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Immunology |
Departments of Immunohematology and Blood Transfusion [S. Z., M. P. M. V., S. C. F. M., J. A. H., M. E. O., R. P. M. S., K. L. M. C. F., H. W. N., F. O., S. H. v. d. B., R. O., C. J. M. M.] and Surgery [S. Z.], Leiden University Medical Center, 2333 ZA Leiden, the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% of human cancers, making it an attractive target antigen for immunotherapy of cancer. Research in this area has thus far primarily focused on p53-specific CTLs. Although these CTLs were shown to be highly effective against p53-overexpressing tumors in vivo, immunological tolerance seems to strongly restrict the spectrum of the p53-specific CTL repertoire in p53+/+ subjects. In view of the emerging role of CD4+ Th (Th) cells in the antitumor response, we investigated the specificity and antitumor efficacy of the p53-specific Th response in mice. Our data show that high affinity Th cells against the naturally processed epitope p53108–122 can be elicited in both p53-/- and p53+/+ mice, indicating that the p53-specific T-cell response is not affected by tolerance at the Th level. Furthermore, p53108–122-specific Th cells were effective in enabling p53-specific CTLs to control the growth of p53-overexpressing tumors in vivo. Therefore, exploitation of the p53-specific Th response appears to be a highly useful aspect of immunotherapeutic strategies against cancers. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50% of all human malignancies (1)
. The aberrant expression of p53 in a wide variety of tumors, mainly caused by mutations in the p53 gene, and its direct involvement in the malignant transformation of those tumors make it an attractive target for immunotherapy. Studies aiming at evaluating p53 as a target for immunotherapy were mainly focused on the identification of p53-derived MHC class I presented peptides, which are recognized by CD8+ CTLs (2, 3, 4, 5, 6)
. In several studies p53-specific vaccination in p53+/+ mice was shown to induce protective immunity against a subsequent challenge with p53 overexpressing tumors (3
, 7, 8, 9)
. Although this antitumor effect was attributed to the presence of p53-specific CTLs, in only one of these studies limited evidence was provided to support the notion that CTLs were actually involved (3)
. Recent experiments demonstrated that in p53+/+ mice a considerable degree of tolerance for p53 exists at the CTL level and that only low avidity CTLs can be found against p53 in the periphery (10, 11, 12)
. Several efforts to raise the CTL response, using CTLA-4 blockade or CD40 activation, did result in higher cell numbers, but not in higher avidity (10
, 13)
. These data suggest that immunization strategies exclusively aiming at induction of the CD8+ arm of the p53-specific T-cell response are unlikely to result in effective antitumor immunity. Numerous studies show that anti-p53 IgG antibodies can be found in the serum of cancer patients (14, 15, 16, 17, 18, 19, 20, 21, 22) . Because isotype switching from IgM to IgG antibodies is Th4 cell dependent, this demonstrates a role for CD4+ cells in inducing p53-specific responses (23) . Moreover, as opposed to CTLs, these findings indicate that tolerance against wt.p53 is incomplete at the level of antibodies and CD4+ cells. Recent literature underscores the importance of CD4+ cells as regulators of the antitumor immune response (24, 25, 26, 27) . Several studies demonstrate that Th cells display a unique ability to orchestrate an immunological attack by mobilizing multiple effector arms of the immune system. One such an effector mechanism of CD4+ cells, following specific recognition of antigen presented on APCs in the context of MHC class II, involves the induction of CTL responses by activating the APC through CD40-CD40-ligand interaction (28, 29, 30) . Other recent findings demonstrate the specific nature of T-cell help in the eradication of MHC class II negative tumors (24) . This encompasses not only the induction of CTL via cross-priming, but also the recruitment of MHC nonrestricted effector cells with tumoricidal activities, such as eosinophils and macrophages (25) .
In this study we identified four murine wt.p53-derived peptides against which specific Th responses could be elicited after vaccination of p53-/- mice. Th cells against one of these peptides, p53108–122 (LGFLQSGTAKSVMCT), not only recognized peptide loaded DCs, but also p53 protein loaded DCs, arguing that these T cells recognized a naturally processed epitope. The antitumor efficacy of the p53108–122-specific Th cells, as well as the fact that such Th cells can also be elicited in normal p53+/+ mice, point at the potential of p53-specific Th immunity for immunotherapy of cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peptides.
