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,
*
University of Pittsburgh Cancer Institute and
Departments of Pathology and
Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and
National Cancer Institute, Bethesda, MD 20892
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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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The ability to induce CTL recognizing wt p53 epitopes has established a basis for future development of a broadly applicable p53-based immunotherapy (7, 8, 9, 10, 11, 12, 13, 14). The two HLA-A2.1-restricted, human wt p53 epitopes most often used in these studies are p53149–157 and p53264–272. We and others have previously reported on the generation of CTL recognizing the wt p53264–272 epitope from PBMC obtained from healthy donors (7, 13, 14, 15, 16, 17, 18). However, it is unconfirmed whether similar effector cells can be generated from PBMC of patients with cancer. Although it is reasonable to expect that patients with SCCHN, especially those who accumulate p53, have a higher frequency of p53-responsive precursor T cells than normal donors, it is equally probable that these T cells are neither present nor functional. Patients with SCCHN are known to be immunosuppressed (19, 20, 21), their T cells have signaling defects (21), and a higher proportion of apoptotic T cells has been detected in the peripheral blood of these patients compared with that in healthy individuals (22). Therefore, it is important to determine whether stimulation of PBMC with the wt p53264–272 leads to the generation of p53-specific CTL in patients with SCCHN.
The status of p53 has been shown to be critical for the ability of the tumor to process and present the wt p53264–272 epitope. To date, most tumor cell lines shown to be sensitive to CTL specific for wt p53264–272 accumulate mutant p53 molecules, whereas tumor cells expressing wt p53 (exons 5–8) with no p53 accumulation are not sensitive to these CTL (17). In this report, we show that CTL specific for the wt p53264–272 epitope could be generated from PBMC of patients with SCCHN using peptide-pulsed autologous dendritic cells (DC) for in vitro sensitization (IVS). The frequency and activity of these T cells were evaluated relative to the presence of p53 gene mutations and protein expression in the patients’ tumors and to the presence of anti-p53 Abs in their sera.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HLA-A2+ SCCHN cell lines SCC-4 and SCC-9 were obtained from the American Type Culture Collection (Manassas, VA). The SCC-4 cell line expresses and accumulates p53 with a missense mutation/point mutation in position 151, but does not present the wt p53264–272 epitope (16). The SCC-9 cell line expresses, but does not accumulate, p53 molecules, in which codons 274–285 have been deleted, and presents the wt p53264–272 epitope (16). The SCCHN cell line PCI-13 was established in our laboratory (23). PCI-13 was previously described to express a p53 missense mutation in codon 286 (Glu to Lys) and to present the wt p53264–272 epitope (18). The p53-null osteosarcoma cell line, SaOS-2, was purchased from American Type Culture Collection, and the p53+ cell line SaOS-2cl3 was derived by transduction of p53-null SaOS-2 cells with p53 cDNA expressing a p53 missense mutation in codon 143 (9, 18). Tumor cells were cultured in plastic culture flasks (Costar, Cambridge, CA) under conditions described previously (18), using DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 µg/ml streptomycin, and 50 IU/ml penicillin (all from Life Technologies, Grand Island, NY). For subculturing, cells were detached from plastic using 0.05% trypsin/0.02% EDTA solution (Life Technologies). The hybrid (TxB) T2 cell line (24), which is deficient in TAP protein, was obtained from American Type Culture Collection and maintained in RPMI 1640 (Life Technologies) containing 10% heat-inactivated FBS, 2 mM L-glutamine, and antibiotics. The cultures were routinely tested and found to be free of mycoplasma contamination (Gen-Probe, San Diego, CA).
p53264–272 peptide
The HLA-A2.1-binding peptide, LLGRNSFEV (7, 8), corresponding to p53264–272, was synthesized by standard F-moc methodology, purified, and stored as a lyophilized preparation. The peptide was purified by reverse phase HPLC, and its amino acid sequence was confirmed by mass spectroscopy. The peptide was dissolved in DMSO (Fisher Scientific, Pittsburgh, PA) at 1 mg/ml and diluted with PBS just before use.
