(Received for publication, November 17, 1994; and in revised form, January 13, 1995)
From the
The antigen presentation pathway yields peptide-MHC class I complexes on the antigen presenting cell (APC) surface for recognition by appropriate T-cells. Expression of the peptide-MHC complex on APC surface is preceded by several steps that include the generation of peptide fragments in the cytoplasm and their assembly with MHC molecules in the endoplasmic reticulum. It is now clear that MHC binding to optimally processed peptides in the endoplasmic reticulum is obligatory for their stable expression on the cell surface. However, whether a similar obligatory relationship exists between generation of processed peptides and their expression as peptide-MHC on APC surface is not known. Here, we addressed this question by analyzing the processing of ovalbumin (aa257-264, SL8) or influenza nucleoprotein (aa366-374, AM9) analogs. We examined the generation of naturally processed peptides using precursors that did, or did not, contain residues flanking the optimal MHC-binding peptides. By characterizing the peptides generated from these precursors by T-cell stimulation assays and by high performance liquid chromatography analysis, we established that intracellular assembly of peptide-MHC complexes and their expression on the cell surface can occur with peptides that lack flanking residues. The presentation of these endogenously synthesized perfect fit peptides demonstrates that the cleavage of precursor polypeptides is an independent step in the antigen presentation pathway.
Antigen presentation is the mechanism by which peptide-MHC class I complexes are displayed on the cell surface for
recognition by appropriate T-cells(1, 2) . The
peptides displayed by MHC on the cell surface represent precisely
cleaved proteolytic fragments of intracellular
proteins(3, 4) . From the analysis of naturally
processed peptides, a clear picture of the general structural features
of these peptide products (5) and how they bind to the MHC
molecules has emerged(6, 7) . This taken together with
discoveries of the transporter and proteasome genes within the MHC has
suggested an outline of how endogenous proteins yield peptide-MHC
complexes on the cell surface(8) .
At least four distinct events are necessary for generating peptide-MHC complexes on the APC surface. (a) Intracellular proteins are fragmented, and their peptide products are (b) transported into the ER wherein (c) they are assembled into peptide-MHC complexes that (d) travel to their final destination on the cell surface(8, 9, 10) . The constitutive nature and remarkable efficiency of this process (11, 12, 13, 14, 15) raises the question of whether these steps occur independently, or whether each step is dependent upon successful completion of the preceding step, i.e. the peptide-MHC complexes are generated by a series of concerted steps. Indeed, the last three steps (b-d) of this pathway are concerted. Loss of either one or both subunits of the TAP1/2 transporter prevents assembly and surface expression of peptide-MHC complexes(16, 17, 18, 19, 20) . Likewise, empty MHC class I molecules are physically associated with the TAP heterodimer as well as with calnexin and are retained in the ER until they bind peptides(21, 22, 23) . Thus, peptide transport, assembly of peptide-MHC complexes, and their expression on the cell surface are sequential and concerted steps. Because all known naturally processed peptides represent proteolytic products of cellular proteins, existence of a similar obligatory relationship, if any, between the requirements for proteolysis and display of their products on the cell surface is not known. For example, it is conceivable that, under physiological conditions, TAP may transport only those endogenous peptides that were produced by the proteasome.
Current evidence favors the 26S proteasome, containing
the MHC-encoded (24, 25, 26, 27) ,
-interferon-inducible (28, 29) LMP-2 and LMP-7
polypeptides, as being responsible for antigen
processing(8, 10) . Whether this is the only mechanism
for generating processed peptides has been questioned first by the
intact antigen presenting ability of cell lines lacking both LMP-2 and
LMP-7(30, 31, 32) , and more recently by the
dramatically distinct phenotypes of peptide-MHC expression between TAP1
knock-out mice (20) and LMP-7 (33) or LMP-2 knock-out
mice(34) . TAP1 mutant mice are virtually devoid of MHC
molecules on the cell surface, fail to present endogenous peptide-MHC
class I complexes, and as a consequence are severely depleted in their
CD8+ T-cell subset in the thymus and
periphery(20, 35, 36) . By contrast, LMP-7 or
LMP-2 mutant mice express significant to normal levels of MHC and
CD8+ T-cells and show only selective defects in antigen
presentation to T-cells(33, 34) . Whether the
incomplete defects in peptide-MHC expression in LMP-2 and LMP-7 mutant
mice can be attributed to a reduced or abnormal supply of naturally
processed peptides and/or to inefficient peptide transport by
disruption of LMP-2 and/or LMP-7-dependent link between the proteasome
and the TAP complex is presently unclear. Thus, while it is established
that efficient supply of cytoplasmic peptides to the MHC in the ER
depends upon proteolysis (37) , as well as TAP-mediated
transport(20) , the mechanism(s) that generate naturally
processed peptides and deliver them to TAP remain poorly understood.
