Immune System and Cancer

J.club

07/29/03

What are NK cells?

A part of the native immune system, share a common early progenitorwith T cells but do not develop in the thymus

~ 10% of blood lymphocytes, defined by surface markers (ex. CD56, NK 1.1, CD2, CD16)

Activated by IFN, IFN and IL-12(IL-12 commonly used to activate NK cells in vitro)

Involved in early response to infection with certain viruses and intracellular bacteria

(first line of defense, giving CTLs time to differentiate)

“Natural Killers”

NK discovered as tumor killer cells:

Mice were immunized against a tumor, then their lymphocytes tested for ability to kill tumor cells in vitro.

But negative controls – lymphocytes from unimmunized mice or mice Immunized against a different tumor – also killed tumor cells very well!

NK assay: 51Cr release from YAC-1 cells

MHC/HLA review

KIR CD94+a/b +c, e/h, f

NKG2dNCR

NK receptor overview

NK receptors

Most inhibitory receptors recognize MHC I molecules

2 groups:

•Killer Immunoglobulin Receptors (KIRs)•C-type lectin receptors (CD94+NKG2 family member)

Both have activating counterparts, but inhibitory signals dominant. Activation/inhibition depends on:

•KIRs - long cytoplasmic tail with ITIMs vs. short tails + adaptor with ITAMs•C-type lectin receptors - NKG2 member: a/b activating, c, e/h, f inhibitory

NKG2d human and mouse ligands

RAE-1/H60 evidence:

•Tumors expressing RAE-1 or H60 are rejected, NK cell-dependent.

•Mice immune to re-challenge with the same tumor cells, even if RAE-1/H60 are no longer expressed – a role for adaptive immune system

New activating receptor: NKG2d

•Low homology to other NKG2 receptors, an activating receptor conserved between humans, mice and rats

•Expressed on NK cells, macrophages, / and / CTL’s

•Homodimer, forms an activating complex with DAP-10, which contains SH2 domains and recruits PI3K

•Can override inhibitory signals from KIRs and C-type lectins

Killing pathways (from Takeda et al, 2002)

Adhesion molecules may help NK get to tissue

•Human and rat NK cells synthesize fibronectin, mAb’s against FN block NK cytotoxicity against YAC-1

•NK cells express 41 and 51 integrins, mediate adhesion to FN in an in vitro assay

•NK cells express L-selectin, its expression is upregulated by IFN, IL-10 and IL-12

•IL-12 also promotes NK adhesion to P and E (endothelial) selectins under flow conditions

•LFA-1

•N-CAM

Immune surveillance idea and NK cells

In 1909, Paul Ehrlich proposed that the immune system could repress carcinomas. Idea was extended in 1957 by Burnet/Thomas – “immunesurveillance” as a way of maintaining tissue homeostasis.

NK cells a good candidate:

•Many virally infected and tumor cells express less MHC I, escape CTL detection/killing

• NK cells kill MHC I – cells in vitro

•NK reject MHC I – tumor cells, not same cells MHC I+

(same experiment for mets.)

•Irradiated mice get MHC I – lymphocytes, rapid NK-mediated disappearance

•Atomic bomb/ Chernobyl survivors have mutations in many differentgenes, except HLA loss in T cells

Tumor/NK evidence in mice

Direct:

•NK cells kill MHC I - tumor cells in vitro

•Eliminate tumor cells from circulation of mice/rats

•Protect mice from MCA skin carcinogenesis

•CTL knockouts have OK control of carcinogen-induced sarcoma growth,perforin knockouts (no CTL or NK activity) have deficient control

Correlative:

•A/J mice have low NK activity and high rate of lymphoma, C57/BL6 mice have high NK cell activity and low rate of lymphoma

•Mice selected for low acute inflammatory response (AIR) are moresusceptible to skin carcinogenesis by DMBA/TPA, and have more lung metastases than wt. or mice selected for high AIR

Tumor/NK evidence in mice

Drawbacks:

•No good data on protection from spontaneous tumors (except MCA)

•Good stimulation by blood cells but few other (ex. Not by low MHC I liver)

•In vitro models often activated by cytokines at far above physiological levels

•NK cells require homing signals (MIP-1 for homing to CMV foci in liver)

•No good mouse model lacking NK cells

(until very recently: a group was using a granzyme A promoter to express Ly49A cDNA, and got a mouse that’s specifically NK-deficient, both by FACS and by functional assays

The mouse has impaired control of tumor growth/mets., confirmed NK role by adaptive transfer)

Tumor/NK evidence in people

Direct:

•In vitro, IL-12 activated human NK cells are capable of killing MHC-

tumor cells

•Chediak-Higashi syndrome – impaired NK degranulation, susceptible to highly metastatic lymphomas

Correlative in vivo:

•Patients on immunosuppressants get more blood tumors

•People with congenital or acquired immunodeficiencies have a significantly higher incidence of malignancies (viral infection?)

•NK is impaired in cancer patients, by in vitro studies on YAC-1 andIFN response

•High peripheral blood NK activity (in treated, apparently disease-free patients) correlates with longer metastasis-free survival

NKT cells

A recently discovered subpopulation of T cells that express NKmarkers (ex. NK 1.1, Ly49 in mice), CD44, Ly6C

Originate in bone marrow, differentiate in the thymus

Express a limited set of T cell receptors, CD4+/DN (60%/40%)

CD1-dependent activation (MHC I – like proteins conserved in mammals)

Implicated in immunoregulation and tumor growth, althoughnot clear if alone or NK-regulated

CD1 ligand details

Sites of constitutive expression in mouse: thymus, liver, spleen, lung

NKT recognize CD1 bound to glycolipid(experimentally used - -galactosylceramide, -GalCer)

NKT function

Specialized regulatory component of immune system?