Wt.p53 peptides were generated as described before (31)
. The purity of the peptides was determined by reverse-phase high-performance liquid chromatography and was found to be routinely >90% pure. Mass spectrometry analysis (Lasermat, Finnigan MAT, United Kingdom) showed that their molecular masses were within 0.1% of the calculated values. Peptides were dissolved in 0.5% DMSO in PBS and, if not used immediately, stored at -20°C. The nomenclature concerning the p53-derived peptides refers to the numbers of the first and last amino acid. The peptides p53153–171 (RVRAMAIYKKSQHMTEVVR) and p53119–134 (VMCTYSPPLNKLFCQL) were selected because they contain putative CTL epitopes (Table 1B)
. The peptide p53108–122 (LGFLQSGTAKSVMCT) and peptide p53135–150 (AKTCPVQLWVSATPPA) were selected on the basis of the presence of an I-Ab motif sequence (Table 1A)
. The peptide p53100–131 (YQGNYGFHLGFLQSGTAKSVMCTYSPPLNKLF) is a wt.p53-derived 32-mer containing p53108–122 with its natural flanking sequences. The wt.p53-derived 32-mer-peptide p53146–167 was used as a negative control. Peptide env-H19 (EPLTSLTPRCNTAWNRLKL; amino acid120–138) is the immunodominant helper epitope from the R-MuLV env-gp70 protein and was used as a control peptide (24)
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Immunization Strategies.
p53-/- and C57BL/6 male mice were used for s.c. immunization. The two peptides containing potential CTL epitopes were administered at a dose of 60 µg dissolved in PBS and emulsified in 50% IFA. Mice were boosted after 2 weeks with the same dose. The MHC class II I-Ab motif-bearing peptides were administered in mixes of three peptides at a final dose of 60 µg/peptide/mouse emulsified in 50% IFA. Mice were boosted after 2 weeks with identical mixtures.
T-Cell Cultures.
CD4+ T cells were obtained from immunized mice by culturing spleen cells (2.5 x 106 cells/well of a 24-well plate) in the presence of 5–10 µg/ml peptide or protein in complete medium. Complete medium consists of Iscove’s Modified Dulbecco’s Medium (BioWhittaker, Walkersville, MD) supplemented with 8% FCS, 100 IU/ml penicillin, 2 mM glutamine (ICN, Costa Mesa, CA), and 30 µM 2- mercaptoethanol (Merck, Darmstadt, Germany). Cultures were maintained at 37°C in humidified air containing 5% CO2. No exogenous IL-2 was added. On day 4 dead cells were removed from the culture by centrifugation over a Ficoll density gradient and seeded in 24-well plates at 1 x 106 cells/well together with 30 IU IL-2/ml (Chiron BV, Amsterdam, the Netherlands). On day 7 the T-cell cultures were stimulated with 1 x 106 irradiated B6 spleen cells/well in the presence of 5–10 µg/ml peptide or protein. The responding cells were Ficolled again on day 11 and seeded in 24-well plates at 0.5 x 106 cells/well in the presence of 30 IU IL-2/ml. Established Th lines and clones were cultured at 1 x 105 cells/well and stimulated as described above.
Cell Lines and Antibodies.
MECs of C57BL/6 origin (B6MEC) were transformed by transfection with the oncogenes mut.p53 + H-ras (tumor cell line 4J) as described previously (33)
. The tumor cell line 4J expresses high levels of mutant p53 as determined by immunoprecipitation and cytospin (34)
. The p53-/- MEC cell line is a MEC line obtained from p53-/- mice and does not express p53. 3A12 is a Th clone specific for the env-H19 peptide derived from the R-MuLV envelope protein (24)
. 1H11 is a wt.p53-specific CTL line (35)
obtained from p53-/- mice. 9.5 is a HPV16 E7-specific CTL line. D1 cells are long-term growth factor-dependent immature splenic DCs derived from C57BL/6 (H-2b) mice and were cultured as described (36
, 37) . Before use, D1 cells were incubated for 48 h with peptides, protein, or tumor cell lysates. Subsequently, D1 cells were activated by adding lipopolysaccharide (10 µg/ml) for 6 h and then thoroughly washed and irradiated. Tumor cell lysates were prepared by three freeze/thaw cycles of cell suspension (1 x 106 cells/ml) in complete medium. The equivalent of 2.5 x 106 cells was added to the D1 cultures. The p53-specific IgG2b antibody pAb 122 was added to cell lysates at a concentration of 10 µg/ml. APC-labeled antimouse CD8a (LY-2; 53–6.7), APC-labeled antimouse CD4 Ab (L3T4; clone RM4–5) and FITC-labeled antimouse CD3e Ab (CD3
chain; 145–2c11) were used for FACS analysis.