Generation of anti-p53 CTL using peptide-pulsed autologous DC
Peripheral blood or a leukapheresis product was obtained from HLA-A2+ SCCHN patients or normal donors, respectively, and PBMC were isolated by sedimentation over Ficoll-Hypaque gradients (Amersham Pharmacia Biotech, Piscataway, NJ). The study was approved by the institutional review board at the University of Pittsburgh, and written informed consent was obtained from each individual. Human DC were generated according to a modified method of Sallustro and Lanzavecchia (25). Briefly, PBMC were incubated for 1 h at 37°C in AIM-V medium, and nonadherent cells were removed by gentle washing with warm medium. The remaining (adherent) cells were incubated in AIM-V medium containing 1000 U/ml GM-CSF (Immunex, Seattle, WA) and IL-4 (Schering Plow, Kennilworth, NJ). The cultures were supplemented with additional IL-4 and GM-CSF on day 4 of culture. DC were harvested on day 6 using cold Hanks’ solution (Life Technologies) and were used as APCs. DC were resuspended at the concentration of 2 x 106 cells/ml in AIM-V medium containing 10 µg/ml peptide and incubated at 37°C for 4 h. Subsequently, the peptide-pulsed DC were cocultured with autologous PBMC in 24-well tissue culture plates (Costar, Corning, NY) in a final volume of 2 ml/well of AIM-V medium supplemented with 10% human AB serum (Pel-Freeze, Brown Deer, WI) and 25 ng/ml of IL-7 (Genzyme, Cambridge, MA) for the first 72 h and additionally with 20 IU/ml IL-2 (Chiron/Cetus, Emeryville, CA) for the remaining time in culture. The lymphocytes were restimulated weekly with the peptide-pulsed autologous DC. Irradiated (3000 rad) autologous PBMC were used as APCs after the second round of restimulations. The reactivity of generated T cells was tested against various targets in 24-h enzyme-linked immunospot (ELISPOT) assays as well as cytotoxicity assays. The specificity was determined in Ab blocking experiments and was confirmed by tetramer staining (see below).
ELISPOT assay for IFN-
The ELISPOT assay was performed in 96-well plates with nitrocellulose membrane inserts (Millipore, Bedford, MA) exactly as previously described by us (26). The capture and detection Abs were purchased from Mabtech (Nacka, Sweden). The spots were counted by computer-assisted image analysis (ELISPOT 4.14.3; Zeiss, Jena, Germany). For Ab blocking experiments, target cells were preincubated with anti-HLA class I-specific mAb, W6/32 (HB95; American Type Culture Collection), anti-HLA-A2-specific mAb, BB7.2 (HB82; American Type Culture Collection), or the respective IgG isotype controls (IgG2a and IgG2b, respectively, both from PharMingen, San Diego, CA) for 30 min. The assay reproducibility was controlled using PBMC obtained from a normal donor, cryopreserved in a series of vials, and was tested each time the assay was performed after stimulation with PMA (1 ng/ml) and ionomycin (1 µmM; both from Sigma, St. Louis, MO). The interassay reproducibility of the assay was acceptable, with a coefficient of variation of 15% (n = 30).
Cytotoxicity assay
The 4-h 51Cr release assay was performed at four E:T cell ratios as previously described (27). Briefly, sensitized targets were labeled with 51Cr for 45 min at 37°C, washed, and added to wells of 96-well plates (1 x 104 cells/well). Effector T cells were then added to give various E:T cell ratios. When Ab blocking experiments were performed, target cells were incubated with anti-HLA class I Ab or anti-HLA-A2 Ab at a final concentration of 9 µg/ml for 30 min before adding effector cells. The percent specific lysis was calculated according to the formula: percent specific lysis = (experimental cpm - control cpm)/(maximal cpm - control cpm) x 100.
Tetrameric peptide/HLA-A2.1 complexes (tetramers)
The streptavidin-PE-labeled tetramers used in this study were obtained from the tetramer core facility at the National Institute of Allergy and Infectious Disease (Atlanta, GA). Three-color flow cytometry assays (FACScan; Becton Dickinson, San Jose, CA) were performed with anti-CD3-peridinin chlorophyll protein, anti-CD8-FITC, and tetramer-PE. The specificity of the p53264–272/HLA-A2.1 tetramer was confirmed by staining of a CTL line specific for p53 (18) and by the lack of staining of irrelevant CTLs or HLA-A2-negative PBMC of healthy donors. Additionally, the streptavidin-PE-conjugated HIV peptide (ILKEPVHGV)/HLA-A2.1 tetramer was used as a negative control. Cells were considered positive for tetramer staining when populations clustered and were at least 1 log above the mean fluorescence of the negative population. For each sample, 75,000 events were collected progressively after live gating on lymphocytes by forward and side scatter.
