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,
Departments of
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Medicine,
Microbiology and Immunology, and
Biochemistry and Biophysics, University of North Carolina School of Medicine, and Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the difficulties in this area is the lack of animal models to evaluate DC function. Class I MHC proteins bind peptides that are typically 8–10 aa in length and generated by degradation of cytosolic proteins by the proteosome (17, 18, 19, 20, 21, 22). MHC molecules from different animal species bind different sets of peptides. Thus, studies to optimize DC activity in mice using mouse MHC proteins may have little applicability to enhancing CTL responses to epitopes derived from human tumors.
HER-2/neu is a proto-oncogene that is overexpressed in 
33% of
patients with breast cancer (23, 24). Previous
investigators have shown that aa 654–662 from HER-2/Neu (termed GP2 in
this manuscript) can bind to HLA-A2 and initiate a T cell response
against adenocarcinoma cells of the breast, ovary, and pancreas
(25, 26). To overcome the problems associated with both
human epitopes not binding to mouse MHC molecules and mouse
CD8+ T cells not interacting well with the 
3
domain of human MHC proteins, we generated transgenic mice expressing
the 1 and 2 domains of HLA-A2 and the 3 domain of
H2Kb. Using these mice, we have explored the
influence of the type of Ag and the route and timing of delivery of
autologous DCs in the generation of CTL responses against the GP2
epitope from HER-2/Neu.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T2 (174 x CEM.T2), HmyC1R-neo (C1R-neo), and HmyC1R-B*0702
(C1R-B7) cell lines were provided by Dr. Peter Cresswell (Yale Medical
Center, New Haven, CT) (27). HmyC1R-A*0201 (C1R-A2) cells
were described previously (28). All cells were grown in
RPMI 1640 supplemented with 10% (v/v) FBS, 5 mM
L-glutamine, and 5 x 10-5 M
2-ME (R10). DCs were generated from mouse bone marrow by flushing the
femur and tibia of individual mice with R10 and culturing the isolated
cells in low adherence plates (Costar, Cambridge, MA) in the presence
of GM-CSF (10 ng/ml), and IL-4 (1000 U/ml; Endogen, Woburn, MA). GM-CSF
and IL-4 were added to the medium at the same concentration on days 3
and 7. Additionally on day 7, TNF- (10 ng/ml; Endogen) was added to
the medium, and the nonadherent cells were collected on day 12 and
analyzed by flow cytometry before use in vaccination protocols. Human
EBV-lymphoblastoid cell lines (EBV-LCL) were generated as previously
described (29). HLA A2+ and
B7+ EBV-LCL were used in these experiments.
Flow cytometry
On day 12, expanded DCs were characterized for the expression of
specific surface markers by flow cytometry as described previously
(30). Cells were stained with the following Abs:
FITC-CD3
(clone 145-2C11 IgG1), FITC-CD4 (clone GK1.5 IgG2b),
FITC-CD8 (clone 53-6.7 IgG2a), PE-CD11b (clone M1.70 IgG2b),
FITC-CD11c (clone HL3 IgG1), FITC-CD14 (clone rmC5-3 IgG1), PE-CD80
(clone 16-10A1 IgG2), PE-CD86 (clone GL1 IgG2a),
FITC-I-Ab (clone AF6-120.1 IgG2a), and FITC-Mac-1
(all Abs from PharMingen, San Diego, CA). DCs were analyzed using a
FACScan (Becton Dickinson, Franklin Lakes, NJ) with Cicero software
(Cytomation, Fort Collins, CO). Mature DCs were characterized by a
100-fold increased expression of CD80 or CD86 and class II compared
with adherent macrophages, and in addition >90% of the cells had to
express both CD11b and CD11c. For the vaccination protocols, 75–85%
of the cells injected were DCs by the previous characterization.
Preparation of HLA-A2/peptide complexes
Residues 1–275 of HLA-A2 and human ß2m were expressed in Escherichia coli as inclusion bodies, purified, and folded as described previously (31).
Synthetic peptides
All peptides were synthesized by the Peptide Synthesis Facility at the University of North Carolina (Chapel Hill, NC). The sequence of the wild-type peptide (GP2) is IISAVVGIL. Substitutions made at peptide positions 2 and 9 are given in single-letter code abbreviations. The peptides were purified to >95% purity by reverse phase HPLC, and identity was confirmed by mass spectrometry. All peptides were dissolved in 100% DMSO at 20 mg/ml by weight.
