From the
Departments of Genetics and
¶Pathology and Immunology, Washington University
School of Medicine, St. Louis, Missouri 63110
Received for publication, April 9, 2003 , and in revised form, May 5, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies of the structure and function of several different oligomeric
immune receptors have been facilitated by the covalent attachment of various
subunits via flexible linkers. This is particularly true in the case of MHC
class II molecules. The peptide ligand-binding site of MHC class II molecules,
in contrast to class I molecules, is formed by the interaction of two separate
membrane-bound chains designated and
. To produce MHC class II
molecules occupied by homogeneous peptides, constructs were made whereby
different peptides were attached to the N terminus of the class II
-chain using flexible linkers
(4). Class II molecules with
covalently bound single peptides have been very informative in studies of
class II assembly and importance of chaperone molecules in optimal class II
peptide loading
(46).
Furthermore, such class II molecules with covalently attached peptides have
provided insights into CD4 T cell development and the specific role of the
peptide bound to class II
(7).
Based on these successes, it would be very attractive to tether a peptide
to an MHC class I molecule. In the case of the class I molecule, the
ligand-binding site is formed between two outer domains (1
and
2) of the class I heavy chain in a manner independent of
the heavy chain
3-domain
(8). Whereas the structure of
the class II molecule accommodates peptides protruding from the ends of its
ligand-binding groove, the class I structure optimally accommodates peptides
of defined length that do not extend beyond the binding groove
(9,
10). In fact, interactions
between the peptide termini and conserved class I heavy chain residues at the
ends of the binding groove are critical for stable peptide anchoring
(11). Therefore, a linker that
extends from the C-terminal residue of the peptide might be predicted to
interfere with strong peptide binding. Indeed, attempts to engineer class I
heavy chains with tethered peptides at the N terminus have been successful
only for select peptide·class I complexes
(12,
13).2
By contrast, linking
2m to the heavy chain using a flexible
linker appears to be universally applicable for different mouse and human
class I molecules
(1416).
More recently, others have produced constructs with the peptide covalently
attached to free
2m
(1719).
Although MHC class I heavy chains (H-chains) can associate with these
peptide·
2m molecules and present the peptides to T
cells, it remains unclear the extent to which these molecules exclude the
binding of competing free peptide ligands.h
Due to the limitations described above, we
(21) and others
(20,
22) have investigated
strategies to produce completely assembled class I molecules whereby all three
components (heavy chain, 2m, and peptide) are attached by
flexible linkers. By ordering the components in the format
peptide-spacer-
2m-spacer-heavy chain and by optimizing linker
lengths, we were able to produce single-chain trimers (SCTs) of class I. This
format appears to be widely applicable for different mouse and human H-chains
and their respective peptide ligands
(2022).
We have focused our initial characterizations on the Kb SCT
containing the ovalbumin peptide (OVA) due to the availability of a
three-dimensional structure and well characterized peptide-specific monoclonal
antibodies (mAbs) and T cells
(2325).
Thus far, our investigations have revealed that antibodies and T cells
recognize cognate SCTs very similar to normal class I molecules bound with
noncovalently attached peptides (referred to here as native class I
molecules). Furthermore, SCTs display rapid assembly in the ER and have very
stable expression at the cell surface, properties consistent with their
covalent nature (21) (data not
shown). As a likely reflection of their surface stability, we have shown that
the OVA·2m·Kb SCTs are potent
stimulators of antibodies and CTL specific for native OVA·Kb
peptide complexes (21). The
surface stability of SCTs was surprising in light of the aforementioned notion
that the class I binding groove appears to be "closed" and the C
terminus of the peptide interacts with conserved H-chain residues prominent in
anchoring the peptide. However, it is noteworthy that peptides with C-terminal
extensions have been eluted from class I heavy chains isolated with unique
antibodies (26,
27). Furthermore, the
structure of a class I molecule with a C-terminally extended peptide suggested
a potential mechanism to accommodate such extensions through unique
arrangements of heavy chain residues
(28). Thus, we considered it
important to probe the mechanism of peptide binding by SCTs to better exploit
them for future applications, including the in vivo and ex
vivo stimulation of T cells to treat human diseases.