p53 mutation analysis, immunohistochemistry, and detection of p53 Abs
All cases of SCCHN included in this study were available as paraffin blocks archived at the University of Pittsburgh Medical Center. The histology of each case was reviewed by a pathologist (S.D.F.), and representative tissue sections containing areas of invasive SCCHN were selected for microdissection. Normal-appearing salivary gland tissue or skeletal muscle was microdissected separately to serve as an internal nontumor control. Using 4-µm-thick recut unstained histologic sections, normal and malignant tissue samples were removed under stereomicroscopic observation. Sufficient material was collected from a single histologic section to afford replicate analysis. Samples were treated with proteinase K at a final concentration of 100 µg/ml for 2 h and then boiled for 5 min to remove protease activity. Sets of amplification primers flanking exons 5 through 8 of the p53 gene were used in four separate PCR (28). Amplified DNA from microdissected tissues also included splice sites. PCR products were electrophoresed in 4% agarose, and the ethidium bromide-stained bands were excised and then isolated with glassmilk. DNA sequencing used antisense PCR primers for each exon with [33P]dATP as the reporter molecule, and sequence analysis was read from overnight-exposed autoradiograms of 6% polyacrylamide gels.
For p53 immunohistochemistry, Formalin-fixed, paraffin-embedded tumor tissues were sectioned (3–5 µm), air dried overnight at 37°C, deparaffinized, dehydrated, and stained with a mAb against p53, D0-7 (Dako, Carpinteria, CA), which recognizes an epitope in the N terminus between aa 35 and 45 and reacts with wt and most mutant forms of p53 protein. The avidin-biotin-peroxidase method was used to visualize the p53, according to the instructions supplied by the manufacturer (Dako). The immunostained slides were evaluated by light microscopy for p53 accumulation. The tumor was considered p53 positive when >25% of the tumor cells showed staining intensity of 2+ and higher on a scale of 0–4+. IgG isotype mAb used at the same concentration as the primary mAb served as a negative control.
Ab to p53 in the patients’ and control sera was detected by an ELISA
purchased from PharmaCell Immunotech Coulter (Miami, FL) using
microtiter plates coated with recombinant human wt p53 protein.
Peroxidase-conjugated goat anti-human IgG was used for detection of
human anti-p53 Ab by a colorimetric reaction. Staining intensity
was compared with a standard curve, and anti-p53 levels 
1.1 U/ml
were considered positive. Assays were performed twice in triplicate and
included sera obtained from seropositive as well as seronegative
individuals as internal positive/negative controls.
HLA-A subtyping
PBMC or tumor cell lines were phenotyped for expression of HLA-A2 molecules by flow cytometry using the anti-HLA-A2 mAb, BB7.2, and IgG isotype as a control. Verification of the A0201 subtype was performed using PCR with sequence-specific primers as previously described (29). Briefly, DNA was obtained from PBMC using SDS and proteinase K, and after removal of protein contaminants with a saturated salt solution, DNA was precipitated using 2 vol of ethanol. Following washing and drying, the DNA pellet was reconstituted and quantitated by reading OD at 260 nm. The extracted DNA was subjected to PCR with sequence-specific primers (Dynal, Oslo, Norway) with primers that distinguish the specific allelic polymorphisms. Each primer set was designed to give an amplified fragment of a specific size, which was detected by gel electrophoresis and ethidium bromide staining. Patterns of positive and negative amplifications yielded the relevant genotype.
Statistical analysis
A two-tailed Wilcoxon rank sum test was performed to analyze ELISPOT data. Differences were considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC populations generated from the patients’ adherent mononuclear
cells in the presence of GM-CSF and IL-4 were routinely phenotyped by
flow cytometry and found to be CD14-,
CD40+, CD80+,
CD83-, CD86+,
DR+, HLA class I+ (data not
shown). The phenotypes and yields of DC were comparable to those of DC
generated from PBMC of normal donors using the same procedure. The DC
were pulsed with the wt p53264–272 peptide and
tested for the ability to stimulate autologous T cells in IVS. The
outgrowing T cells were evaluated for anti-p53 epitope activity
against various targets in ELISPOT and 4-h 51Cr
release assays and were found to be specific and HLA-A2 restricted. The
ELISPOT data obtained for one representative patient (patient 2) are
shown in Fig. 1
A. The
generated T cells were HLA class I restricted and reacted against T2
cells pulsed with p53264–272 peptide and, to a
lesser extent, against an HLA-A2.1-matched sarcoma cell line,
SaOS-2Cl3, as well as against a SCCHN cell line, SCC-9, both of which
naturally present the epitope (Fig. 1B). It is generally
observed that the reactivity of CTL is greater against peptide-pulsed
targets than tumor cells, presumably because peptide-pulsed target
cells are presenting the epitope at physiologically abnormal high
levels compared with levels of the epitope naturally presented by
tumors.
