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*
Department of Immunohematology and Bloodbank, Leiden University Medical Center, The Netherlands;
Department of Ophthalmology, Leiden University Medical Center, The Netherlands; and
Virogenetics Corporation, Troy, NY 12180
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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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50%) in the
Caucasian population (1). Vast numbers of peptides
representing proven or potential HLA-A*0201-restricted tumor epitopes
have been tested for their immunogenicity in such mice (e.g., Refs.
2, 3, 4, 5, 6, 7, 8, 9, 10). In most of these studies, the immunogenicity of
the peptides was related to the capacity of splenocytes from
peptide-immunized mice to display a cytotoxic T cell response against
target cells in vitro. Two recent reports addressed the capacity of in
vitro-expanded, adoptively transferred HLA-transgenic CTL to eradicate
established tumors in vivo. CTL isolated from HLA-transgenic mice that
were immunized with human p53 or Her-2/neu peptides could partially
control the growth of human tumor xenografts expressing these epitopes
in SCID mice (11, 12). In the present study, we isolated HLA-A*0201-restricted CTL specific for the human melanoma Ag gp100 from HLA-A*0201/Kb (A2/Kb)3-transgenic mice (13) and examined the capacity of these CTL to eradicate human melanoma cells in vitro as well as in vivo. We were particularly interested in the in vivo eradication of intraocular human uveal melanomas. This type of melanoma is the most common primary intraocular malignancy in adults. Although treatments of the primary tumor that allow preservation of the eye have progressed greatly, these approaches have severe side effects. Gunduz et al. (14) recently showed that 42% of eyes successfully treated with radiation subsequently developed radiation retinopathy within the first 5 years after treatment. As a result of the high incidence of metastases, which are largely refractory to conventional treatment, uveal melanoma has a high mortality rate (reviewed by Pyrhönen in Ref. 15). Because gp100 was found expressed in the majority of human uveal melanomas (16, 17, 18), this Ag would constitute an appropriate target for immunotherapy of such tumors. A potential hurdle for successful immunotherapy of such tumors is the fact that the eye is, at least to a certain extent, an immune-privileged site (19, 20). Furthermore, melanoma cells have been reported to exert lymphocyte-inhibitory properties within the ocular microenvironment (21). To test the feasibility of immunotherapy of intraocular human uveal melanomas, we engrafted human HLA-A*0201+, gp100+ uveal melanoma cells in the anterior chamber of the eye of A2/Kb-transgenic mice. The xenogeneic melanoma cells, which are rapidly eliminated in such mice when grafted into other sites of the body, were capable of forming tumors in this site. We employed this tumor model to test whether systemic administration of gp100-specific CTL, isolated from A2/Kb mice and expanded in vitro, would result in elimination of these intraocular tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M1B-gp100 was derived from a nontransformed mouse embryo cell line of C57BL/6 (B6) origin through transfection with the genes for murine B7.1, HLA-A*0201/Kb (A2/Kb), and human gp100. M1B-MAGE was generated in a similar manner by transfection with the gene for human MAGE-2. BA-gp100 and BA-MART were derived from murine melanoma cell line B16-F10 through transfection of the genes for A2/Kb and human gp100 or human MART-1, respectively. Previously described cell lines in these experiments included Jurkat A2/Kb (13), human melanoma cell lines BLM, BLM-gp100 (22), Mel 397 (23), and Mel 397 A2/Kb (kindly provided by G. J. Adema, University Hospital, Nijmegen, The Netherlands), human uveal melanoma cell lines OMM-1 (17), OCM-3, and 92–1 (24), and human osteosarcoma cell line SAOS (25). Infection of SAOS with recombinant ALVAC (described below) was performed by infection of 106 cells in 0.5 ml serum-free medium with a multiplicity of infection of 10 during 3 h at 37°C, followed by washing of the cells and culturing for 2 days. Murine CTL clone 100B6 specifically recognizes a peptide epitope derived from the adenovirus type 5 E1B protein (26). All cell lines were maintained in IMDM (BioWhittaker, Walkersville, MD), supplemented with 8% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 µM 2-ME. Murine CTL lines and clones were cultured in IMDM supplemented with 8% heat-inactivated FCS, 10 cetus units IL-2/ml, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 µM 2-ME, and 2 mM L-glutamine. WEHI 164 clone 13 cells were cultured in RPMI 1640 supplemented with 8% heat-inactivated FCS, penicillin, L-glutamine (216 mg/ml), L-asparagine (36 mg/ml), and L-arginine-HCl (116 mg/ml).
Immunization of HLA-A*0201/Kb-transgenic mice
HLA-A*0201/Kb
(A2/Kb)-transgenic mice were kindly provided by
Dr. L. Sherman (Scripps Laboratories, San Diego, CA). These mice
express a chimeric class I MHC molecule composed of the 
1 and 2
domains of HLA-A*0201 and the 3, cytoplasmic, and
transmembrane domains of the mouse H-2Kb molecule
(13). A2/Kb mice were vaccinated
with different recombinant ALVAC canary poxviruses (Virogenetics, Troy,
NY). ALVAC-gp100 encoded the full-length human gp100 sequence.