In this study, we explore immunological recognition of SCTs that have been engineered with a mutation of the heavy chain residue Tyr84, a conserved residue that creates part of the F pocket of the peptide-binding groove. We further compare the accessibility of SCTs with and without this mutation for binding exogenous free peptides. Our findings suggest that the enhanced surface stability of SCTs is rendered by their ability to continuously rebind the attached peptide. In addition, we report the production of recombinant SCT molecules and their use to generate tetramers for enumeration of antigen-specific T cells. This latter finding may result in improved tetramers for peptide·class I peptide complexes that are prone to dissociation.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Lines, Antibodies, and PeptidesL cell line LM1.8 (H-2k) (29) was a gift from Dr. Phillipe Kourilsky (INSERM, Institut Pasteur, Paris, France). The mAbs used in this study include the following: 64-3-7, which recognizes open (peptide-free) forms of class I molecules tagged with this epitope (30); B8-24-3, which recognizes folded Kb (American Type Culture Collection, Manassas, VA); 25-D1.16 (a gift of Dr. Jonathan Yewdell, National Institutes of Health), which recognizes Kb + the SIINFEKL peptide (24); and Y3, which recognizes folded H-2K molecules (31). The OVA-derived peptide (SIINFEKL) (32) and SIYR peptide (SIYRYYGL) (33) were synthesized on an Applied Biosystems Model 432A peptide synthesizer.
DNA ConstructsConstructs were generated using standard
techniques and were confirmed by DNA sequence analysis. The generation of mAb
64-3-7 epitope-tagged Kb (Kb R48Q/R50P) and the
OVA·2mb·Kb
(OVA·Kb) SCT has been described
(21). The
OVA·Kb SCT consists of, beginning at the N terminus, the
leader sequence of
2mb followed immediately by the
SIINFEKL sequence and then a linker of 15 residues
((G4S)3). This first linker is followed by the mature
2mb sequence, the second linker of 20 residues
((G4S)4), and then the mature Kb sequence.
The Y84A mutation in Kb and the OVA·Kb SCT was
created using PCR mutagenesis. Constructs were expressed from the pIRESneo
vector (Clontech). For expression of SCT constructs in Escherichia
coli, the leader sequence was replaced by a methionine immediately
upstream of the SIINFEKL sequence. Furthermore, the Kb H-chain
(without the 64-3-7 epitope) was truncated at residue 280, just upstream of
the transmembrane domain, and a BirA biotinylation sequence
(GGGLNDIFEAQKIEWHE) was added. These constructs were all expressed from the
pET21a vector (Novagen, Madison, WI).
CTLThe OVA·Kb (SIINFEKL) CTL were
generated from OT-1 mice. Freshly explanted splenocytes (2.5 x
106) were cultured with 2.5 x 106 irradiated
splenocytes (2000 rads) in the absence of interleukin-2 and in the presence of
5 x 10-6 M SIINFEKL peptide. After 5 days, the
cells were harvested and used in a 51Cr release assay. Target cells
(23 x 106) were labeled for 1 h with 150 µCi of
51Cr (Na51CrO4, PerkinElmer Life Sciences; 1
Ci = 37 GBq) in 200 µl of RPMI 1640 medium and 10% bovine calf serum at 37
°C in 5% CO2. OT-1 T cells were plated at various
concentrations onto 96-well microtiter plates, and washed target cells (2
x 103/well) were added. For some groups, the SIINFEKL peptide
was present during the assay at a final concentration of 5 µM.