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A.
Among tumor cell lines tested in 4-h 51Cr release
assays were the sarcoma lines SaOS-2 and SaOS-2Cl3 and the SCCHN cell
lines SCC-9 and PCI-13, which naturally present the wt
p53264–272 epitope. All of these cell lines were
lysed by the effector cells (Fig. 2B), and this lysis was
blocked by anti-HLA-A2 mAb. In contrast, the SCCHN cell line SCC-4
and the sarcoma cell line SaOS-2, which do not express the epitope,
were only minimally lysed, and their killing was not blocked by
anti-HLA-A2 mAb. These data demonstrate that CTL generated ex vivo
in response to the wt p53264–272 peptide were
able to eliminate tumor cells expressing this epitope, but not the
targets unable to present it.
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summarizes the results obtained
with PBMC of patients with SCCHN and of normal donors after IVS with
the p53264–272 peptide. T cells of three of
seven patients with SCCHN were found to be reactive against
peptide-loaded T2 cells as well as against tumor cells naturally
presenting the wt p53264–272 epitope. This
reactivity was HLA class I restricted, as it was blocked by
anti-HLA class I or anti-HLA-A2 mAb. Similar responses were
obtained in two of seven healthy donors (data not shown). Two
additional normal donors who did not show any response after the fourth
stimulation with autologous DC pulsed with
p53264–272 peptide were found to express the
HLA-A2.7 allele and were excluded from the study.
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Tetrameric p53264–272/HLA-A2.1 complexes
were used to confirm the anti-p53264–272
specificity of CTL present in bulk IVS cultures. Fig. 3 shows representative results of
three-color flow cytometry performed with T cells generated from PBMC
of patient 2 after two to four successive restimulations. Only 0.03%
tetramer-positive CD8+ T cells of gated
CD3+ lymphocytes were present in fresh PBMC
obtained from this patient. After four IVS cycles, up to 16.4% of all
CD3+ lymphocytes were
CD8+/tetramer+. Similarly
robust responses were observed with cells obtained from healthy donor 2
and, to a lesser extent, patients 1 and 3 and healthy donor 1 (Table I
). The frequencies of p53264–272-specific
precursor CTL in unstimulated lymphocytes obtained from patients or
healthy donors were above background, as established in
HLA-A2.1-negative individuals or by using a tetramer with an irrelevant
peptide (HIV-1 reverse transcriptase peptide, pol 476–484). The
frequencies ranged between 0.005 and 0.04% of the
CD3+ T cells in all samples analyzed. They were
higher for SCCHN patients 1 (0.04%), 2 (0.03%), and 3 (0.02%) than
for the nonresponding patients or the nonresponding normal controls,
both of which had a mean of 0.01%. The tetramer-based method appeared
to be more sensitive for detection of responder T cells than the
ELISPOT assay. As shown in Figs. 1 and 3, a higher frequency of
tetramer-positive cells (6.6% of CD3+ T cells)
was obtained after the third stimulation compared with that of
IFN--secreting T cells measured by ELISPOT (
300/20,000 T
cells = 1.5%), as also reported by others (30).
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Although most HLA-A2+ tumor target cells
sensitive to lysis by CTL recognizing the wt
p53264–272 epitope do accumulate mutant p53,
this phenotype is not an absolute prerequisite for their
recognition by the CTL. In particular, a mutation at codon 273
is known to prevent the processing and presentation of the
p53264–272 epitope due to interference
with the proteasome pathway (31). Immunohistochemistry for
p53 as well as sequencing of the p53 gene in the patients’ tumors were
performed, therefore, to investigate a possible association between the
presence of a CTL response specific for the
p53264–272 epitope and the p53 status of the
tumor. Tumor samples were available for all patients studied, and the
results of the analysis are shown in Table I.
