHC Class II Cells Undergoing Apoptosis in 9L
Tumors
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  Table 4. Apoptosis of OX62 Dendritic Cells in
9L Tumors   Total Cells OX62
Cells OX62/VAD-FMK Apoptotic Cells  
608a 39 11
28.0 627 55 10
18.0 666 47
4 8.5 666 66
3 4.5 635 32
5 15.6 818 36
4 11.1 564
30 11 36.7 695
12 5 41.6  
4015.5 2112.7 aTotal
cells tabulated using Hoescht staining of intact
nuclei.
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What Receptor Ligand Interactions Are
Responsible for Induction of Apoptosis of DC?

• Soluble Factor Produced by Tumor Cells?
• Tumor Cell Surface Receptor?
• Extracellular Matrix Proteins?
• Combination of Factors?

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Ethidium bromide stained RT-PCR products using
iNOS specific primers
Southern blotting of iNOS RT-PCR products
Western blot using iNOS specific mAb
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Induction of Apoptosis by HA of DC
TUNEL Assay and FITC-VAD-FMK
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Induction of DC Apoptosis is Based Upon CD44HA
Interactions
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What can we conclude from these experiments?

• Intra-tumoral delivery of DC did not increase
  survival of 9L bearing rats even when RS was used
  to induce tumor apoptosis
• DC in 9L undergo apoptosis as a consequence of
  iNOS production induced via CD44HA interactions
• Local expression of IL12 decreases DC apoptosis
  in 9L

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Co-culture of NK Cells and DCs Results in Their
Reciprocal Activation Which Induces Enhanced
Anti-tumor Lytic Function
Tu
Fernandez et al. Nature Medicine 5405-411
(1999). Ferlazzo G. et al, J. Exp. Med.
195343-51, 2002. Piccioli D. et al, J. Exp.
Med. 195335-41, 2002. Gerosa F. et al, J. Exp.
Med. 195327-33, 2002
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Co-incubation of NK Cells and iDC Results in
Enhanced Tumor Cell Lysis



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Co-incubation of NK Cells and DC Does Not Result
in Enhance Lysis of 9L Gliosarcoma

Additionally IL4 has no effect Anti-Class I has
no effect
Anti-Class I - IL4 -
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Based on these data, we are posing three
questions1) What NKDC receptorligand
interactions promote enhanced tumor cell
lysis?2) Are those interactions important for
promoting non-adaptive and adaptive immunity?3)
Are expression and function of those receptors
and ligands affected locally and/or systemically
by the immunosuppressive effects of tumors?
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Potential receptorligand interactions regulating
NKDC reciprocal activation?

• NK Cell DC
• CD2 CD58
• CD11a CD50/CD54/CD102
• CD11b/CD18 CD54
• CD47 CD172
• CD56 CD171
• TNFfl TNFfr CD120a, -b
• CD158 MHC Class I
• CD159/CD94 MHC Class I
• CD161b,d/f Clrb/g
• Ly49 MHC Class I
• NKp30 ?

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Activation and inhibition receptors on NK cells
that bind Ligands on DCs Do they play a role
in inducing cytotoxicity?


NK

DC
Tumor


Charged residue in the TM region for docking
of adaptor proteins with ITAM
ITIM I/VXYXXL
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Activation and inhibition receptors on NK cells
that bind Ligands on DCs Do they play a role
in inducing cytotoxicity?


Clrg
CD161F
?
NK
NKp30
DC
Tumor
Clrb
CD161A
CD161B,D


Charged residue in the TM region for docking
of adaptor proteins with ITAM
ITIM I/VXYXXL
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Are CD161-Clr Interactions Involved in the
Enhanced Tumor Cell Lysis Induced NKDC
Co-culture?