The plates were centrifuged at 50 x g for 1 min and incubated
for 4 h at 37 °C in 5% CO2. Radioactivity in 100 µl of
supernatant was measured in an Isomedic -counter (ICN Biomedicals,
Huntsville, AL). The mean of triplicate samples was calculated, and percentage
51Cr release was determined according to the following equation: %
51Cr release = 100 x ((experimental 51Cr release -
control 51Cr release)/(maximum 51Cr release - control
51Cr release)), where experimental 51Cr release
represents counts from target cells mixed with effector cells, control
51Cr release represents counts from target cells in medium alone,
and maximum 51Cr release represents counts from target cells lysed
with 5% (v/v) Triton X-100 (Sigma).
Production of Recombinant Proteins and TetramersRecombinant
OVA·Kb and OVA·Kb Y84A SCTs were produced
as insoluble inclusion bodies following induction with 1 mM
isopropyl--D-thiogalactopyranoside in E. coli
BL21(DE3) Codon Plus RIL cells (Stratagene). These inclusion bodies were
purified and then dissolved in 6 M guanidine, 10 mM Tris
(pH 8.0), and 20 mM
-mercaptoethanol. N-terminal sequencing
of each of these proteins revealed that the N-terminal fMet residue had been
removed during expression in E. coli. Protein refolding was
accomplished using a standard MHC class I refolding protocol. Briefly, three
injections of protein, each of which raised the total concentration of protein
in refolding buffer (400 mM L-arginine, 100 mM Tris (pH
8.0), 2 mM NaEDTA, 0.5 mM oxidized glutathione, and 5
mM reduced glutathione) by 1 µM, were added over 3
days to slowly stirred refolding buffer at 4 °C. Refolding reactions were
concentrated and subjected to gel filtration chromatography using a Superdex
200 16/60 column (Amersham Biosciences) and in vitro biotinylated for
12 h at 20 °C in the presence of 15 µg of BirA (Avidity, Boulder, CO),
80 µM biotin, 10 mM ATP, 10 mM MgOAc, 20
mM Bicine, and 10 mM Tris-HCl (pH 8.3). To remove free
biotin, monomeric complexes were again purified by gel filtration, tested for
biotinylation by Western blotting, and then tetramerized by addition of
phycoerythrin-labeled streptavidin (BD Biosciences) at a molar ratio of 4
molecules of SCT monomers to 1 molecule of phycoerythrin-labeled
streptavidin.
Enzyme-linked Immunosorbent Assay (ELISA)Purified recombinant OVA·Kb and OVA·Kb Y84A SCTs were bound to Nunc Maxisorp immunoplates (Nalge Nunc, Rochester, NY) overnight at 4 °C in phosphate-buffered saline at a concentration of 2 µg/ml. Antibody incubations were carried out for 1 h at 4 °C in blocking buffer consisting of 1% bovine serum albumin and 0.3% Tween 20 (both from Sigma) in phosphate-buffered saline. Alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as a secondary detection reagent. Colorimetric detection was achieved using Sigma Fast p-nitrophenyl phosphate substrate, and absorbance at 405 nm was analyzed using a Bio-Rad Model 550 plate reader.