Proliferation Assay.
Responder cells were seeded at a concentration of 2 x 104 cells/well (T-cell cultures) or 1 x 104 cells/well (T-cell lines) in a 96-well U-bottomed plate. Irradiated C57BL/6 spleen cells were incubated with either peptide or protein (5–10 µg) and added as stimulator cells at a concentration 1 x 105 cells/well. Alternatively, irradiated and lipopolysaccharide-activated D1 cells were used as stimulators at a concentration of 1250 cells/well. No exogenous IL-2 was added. After 3 days at 37°C the cultures were pulsed with [3H]thymidine (NEN; 0.5 µCi/well); incubated for 18 h and then harvested. Thymidine incorporation was measured on a Beta-plate liquid scintillation counter (Wallac, Turku, Finland). Depicted in the figures are cpm.
Limiting Dilution.
Cells of specifically proliferating T-cell cultures were titered to concentrations of 100, 30, 10, 3, or 1 cells/well in complete medium. The cells were stimulated weekly with 5 x 104 irradiated spleen cells/well in the presence of 5–10 µg/ml peptide or protein and supplemented with 30 IU IL-2/ml. Proliferating clones were analyzed for antigen specificity.
Cytokine Assay.
Supernatant of specifically stimulated T-cell cultures was harvested after 3 days. Production of IFN-
by the T cells was measured by a sandwich ELISA performed in maxisorp plates (Nunc, Roskilde, Denmark) using antimouse-IFN--specific antibodies [clones R4–6A2 (capture) and biotinylated XMG1.2 (detection); PharMingen, San Diego, CA], streptavidin conjugated poly-horseradish peroxidase (CLB, Amsterdam, the Netherlands), and ABTS [2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid; Sigma, St. Louis, MO] as a substrate. Absorbance was measured at 415 nm using kineticalc 2.12 software in an EL312e Biokinetics ELISA plate reader (Biotek Instruments, Winooski, VT). The same protocol for measuring cytokines was used for IL-2 [clones JES6–1A12 (capture) and biotinylated JES6–5H4 (detection); PharMingen], IL-4 [clones 11B11 (capture) and biotinylated BVD6–24G2 (detection); PharMingen], and IL-10 [clones JES5–2A5 (capture) and biotinylated SXC-1 (detection); PharMingen].
Adoptive Transfer of Th Cells and CTL.
Female, nude T-cell-deficient C57BL/6 mice were challenged s.c. with 10 x 106 4J tumor cells. After 16 days the wt.p53-specific Th line D2.p53, wt.p53-specific CTL clone 1H11, and control Th line 3A12 were injected i.v. in various combinations at numbers of either 4 x 106 or 8 x 106 cells per mouse. If indicated, 6 x 105 IU rIL-2 were added s.c. in 50% IFA on the day of adoptive transfer and 7 days later.
FACS Analysis.
FACS analysis was performed as described elsewhere (31)
with modifications.
Statistical Analysis.