Of the three patients who showed CTL responses (patients 1, 2, and 3), the tumors of patients 1 and 2 had the wt epitope in exons 5–8 of the p53 gene and no p53 accumulation, whereas that of patient 3 accumulated p53 expressing a missense mutation at codon 273. All three tumors were, therefore, unlikely to present the wt p53264–272 epitope. In contrast, mutations in p53 exons 5–8 were detected in all tumors obtained from the four patients who did not show CTL responses to wt p53264–272 epitope (patients 4–7). The tumors of patients 4, 5, and 6 accumulated p53 and could present the epitope, whereas the tumor of patient 7 had a mutation in codon 213 (exon 6), resulting in a stop codon, and no detectable p53 protein. Therefore, it would appear that the presence of a CTL response to the epitope under study is more prevalent in patients bearing a tumor unable to present the epitope.
It is important to note that CTL responses were also generated in two
of seven HLA-A2.1+ normal donors, but not in two
donors who were found to express the HLA-A2.7 subtype. This observation
is consistent with the finding that most HLA-2.1-restricted peptides do
not bind to HLA-A2.7 molecules (32). None of the patients
with SCCHN or normal controls included in this study was p53
seropositive (Table I), and thus no insights were obtained into
possible interactions between humoral and cellular anti-p53 immune
responses in these patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, in this as well as our previous study (18), we noted that CTL responses to wt p53 epitopes could not be induced from PBMC obtained from all the normal donors or patients with SCCHN tested. The failure to induce CTL recognizing wt p53 epitopes in certain individuals might be due to a variety of reasons, including technical limitations of the methods used to induce and detect the CTL as well as their HLA-A2 subtype. However, biological events, such as clonal deletion or anergy of T cells specific for self-epitopes, appear to be primarily responsible for the paucity of these effector cells (33, 34, 35). The estimated precursor frequencies of CTL specific for p53264–272 peptide ranged from 1:33,000 to <1:300,000 in PBMC of three normal donors tested by limiting dilution (15). In the current study, we applied for the first time tetramer technology to detect human T cells specific for the wt p53264–272. The use of tetramers is particularly attractive, because it allows a direct quantification of Ag-specific T cells from blood without the need for their in vitro expansion (30, 35). Using three-color flow cytometry, we detected relatively high frequencies (up to 1:2500) of T cells specific for the epitope in patients with SCCHN who responded to IVS. Using the tetramer technology, we are currently involved in determining the proportions of anti-p53 precursor T cells in the circulation of a larger number of HLA-A2.1+ SCCHN patients compared with healthy donors by four-color flow cytometry (T. K. Hoffmann, A. D. Donnenberg, T. L. Whiteside, and A. B. DeLeo, manuscript in preparation). The preliminary data confirm our previous observations, indicating that the precursor frequencies are higher in patients with SCCHN who responded to IVS with the wt p53264–272 peptide than in those who did not respond or in normal donors. Studies by Sherman’s group (34, 35) demonstrated that tolerance to self-p53 epitopes in mice is associated with deletion of high-avidity T cells and retention of low to intermediate affinity T cells. In humans, comparable anti-self-p53 CTL have been isolated following IVS (18). Based on our detection of nonexpandable tetramer-positive anti-p53264–272 T cells in PBMC of non-IVS responders, these cells can be considered to have been tolerized. Presently, however, one can only speculate as to the mechanism(s) contributing to their anergy. It is possible that, due to their inherited repertoire, the TCRs capable of recognizing this self-p53 epitope (tetramer positive) in nonresponding individuals have a lower affinity for the epitope and/or a lower density than the TCRs expressed in responding individuals, which could prevent their appropriate stimulation and expansion (33). An area under investigation, therefore, is a comparative analysis of the tetramer binding affinity of precursor CTL in PBMC obtained from nonresponding and responding individuals.
In the current study, we observed that CTL specific for the wt
p53264–272 epitope were generated only from PBMC
of SCCHN patients with tumors unable to present this epitope (Table I).
The tumors either expressed a wt p53 genotype (exons 5–8) with no p53
accumulation or accumulated mutant p53 molecules with a point mutation
(R to H) at codon 273. This mutation, which occurs in 12% of human
cancers and is considered at a hot spot, has been shown to block
processing of the p53264–272 epitope
(31). It is tempting to speculate, therefore, that tumor
cells harboring the codon 273 mutation might be able to evade
recognition by CTL in HLA-A2+ individuals and
thus have a competitive edge for growth. The presence of
epitope-specific CTL precursors in PBMC of such individuals might well
facilitate the outgrowth of the epitope-loss tumors. This type of
immunoselection could lead to the development of tumors that
successfully escape the host immune system (36, 37). The
results of our analysis of the protective and therapeutic effects of
p53-based vaccines in mice exposed to the chemical carcinogen,
methylcholanthrene, are supportive of this concept. We observed a
higher incidence of epitope-loss tumors in animals immunized against a
single wt p53 epitope than in control groups of mice (V. Cicinnati, G.