•Secrete large amounts of Th1 and Th2 cytokines upon stimulation, fast

Th1 – inflammatory, IFN main cytokine, involves CTLs and macrophagesTh2 – humoral, IL-4 main cytokine, stimulates T-helper cells and Ab production

•Can rapidly stimulate T and B cells in antigen-nonspecific manner

•Activate NK cells, macrophages, recruit dendritic cells

•Can induce Fas-mediated killing of CD1+ thymocytes

Experimentally activated by anti-CD3

NKT evidence in cancer

•Rag -/- mice (lack NKT, T and B cells) get more metastases than wt micewith low IL-12 stimulation. Corrected by adoptive transfer of NKT cells.

-GalCer is beneficial in preventing tumor growth/mets. in mice(stimulation of dendritic cells to release IL-12 and activate NK?)

•IFN release also important (stimulate TRAIL expression on NK?)

•Important in resistance to MCA-induced fibrosarcomas (no exogenous-GalCer or IL-12 stimulation)

•Purified NKTs cytotoxic to syngeneic MCA-induced tumor line

NK/NKT big picture:

• Innate Immune Responses -- components:

Macrophage, Dendritic cells, Neutrophils,

Mast cells, Eosinophils, Basophils

NK cells, NKT cells

Complement system

Immune System

Acquired Immune Responses

• B cells

• CD4+ T cells

• CD8+ T cells

Cancer Immunosurveillance Hypothesis

• “It is an evolutionary necessity that there should be some mechanism for eliminating or inactivating such potentially dangerous mutant cells and it is postulated that this mechanism is of immunological character” -- Macfarlane Burnet and Lewis Thomos (1957)

• Data disfavored the hypothesis: studies using nude mice

1) Osias Stutman used CBA/H background, look atMCA

carcinogen-induced tumor

wt/nude nu/nu

7/39, 95days 5/27, 90days

2)10,800nu/nu ~ wt mice in spontaneous tumor develp.

Cancer Immunosurveillance Hypothesis

Data supporting the hypothesis • INF-γ data: block, KO, DN IFNGR1,or

STAT-1 KO mice have more spon. Or induced tumors ;

• Perforin-/- more prone to MCA-tumor• Rag2-/- increased rate of spontaneous

tumor in aged mice • Other KO mice researches

Cancer Immunosurveillance Hypothesis

Correlative data supporting the hypothesis:• Immune-suppressed patients have higher

incidence of melanoma, lung cancer;• Positive correlation between tumor infiltrating

lymphocyte response and increased survival (melanoma, breast, colon, prostate…)

Cancer Immunoediting

Acquired Immune System?

Not enough!• CD8-/- mice seems to have

similar rate of MCA induced tumor to WT; (low MHC I!!)

• CD4-/- mice can reject syngeneic tumor graft while CD8-/- can’t;

• Rag2-/-• In PND patients, CTLs targeting neuornal

antigen cdr2 seem to protect the patients from tumor growth.

Innate Immune Responses and Cancer--- Inflammation and Cancer

• Again, the hypothesis that “tumor: wounds that fail to heal” is around for a long time. Virchow hypothesized that the origin of cancer was at sites of chronic inflammation. –1863

• But, is it true? Is inflammation helping or hindering tumor growth?

Innate Immune Responses and Cancer--- Inflammation and Cancer

•Again, like everything else, two opposite views:

1) Inflammatory infiltrations contribute to tumor growth by inducing DNA damage, providing growth and surviving factors, angiogenic/ lymphangiogenic factor, and proteases; -- “Foes”

2) Inflammatory infiltrations help to kill transformed cells, therefore limiting the growth of tumor. – “Friends”

Innate Immune Responses and Cancer--- Inflammation and Cancer

Coussens LM and Werb Z, Nature, 2002, 420:860Balkwill F and Mantovani A, Lancet, 2001, 357:539

• Data supporting “Foes”1) Association between chronic inflammation and cancer risk

Malignancy Inflammatory stimulusBladder schistosomiasisCervical papillomavirusColorectal inflammatory bowel diseasePancreatic chronic pancreatitisLung bronchitisetc…

Inflammation and Cancer

Cellular components

Polymorphonucleates (PMNs)

MMPs

ECM remodeling, Facilitate migration

Chemokines (IL-8, IP-10, MIG, MIP-1a,b etc)

Recruit TAMs

VEFG

Angiogenesis

Mast cells

TFN-a, VEGF, FGF-2, IL-8

Angiogenesis

TryptaseChymase

IL-2IFN-gIL-12

Kill tumor cells CTLs

IL-10 (tumor as well)

TGF-b1PDGbFGFTGF-aIGF-I/II

Tumor growth

TNF-aIL-1

Thrombospondin-1Angiogenesis

MMPs, uPA

ECM remodeling, Facilitate migration

Macrophages (TAMs)

Inflammation and Cancer

• Data supporting “Friends”

1) Individual cytokines been shown to mediate tumoricidal activity (TRAIL);

2) Activated macrophages mediate tumor rejection;

3) Some report says TAM positively correlate disease-free probability after surgery (while others report not informative, both prostate cancer);

Why all these conflicting data?• They are looking at different stages;

Csf1op/csf1op(do not express CSF1 which recruits MAPs): does not affect the incidence or the growth of the primary tumors, but delayed the dev. to invasive, metastatic carcinomas. 2 stages: CSF-1 promote the later stage. - Lin EY et al., 2001

Why all these conflicting data?

• Depend on the type of tumor and the stage of tumor– secrete cytokines, chemokines to attract leukocytes,actively involved in the modulation of immune responses (Th1 vs. Th2 etc..)