ALVAC-AAA (PE03) encoded the four epitopes
(MART-127–35,
gp100154–162,
gp100209–217, and
gp100280–288) separated by a triple alanine
spacer (AAA-AAGIGILTV-AAA-KTWGQYWQV-AAA-ITDQVPSFV-AAA-YLEPGPVTA-AAA).
This spacer sequence has successfully been employed for other
multiepitope constructs (27). ALVAC-NKRK (PE02) contained
the epitopes separated by an NKRK spacer
(NKRK-AAGIGILTV-NKRK-KTWGQYWQV-NKRK-ITDQVPSFV-NKRK-YLEPGPVTA-NKRK).
This spacer sequence is predicted to contain three trypsin cleavage
sites. Trypsin-like activity has been associated with the proteasome.
The parental ALVAC virus was used as a negative control.
Mice received three i.v. doses of 107 PFU of the same ALVAC. Ten days after the last vaccination, the mice were sacrificed, and splenocytes were isolated. Different irradiated stimulator cells, sharing expression of A2/Kb and gp100, were used in the following order: 1) A2/Kb LPS blasts loaded with the three human gp100 peptides (to prevent competition between these peptides for binding to A2/Kb, three portions of LPS blasts were separately loaded with the different peptides and washed three times, after which they were pooled); 2) Mel 397 A2Kb; and 3) MIB-gp100. For anti-tumor vaccination studies, mice received two i.v. doses of 107 PFU with a 10-day interval. Ten days after the last vaccination, the mice were challenged with 3 x 105 OMM-1 cells in the anterior chamber of the eye, and tumor development was monitored.
Analysis of CTL activity in vitro
Cell-mediated lymphocyte cytotoxicity was measured with a
51Cr release assay as described previously
(2). The mean percentage of specific lysis in triplicate
wells was calculated as follows: % specific lysis = [(cpm
experimental release - cpm spontaneous release)/(cpm maximum
release - cpm spontaneous release)] x 100. For TNF- release
assays, Jurkat A2/Kb cells were loaded with
peptide (10 µg/ml) for 1 h at 37°C, washed three times, and
used as stimulator cells. In short, 5 x 103
murine T cells were added to 104 peptide-loaded
Jurkat A2/Kb cells. After 24 h, the
supernatant was harvested, and its TNF content was determined by
its cytolytic effect on mouse fibrosarcoma cell line WEHI 164 clone
13, measured by the intensity of vital staining on an ELISA plate
reader. The maximum cytolytic effect was estimated by adding 500 pg/ml
human rTNF-.
Adoptive immunotherapy experiments
A modified quantitative technique for deposition of a definitive
number of tumor cells into the anterior chamber of the mouse eye was
used (28). Mice were deeply anesthetized with a mixture
(ratio 1:1) of xylozine (Rompun 2%; Bayer, Leverkusen, Germany) and
ketamine hydrochoride (Aescoket, Aesculaap BV, Boxtel, The Netherlands)
given i.p. The eye was viewed under the low power (8x) of a dissecting
microscope, and a sterile 30-gauge needle was used to puncture the
cornea at the corneoscleral junction, parallel and anterior to the
iris. A glass micropipet (80 µm in diameter) was fitted into a
sterile infant-feeding tube, which was mounted onto a sterile 0.1
Hamilton syringe (Hamilton, Whittier, CA). The pipette, loaded with
OMM-1 cell suspension (7.5 x 107/ml), was
introduced through the puncture site of the cornea, and 4 µl of the
OMM-1 cell suspension was delivered into the anterior chamber. The eyes
were examined three times per week with a dissecting microscope to
observe and document tumor growth. Subcutaneous induction of tumors, as
well as i.v. administration of CTL clones, was performed as described
previously (28, 29). Tumor-challenged mice were assigned
randomly to treatment protocols with relevant CTL or control CTL. All
CTL treatments were performed in combination with a s.c. injection of
105 Cetus units of rIL-2 in an IFA depot.
Histological analysis of eyes of treated and control mice was performed
as described previously (28).