Flow CytometryViable cells, gated by forward and side scatter, were analyzed on a FACSCalibur (BD Biosciences), and data were analyzed using CELLQuest software (BD Biosciences). Staining with anti-class I mAbs was visualized using phycoerythrin-conjugated goat anti-mouse IgG (BD Biosciences). For tetramer analysis, cells were stained concurrently with fluorescein isothiocyanate-labeled anti-CD8 antibody (Caltag Laboratories, Burlingame, CA) and phycoerythrin-labeled tetramers (conventional or SCT-based). Conventional OVA·Kb tetramers (noncovalent) were obtained from the NIAID MHC Tetramer Core Facility (Atlanta, GA). Brefeldin A (BFA) turnover was performed as described (34).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Native Kb H-chains with the Y84A Mutation Exhibit Relatively Poor Peptide BindingTo explore the importance of Tyr84 for peptide binding, we introduced the Y84A mutation into Kb and the OVA·Kb SCT. In addition, to better assess the quality of peptide binding, the epitope recognized by mAb 64-3-7 was also introduced into all of the constructs. We have shown previously, with several different mouse and human class I molecules (including Kb), that this epitope tag permits the discrimination of peptide-occupied (folded) versus peptide-empty (open) H-chain conformers without altering the peptide binding preferences of each respective molecule (30, 34, 38, 39). Initially, we compared the quality of peptide binding between native Kb (no covalent attachments) versus Kb Y84A molecules expressed on L cells (H-2k). This was accomplished serologically using mAb B8-24-3 to detect peptide-occupied surface Kb and mAb 64-3-7 to detect peptide-empty surface Kb. Using this approach, we estimated that 21% of native Kb molecules were peptide-empty at steady state, whereas 59% of Kb Y84A molecules were peptide-empty (Fig. 2A, upper panels, and Table I). Given that peptide occupancy by class I is a requirement for ER exit (1, 2), these data imply that the Y84A mutation renders Kb molecules less able to retain their bound peptides. Consistent with this idea, overnight incubation of Y84A mutant molecules with exogenous OVA peptide substantially increased surface expression of folded molecules (3.6-fold increase), whereas native Kb exhibited only modest induction (1.3-fold) (Fig. 2A and Table I). This indicated that exogenous peptides could more easily displace the peptides acquired by the Y84A mutants in the ER. Together, these results confirm that Tyr84 plays an important role in peptide binding to the H-chain.
Effects of the Y84A Mutation on the SCT BackgroundWe next performed a similar comparison of OVA·Kb SCTs with and without the Y84A mutation. As shown in Fig. 2B and summarized in Table I, the OVA·Kb SCT displayed very few peptide-empty forms at the cell surface (12%), regardless of the presence of the Y84A mutation. The paucity of peptide-empty forms of the Y84A SCT molecules contrasted with the poor peptide occupancy we observed for native Kb with the Tyr84 mutation (Fig. 2A). This disparity indicates that having the peptide covalently attached in the SCT overcomes the otherwise detrimental effects of the Y84A mutation.
Comparison of the wild-type versus Y84A SCT molecules using the OVA·Kb-specific mAb (25-D1.16) revealed that this mutation improved the interaction of mAb 25-D1.16 with the SCT. mAb 25-D1.16 recognizes the OVA·Kb SCT well, but not quite as efficiently as native OVA·Kb complexes (determined by comparing the ratio of OVA·Kb-specific staining to total folded Kb staining using mAb B8-24-3) (Fig. 2 and Table I). However, mAb 25-D1.16 staining of the SCT with the Y84A mutation was essentially equivalent in comparison with mAbs 25-D1.16 and B8-24-3 and matched very closely the staining observed with native OVA·Kb complexes. Importantly, this mAb binds to the OVA·Kb complex along a surface near the C terminus of the peptide (35). Based on these observations, it seems likely that the linker extending from the peptide in the SCT partially impairs mAb 25-D1.16 binding, but mutation of Tyr84 restores full antibody binding by "opening" the binding groove of the H-chain to permit a better fit of the linker. An alternative possibility is that suboptimal mAb 25-D1.16 recognition of the wild-type SCT is due to binding of endogenous peptides in place of the covalent peptide in a fraction of the SCT molecules, and mutation of Tyr84 reduces the binding of endogenous peptides with a corresponding increase in mAb 25-D1.16 staining. Data presented below argue against this possibility, however, and instead support the model of less hindrance from the linker.