A different approach to addressing the role of proteolysis in
antigen presentation is to focus on the antigen rather than the
protease(s). By examining the processed products of antigen precursors
that do or do not contain residues flanking the optimal MHC-binding
(``perfect-fit'') peptides, we can determine whether peptide
cleavage had occurred and whether it was an obligatory step in the
expression of peptide-MHC complexes on the cell surface. Although
conceptually straightforward, this approach has been difficult to
implement. First, only vanishingly small amounts of processed peptides
are present in the APC, and individual peptides are detectable in
complex cell extracts only because of highly sensitive T-cell
activation assays(3, 4, 38) . Second, a more
profound problem is the fact that efficient synthesis of precursor
polypeptide requires presence of the translational initiation
(``ATG,'' methionine) codon(15, 39) . Thus,
endogenously synthesized perfect-fit precursors must contain methionine
as the first residue of the processed peptide. Recently, we showed that
cells expressing minimal analogs of the ovalbumin octapeptide,
aa257-264 (SIINFEKL, SL8), serve as efficient APCs for
SL8/K-specific T-cells as well as allow analysis of the
processed peptides in HPLC-fractionated cell
extracts(14, 40) . By comparing extracts of cells
expressing different MHC molecules, we showed that appropriate MHC
molecules are essential for stabilizing otherwise rapidly degraded
processed peptides. This extreme instability of processed peptides
ruled out their direct analysis in cells lacking MHC. However, we
noticed one instance where the Met-SL8 (MSL8) peptide was actually
present in D
cells expressing the minigene encoding the
MSL8 precursor. The fortuitous discovery of the perfect-fit MSL8
peptide that could be endogenously translated as well as be detected as
such in cell extracts prompted us to use this model system to address
the question of whether cleavage of flanking residues from endogenously
synthesized polypeptides is an obligatory step in the antigen
presentation pathway.
We compared the naturally processed peptides
that were generated from N terminally extended precursors or from
perfect fit peptides. DNA constructs encoding ovalbumin
(OVA257-264, SL8) or influenza nucleoprotein (NP366-374,
AM9) analogs were used as model antigens for generating peptide/K or D
MHC complexes. MHC-bound peptides were extracted
from transfected cells and were characterized by their HPLC elution
profiles. Here we show that sequences flanking the optimal MHC-binding
peptides were unfailingly cleaved from endogenously synthesized
precursors indicating that they were processed. However, perfect fit
precursors that exactly matched the optimal MHC-binding peptides were
presented on the cell surface as such with comparable efficiency. These
results show that cleavage of flanking residues is not obligatory for
the intracellular assembly or for cell surface expression of
peptide-MHC complexes and establish that proteolytic cleavage of
antigenic precursors can be segregated from other steps in the antigen
presentation pathway.
For
separation of peptides associated with intracellular and cell surface
MHC molecules, cells were first incubated with B22.249
(anti-D) ascites and thoroughly washed before lysis. The
supernatant fluid of the centrifuged lysate was added to protein
A-Sepharose beads to isolate cell surface MHC molecules. The
supernatant fluid of the lysate containing intracellular MHC molecules
was then subject to another round of anti-D
immunoprecipitation. Peptides from these MHC immunoprecipitates
were eluted and assayed as described above. Data shown are
representative of three independent experiments.
Figure 1:
Schematic diagram showing the
precursor-product relationships for the antigens and MHC molecules
used. Precursor peptides and their abbreviations are shown on the left
with boxed peptides representing the products associated with
the K or D
MHC class I molecules. One, two, or
no N-terminal amino acids are cleaved to result in the peptide products
shown with their abbreviations on the right. Peptide sequences with xs
represent the MHC allele-specific consensus motifs for bound peptides. A, SL8/K
-specific B3Z T cell hybrids were used to
analyze the depicted precursor-product model systems. B, AM9
or MM9/D
-specific DBFZ.25 T cell hybrids were used to
analyze this depicted precursor/product model
system.
