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What the T-cell sees

What the T-cell sees
We have on several occasions alluded to the fact that the αβ T‐cell receptor sees peptide antigen associated with an MHC class I or II molecule on the surface of cells. Now is the time for us to go into the nuts and bolts of this relationship.

Haplotype restriction reveals the need for MHC participation
It has been established in “tablets of stone” that T‐cells bearing αβ receptors, with some exceptions, only respond when the antigen‐presenting cells (APCs) express the same MHC haplotype as the host from which the T‐cells were derived (Milestone 5.1). This haplotype restriction on T‐cell recognition tells us unequivocally that MHC molecules are intimately and necessarily involved in the interaction of the antigen‐bearing cell with its corresponding antigen‐specific T‐lymphocyte. We also learn that, generally, cytotoxic T‐cells recognize antigen in the context of class I MHC, and helper T‐cells interact when the antigen is associated with class II molecules. Accepting, then, the participation of MHC in T‐cell recognition, what about the antigen

Milestone 5.1 MHC restriction of T‐cell reactivity

MHC was known to be a dominant controlling element in tissue graft rejection, but could this really be its main function?
A dramatic Nobel prize‐winning revelation by Peter Doherty and Rolf Zinkernagel was that cytotoxic T‐cells taken from an individual recovering from a viral infection would only kill virally infected cells that share an MHC haplotype with the host. They found that cytotoxic T‐cells from mice of the H‐2d haplotype infected with lymphocytic choriomeningitis virus could kill virally infected cells derived from any H‐2d strain but not cells of H‐2k or other H‐2 haplotypes. The reciprocal experiment with H‐2k mice shows that this is not just a special property associated with H‐2d (Figure M5.1.1a). Studies with recombinant strains (see Table 4.4) pin‐pointed class I MHC as the restricting element and this was confirmed by showing that antibodies to class I MHC block the killing reaction.
The same phenomenon has been repeatedly observed in the human. HLA‐A2 individuals recovering from influenza have cytotoxic T‐cells that kill HLA‐A2 target cells infected with influenza virus, but not cells of a different HLA‐A tissue‐ type specificity (Figure M5.1.1b). Note how cytotoxicity could be inhibited by antiserum specific for the donor HLA‐A type, but not by antisera to the allelic form HLA‐A1 or the HLA‐DR class II framework. Of striking significance is the inability of antibodies to the nucleoprotein to block T‐cell recognition even though the T‐cell specificity in these studies was known to be directed towards this antigen. As the antibodies react with nucleoprotein in its native form, the conformation of the antigen as presented to the T‐cell must be quite different.
 In parallel, an entirely comparable series of experiments has established the role of MHC class II molecules in antigen presentation to helper T‐cells. Initially, it was shown by Ethan Shevach and Alan Rosenthal that lymphocyte proliferation to antigen in vitro could be blocked by antisera raised between two strains of guinea‐pig that would have included antibodies to the MHC of the responding lymphocytes. More stringent evidence comes from the type of experiment in which a T‐cell clone proliferating in response to ovalbumin on antigen‐presenting cells with the H‐2Ab phenotype fails to respond if antigen is presented in the context of H‐2Ak. However, if the H‐2Ak antigen‐presenting cells are transfected with the genes encoding H‐2Ab, they now communicate effectively with the T‐cells (Figure M5.1.2).
Figure M5.1.1 T‐cell killing is restricted by the MHC haplotype of the virus‐infected target cells. (a) Haplotype‐restricted killing of lymphocytic choriomeningitis (LCM) virus‐infected target cells by cytotoxic T‐cells. Killer cells from H‐2d hosts only killed H‐2d‐infected targets, not those of H‐2k haplotype and vice versa. (b) Killing of influenza‐infected target cells by influenza nucleoprotein (NP)‐specific T‐cells from an HLA‐A2 donor. Killing was restricted to HLA‐A2 targets and only inhibited by antibodies to A2, not to A1, nor to the class II HLA‐DR framework or native NP antigen.
Figure M5.1.2 The Tcell clone only responds by proliferation in vitro when the antigenpresenting cells (e.g., macrophages) pulsed with ovalbumin express the same class II MHC.