SCTs with Y84A Are Stable at the Cell SurfaceThe above
findings indicate that the Y84A SCT molecules retain binding of the covalent
peptide and, in fact, appear to better tolerate the linker between the peptide
and 2m. To determine whether this mutation affects the
stability of the molecules, their cell-surface turnover was analyzed in the
presence of BFA. BFA blocks the secretory pathway to prevent the arrival of
newly synthesized class I molecules at the cell surface, permitting a
determination of the half-life of pre-existing surface molecules
(40,
41). We have previously shown
that OVA·Kb SCTs are more stable under these conditions than
native OVA·Kb complexes
(21). Here, cells expressing
Kb, Kb loaded with exogenous OVA peptide, or SCT or Y84A
SCT molecules were treated with BFA, and their respective turnover rates were
monitored by flow cytometry. As shown in
Fig. 3, SCTs with or without
the Y84A mutation were remarkably stable at the cell surface. Whereas normal
Kb had a half-life of
8 h, both SCT forms displayed little
turnover during the 16-h time course (as detected with either mAb B8-24-3 or
25-D1.16). Furthermore, as indicated by the mAb 25-D1.16 staining, both SCTs
were more stable than Kb molecules fed the OVA peptide overnight.
Thus, the Y84A mutation did not affect the remarkable cell-surface stability
of the SCTs, consistent with their high peptide occupancy determined
serologically (Fig. 2).
|
Effect of the Y84A Mutation on Peptide DisplacementWe
envisioned two models consistent with the stable nature and pronounced peptide
occupancy of the SCT. Either the spacer somehow strengthens the interaction of
the peptide with the H-chain, or the covalent linkage permits the peptide to
rapidly rebind after dissociation such that the trimeric structure is
maintained. To shed light on this issue, we compared the displacement of the
OVA peptide from native OVA·Kb complexes versus the
SCT molecules. Cells expressing Kb (loaded with exogenous OVA), the
OVA·Kb SCT, or the OVA·Kb Y84A SCT were
incubated with various concentrations of a competing Kb-binding
peptide (SIYRYYGL). The SIYR peptide is a known Kb ligand of
comparable affinity to the OVA peptide
(33). After overnight
incubation, displacement of the OVA peptide by the SIYR peptide was revealed
as a decrease in mAb 25-D1.16 staining intensity.
Fig. 4 demonstrates that the
OVA peptide linked to either the SCT or the Y84A SCT was displaceable using
high concentrations of the competing peptide. By contrast, native
OVA·Kb complexes were unaffected under these conditions,
even at 500 µM SIYR peptide. This implied that the cell-surface
stability and high level of peptide occupancy of the SCTs were conferred by
their ability to efficiently rebind the attached peptide. Interestingly, the
SCT with the Y84A mutation was 5 times more refractory to peptide
displacement. This effect could result from decreased binding of competing
peptides to the mutant SCT and/or fortuitous stabilizing interactions with the
covalent linker that lead to improved association of the tethered peptide, a
phenomenon that has been suggested for class II molecules with covalent
peptides (36).
|
CTL Detection of SCTs with and without Y84AWe previously showed that targets expressing the OVA·Kb SCT are recognized by cognate CTL (OT-1) to a similar degree as peptide-fed native Kb targets (21). Although Y84A SCT molecules are recognized very well by mAb 25-D1.16, it was important to determine whether this mutation alters CTL detection. As shown in Fig. 5, OT-1 CTL efficiently recognized target cells expressing the OVA·Kb Y84A molecules. Indeed, lytic activity against these targets was comparable to that against peptide-fed targets and somewhat better than that against SCT without the Y84A mutation. Improved CTL detection of the SCTs with the Y84A mutation is consistent with their improved detection using mAb 25-D1.16. Furthermore, these observations indicate that mutation of Tyr84 does not affect recognition of OVA·Kb by OT-1 T cells. It is noteworthy that this residue can also be mutated in HLA A2 without loss of CTL detection (42).
|
Characterization of Recombinant OVA·Kb SCTsBecause our data revealed that SCTs are quite stable at the cell surface and make excellent CTL targets, we thought that recombinant SCTs might be useful for a variety of applications. To this end, versions of the OVA·Kb SCTs lacking the leader sequence (replaced by a Met for initiation) and the transmembrane domain were expressed in E. coli. Recombinant material from purified inclusion bodies exhibited the expected molecular mass upon SDS-PAGE, and N-terminal sequence analysis revealed that the initiator Met was efficiently removed by the bacteria, exposing the desired N terminus (Ser) (data not shown). This material was subjected to refolding under standard class I refolding conditions. Soluble refolded material was obtained with efficiency comparable to what we routinely observed for conventional class I refolding.