Determination of thermal stability
HLA-A2/peptide complexes were exchanged into a 10 mM KH2/K2HPO4 buffer, pH 7.5, and adjusted to a final protein concentration of 4–12 µM. The change in circular dichroic signal at 218 nm was measured as a function of temperature from 4–95°C on a Peltier temperature-controlled AVIV 62-DS spectropolarimeter (Aviv Associates, Lakewood, NJ). The final melting curve was the average of at least three experiments for each HLA-A2/peptide complex. Tm values were calculated as the temperature at which 50% of the complexes were denatured using a two-state denaturation model (32).
Cell surface half-life assay
The determination of cell surface half-lives (t1/2) of A2/peptide complexes was performed as described previously (33). Briefly, 2.5 x 106 T2 cells were incubated overnight in AIM V serum-free medium at 37°C in 5% CO2 in the presence of 50 µM peptide. To block the egress of new A2 molecules to the surface, cells were incubated at 37°C in 5% CO2 in RPMI 1640, 15% FCS, and 10 µg/ml brefeldin A (BFA; Sigma, St. Louis, MO). This concentration of BFA is toxic to the cells; therefore, after 1 h the cells were transferred to RPMI 1640, 15% FCS, and 0.5 µg/ml BFA. At the indicated time points, 2.5 x 105 cells were removed, incubated with BB7.2, and analyzed by flow cytometry as described above for cell surface stabilization assay. Each time point is evaluated as the mean fluorescence with peptide minus the mean fluorescence without peptide and normalized to the maximal level of fluorescence (at time zero) for each peptide.
Construction of A2Kb transgenic mice
An HLA-A*0201/H2Kb (A2Kb) fusion gene was constructed from a genomic clone of HLA-A*0201 and H2Kb. A HindIII-BglII fragment encoding the promoter sequence, leader sequence, and exons 1–3 of HLA-A*0201 was ligated to a BamHI fragment encoding exons 4–7 and 5'-flanking sequences of H2Kb. The fusion construct was sequenced for verification and microinjected into embryos derived from FVB/n mice. Founder mice were assessed by surface expression of HLA-A2 using flow cytometry on peripheral blood cells. All animal work was performed under protocols approved by the Division of Laboratory Animal Medicine at the University of North Carolina.
CTL activity in A*0201 Kb transgenic mice (A2Kb)
DCs were cultured from bone marrow cells isolated from A2Kb transgenic mice in R10. After the addition of recombinant cytokines as described previously, DCs were cultured in six-well nonadherent plates. After 12 days, an aliquot of the expanding cells was analyzed by flow cytometry as previously described. Another aliquot of cells (1 x 106) was incubated with 10 µg of GP2, L9V, or I2L peptide in PBS and 0.5% mouse serum. DCs (5 x 105) were injected either intradermally (i.d.; footpad) or s.c. (base of the tail) or into the lateral tail vein (i.v.) of A2Kb mice in the same manner at specific intervals (weekly or every 3 wk). After the indicated number of vaccinations, mice were sacrificed. For local s.c. or i.d. injections the draining lymph nodes were removed, and lymphocytes were isolated and tested for lysis of peptide-pulsed target cells by a conventional 51Cr release assay. For i.v. inoculation, splenocytes were removed and tested for CTL activity.
The percent specific lysis for CTL assays was determined using/ the following formula: % specific lysis = [(cpmsample - cpmspontaneous)/(cpmtotal - cpmspontaneous)] x 100. Lytic units were calculated using a program provided by G. Ferrari (Duke University, Durham, NC). The LU20 value is the number of lytic units per million cells required to yield 20% specific lysis.
Long-term responses
A2Kb mice were injected s.c. with autologous DCs every week for 3 wk. Subsequently, half the mice were injected weekly s.c. with peptide-pulsed DCs for a total of seven vaccinations. A second cohort of animals was injected weekly s.c. with peptide-pulsed C1R-A2 cells. Following the sixth and seventh injections, mice were sacrificed, and CTL assays were performed on isolated splenocytes. A third cohort of A2Kb transgenic mice was treated every 3 wk s.c. with peptide-pulsed DCs for a total of six treatments. CTL responses were analyzed after s.c. vaccination from the draining lymph nodes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Tm, which is the temperature
at which 50% of protein is denatured, has been shown previously to be
proportional to the free energy of peptide binding to class I MHC
molecules (32, 34). Initially we examined the binding
affinity of GP2 to the class I heavy chain HLA-A2. As shown in Fig. 1
, the
Tm for the GP2 peptide was 36.4°C.