The effect of the various therapies on measured tumor size in mice was statistically analyzed using the Mann-Whitney test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Refs. 38
, 39
). Furthermore, two peptides were selected based on the notion that in other antigens sequences of natural CTL and Th epitopes were found to overlap. This phenomenon of epitope linkage was, among others, found for epitopes in the HPV16 E7 protein (31
, 40)
, the HIV-1 envelope protein (41)
, and the insulin-dependent diabetes mellitus-associated GAD65 protein (42)
. A potential advantage of epitope linkage lies in the increased chance for simultaneous presentation of both the MHC class I and the class II-restricted epitopes on the surface of a single APC, thereby facilitating the delivery of cognate T-cell help to CTL priming (43)
. Artificial linkage of physiologically nonoverlapping CTL and Th epitopes has proven to be advantageous as shown in studies with HIV and measles (43
, 44)
. Peptide p53153–171 was generated by extending the known wt.p53-derived H-2Kb-restricted CTL epitope p53158–166 (AIYKKSQHM) with five amino acids at both ends with its natural flanking sequences (35)
. Peptide p53119–134 was selected because it comprises 4 additional wt.p53-derived peptides that were reported previously to bind to either H-2Kb or H-2Db (35)
. The immunogenicity of the set of 11 p53-derived peptides was examined by analyzing their capacity to induce peptide-specific proliferative responses. Initially, we vaccinated p53-/- mice to circumvent possible influences of p53-specific tolerance on the induction of peptide-specific immunity. The mice received two subsequent vaccinations with 60 µg of peptide, after which spleen cells were stimulated with the relevant peptide in vitro. After 2 weeks of in vitro stimulation the various T-cell cultures were tested for peptide-specific proliferation. Specific responses were obtained against 4 of the 11 peptides (Table 1
; Fig. 1, A–D
), whereas long-term T-cell cultures could be maintained for 3 peptides (Fig. 1, E–G)
. Notably, the long-term cultures showed increased peptide specificity as compared with the early T-cell cultures, which is conceivably because of the selection of peptide-specific T cells in the former cultures.
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, whereas additional experiments demonstrated that the peptide-specific response was MHC class II restricted (Fig. 2B)
. Moreover, this Th line proved to be clonal as demonstrated by the unanimous expression of the Vß14 TCR chain on all of the cells using a Vß Screening Panel (PharMingen; data not shown). Th cultures capable of expanding in the presence of either protein or peptide antigen are likely to possess the capacity of recognizing physiological quantities of naturally processed epitope. To examine this, we compared the proliferative capacity of the p53108–122-specific Th line D2.p53 against APCs pulsed with the p53108–122 peptide, a longer 32-mer peptide (p53100–131) comprising this p53108–122 epitope, as well as a full-length p53 protein. The Th line responded strongly to either of the three antigen formulations, but not to the control peptides and control protein (Fig. 2C)
. Because the Th line did not respond to a control protein derived from the same expression system, but did react against recombinant p53 derived from a baculovirus expression system, the reactivity against p53 represented indeed true antigen-specific responsiveness. Importantly, although reactivity against APC pulsed with the 15-mer peptide p53108–122 is probably largely the result of recognition of peptides that have been loaded exogenously onto surface I-Ab molecules, reactivity against APC pulsed with either the 32-mer peptide and especially full-length p53 requires antigen uptake and processing by the APC. Therefore, our data indicate that peptide p53108–122, as recognized by Th line D2.p53, represents a naturally processed p53-derived Th epitope.
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. A long-term Th line derived from one of these early cultures showed similar reactivity (Fig. 3B)
. The specific reactivity for p53 protein-pulsed APC was more pronounced for this established Th line, in line with the fact that this culture is more enriched for T cells of the relevant specificity. Our data thus demonstrate that Th cell immunity against the wt.p53-derived epitope p53108–122 can be induced in both p53-/- and p53+/+ mice. To further analyze these responses, we determined the sensitivity of the well-defined p53-/- Th clone D2.p53 to its antigen by comparing its capacity to recognize low amounts of peptide with a previously isolated Th clone specific for a peptide derived from a R-MuLV envelope protein (clone 3A12). This clone has been shown to assist CTLs in the eradication of highly aggressive R-MuLV induced RMA tumors.5
Th line D2.p53 responded against APCs loaded with peptide and protein antigen in the 2–5 nM range (Fig. 4, A and B)
, and thereby revealed a sensitivity which was even higher than that of the R-MuLV-specific clone (Fig. 4C)
. Therefore, we conclude that the affinity of Th line D2.p53 is sufficiently high to fulfill the requirements for mediating adequate antitumor responses.
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. Because the p53-overexpressing tumor cell line is MHC Class II negative, this recognition must involve cross-presentation by the D1 cells. Cross-presentation improved if the p53-specific IgG2b antibody pAb 122 was added to the lysate (Fig. 5A)
, consistent with previous reports by others, which indicated that antigens can be more efficiently presented by DCs through Fc receptor-mediated uptake than after fluid phase internalization (45, 46, 47)
. Determination of the lymphokine profile of Th line D2.p53 revealed secretion of considerable levels of IFN- on specific stimulation, whereas only limited amounts of IL-4 and no detectable levels of either IL-10 or IL-2 were found (Fig. 5B)
. Taken together, our data suggest that peptide p53108–122 represents a naturally processed epitope that can be derived, processed, and presented from p53-overexpressing tumor cells. The peptide p53108–122-specific clonal Th line D2.p53 is capable of recognizing physiological quantities of its cognate antigen and, when triggered by its antigen, displays a Th1 type lymphokine profile.