Peptide-MHC complexes expressed on the
cell surface are first assembled in the ER(47) , and the
possibility remained that processed peptides in the trifluoroacetic
acid extracts represented only a subset of processed peptides (e.g. only those peptides present on the cell surface). To establish
that all extra- and intracellular MHC-bound peptides had been cleaved
to the optimal SL8 peptide, we looked for possible SL8 analogs that
could be bound to K MHC in cells lysed with detergents.
First, synthetic SL8, KSL8, or MSL8 peptides were all capable of
stimulating SL8/K
-specific B3Z T-cells (Fig. 2A). The dose-response curves, consistently
within 2-fold of each other, show that picomolar concentrations of
these peptides could be readily detected, and in addition, each of
these peptides could also be readily distinguished by their
characteristic HPLC elution profiles (Fig. 2B). Second,
both MSL8 and MKSL8 DNA constructs allowed generation of
T-cell-stimulating peptide/K
complexes on the cell surface (Fig. 2C). Nevertheless, with both precursors only the
cleaved SL8 octapeptide was bound by K
MHC (Fig. 2D). The HPLC elution profiles of T-cell
stimulating activity of naturally processed peptides in anti-K
(Y3 monoclonal) immunoprecipitates from either MSL8- or
MKSL8-transfected cell extracts corresponded exactly to that of
synthetic SL8. Other possible candidates such as MSL8 or KSL8 peptides
were clearly undetectable and were estimated to represent less than
0.1% of the amount of SL8 recovered. In other experiments (data not
shown), the same profiles were obtained when K
MHC were
immunoprecipitated with polyclonal antiserum specific for the
cytoplasmic tail of K
(48) , ruling out a possible
peptide-specific bias in the subset of K
MHC
immunoprecipitated by the Y3 mAb(49, 50) . We conclude
that in living cells, similar to the native ovalbumin, the same SL8
octapeptide was processed from these 9-10 aa precursors for
presentation by K
MHC. However, SL8 itself cannot be
synthesized within cells without the addition of the methionine residue
for initiating translation(15) . Therefore, to test whether
this proteolytic removal of N-terminal-flanking residues, regardless of
the specific mechanism involved, was obligatory for generation of
peptide-MHC complexes, we analyzed model precursors that exactly
matched the MHC-bound, naturally processed peptides and could be
translated within cells.
Figure 2:
Only
cleaved products are present in K-COS cells transfected
with precursor constructs encoding N terminally extended antigenic
peptides. A, K
-L cells with synthetic peptides,
SL8 (
), KSL8 (
), and MSL8 (
), at the indicated
concentrations were assayed in the exogenous peptide assay to show that
these peptides are efficiently recognized. Medium control (
). B, HPLC chromatograms of the synthetic peptides, KSL8, SL8,
MKSL8, and MSL8, show excellent resolution of KSL8, SL8, and MSL8. C, K
-COS cells were transiently transfected with
vector DNA (
) or DNA constructs encoding MSL8 (
) or MKSL8
(
) at the indicated DNA concentrations. Stimulatory peptide-MHC
complex expression in transfected K
-COS cells was shown by
the endogenous peptide assay. D, precursor peptides are
cleaved to SL8 in K
-COS cells as shown by recovery of only
SL8 in HPLC fractions of total K
-bound peptide extracts
from MSL8 (
) and MKSL8 (
) transfected K
-COS
cells and assay of the fractions in the exogenous peptide assay. Mock
run (+) shows absence of any peptide carryover from previous runs.
One of five similar experiments is shown for each
panel.
Figure 3:
Uncleaved MSL8 is expressed on the cell
surface of D-COS cells. D
-COS cells were
transfected with vector DNA (
) or DNA constructs encoding MSL8
(
) or MMSL8 (
). A, stimulatory peptides present on
cell surface (large symbols) or intracellular (small
symbols) MHC molecules were acid eluted and detected in the
exogenous peptide assay. Medium control (
). B,
identification of stimulatory peptides as cleaved MMSL8 or uncleaved
MSL8 was achieved by HPLC fractionation of total MHC-bound peptide
extracts from MMSL8 (
) and MSL8 (
) transfected
D
-COS cells and assay of the fractions in the exogenous
peptide assay. One of two to three similar experiments is shown for
each panel.
