Selección de las tuberías de revestimiento

After their initial identification, two seemingly paradoxical observations regarding the functions of NK cells were made. Firstly, it had been noted that many tumour cell lines were sensitive to lysis by NK cells, some of which lacked MHC class I expression. Secondly, NK cells were shown to be responsible for the phenomenon in certain inbred strains of mice, of hybrid resistance to bone marrow grafts (379). Hybrid resistance refers to the situation where for example, ( A x B) F1 hybrid offspring can reject grafts from parent A or B (380). Thus, the in vitro cytotoxicity of tumour cells did not appear to show specificity for MHC class I molecules, whereas hybrid resistance clearly did. Finally, this elusive question was answered by the elegant “missing se lf hypothesis first put forward by Klas Karre (381). The basis of this theory was that high levels of a complete set of self MHC class I molecules on target cells were required for their protection from lysis by NK cells. Many tumour cells have a missing or altered expression of class I alleles, while a graft of either A or B origin would be lysed by NK cells from (A x B) hybrids because it did not express the full set of (A x B) MHC molecules (382). In later years, the molecular basis of the missing self hypothesis was explained by the discovery of NK cell inhibitory receptors specific for MHC class I molecules on target cells. Two types of NK cell inhibitory receptors have been identified, belonging either to the immunoglobulin superfamily or to the family of C-type lectin molecules.

In humans, NK cells express a number of inhibitory receptors specific for MHC class I, which are type I integral membrane glycoproteins belonging to the immunoglobulin superfamily. These receptors, which have been termed killer inhibitory receptors (KIR), can be grouped into two subtypes based on their structure. The NK cell inhibitory receptors specific for HLA-C contain 2 immunoglobulin domains and have been designated KIR2DL, while those specific for HLA-A and HLA-B contain 3 immunoglobulin domains and are designated KIR3DL (383). The antibody-mediated masking of the NK cell KIR, or their MHC ligands results in the reversal of the NK cell inhibition (384). Aside from having a

negative effect on NK cell cytotoxicity, KIR interactions with MHC class I on target cells has also recently been shown to disrupt NK cell adhesion to target cells (385). The KIR2DL receptors recognise two subsets of HLA-C allotypes which are determined by alternative amino acid sequence motifs at position 77 and 80 of the a1 helix of the HLA-C molecule (386). Thus, the KIR2DL1 receptor recognises HLA- Cw2, Cw4, Cw5, Cw6, Cw15 and Cw17, while the KIR2DL2 receptor recognises HLA-Cw1, Cw3, Cw7, Cw8, Cw12, Cw14 and Cw16 molecules. This dimorphism could thus lead to allospecific recognition by NK cells of target cells bearing HLA-C (386). The effect of the peptide bound to target cell HLA class I molecules on the inhibitory signal delivered to NK cell KIR is still unclear. However, some degree of NK cell specificity for the peptide bound to certain HLA-B molecules has been demonstrated (387).

In addition to the Ig-type KIR, human NK cells also express a C-type lectin inhibitory receptor complex that is specific for HLA class I molecules. CD94 belongs to the C- type lectin family, and forms a complex with NKG2A molecules (388). However, in contrast to the KIR, which are specific for polymorphic class I molecules, the CD94/NKG2A complex is specific for HLA-E (389). HLA-E is a non-classical class I molecule of limited variability, and its expression on the cell surface depends on the binding of a peptide derived from the leader sequences (amino acids 3-11) of HLA- A, B and C (390). Thus, the CD94/NKG2A receptor complex recognises HLA-E bound to peptides from the signal sequences of polymorphic class I molecules (391).

All of the mouse and human NK cell inhibitory receptors described so far contain intracellular immunoreceptor tyrosine-based inhibitory motifs. These intracellular motifs are responsible for the generation of the inhibitory signal (392). In order to avoid self-reactivity, it is necessary for every NK cell to express at least one inhibitory receptor specific for self MHC class I alleles. Both types of NK cell inhibitory receptors are clonally distributed on NK cells and unlike the KIR, the CD94 receptor complex is more widely expressed on NK cells. The repertoire and usage of KIR on NK cell clones varies between individuals (393). In mice, MHC-specific inhibitory receptors also exist. The best characterised of these is the C-type lectin

Ly-49A receptor, which is specific for H-2^ or H-2"^ molecules (394). Other members of the Ly-49 family exist, and these are likely to also be specific for H-2 molecules. Initially it was thought that, since the human and murine receptors belonged to different families, they must have evolved separately towards the same function (395). However, recently a murine Ig type molecule, namely gp49B1, has been identified that seems a likely candidate for the murine equivalent of the human killer inhibitory receptors (189).

