Natural Killer Cells in the Biology of Melanoma

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Biology of Natural Killer (NK-) Cells (I)

Comprise about 10-15% of total blood lymphoid cells Characterized phenotypically by the presence of CD56 and the lack of CD3

Biology of Natural Killer Cells (II)

Can be divided in two main subclasses with different expression pattern
CD56 Dim CD16 High
10% of blood NK-Cells

CD56 Bright CD16 Low/Neg

90% of blood NK-Cells

Biology of Natural Killer Cells (III)

NK Cells are involved in several process:
Recognize and kill tumor cells and virally infected cells Involved in immune system regulation Involved in angiogenesis Tissue homeostasis

Biology of Natural Killer Cells (IV)

NK subclasses posses different biologic profile:
Proliferative response to IL-2 Intrinsic cytotoxic capacity Activating-inhibitory NK receptor repertoire Adhesion molecule expression Tissue homing Cytokine production Monokine/Chemokine receptor expression

Natural Killer Cells and Melanoma

NK-Cell dysfunction in advanced melanoma patients
Low killing activity in vitro Impaired response to interferons Not efficient in homing to malignant tissues Only few NK-cells are found in MM-metastases which is potentially reversible by treatment with IL-2

Material and Methods

12 patients in melanoma stage IV without previous chemotherapy 6 healthy donors (matched for age and sex) Flow Cytometry analysis for different molecules involved in the regulation of NK-cell killing and tissue homing

Activating Receptors
NKp46 Expressed in resting NK cells Involved in melanoma cell killing Ligand unknown NKp44 Expressed in activated NK cells Involved in melanoma cell killing Ligand unknown

CD44 Receptor for hyaluronic acid with trigger properties

Inhibitory Receptors
CD85j Ig superfamily glycoproteins (ILT/LIR) Specific for a broad range of HLA class I antigens NKG2A C-type lectin glycoproteins Recognizes HLA-E class I glycoprotein Forms heterodimers with CD94 KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1 Ig superfamily glycoproteins that recognize defined groups of polymorphic HLA class I glycoprotein (HLA-Cw3, HLA-Cw4 , HLA-G and HLA-Bw4 respectively) Involved in NK regulation of cell-mediated lysis of melanoma cells

Chemokine Receptors
CXCR1
Expressed by several melanoma cell lines Their ligands IL8 and GROa are produced by different melanoma cell lines

CX3CR1
It s ligand Fractalkine is overexpressed in the brain and blood vessels in case of inflammation Mediates both leukocyte migration and adhesion

CCR7
Homing to lymphoid tissue Expressed by melanoma cell lines

CXCR3
Expressed by melanoma cells Their ligands IP10, I-TAC and MIG have been detected in the tumor microenvironment of human melanoma

Killing Effector Molecules

Granule exocytosis pathway
Perforin Membrane disrupting protein Granzyme A Serine protease

Death receptor pathway

CD95L, CD40L, Trail TNF family ligand Involved in cell target apoptosis signaling

Apoptosis Related Receptors

Trail-R2 Transmembrane protein with cytoplasmatic domain contains a death domain (DD) Elicit an apoptotic death response upon binding of Apo2L/TRAIL Trail-R3 Lacking a cytoplasmatic segment Act mainly as an antagonizing decoy receptor for the death domain containing Apo2L/TRAIL receptors Trail-R4 Contains a truncated death domain which renders it unable to induce apoptosis Can be cosidered as antagonizing decoy receptor

Adhesion Molecules
CD62L Important for homing of NK cell via the high endothelial venules (HEV) to peripheral LN and Peyer's patches LFA-1 Responsable for intercellular adhesion with APC and arrest of lymphocyte rolling on endothelial cells CD2 Involved in cell signaling and CTL target interaction

Results
Dim
NKp44, NKp46 CD44 CD85J NKG2A KIR2DL3, KIR3DL1 KIR2DL1 KIR2DL4 CXCR1, CX3CR1, CCR7 CXCR3 Perforin Granzyme CD40L Trail Trail-R2, Trail-R4 Trail-R3 CD62L LFA-1 CD2 0 0 0 0 ++ ++ 0 0 --++ ++ 0 + 0 0 0

Bright
0 0 ++ 0 0 0 0 -0 -++ ++ 0 ++ 0 -0

Conclusions (I)
In melanoma patients NK cells seems to be activated as indicated by higher expression of CD40L, FasL and Trail NK cell activity is inhibited: low expression of the effector molecules granzyme and perforin Inhibitory receptors (NKG2A, KIRs) are overexpressed

Conclusions (II)
There might be a role of minor HLA-I molecules (HLA-G as the ligand of KIR2DL4, which is upregulated) in NK cell inhibition Low expression of CXCR3, LFA1 and CD44 may impaire homing of NK cells to lymphoid tissues and metastases site