Figure 4:
Uncleaved ML8 octapeptide is expressed on
the cell surface of K-expressing L cells. A and B, K
-L cells with synthetic peptides ML8 (
),
SL8 (
), IL7 (
), and IL6 (
) at the indicated
concentrations were assayed in the exogenous peptide assay. Medium
control (
). BKMZ T cells (A) recognize ML8 and SL8,
whereas B3Z T cells (B) do not recognize ML8. The truncated
peptides are recognized less efficiently by both T cells. C and D, K
-L cells (
) or K
-L
cells stably transfected with ML8 (
) or MSL8 (
) constructs
were used in the endogenous peptide assay. BKMZ T cells (C)
recognize both ML8- and MSL8-expressing K
-L cells, but B3Z
T cells (D) recognize only MSL8-expressing K
-L
cells. One of three similar experiments is shown for each
panel.
As the third and completely independent model,
we used T-cells specific for the influenza nucleoprotein peptide AM9
(ASNENMETM)-D complex (Fig. 1B). The
DBFZ.25 T-cells recognize both AM9 and MM9 (MSNENMETM) synthetic
peptides with superimposable dose-response curves indicating that the
Ala to Met substitution does not affect D
binding or T-cell
receptor specificity for the peptide-D
complexes (Fig. 5A). Because the presence of methionine as the
first residue allows translation, D
-COS cells were
transfected with DNA constructs encoding MM9 or MAM9 and were tested
with DBFZ.25 T-cells. Significantly, the T-cell response to cells
transfected with either DNA construct was superimposable suggesting
that comparable amounts of peptide-D
complexes were
expressed on the cell surface (Fig. 5C). The naturally
processed peptides from D
-COS cells expressing either MAM9
or MM9 peptides were extracted and analyzed by HPLC. This was important
to establish their identity as well as to test the formal possibility
that both MAM9 and MM9 were giving rise to a common cleaved peptide
fragment (e.g. the octamer SNENMETM) that was responsible for
stimulating the DBFZ.25 T-cells. The HPLC elution profiles of extracted
peptides produced in either MAM9- or MM9-transfected cells yielded
single activity peaks with distinct retention times (Fig. 5D). Furthermore, the elution profiles of these
peaks were identical to those obtained with synthetic AM9 and MM9
peptides (compare Fig. 5, B and D). Similar
results were also obtained with D
-L cells stably
transfected with MAM9- or MM9-encoding DNA constructs ruling out any
potential artifacts in the transient COS cell expression system (data
not shown). Thus, as observed above for the MSL8-D
and the
ML8-K
complexes, the MM9 peptide also entered the antigen
processing pathway to yield the MM9-D
complex on the cell
surface. In summary, our data unequivocally show that cleavage of
flanking residues that necessarily occurs for all known cellular
proteins is not obligatory for peptide-MHC expression on the cell
surface.
Figure 5:
Uncleaved MM9 nonapeptide is expressed on
the cell surface of D-COS cells. A,
D
-L cells with synthetic peptides, AM9 (
) and MM9
(
), at the indicated concentrations were assayed in the exogenous
peptide assay to show that both peptides were recognized by DBFZ.25 T
cells. Medium control (
). B, HPLC chromatograms of the
synthetic peptides, AM9 and MM9, show excellent resolution. C,
D
-COS cells were transiently transfected with vector DNA
(
) or DNA constructs encoding MM9 (
) or MAM9 (
) at the
indicated DNA concentrations. Stimulatory peptide/D
expression in the transfected D
-COS cells was
indicated by comparable T cell response. D, identification of
stimulatory peptide as cleaved MAM9 or uncleaved MM9 was achieved by
HPLC fractionation of trifluoroacetic acid peptide extracts from MAM9
(
) and MM9 (
) transfected D
-COS cells and assay
of the fractions in the exogenous peptide assay. Mock runs (+)
show absence of peptide carry over between samples. One of four or five
similar experiments is shown for each
panel.
The display of precisely cleaved peptides from cellular proteins has emerged as a defining feature of the peptide-MHC class I presentation pathway. Here, we establish for the first time that the precise cleavage of flanking residues that invariably occurs during the generation of optimal MHC-binding peptides is not obligatory for presentation of endogenously translated precursors.
Several key
steps regulate the MHC class I antigen presentation pathway. The best
characterized of these is the translocation of antigenic peptides from
the cytoplasm to the ER by the TAP1/TAP2 (TAP) transporter. Compelling
evidence is now available that TAP-mediated translocation of synthetic
peptides into the ER is ATP-dependent and is selective for peptide
length (between 8-25 residues) and upon the nature of the
C-terminal
residue(20, 51, 52, 53, 54) .
