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 T‐cell clone only responds by proliferation in
vitro when the antigen‐presenting cells (e.g.,
macrophages) pulsed with ovalbumin express the same class II MHC.
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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 T‐cell 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 T‐cell 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.
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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.