In comparison, the Tm for a high
affinity peptide such as the A2 binding peptide from the signal
sequence of calreticulin is 54°C (Fig. 1). Because GP2 lacked the
preferred amino acids found in the anchor positions of HLA-A2, we
prepared peptides substituted with the preferred amino acids.
Substitutions of amino acids in the anchor positions of the peptide
stabilized the complex, as shown by an increase in the
Tm of 2 and 6oC
for the substituted peptides L9V and I2L, respectively. An increase of
6°C is significant and suggests a large increase in the half-life of
the complex.
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We next measured the half-life of HLA-A2 complexes incubated with
GP2, the APLs, and a strong binding peptide from calreticulin (ML) on
T2 cells. This cell line has a large deletion of genetic material on
chromosome 6, which encompasses the TAP1 and TAP2 genes
(27). Thus, the T2 cell line does not efficiently present
endogenous nonsignal sequence peptides, and the MHC complexes that
egress to the surface readily disassociate in the absence of peptide.
However, these complexes can be stabilized by addition of exogenous
peptide. We incubated T2 cells in serum-free medium overnight with 50
µM of the individual peptides and measured the half-life of the
HLA-A2 complex by flow cytometry. The calculated half-lives were 24
min, 42 min, 1.8 h, and 19.5 h, respectively, for GP2, L9V,
I2L, and ML (Fig. 2). Thus, we confirmed
that substitutions in the anchor positions of the GP2 peptide greatly
enhanced the stability of MHC complexes in vitro.
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To evaluate whether the enhanced immunogenicity found in vitro
using the APLs affected in vivo activity, peptide-pulsed DCs were
administered to A2Kb mice i.v., and CTL activity
was determined from isolated splenocytes. Initially, the nature of the
CTL response was evaluated using the wild-type peptide, GP2, or the
altered peptide, I2L, pulsed onto DCs, C1R-A2 transfectants, or an
A2+ EBV-transformed B cell line. For these
experiments, DCs, human C1R-A2 transfectants, or human
HLA-A2+ EBV-LCL cells pulsed with either the
wild-type peptide, GP2, or the altered peptide, I2L, were adoptively
transferred i.v. I2L was chosen, as this altered peptide showed the
greatest ability to stabilize MHC complexes in vitro. Mice were
sacrificed after the fourth cell transfer, splenocytes were isolated,
and CTL assays were performed. We were unable to detect T cell
responses from the spleen after i.v. administration of DCs pulsed with
the wild-type peptide GP2 or after stimulation with either GP2 or I2L
pulsed onto EBV-LCLs or C1R-A2 cells (Fig. 3). In contrast to GP2, I2L-pulsed DCs
were able to induce significant CTL activity after four in vivo
vaccinations (Fig. 3). Similarly, we did not find CTL responses in
A2Kb mice after administration of GP2-pulsed DCs
injected either i.d. or s.c. (data not shown). Therefore, DCs pulsed
with GP2 are unable to induce CTL responses in
A2Kb mice, and this lack of response is not route
dependent. Furthermore, the lack of a CTL response observed after the
administration of I2L-pulsed C1R-A2 transfectants or
A2+ EBV-LCL suggests that the CTL response is not
due to cross-priming and presentation of the epitope by mouse
APCs.
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). Interestingly, there was an increase
in CTL activity in animals that received DCs pulsed with the poorer
binding epitope L9V compared with that in animals pulsed with I2L.
Thus, while the initial CTL response is strongly influenced by the
stability of the peptide MHC complex, the small degree of enhanced
stability of HLA-A2 found using I2L compared with L9V did not augment
the CTL response in vivo.
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The CTL response after DC vaccination differs depending on the
route of administration (35). There are conflicting data
as to whether the efficacy of DC-based vaccines differs depending on
the route used. To investigate this, A2Kb mice
were injected with I2L-pulsed DCs given i.v., s.c. (in the flank), or
i.d. (footpad). After injection of peptide-pulsed DCs, we evaluated
whether the anatomic site of activity and the timing and magnitude of
the CTL response differed. Significant CTL activity was detected in
cultures prepared from lymphocytes isolated from the draining lymph
nodes after two i.d. injections and three s.c. injections. We did not
detect significant CTL activity from mesenteric lymph nodes or lymph
nodes isolated from the base of the tail after four i.v. injections.