Loss of TAP function, with a few
exceptions(56, 57, 58) , effectively disrupts
assembly of peptide/MHC complexes in the ER and as a consequence causes
dramatic loss of the ability to present endogenous peptide-MHC
complexes on the cell
surface(19, 20, 57, 59) . Because
endogenous peptide-MHC class I expression in the thymus is essential
for positive selection, TAP knock-out mice also lack CD8+
T-cells(20) . In vitro assays have shown that TAP
binds synthetic peptides (54) and is associated with empty MHC
class I molecules(21, 22) , thus providing the
structural basis for peptide transport and for efficient loading of
these peptides onto MHC molecules in the lumen of the ER. In addition
to physical association with TAP, empty MHC molecules interact with the
chaperone calnexin in the ER that regulates assembly of MHC heavy
chains with 2-microglobulin and their exit from the
ER(23, 60) . Therefore, the availability of peptides
in the ER and their loading onto the MHC molecules are key steps that,
if disrupted, can severely compromise the exit of peptide-MHC complexes
from the ER en route to the cell surface.
By contrast to the
overwhelming evidence for the key role of TAP in the antigen
presentation pathway, the mechanism by which peptides are generated and
supplied to TAP remain poorly defined. The hypothesis that precisely
cleaved peptides are generated by the proteasome in the cytoplasm and
are the natural substrates for TAP transport remains to be proven.
Clearly the 26S cytoplasmic proteasome is involved in the antigen
presentation pathway as elegantly demonstrated with selective
inhibitors by the Rock laboratory(37) . However, whether the
physiological products of this proteasome are optimal peptides as
suggested by its in vitro activity is not yet
clear(61) . Indeed, MHC-associated peptides considerably longer
(up to 33 aa) than the final 8-10 residue peptides have been
found in cells (62, 63, 64) . Although it is
not known whether these longer peptides are intermediates in the
antigen presentation pathway, the expression of the HLA-B27-associated
peptides was shown to be TAP-dependent(63) . In addition, two
examples have provided evidence consistent with the notion that
cleavage of longer precursors to the final products can occur in the
ER(40, 65) . These observations have raised questions
concerning the identity of the natural TAP substrates and the temporal
order of peptide cleavage steps, i.e. before and/or after
transport. In this context, presentation of the perfect-fit peptides
used here can serve as the first definitive examples of an endogenous
antigen (MSL8, ML8, or MM9) that remain unmodified from synthesis to
cell surface expression as peptide-MHC complexes. Furthermore, because
efficient presentation of cytoplasmic peptides depends upon TAP, as
shown by Bacik et al.(66) and in our own unpublished
studies, ()strongly suggests that TAP can transport
perfect-fit peptides in living cells. It is important to emphasize,
however, that while our results suggest that perfect-fit peptides are
transported by TAP, the identity of processed peptides of normal
cellular proteins and the cleavage events that occur before or after
transport remain to be established. We emphasize that our results do establish that none of these cytoplasmic or ER cleavage
events are obligatory for cell surface expression of the peptide-MHC
complex.
The presentation of perfect-fit peptides shows that proteolytic cleavage steps can be dissociated from other concerted steps in the antigen presentation pathway. Both endogenously synthesized and cytoplasmically loaded native proteins yield peptide-MHC complexes on the cell surface. Except for the signal sequence-associated peptides (56, 57, 66) and rare exceptions(58) , presentation of endogenous peptides occurs in a TAP-dependent manner. Whether a single obligatory cleavage mechanism (such as the LMP2/LMP7 containing proteasome) is the sole source of processed peptides or whether multiple mechanisms converge to feed into the subsequent transport step is an important unresolved question. That perfect-fit peptides bypass the cleavage steps for presentation on the cell surface allows the possibility that there could be more than one mechanism supplying processed peptides. It is interesting that redundancy in the proteolytic mechanisms was also indicated by the antigen presentation function of cells lacking LMP2 and/or LMP7(30, 31, 32, 33, 34) . On the other hand, it is formally possible that perfect-fit peptides are presented because they pass through the proteolytic mechanism but without removal of any residues. Further biochemical analysis of the fate of precursor proteins and their products is required to elucidate the nature of these peptide cleavage and delivery mechanism. The model systems described here provide the tools for such analysis.