Dworacki, and A. B. DeLeo, unpublished observations).
Although based on an analysis of a limited number of patients, the observation that CTL recognizing the wt p53264–272 epitope could not be generated from PBMC of patients with tumors likely to present this epitope suggests that another form of immune evasion might have taken place in vivo. By preventing CTL generation or by elimination of p53-specific precursor T cells, these tumors could have escaped from immune intervention. The capability of SCCHN and other human tumors to disarm the host immune system by inducing dysfunction or apoptosis of effector cells has been well recognized (21, 22, 38, 39, 40) and is under extensive investigation in our laboratories. However, for patient 7 no inverse relationship between IVS responsiveness and the p53 status of the tumor was observed. This patient’s PBMC did not respond to IVS, yet this individual’s tumor can be considered to be p53-null (one p53 allele was deleted, and the other had a stop codon at 213). Until other similar cases are found, it is difficult to speculate on the possible basis for this observation. Unlike the other patients studied, however, this patient recently underwent extensive tumor resection and adjuvant postoperative therapy. As a result, this individual may be immunocompromised to a greater extent than any of the other patients studied.
We anticipate that the results of the analysis of PBMC obtained from a larger population of patients with SCCHN using tetramers will provide further insights into the intriguing possibility that the frequency of p53264–272 CTL precursors in the peripheral blood of patients with SCCHN, particularly those whose tumors have a low potential of presenting the epitope, is significantly different from that in healthy donors. If a confirmation of this emerging pattern is obtained, it would suggest that the wt p53264–272 epitope is immunogenic in some HLA-A2+ individuals, and that CTL specific for this epitope may well have influenced the outgrowth of epitope-loss tumors that are able to avoid these effector cells. The possibility that immunoselection of such tumor cells might occur during p53-based immunotherapy merits consideration in designing future clinical trials. It implies that the use of vaccines capable of targeting multiple p53 epitopes expressed on different class I HLA molecules might be necessary to prevent tumor escape from the immune system.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Theresa L. Whiteside, University of Pittsburgh Cancer Institute, W1041 Biomedical Science Tower, 211 Lothrop Street, Pittsburgh, PA15213.
3 Abbreviations used in this paper: SCCHN, squamous cell carcinoma of the head and neck; DC, dendritic cell; ELISPOT, enzyme-linked immunospot; IVS, in vitro sensitization; wt, wild-type sequence.
Received for publication May 15, 2000. Accepted for publication August 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain expression in peripheral blood lymphocytes of patients with head and neck cancer. Clin. Cancer Res. 5:329.This article has been cited by other articles:
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T. K. Hoffmann, J. Muller-Berghaus, R. L. Ferris, J. T. Johnson, W. J. Storkus, and T. L. Whiteside Alterations in the Frequency of Dendritic Cell Subsets in the Peripheral Circulation of Patients with Squamous Cell Carcinomas of the Head and Neck Clin. Cancer Res., June 1, 2002; 8(6): 1787 - 1793. [Abstract] [Full Text] [PDF]
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T. K. Hoffmann, D. J. Loftus, K. Nakano, M. J. Maeurer, K. Chikamatsu, E. Appella, T. L. Whiteside, and A. B. DeLeo The Ability of Variant Peptides to Reverse the Nonresponsiveness of T Lymphocytes to the Wild-Type Sequence p53264-272 Epitope J. Immunol., February 1, 2002; 168(3): 1338 - 1347. [Abstract] [Full Text] [PDF]
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M. Schnurr, P. Galambos, C. Scholz, F. Then, M. Dauer, S. Endres, and A. Eigler Tumor Cell Lysate-pulsed Human Dendritic Cells Induce a T-Cell Response against Pancreatic Carcinoma Cells: an in Vitro Model for the Assessment of Tumor Vaccines Cancer Res., September 1, 2001; 61(17): 6445 - 6450. [Abstract] [Full Text] [PDF]
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