CTL activity was found in cultures prepared from lymphocytes isolated
from the spleen after four i.v. injections (see Fig. 5). Thus, peptide-pulsed DCs given i.d.
resulted in the most rapid CTL response, which occurred in the local
draining lymph nodes. Adoptive transfer of DCs given i.v. induced
measurable CTL responses only in the spleen. However, while the time to
onset of the CTL response was different, the magnitude of the response
did not differ depending on the route of administration. Regardless of
the route of administration, CTL activity was induced after
peptide-pulsed DC administration.
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For DC-based immunotherapy in patients (5, 10, 14, 15), multiple vaccinations are administered to generate CTL
responses. To investigate whether the injection schedule impacted on
the timing and magnitude of a CTL response, peptide-pulsed DCs were
injected s.c. into A2Kb mice either weekly or
every 3 wk for seven treatments. CTL responses were determined from the
draining lymph nodes after the third to seventh vaccination. We found
that CTL responses peaked after the third injection, but then,
unexpectedly, diminished after the sixth and seventh injections in mice
that were administered I2L-pulsed DCs weekly (Fig. 6). This reduction in CTL activity was
not observed in mice that received DC vaccinations every 3 wk.
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Another possible explanation for the diminished CTL activity found
after the administration of peptide-pulsed DCs weekly is enhanced
apoptosis of activated CTLs by DCs. To evaluate this, we determined
whether the administration of an APC that could not induce CTL
responses, such as peptide-pulsed C1R-A2 cells, led to a similar
reduction in CTL activity. All mice received I2L-pulsed DCs s.c. weekly
for three vaccinations. After this initial priming, half the mice
received the same treatment for three more injections. The other half
of the cohort received s.c. injections with C1R-A2 cells pulsed with
the I2L peptide (Fig. 7). Again, after
vaccination with I2L-pulsed DCs given weekly, we observed a decrease in
the CTL response after the sixth injection. By comparison, mice that
received three weekly injections of peptide-pulsed DCs followed by
weekly injections with C1R-A2 transfectants maintained CTL responses
over the entire protocol.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous investigators have shown that the dissociation rate of a peptide for the class I heavy chain complex is a strong determinant of the immunogenicity of that peptide (20, 33, 36). Of the 50 peptides we have evaluated that bind to class I molecules, the GP2 peptide has the poorest measurable binding profile (E. J. Collins, unpublished observations). Thus, this is a natural candidate epitope to alter in an attempt to improve its immunogenicity. We have found that there may be a threshold dissociation rate that is critical in the initiation of T cell responses using peptide-pulsed DCs. We were unable to generate responses regardless of the mode of delivery of the peptide using the wild-type peptide, which has a half-life of 24 min. Above this threshold, we found that a small enhancement of binding, as shown using the I2L peptide (half-life, 1.8 h) compared with L9V (half-life, 42 min), did not improve CTL responses after peptide-pulsed DC vaccination.
It is interesting that we and others have been unable to generate
responses to the GP2 peptide in HLA-A2Kb
transgenic mice. The addition of the Kb 3
domain should have no effect on peptide binding. Lustgarten et al.
(37), using mice transgenic for both human CD8 and HLA-A2,
were able to generate responses to several different epitopes from
HER-2/Neu, but not to the GP2 peptide. Similarly, we have been unable
to generate responses to the GP2 peptide after treating mice with
peptide in IFA, peptide alone (J. S. Serody, E. J. Collins, and J.
A. Frelinger, unpublished observations) or peptide-pulsed DCs.
Previously, investigators have shown that A2 transgenic mice have a
similar T cell repertoire as humans (38). The
absence of a response in these mice to the GP2 peptide suggests
differences in the fine specificity of T lymphocytes between species.
Alternatively, responses to GP2 from patients could be due to
stimulation previously by cross-reacting epitopes.