Table 1-1 NK cell inhibitory receptors and their ligands

NK Cell Receptor Receptor Type^ Ligand

Human NK Cells

CD94/NKG2A C-type lectin HLA-E^

KIR2DL1 Ig superfamily HLA-CW2, 4 ,5 , 6, 15, 17 KIR2DL2, 3 Ig superfamily HLA-Cwl, 3, 7, 8, 12, 14, 16

KIR3DL1-5 Ig superfamily HLA-A, HLA-B

Murine NK Cells:

Ly49A C-type lectin _{H-2^, H-2''}

Gp49B1 Ig superfamily H-2

NK cell receptors belong either to the family of C-type lectin molecules, or to the immunoglobulin superfamily

The CD94/NKG2A complex binds to HLA-E molecules in complex with a peptide derived from the leader sequence of HLA-A, -B or -C antigens

1.7.6 NK cell signalling

The signalling events following the ligation of the FcyRIIIA receptor on NK cells have been partly defined. The ligation of FcyRIIIA on the NK cell results in recruitment of protein tyrosine kinases by the immuno-tyrosine activatory motif region of the receptor. This leads to the activation of protein tyrosine kinases, which trigger multiple downstream signalling events. These include the phosphorylation of phospholipase 0, which then cleaves phosphoinositide, and the ultimate mobilisation of intracellular calcium (396). Finally, this results in the release of cytotoxic granules by NK cells and target cell lysis.

The signalling events following NK cell-mediated natural killing are less well defined, although some similarities to FcyRIIIA signalling events have been noted. Phosphotyrosine kinase activation, phospholipase C-catalysed release of phosphoinositides, and elevations in intracellular calcium levels usually occur during most forms of natural killing (397). However, the natural killing of K562 tumour target cells requires protein kinase C, while antibody-dependent cellular cytotoxicity appears to be independent of protein kinase C (398). Furthermore, some virus- infected target cells trigger NK cell cytotoxicity in the absence of detectable phospholipase C-dependent calcium signalling (399).

The positive signalling events arising from the ligation of FcRylllA or receptors involved in natural killing can be countered by negative signals resulting from the interaction of NK cell inhibitory receptors with MHC class I molecules. The effects of both lectin and immunoglobulin-type NK cell inhibitory receptors are mediated via a

common inhibitory mechanism. This mechanism involves the tyrosine

phosphorylation of the conserved inhibitory tyrosine inhibitory motif in the cytoplasmic tail of the receptors. This results in the association of the receptor with the Src homology 2 domain-containing tyrosine phosphatase (SHP-1), and the inhibition of proximal tyrosine kinases by SHP-1 (397). Thus, by interrupting the tyrosine phosphorylation of key molecules, inhibitory receptors can inhibit NK cell activation. Significantly, these receptors can only exert their inhibitory effect on NK cell activation when they are co-aggregated with an activating receptor (400). Presumably, this reflects the need for SHP-1 to be in close proximity to the tyrosine

kinases in order to prevent their activation. Furthermore, there is evidence to suggest that the inhibitory effect of killer inhibitory receptors is spatially restricted, and occurs at a local rather than a global level. It has been recently demonstrated that an NK cell already bound to a resistant target cell can simultaneously bind and kill a susceptible target cell (401).

f.7.7 Balancing the opposing forces

The capacity of an NK cell to mediate killing or secrete cytokines is though to be determined by a balance of such positive and negative signalling events as those described above. Precisely how these opposing events are weighted is not clear, however both quantitative and qualitative factors may be involved. The expression of MHC class I frequently protects target cells from lysis and the loss of class I renders them sensitive to lysis. However, certain targets with decreased MHC class I expression are not sensitive to NK cell cytotoxicity, suggesting that even in the absence of inhibitory signals a triggering event is required (363). Conversely, certain target cells such as the highly NK-sensitive Jurkat, K562 or YAC-1 cells are such potent activators of NK cells, that MHC expression does not inhibit their killing (402).

NK CELL