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, we analyzed whether D2.p53 can provide help to tumor-specific CTLs in vivo. We have shown previously that adoptively transferred CTLs directed against the wt.p53 peptide p53158–166 are highly effective in eradicating p53-overexpressing tumors in vivo, provided that the tumor-challenged mice also receive a s.c. depot of recombinant IL-2 (35)
. We now tested whether coadministration of p53-specific Th cells could replace the need for exogenous IL-2. Previous experiments by our laboratory have shown that Th cells with a relevant antigen specificity can effectively assist adoptively transferred CTLs in eliminating Sendai virus infections and R-MuLV-expressing tumors (49)
.5
Nude mice bearing palpable p53-overexpressing tumors received p53-specific CTLs (clone 1H11) in combination with either IL-2, p53-specific Th cells (line D2.p53) or R-MuLV-specific Th cells (clone 3A12). In accordance with data described previously (35)
, p53-specific CTLs in combination with IL-2 resulted in complete tumor regression, whereas the CTL failed to eradicate the tumors in the absence of exogenous IL-2 (Fig. 6)
. Importantly, the p53-specific Th cells provided help to the CTLs, as opposed to the R-MuLV-specific Th cells, which recognize an antigen that is not expressed by the tumor concerned. The same R-MuLV-specific Th cells had clear cut antitumor activity against R-MuLV-induced RMA tumors.6
The capacity of Th line D2.p53 to provide antigen-specific help to the p53-specific CTL was equally clear in a second, independent experiment. This latter experiment also showed that administration of p53-specific Th cells alone, without the p53-specific CTL, did not have any effect on tumor growth, indicating that these Th cells exert their effect by providing help to the wt.p53-specific CTL (Fig. 7)
.
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and 7
). Consequently mice treated with the latter combination, despite displaying prolonged survival, eventually developed progressive tumor growth. Raising the number of transferred Th cells did not increase the antitumor effect of the combination of D2.p53 and the CTL (Fig. 7)
. This is most likely related to the fact that the p53-specific Th line D2.p53, in contrast to previously studied Th clones against Sendai virus and R-MuLV (49)
,5
does not secrete measurable quantities of IL-2 on antigenic stimulation (Fig. 5B)
. The absence of antitumor activity by the Th line D2.p53 alone (Fig. 7)
may also be a result of this meager IL-2 secretion. However, in additional experiments no difference in tumor development was detected after treatment with either the Th clone D2.p53 alone or in combination with exogenously added IL-2 (data not shown). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-producing Th cells recognizing the naturally processed epitope p53108–122 can be found in the repertoire of both p53-/- and p53+/+ mice. Moreover, we show here that a p53-/- derived wt.p53 Th line (D2.p53) can effectively assist p53-specific CTLs in delaying the growth of p53-overexpressing tumors in vivo. The absence of immunological tolerance for the ubiquitously expressed wt p53 auto-antigen at the CD4+ Th level was predicted by the finding of natural IgG class anti-p53 antibodies and Th responses in cancer patients and tumor-bearing mice (14, 15, 16, 17, 18, 19, 20, 21, 22 , 50, 51, 52) . This stands in remarkable contrast to the considerable degree of tolerance for p53 at the CD8+ CTL level. Although some indications have been found for high affinity p53-specific CTLs in p53+/+ mice as well as in human subjects, only few studies provided direct evidence for such CTLs (3 , 5) , whereas other studies indicate that the p53-specific CTL repertoire in p53+/+ subjects is highly restricted (10, 11, 12) . In accordance with these latter studies, numerous attempts using a variety of vaccine formulations never resulted in the induction of detectable CTLs against the p53158–166 epitope in p53+/+ B6 mice, although these CTL were reproducibly found in appropriately immunized p53-/- mice (35) .7 In our opinion, this case of split-tolerance can be explained by taking into account the metabolism of wt p53. In normal somatic cells, this protein has a very high turnover, as it becomes rapidly degraded via the ubiquitin- and proteasome-dependent pathway (53) . It is highly conceivable that this degradation pathway efficiently feeds p53-derived peptides into the class I MHC presentation pathway, enabling normal somatic cells including thymic APCs to present these peptides at their surface. In contrast, the dominance of the proteasome-dependent degradation will not leave considerable amounts of p53 available for use by the class II MHC processing pathway. Furthermore, this prevents accumulation of wt p53 to steady-state levels that would suffice for cross-presentation by professional APCs. Taken together, these circumstances make it very unlikely that, in a healthy subject, p53-derived peptides are presented in the context of class II MHC, either in the thymus or in the periphery, thereby explaining the lack of tolerance at the Th level (54) . Indeed, as noted before, very similar Th lines against wt.p53 peptide p53108–122 were generated by us in both p53-/- and p53+/+ mice. However, it should be mentioned that no affinity tests were performed with the p53+/+-derived Th line, hampering a full comparison between the two cell lines. Therefore, the possibility that a limited level of tolerance is present at the Th level cannot be totally excluded.