Recombinant SCT molecules were prepared for OVA·Kb and OVA·Kb Y84A and were initially compared using ELISA. Equivalent amounts of both SCTs were bound to ELISA plates and then probed with various mAbs. Fig. 6 gives the results from a representative experiment demonstrating that the recombinant molecules reacted strongly with each mAb tested, including mAb 25-D1.16. Importantly, when comparing the SCT and Y84A SCT, the ratio of the signals obtained using mAb 25D-1.16 versus B8-24-3 was remarkably similar to what we observed for the corresponding molecules expressed in mammalian cells (compare with Fig. 2 and Table I). In other words, the OVA·Kb signal (mAb 25-D1.16) was improved relative to the total folded Kb signal (mAb B8-24-3) for the Y84A SCT molecules. This finding supports the model that the improved mAb 25-D1.16 recognition of the Y84A SCT mutant is due to better tolerance of the linker. Indeed, one explanation mentioned above for the suboptimal mAb 25-D1.16 recognition of the wild-type SCT expressed in mammalian cells could be that some endogenous peptides are bound in place of the covalent peptide, thus reducing the mAb 25-D1.16 staining. This is clearly not the case because the recombinant molecules showed the same pattern of suboptimal mAb 25-D1.16 staining that was improved with the Y84A mutation, yet these molecules were refolded in the absence of any exogenous peptides.
|
Production of Tetramers Using SCTsThe ELISA data obtained
using the recombinant SCTs indicated that they assumed the correct
conformation. Therefore, we were interested in determining whether SCTs could
be used to generate tetramers for enumerating antigen-specific T cells. Given
the stability of the OVA·Kb SCT at the cell surface, we
reasoned that tetramers made with SCTs might prove advantageous under certain
circumstances. To test the feasibility of using SCTs for tetramers, we
produced recombinant OVA·Kb Y84A molecules that included a
C-terminal biotinylation target sequence. After enzymatic biotinylation of
refolded molecules, tetramers were generated by the addition of
phycoerythrin-labeled streptavidin. The tetramers were tested by staining of
splenocytes from OT-1 transgenic mice (positive control) or C57BL/6 mice
(negative control) and compared with staining using native
OVA·Kb tetramers prepared using conventional methods.
Analysis of cells stained for CD8 versus the tetramers is shown in
Fig. 7. The SCT-based tetramers
were clearly functional, as they readily stained CD8+ cells from
the OT-1 mice, but not from naive B6 animals, similar to the conventional
tetramers. Thus, SCTs can be used to generate tetramers, and the unique
properties of SCTs may offer improvements over conventional tetramers. In
support of this conclusion, Greten et al.
(20) recently reported the
construction of bivalent staining reagents consisting of human HLA A2
molecules with covalently linked peptide and 2m.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The continuous rebinding of peptide does not appear to affect CTL or antibody detection of the OVA·Kb SCT. Furthermore, it is unlikely that peptide displacement would be a significant problem in vivo because high concentrations of competing peptides of the required length and sequence are unlikely to be encountered under normal conditions. Certainly, the presence of naturally processed peptides in the ER of cells expressing SCTs is insufficient to result in significant displacement of the covalent peptide in the SCT. Rather, we have found that cells expressing OVA·Kb SCTs are potent stimulators of CTL both in vitro and in vivo (21) (data not shown). Furthermore, we have now made SCTs of several different mouse and human H-chains bound by various of their known respective ligands, and all SCTs tested thus far are superior targets for CTL (data not shown). It is less clear, however, whether incorporation of the Y84A mutation into the SCT will improve T cell interaction for all peptide·class I complexes. As noted above, mAb 25-D1.16 binds the OVA·Kb complex toward the C-terminal end of the peptide (35) and thus would be expected to be influenced by the linker more than T cells. Although we have observed a trend toward somewhat better CTL recognition of the OVA·Kb Y84A molecule (Fig. 6) (data not shown), our preliminary analysis of the HLA A2 SCT suggests that there is little difference in T cell recognition afforded by the Y84A mutation with this particular H-chain (data not shown). Thus, the Y84A mutation has provided important mechanistic insights into the nature of peptide binding by the SCT, but may not be a universal requirement for making SCTs with optimal T cell recognition.
