Recognition of tumor cells, like that of other types of targets, by CTL is mediated by a complex resulting from the loading of HLA class I molecules with antigenic peptides. This complex is generated by the HLA class I APM through four steps: (i) protein cleavage into peptide, (ii) transport of the resultant peptides into the endoplasmic reticulum (ER), (iii) assembly of the peptides with HLA class I heavy chains and 02 m and (iv) transport of the HLA class I antigen-peptide complexes to the cell membrane (Fig. 2.2). Each of these steps can be modulated by cytokines. We have recently described these steps in detail and we refer the interested reader to our paper also as a source of references . Here, we will briefly present the most important information.
Peptide ligands for association with HLA class I molecules are generated from proteins by proteasomes which are comprised of a barrel shaped 20 S core particle with caps on the ends to regulate the entry of substrate proteins. The 20 S core proteasome consists of four heptameric rings stacked atop of one another, with the inner two rings containing the three proteolytic subunits MB1, delta and zeta which are responsible for chymotryptic, tryptic and post-glutamyl cleavage. Proteasome composition is modulated by IFN-y which induces the replacement of MB1, delta and zeta subunits with Low Molecular Mass Polypeptides (LMP) 7, LMP2 and LMP10, respectively, and also induces expression of the proteasome activator subunits, PA28a and PA28a. The latter replace the constitutive 19 S caps. This change of subunits results in the formation of an "immunoproteasome" which generates a different spectrum of peptides than the constitutive proteasome. As a result, the epitopes expressed by the peptides generated by proteasomes and immunoproteasomes from a given tumor antigen may be different.
After degradation by proteasomes or immunoproteasomes into relatively large peptides (12-20 amino acids in length), the N-terminus of most peptides is further trimmed by aminopeptidases. Peptides of the appropriate size are transported into the ER lumen by an IFN-y inducible member of the ATP-Binding Cassette (ABC)-family of transporters termed the Transporter Associated with Antigen Processing (TAP) which is composed of the two subunits TAP1 and TAP2. In the absence of TAP, HLA class I antigens are not loaded with peptides. As a result, they are poorly exported from the ER and have a low surface expression. This appears to be the rule for all of the allospecificities that have been tested with the exception of HLA-A2. The latter alloantigen can bind peptides derived from signal sequences whose presence in the ER is independent of TAP function. Therefore, TAP deficient cells exhibit a limited supply and repertoire of peptides that can be presented to CD8+ T cells. On the other hand, when TAP is downregulated rather than lost, the level of HLA class I antigen expression may not be affected as dramatically, but the repertoire of presented peptides may still be reduced leading to ineffective recognition of target cells by CTLs. This is clearly shown by the resistance to CTL killing of head and neck squamous cell carcinoma (SCCHN) cell lines in spite of HLA class I antigen expression. The lack of recognition of SCCHN cells by CTL is associated with downregulation of some APM components, such as LMP2, TAP1, TAP2 and tapasin in tumor cells. Furthermore, recognition of SCCHN cells by CTL can be restored by transfecting tumor cells with TAP1 cDNA .
After transport into the ER by TAP, peptides may be further trimmed by the heterodimeric ER aminopeptidase ERAP1/ ERAP2 before they bind to HLA class I antigens. The loading of peptides onto HLA class I antigens is facilitated by the chaperones calreticulin, ERp57 and tapasin which are members of the peptide-loading complex. The lack of their expression causes a reduction in the repertoire of peptides and/or HLA class I antigen expression at the cell surface, leading to impaired CTL recognition. Once assembled with peptides, HLA class I molecules dissociate from TAP and travel to the cell membrane and present peptides to CTL.
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