This ppt contains a detailed explanation of NK cells physiology and ADcc and its association with tumour immunity.

2. Overview
• What are Natural Killer (NK) Cells?
• Definition of NK Cells
• Development of NK Cells
• NK cell subsets
• Structure of Natural Killer (NK) Cells
• Cytotoxic and Effector Immune Response of NK Cells
• Functions of Natural Killer (NK) Cells
• Role of NK cells in various diseases
• ADCC: Mechanism
• Medical applications of ADCC

3. What are Natural Killer (NK) Cells?
• A group of innate lymphocyte cells with cytolytic activity against virus-
infected cells and tumor cells
• Identified by the presence of CD56 (CD56+)
• About 5-20% of all the circulating lymphocytes in the blood
• Initially cytotoxic activity against tumor cells described
• Cytokine-producing effector functions (interferon-γ, TNF-α,
chemokines)
• The name natural killer cells derived from the fact that these cells can
target cells without the need for prior activation

4. Development of NK cells
• Initially thought to develop in bone marrow
• More recent studies have demonstrated that these can develop
and mature in secondary lymphoid tissues like the spleen and
tonsils
• The development progresses through various stages of
maturation, expansion, and acquisition of receptors

5. NK cell subsets
• CD56bright NK cells constitute the
majority of NK cells, being found
in bone marrow, secondary
lymphoid tissue, liver, and skin
• CD56bright NK cells release
cytokines (similar to TH cells)
• CD56bright can change into
CD56dim by acquiring CD16
NK cells
CD56Bright
CD56Dim
• CD56dim NK cells are primarily found
in the peripheral blood and are
characterized by their cell killing
ability
• CD56dim NK cells are always CD16
positive
(CD16 is the key mediator of ADCC)
CD56+

6. Structure of Natural Killer (NK) Cells
• Larger in diameter than other similar lymphocytes.
• Staining: Wright-Giemsa-staining.
• The granules: azurophilic and are filled with hydrolytic and
digestive enzymes
• Also express microvilli
• The granules in these cells consist of two distinct
compartments.
The outer compartment: lysosome-associated
phosphatase acid enzymes and trumetaphosphatase.
The inner compartment: structural components and has no
enzymatic activities associated with it

7. Structure contd.
• The cytoplasm also contains mitochondria and polysomes
• The nucleus: convoluted with distinct polarity against dense
granules and pseudopodia

8. NK cell Receptors
Receptors
Activating
Inhibitory

9. Inhibitory Receptors
• CD94/NKG2: conserved in both rodents and primates
and identifies nonclassical MHC I molecules such
as HLA-E.
• ILT or LIR (immunoglobulin-like receptor): Recently
discovered members of the Ig receptor family
• Ly49: have both activating and inhibitory isoforms.
Ly49 are receptor for classical (polymorphic) MHC I
molecules.

10. • The interaction of NK cell inhibitory receptors
natural-killer group 2, member A (NKG2A) and
Ly49A with its cognate ligand leads to
phosphorylation of immunoreceptor tyrosine-
based inhibitory motif (ITIM) in their
cytoplasmic tails.
• Phosphorylated ITIM recruits phosphatases
such as Src homology domain-containing
tyrosine phosphatase (SHP) and SH2
domain-containing inositol-5-phosphatase
(SHIP) that dephosphorylate signaling
molecules such as Lck, Fyn, Syk, Zap70, and
Vav1, thereby terminating activating receptor
signaling in NK cells.
Schematic representation of natural killer
(NK) cell inhibitory receptor signaling.

11. Activating Receptors
• Ly49: the receptor for classical (polymorphic) MHC
I molecules
• NCR: (natural cytotoxicity receptors) They bind viral
ligands such as hemagglutinins and hemagglutinin
neuraminidases
• CD16: plays a role in ADCC; in particular, they
bind Immunoglobulin G

12. • Interaction of activating receptor NKG2D and
Ly49D with their cognate ligand leads to
phosphorylation of YINM motif or immunoreceptor
tyrosine-based activation motifs (ITAMs) present in
the cytoplasmic tails of associated adapter protein
such as DAP10 and DAP12.
• Phosphorylated ITAM or YINM motif recruits
Syk/Zap70, PI3K, and Grb2/Vav1/SLP-76 complex.
• Grb2/Vav1/SLP-76 pathway activation leads to
downstream activation of MEK/extracellular signal-
regulated kinase (ERK) pathway.
• Phosphorylated Syk recruits PLC-γ which in turn
activates inducible protein-3 (IP-3) and DAG
pathway leading to activation of transcription factors
NF-κB and NFAT.
• The net result of this signaling is the release of
cytokines and chemokines as well as cytotoxic
Schematic representation of natural
killer (NK) cell activating receptor
signaling.