T-cells recognize a linear peptide sequence from the antigen
In Milestone 5.1, we commented on experiments involving influenza nucleoprotein‐specific T‐cells that could kill cells infected with influenza virus. Killing occurs after the cytotoxic Tcell adheres strongly to its target through recognition of specific cell surface molecules. It is curious then that the nucleoprotein, which lacks a signal sequence or transmembrane region and so cannot be expressed on the cell surface, can nonetheless function as a target for cytotoxic T‐cells, particularly as we have already noted that antibodies to native nucleoprotein have no influence on the killing reaction (see Figure M5.1.1b). Furthermore, uninfected cells do not become targets for the cytotoxic T‐cells when whole nucleoprotein is added to the culture system. However if instead we add a series of short peptides with sequences derived from the primary structure of the nucleoprotein, the uninfected cells now become susceptible to cytotoxic T‐cell attack (Figure 5.13).

Figure 5.13 Cytotoxic T‐cells, from a human donor, kill uninfected target cells in the presence of short influenza nucleoprotein peptides. The peptides indicated were added to 51Cr‐labeled syngeneic (i.e., same as T‐cell donor) cells and cytotoxicity was assessed by 51Cr release with a killer to target ratio of 50 : 1. The three peptides indicated in red induced good killing.

Thus was the mystery of T‐cell recognition of antigen revealed. T‐cells recognize linear peptides derived from protein antigens, and that is why antibodies raised against nucleoprotein in its native three‐dimensional conformation do not inhibit killing. Note that only certain nucleoprotein peptides were recognized by the polyclonal T‐cells in the donor population and these peptides therefore constitute the Tcell epitopes. When clones are derived from these T‐cells, each clone reacts with only one of the peptides; in other words, like B‐cell clones, each clone is specific for one corresponding epitope.
Entirely analogous results are obtained when T-helper clones are stimulated by antigen‐presenting cells to which certain peptides derived from the original antigen have been added. Again, by synthesizing a series of such peptides, the T‐cell epitope can be mapped with some precision.

The conclusion is that the T-cell recognizes both MHC and peptide and we now know that the peptide lies along the groove formed by the α‐helices and the β‐sheet floor of the class I and class II outermost domains (see Figure 4.19). Just how are the peptides produced? The answer lies in a step referred to as antigen processing in which the proteases that are present within cells, either assembled into a structure called the proteasome that is present in the cytosol (Figure 5.14a) or located in endosomal vesicles (Figure 5.14b), break down intact protein into peptides. Various molecules are then involved in inserting the peptides into the binding groove of the MHC molecule prior to antigen presentation of the peptide to the TCR on T‐cells. Let’s now look in a little more detail at antigen processing.

Figure 5.14 Antigen processing and presentation. In order to be recognized by T‐cells bearing an αβ receptor, protein antigen (polypeptide) must be broken down (processed) into short peptides by proteolytic enzymes. (a) Antigens (e.g., viral proteins) present within the cytoplasm of a cell are referred to as endogenous antigen and are processed by enzymes that are organized into a structure called the proteasome. The resulting peptides are then moved from the cytoplasm into the ER through the transporters associated with antigen processing (TAP), and subsequently loaded into a newly synthesized MHC class I molecule. (b) In contrast, antigens taken up by endocytosis or phagocytosis from outside of the cell are described as being exogenous antigen and are degraded into peptides by a different set of proteases that are present in the endocytic/phagocytic vacuoles. The newly synthesized MHC class II molecules need to be transported out of the ER in vesicles that subsequently fuse with the peptide‐containing vacuoles. In order to prevent peptides present in the ER binding to the MHC class II molecules (rather than to the intended class I) a “molecular stopper” called invariant chain (Ii) is put into the class II groove. The Ii is later degraded into a fragment called CLIP which is then subsequently exchanged for the peptide. MHC class I and II molecules containing peptides are ultimately taken to the cell surface for antigen presentation to the TCR. MHC class I presents peptides to CD8+ T‐cells whereas MHC class II presents peptides to CD4+ T‐cells.