• Strategy RNA interference
• siRNA design Dharmacon siDesign Center
• 
  Motifs 5-AA(N19)UU
  
  
• 
  5-AA(N21)
• 
  5NA(N21)
• Transfection method Oligofectamine
• 
  Nucleofection
• Detection Flow cytometry
  
  
• 
  Northern blot/Realtime PCR
• 
  Cytotoxicity Assays

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5-AA(N19)UU Motifs for Primers for siRNA
for CD161s, Clrb and
NKp30   Receptors Targeted Sequence Location G/C
Content   CD161A
AAGCACGTGTCTACCTCAGTT
26-44 53 CD161A
AAGACTGCCGCGGGGGCTCAG 56-76 71  
CD161B AAGTGGTCTATGCGGACTTAC
199-217
53 CD161B AAGCGCGAGCCACCTCCATCT
(Invitrogen) 241-261 62 CD161A -B
AAAACAGGTAGTCCAGCTAAG
269-289 43 CD161A -B
AAGGAGCCACGTTGCTGCTCG 560-582
56 CD161A -B AAGTGGATAAACGGCTCGACT
682-704 43 CD161F
AAAGAGTCTATGGTAATGTAA
13-31 31 CD161F
AATGCTTAATTATTTCTCAAA 311-333 17 CD161F
AAGAAGCCACTTTGTTGATCA 374-396
35 CD161F AAAATGAAGAAGAACTGAAGT
398-420 26 CD161F
AACTGAAGTTTGTGCAGAACA 410-432 35 CD161F
AAAGGGAAGACAGCAGTTATT 435-457
35 CD161F AAGTGGATAAATGGCTCTGTT
496-518 35 Clrb/Ocil
AACTGCTGCTACGTAGTGATC
411-429 53 Clrb/Ocil
AACCATAAATACTTATGCTGC 498-520
30 Clrb/Ocil AAGGGGCCGAGCTAGCACGAT
614-636 56 Clrb/Ocil
AAAGGGAGTTCAGGTTACTGG 673-695 43 NKp30
AAGGTGCTGTTGATCGTCTTC
312-330 53
NKp30 AAGGACATCCAAAGCCATGAC
591-609 53 NKp30
AACCGGCAGTGTCATCTATTA 794-812 47 (3 ml
Oligof. 12 ml Opti-MEM) (3 ul 20 mM siRNA
50 ml Opti-MEM) 32 ml Opti-MEM Added to 500 ml
cells in CM

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Targeted siRNA Treatment of DCs Results in
Reduced Message for Clrb Realtime PCR
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Clrg
CD161F
?
NK
DC
Tumor
Clrb
CD161A
CD161B,D
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Reduction in Expression of CD161B in A-NK Cells
Using siRNA Oligonucleotides
Control
Control
siRNA Control
siRNA CD161B
Cell No.
Mean Fluorescence Intensity
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What can we conclude from these experiments?

• siRNA targeting of Clrb in DCs verified by
  Realtime PCR
• siRNA targeting of Clrb in DCs results in reduced
  enhancement of lytic activity against YAC-1
  following A-NKDC co-culture
• siRNA targeting of CD161s in A-NK cells
  verified by flow cytometry
• siRNA targeting of CD161s in A-NK does not result
  in reduced lysis of YAC-1
• siRNA targeting of CD161B in A-NK cells results
  in reduced enhancement of lytic activity
  following A-NKDC co-culture

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Mechanisms of Immune Evasion by Tumors

• Department of Pathology Department of
  Neurological Surgery
• William H. Chambers, Ph.D. Hideho Okada, M.D.,
  Ph.D.
• Tianbing Yang, Ph.D. Ian F. Pollack, M.D.
• Katy Webb Department of Immunology
• Lorissa Figallo Sean Ryan
• Department of Psychology Molecular Genetics and
  Biochemistry
• Melanie Flint, Ph.D. Paul D. Robbins, Ph.D.
• Dept.Cell Biol. Physiol./CBI Department of
  Neurobiology
• Simon Watkins, Ph.D. Carl Lagenaur, Ph.D.
• NSABP/UPCI Biostatistics Stanford University
• Doug Potter, Ph.D. Sheri Krams, Ph.D.
• John Bryant, Ph.D. Christine Hsieh
•