13. Cytotoxic and Effector Immune
Response of NK Cells

14. Cytotoxic Response of NK Cells
The NK cell cytotoxic response is tightly
regulated in four discrete stages.
• Step 1: Recognition of target cells by NK cell:
reorganization of actin cytoskeleton and the
formation of immunological synapse, and
clustering of CAM such as lymphocyte
function-associated antigen 1 (LFA-1) and
CD2
• Step 2: Microtubule organizing center (MTOC)
and secretory lysosome polarize toward the
immunological synapse
• Step 3: Docking which involves moves close
to the plasma membrane of NK cell at the
synapse
• Step 4: Secretory lysosome fuse with the
target cell plasma membrane and releases
the cytotoxic granules into the target cell

15. Effector Immune Response of NK Cells
• IFN-γ activates antitumor immunity
• The tumor stromal cells control the secretion of effector cytokines
• Signaling through activating receptor NKG2D promotes the
release of IFN-γ
• IL-12 induces the production of IFN-γ in NK cells, which could be
further enhanced by TNF-α, IL-1, IL-10 and IL-18
• TGF-β inhibits the production of IFN-γ, TNF-α, and GM-CSF in
NK cells

16. Cont’d.
• IL-12 treatment inhibits tumor metastasis by the NKG2D
pathway
• IL-21: NK cell activation
• IL-15: activate NK cell function and suppress tumor growth.
• These studies point out that apart from the NK cell cytotoxic
function, cytokines secreted by the NK cells also provide a
significant boost to the antitumor immunity
• The cytokines secreted by other immune cells or stromal cells in
the tumor microenvironment can positively or negatively
influence the antitumor function of NK cells

17. Functions of Natural Killer (NK) Cells
1.Cytolytic activity
2.Antibody-dependent Cell-mediated Cytotoxicity
3.The activated NK cells activate macrophages for
phagocytosis
4.React with antigens immediately without any prior
exposure
5.Immunosurveillance of tumor cells
6.Clear dead and senescent cells
7.Display different adaptive features

18. ADCC

19. Antibody dependent cell mediated cytotoxicity
(ADCC) is a cooperative response involving
1. Binding of antibodies produced by B-cells to antigens
2. Recognition of the constant fragment (Fc) by effector cells
3. Release of damaging substances by effector cells
4. Destruction of the pathogenic organism

20. ADCC
•ADCC is activated by
•Specific binding of circulating antibodies to antigens using
•IgG antibodies for the majority of responses
•IgE antibodies for invasive helminths and other parasites
•Recruitment of immune cells by the constant fragment (Fc) of the antibody including
•Granulocytes such as
•Neutrophils
•Monocytes
•Eosinophils
•Other cells such as
•Macrophages
•Natural Killer cells
•ADCC is an adaptation of innate immune functions by the adaptive immune system
•The specificity of ADCC is determined by the specificity of the antibody

21. Effector Mechanisms
• After recognition of the coated pathogen a variety of cells are recruited to
the pathogen

22. Medical applications of ADCC
• The effects against solid tumors:
trastuzumab and rituximab monoclonal antibodies: to involve
ADCC as an important mechanism of therapeutic action (mice)
• Multiple myeloma: Daratumumab monoclonal antibody. Studies
with in vitro materials: ADCC is an important mechanism

23. References
• Judith A. Owen, Jenni Punt, Sharon A. Stranford (2013). Kuby
Immunology. Sixth Edition. W. H. Freeman and Company
• https://www.frontiersin.org/articles/10.3389/fimmu.2017.01124/full
• https://www.chegg.com/learn/medicine-and-health/medical-
terminology/natural%20killer%20cells
• https://step1.medbullets.com/immunology/105048/antibody-
dependent-cell-mediated-cytotoxicity-adcc