SCTs could have several applications in basic research as well as in the diagnosis and treatment of human diseases. As reported here, SCTs can be used to make tetramers, which are increasingly being used to monitor the dynamics of antigen-specific CD8+ T cell responses (43). Two limitations of tetramers technology are (i) their dependence on high affinity peptides to initially fold sufficient quantities of class I H-chains and (ii) problems of peptide dissociation rendering assembled tetramers unstable. Indeed, several well characterized antigenic peptides detected by CD8+ T cells have relatively weak binding to class I molecules (e.g. mouse Ld ligands p2Ca and tum-) (4446). Perhaps using SCT approaches will help in the construction and stability of tetramers made with lower affinity peptides. Furthermore, tetramers are more recently being used to modulate CD8+ T cells in vivo to study pathways of CD8+ T cell activation (47). Again, tetramers with covalently attached peptides may be more stable and thus more potent immunomodulators.
Probably the most exciting potential application of SCT technology will be
their use in DNA vaccination protocols. We have found that SCTs can stimulate
antigen-specific CD8+ T cell and antibody responses following DNA
vaccination (21) (data not
shown). Although SCT potency in antibody and CTL stimulation may reflect SCT
surface stability, additional factors warrant consideration. For example, even
though SCTs remain intact at the cell surface, they do have the potential to
multimerize by sharing subunits. Such multimers (probably dimers) may provide
a structure that enhances antigen presentation by display of a more defined
topology relative to monomeric forms. In any case, in vivo expression
of SCTs following DNA vaccination not only stimulates antigen-specific T cell
responses, but should also be resistant to virus-encoded immune evasion
proteins that shut off peptide supply, such as ICP47 of herpes simplex virus
(4850)
and US6 of human cytomegalovirus
(51). In addition, the
pre-assembled nature of SCTs and/or their rapid assembly kinetics may render
them more resistant to other mechanisms of immune evasion used by pathogens
(e.g. -herpesvirus 68 protein mK3)
(52).
In summary, we have shown here that the unusual stability of SCTs can be explained by their ability to efficiently rebind the covalent peptide, thus prolonging their half-life on the cell surface. The net result is that SCTs maintain a high steadystate level of peptide occupancy and the structural and conformational integrity required for immune recognition.
![]() |
FOOTNOTES |
---|
Present address: Surgery Branch, NIH, Bldg. 10, Rm. 2B02, 9000 Rockville
Pike, Bethesda, MD 20892.
|| To whom correspondence should be addressed: Dept. of Genetics, Washington University School of Medicine, P. O. Box 8232, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-2716; Fax: 314-362-4137; E-mail: hansen{at}genetics.wustl.edu.
1 The abbreviations used are: MHC, major histocompatibility complex;
2m,
2-microglobulin; ER, endoplasmic
reticulum; CTL, cytolytic CD8 T cell(s); H-chain, MHC class I heavy chain;
SCT, single-chain trimer; OVA, ovalbumin-derived peptide SIINFEKL; mAb,
monoclonal antibody; Bicine, N,N-bis(2-hydroxyethyl)glycine; ELISA,
enzymelinked immunosorbent assay; BFA, brefeldin A.
2 L. Lybarger, Y. Y. L. Yu, M. J. Miley, D. H. Fremont, N. Myers, T. Primeau,
S. M. Truscott, J. M. Connolly, and T. H. Hansen, unpublished data.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|