The p53-/- D2.p53 Th cell line displayed clear in vivo antitumor activity when combined with the p53-specific CTL. Yet, no complete remission could be achieved, contrary to the combination of the CTL with exogenous IL-2. This may be because of the modest IL-2 production by the Th line, but this is difficult to prove. Furthermore, it is possible that other mechanisms, such as CD40-CD40L interactions of the Th cells with APC, also play a role.
Importantly, the use of p53-specific Th activity is not restricted to the combination with p53-specific CTL, used as a read-out in the current paper, because such Th cells will also be capable of providing "help" to CTL against other antigens expressed by the same tumor (24)
. On the basis of this principle, we envisage immunotherapeutic approaches against colorectal cancer that use the combination of p53-specific T-cell help with CTL against other antigens expressed by this type of tumor such as carcinoembryonic antigen and epithelial cell adhesion molecule (55, 56, 57)
. Because the key prerequisite for efficacy of p53-specific help in antitumor immunity, overexpression of p53, is found in 50% of all human cancers, p53-specific Th responses appear to be a highly versatile tool for immunotherapy of cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Dutch Organization for Scientific Research (Grant 920-03-094) and by the Dutch Cancer Society (Grants 961352, 961354, 971449, and 971451). 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Department of Immunohematology and Blood Transfusion, Room L3-32, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Phone: 31-71-5265129; Fax: 31-71-5216751; E-mail: cvdmeij{at}lumc.nl
4 The abbreviations used are: Th, T helper; APC, antigen-presenting cell; DC, dendritic cell; IFA, incomplete Freund’s adjuvant; IL, interleukin; MEC, mouse embryo cell; R-MuLV, Rauscher murine leukemia virus; Ab, antibody; FACS, fluorescence-activated cell sorter; cpm, counts per min; wt, wild-type.
5 F. Ossendorp, unpublished observations.
6 F. Ossendorp and C. J. M. Melief, unpublished observations.
7 S. Zwaveling and M. P. M. Vierboom, unpublished observations.
Received 5/ 8/02. Accepted 8/23/02.
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receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med., 189: 371-380, 1999.This article has been cited by other articles:
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C. Kudo-Saito, J. Schlom, K. Camphausen, C. N. Coleman, and J. W. Hodge The Requirement of Multimodal Therapy (Vaccine, Local Tumor Radiation, and Reduction of Suppressor Cells) to Eliminate Established Tumors Clin. Cancer Res., June 15, 2005; 11(12): 4533 - 4544. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, C. N. Coleman, K. Camphausen, J. Schlom, and J. W. Hodge External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing Cancer Res., June 15, 2004; 64(12): 4328 - 4337. [Abstract] [Full Text] [PDF]
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); the Rauscher-specific Th line 3A12 combined with the CTL (
); the CTL with IL-2 (
); the CTL alone (
); no treatment (
). The effect of IL-2 alone on the p53-overexpressing tumor cell line had been tested before and had no influence on tumor growth. The effect of the Rauscher-specific Th line 3A12 alone on tumor growth had also been studied before and was found to be absent. Because of extensive tumor load the mice in the control groups had to be killed after four weeks (
). Bars, ±SE. Statistical analysis was performed using the Mann-Whitney test, revealing that the groups receiving either the combination of the Th line D2.p53 and the CTL or receiving the combination of IL-2 and the CTL showed significant delay in tumor growth compared with the untreated mice, whereas the other groups did not (P < 0.05).