User:Kinkreet/Immunology/Cytotoxic Lymphocytes

Cytotoxic lymphocytes are cells which are specialised in killing cells. Usually the cells are infected with bacteria or viruses, but it can also be a cancer or tumour cell, and even parasites such as worms. There are two major types of cytotoxic lymphocytes - cytotoxic T lymphocytes (CTLs) and Natural Killer (NK) cells. CTLs are part of the adaptive immune system, whereas NK cells are part of the innate immunity, which means it can kill cells without prior exposure to the antigen; they both have the same mode of killing, the major difference is in their recognition.

CTL expresses T-cell receptors (TCRs) and CD8; the TCRs recognises and binds with low affinity to antigens presented on Major Histocompatibility Complexes (HMC) molecules, this binding is strengthened by the simultaneous binding of a co-receptor CD8 (CD4 on helper T cells), which binds specifically to MHC Class I (Class II in CD4+ cells). CTLs must also be activated by the co-stimulatory pathway.

NK cells expresses no TCRs, CD4 or CD8, but a set of germ-line encoded activating (stimulates killing) and inhibitory (block killing) receptors. There are many types of activating and inhibitory receptors, and each binds to different ligands, which are produced during different situations.

By Natural Killer (NK) Cells
NK cells were originally named because they exhibit the ability to kill tumour cells without prior stimulation. They express and present activating and inhibitory receptors on the cell surface. Both activating and inhibitory are present on a normal cell, but the inhibitory signal dominates the activating signal, and thus no cytotoxicity is observed. The two major inhibitory receptors in humans (KIR and NKG2A/CD94 complex) both bind to MHC Class I molecuels as the ligand, and so if MHC class I is presented on the cell, it will not be killed by the NK cell. Every nucleated cell in the body displays MHC Class I, and so can be viewed as a self molecule; when a virus infects a cell, it will attempt to down-regulate MHC Class I production so its antigen will not be presented to CTLs; so in virus-infected cells, the expression of MHC I molecules are usually reduced. MHC I molecules are also down-regulated in cancer and tumour cells. So in these cells, the inhibitory signal is removed, leaving only the activating receptor ligands bound, and this provides the signal for the NK cell to kill the cell.

NKG2D
When a cell is stressed, it releases stress-induced molecules. One such molecule is MICA, a distant homolog of MHC class I which resembles the α units of MHC I, which is expressed primarily in epithelial tumours, as well as other tumour and stressed cells. NKG2D is a receptor for MICA which can be found on most CD8+ cells, γδ T cells, αβ T cells and NK cells.

In NK cells, NKG2D is presented as a homodimer linked by a disulphide bond. When MICA binds to NKG2D, it activates adaptor proteins DAP10 and DAP12, which in turn activates PI3 kinase

The set of ligands which activates NKG2D is not well defined, and other ligands besides MICA and MICB (a close relative of MICA) exist, most of them are induced by DNA damage, cellular and metabolic stress.

CD16
CD16 is a group of low-affinity receptor which binds the Fc region of complexed IgG. There are two types of CD16 - FcγRIIIa (CD16a) and FcγRIIIb (CD16b). Once IgG is bound, it associates with the Fc receptor common γ chain. The cytosolic region of the γ chain contains immunoreceptor tyrosine-based activation motifs (ITAMs). ITAM is a motif found in many common signal transduction molecules; it contains a conserved sequence of YXXLX(7-12)YXXL, which upon interaction of the receptor with the ligand, will be phosphorylated on the tyrosines, and be available as a docking site for downstream proteins to bind to, which will induce signals that kill the cell to which IgG is bound to.

Because the CD16 receptor only recognises the Fc region og IgG, the presence of IgG is a pre-requisite, meaning the adaptive immune system must be activated, or there must be some left-over IgG from the previous infection, in order for CD16-dependent activation to occur. This is known as antibody-dependent cellular cytotoxicity (ADCC), a feature most prominently observed on myeloid cells such as neutrophils and eosinophils, and so in that sense, they are not purely independently innate, as they depend on components of the adaptive immune system.

Natural Cytotoxicity Receptors (NCRs)
Natural Cytotoxicity Receptors (NCRs) are known to recognise and bind to heparan sulfate (with higher affinity if highly charged) on cancer cells to perform tumour targetting It may also bind sialic acids on viral haemagglutinins (NKp46) and other unknown cellular ligands, but evidence shows that most are, or contains, carbohydrates.

There are three types of NCRs - NKp30, NKp44 and NKp46 - named for their relative molecular mass; all of their structures have been determined. NKp30 and NKp46 are expressed on all cells, whereas NKp44 is only expressed when the cell is already activated, and so provides a positive feedback effect.

KIRs
Killer cell Ig-like receptors (KIRs) binds MHC Class I, irrespective of the nature of the peptide that is bound to the cleft of the MHC. It contains 2 Ig-like fold in the extracellular region for ligand-recognition and binding, and multiple immunotyrosine inhibition motifs (ITIMs) on the cytosolic tail for signal transduction. ITIMs have the conserved sequence (S/I/V/L)XYX(I/V/L) which becomes phosphorylated by Src kinases when he ligand bind to the receptor. The phosphorylated ITIM domains provide docking sites for inhibitory downstream enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, or the inositol-phosphatase SHIP.

NKG2A/CD94
NKG2A is presented on the cell surface as a heterodimer with CD94. It recognises and binds to the non-classical Class I MHC HLA-E (in humans) and Qa1 (in mice). Once bound, it phosphorylates its ITIM region in the same way as KIRs.

Killing
The killing process used by both CTLs and NK cells are the same. There are broadly two ways of killing - necrosis and apoptosis. Necrosis is where the damaged cell is destroyed by other cells (often involving complement), which usually leads to rupture of contents; apoptosis is where the damaged cell induces its own destruction, and is often engulfed by phagocytes so the contents will not be released into the surroundings. Previously, it has been debated whether cytotoxic lymphocytes kill by necrosis or apoptosis, it is now clear that cytotoxicity works via an apoptotic pathway.

Killing the target cell occurs in several steps: granule exocytosis, dissociation and recycling. First, once the target cell is recognised, the cytotoxic lymphocyte flattens over the target cell forming a conjugate, then the cytosol of the lymphocyte rearranges, with more microtubules stretching out to the line of contact between the lymphocyte and target, so as to provide a guide for vesicles in exocytosis. Next comes the exocytosis of granules into the space between the lymphocyte and target. The target cell will then die, and the lymphocyte can be recycled and used again.

Perforin/granzyme pathway
One of the components of the granules released is perforin, a 62-66 kDa molecule, which structure is similar to the complement factor C9. When they are released by the cytotoxic lymphocyte (due to an influx of Ca2+ ions), they travel to the plasma membrane of the target cell, and its Membrane Attack Complex Perforin (MACPF) domain punch a hole in the membrane and insert itself into it. <20 monomers aggregate to polymerize into a pore. Other proteins might associate with the perforin pore to give a completed pore, with a diameter of 50-70Å. The perforin pore allows for granzyme B, another component of the granules, to enter into the target cell. There it can activate procaspase-8 to active procaspase-8, which signals further downstream for apoptosis.

Fas pathway
Fas is a member of the tumor necrosis factor receptor superfamily, and is expressed on some immune cells as well as other cells. Fas binds to Fas ligand (FasL), this allows the Fas-Associated protein with Death Domain (FADD) to bridge the Fas receptor with procaspase-8, to form the death-inducing signaling complex (DISC), which activates it to caspase-8. Active caspase-8 then signals to the cell to induce apoptosis, in the same mammer as with the perforin/granzyme pathway.