Previous work in vivo in mice and in humans has suggested that the mode of delivery of DCs is important in eliciting T cell responses (35) (39). DCs given i.v. localized in the reticuloendothelial system and lung, while those given locally homed to the lymphatic system. We found a similar result in the A2Kb mouse. CTL responses were present from the draining lymph nodes after i.d. or s.c. injection. Intravenous injection resulted in responses in the spleen, with little activity in the mesenteric lymph nodes. The route of administration is important in the efficiency of stimulating CTL. The i.d. route was the most effective method, which may be due to the local inflammatory response that occurred after footpad injection. This would result in an increase in the concentrations of both cytokines and chemokines that are critical to migration (40, 41, 42, 43, 44, 45) of DCs from the site and maturation (46, 47). However, we did not find that the route of administration was important in whether a response occurred or the intensity of this response. Additionally, our data suggest that the specific location of tumor cells may be important in the mode of delivery of DCs pulsed with peptides. The i.v. route may be preferable for vaccine administration against leukemia targets, while the i.d. or s.c. route may be preferable for responses to tumors localized in tissues.
As many current vaccine protocols use multiple vaccinations, we were interested in determining whether the schedule of these vaccinations affected CTL activity. Interestingly, we found that weekly administration of DCs s.c. resulted in diminished CTL activity after six treatments. The reasons for the decrease in CTL activity after weekly injection of peptide-pulsed DCs are not entirely clear. We were unable to show CTL activity in the spleen after the sixth s.c. administration of peptide-pulsed DCs, which suggests that the decrease in response is not due to redistribution of the CTL into the systemic circulation.
One possibility may be that weekly administration of peptide-pulsed DCs leads to clonal exhaustion of the responding T cell population. Because DCs are so effective at stimulating T cell responses, the significant T cell expansion associated with the use of DCs for vaccination may result in enhanced activation-induced cell death of the responder population. Using an APC such as C1R-A2 cells, which were unable to induce an initial CTL response, CTL activity was not diminished. This result may be due to decreased CTL activation induced by C1R-A2 cells, which would leave a residual population of peptide-specific T cells after each injection. This hypothesis can be evaluated currently using MHC tetramers to follow the responding population of T cells in vivo. If diminished CTL activity is found after multiple vaccinations using other peptides pulsed onto DCs or DCs transfected with tumor DNA or RNA, clinical studies may need to be modified to include either fewer treatments or the use of less stimulatory APCs. This also suggests that tolerance induced by DCs may be a function of the number of vaccinations of peptide-pulsed DCs and not of the route. If this proves to be true, frequent administration of peptide-pulsed DCs may provide a strategy to treat T cell-mediated autoimmunity.
Our data suggest that the presentation of APLs by autologous DCs can initiate a T cell response against a poorly binding self-epitope. These data also suggest that the use of in vitro models of peptide affinity may be helpful in the design of tumor vaccines. Using the I2L peptide pulsed onto autologous DCs, we have generated CTL responses in three naive individuals that are capable of lysing A2+ ovarian cancer cell lines from two different individuals that overexpress HER-2/Neu (J. S. Serody, unpublished observations). These CTL were not able to lyse an A2+ EBV-transformed B cell line from the patients with ovarian cancer, suggesting that a large number of peptide MHC complexes need to be present on the cell for lytic activity. Thus, self-peptides that bind poorly to MHC molecules are unlikely candidates to induce autoimmunity. Whether this approach results in enhanced killing of tumor cells will require characterization of in vivo CTL activity after clinical vaccination with APL-pulsed DCs. We are currently performing this evaluation.
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Acknowledgments |
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Footnotes |
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2 J.S.S. and E.J.C. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Jonathan S. Serody, Campus Box 7295, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295.
4 Abbreviations used in this paper: DC, dendritic cells; BFA, brefeldin A; i.d., intradermally; Tm, temperature at which 50% of protein is denatured; LCL, lymphoblastoid cell lines; APL, altered peptide ligand.
Received for publication November 15, 1999. Accepted for publication February 22, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain of the heavy chain. Proc. Natl. Acad. Sci. USA 92:1218.This article has been cited by other articles:
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) complex is considerably less stable than the
MLLSVPLLL/A2 (x) complex, as seen by a lower melting
temperature (Tm), 36 vs 54°C. Modification
of anchor residues (P2 and P9) caused an increase in the melting
temperature. Higher Tm values for peptides
with substitutions at P2 (I2L; 
) and P9 (L9V, 
) of GP2 (