Flow cytometry of tumor-infiltrating lymphocytes (TIL)
Eyes were mashed through a nylon filter to obtain single-cell suspensions, passed over a Ficoll gradient, washed, and triple stained with propidium iodide (1 µg/ml), anti-mCD8-FITC (1:200 dilution, PharMingen, San Diego, CA), and HLA-A*0201-allophycocyanin tetrameric complexes harboring the gp100154–162 peptide (1:10 dilution; a kind gift from Dr. H. Spits, The Netherlands Cancer Institute, The Netherlands). Analysis by flow cytometry (FACScalibur; Becton Dickinson, Mountain View, CA) of propidium iodide-negative cells was performed immediately after staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human gp100-specific CTL immunity was induced in HLA-A*0201/Kb (A2/Kb)-transgenic mice through immunization with recombinant canarypox viruses (ALVAC) that encode the three known HLA-A*0201-restricted epitopes gp100154–162 (22), gp100209–217, and gp100280–288 (30). The full-length human gp100 protein is known to harbor, in addition to the three epitopes of interest, at least one and possibly more epitopes that are immunogenic in the context of murine class I MHC (31), while the A2/Kb-transgenic mice coexpress the H-2Db and Kb molecules (13). To skew the CTL response of the mice toward the HLA-restricted epitopes of interest, we immunized not only with ALVAC-gp100, encoding the full-length human gp100 Ag, but also with multiepitope ALVAC, encoding synthetic polypeptides that comprise a string-of-beads arrangement of the three epitopes (see Materials and Methods for details). Similar multiepitope vaccines have previously been employed successfully by several laboratories for the induction of effective anti-tumor CTL immunity in mice (e.g., Refs. 27 and 32).
Mice received three subsequent i.v. doses of the same ALVAC vaccine,
after which the splenocytes were restimulated in vitro in the presence
of cells positive for the three human gp100 epitopes and HLA-A*0201. We
submitted the splenocytes to three sequential rounds of stimulation
with different stimulator cells in the following order:
A2/Kb-transgenic LPS blasts loaded with the three
gp100 epitopes (see Materials and Methods for details), an
A2/Kb-transfectant of the gp100-positive human
melanoma cell line Mel397, and A2/Kb-transgenic
mouse embryo cells transfected with the genes for human gp100 and
murine B7.1 (M1B-gp100). After this three-step restimulation protocol,
the T cell cultures were tested for their reactivity against a panel of
target cells in a TNF- release assay. The T cell cultures responded
against human and murine cell lines expressing both human gp100 and
A2/Kb, but not against the controls (Fig. 1
A). Parallel splenocyte
cultures from mice immunized with control ALVAC did not exhibit
specific responses to these targets (not shown). Reactivity was also
observed against human HLA-A*0201-positive, gp100-negative osteosarcoma
cells (SAOS) that were infected with ALVAC-gp100 (Fig. 1A).
This suggested that the responder cells were capable of recognizing
their target not only in the context of the chimeric
A2/Kb molecule, but also in the context of the
wild-type HLA-A*0201 (see below). Monitoring of reactivity against
peptide-loaded targets revealed that the CTL cultures were exclusively
directed against the gp100154–162 epitope (Fig. 1B). This pattern of reactivity was also observed in
cytolytic assays (not shown).
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After initial analysis of the polyclonal T cell cultures, CTL
clones were isolated through limiting dilution using BLM-gp100 tumor
cells (HLA-A*0201+) as stimulator cells. These
clones showed a reactivity pattern identical with that of the bulk
cultures from which they were derived, in that they recognized human
and murine cells that presented the gp100154–162
epitope in the context of the A2/Kb molecule
(Fig. 2). Importantly, the CTL were also
capable of lysing gp100-positive human melanoma cells expressing the
wild-type HLA-A*0201 restriction element (Fig. 2C). Because
the murine CD8 molecule on these CTL cannot efficiently associate with
the 3 domain of the HLA-A*0201 molecule (13), this
interaction is apparently not required, implying that the TCRs of these
CTL exhibit high affinity for their target Ag. This notion was
confirmed by measuring the reactivity of the
A2/Kb-transgenic CTL clones against titered
amounts of exogenously loaded gp100154–162
peptide. The CTL exhibited efficient lysis of peptide-loaded target
cells, reaching 50% of their maximal lytic activity at peptide
concentrations of 10 ng/ml (Fig. 3A). This sensitivity, which
is similar to that found for TIL1200 cells, a human TIL line
recognizing the same epitope (22), is commonly found for
CTL that are capable of responding to physiological quantities of
naturally processed Ag (33).
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B, and Ref. 22). Taken together, our data
show that immunization of A2/Kb-transgenic mice
with recombinant ALVAC vaccines encoding human gp100 CTL epitopes
can elicit potent HLA-A*0201-restricted CTL immunity against the
gp100154–162 epitope. Gp100-specific CTLs control the growth of intraocular human uveal melanomas in A2/Kb-transgenic mice
At present, no effective treatment is available for metastatic
uveal human melanoma (15). In contrast, a majority of the
human uveal melanomas tested were found to express both gp100 and class
I HLA molecules (16, 17, 18, 34). Furthermore, it has been
shown that uveal melanoma cells can be lysed by tyrosinase and
MAGE-specific CTL clones in vitro (35). Therefore, we set
out to analyze the sensitivity of human uveal melanoma cells for our
A2/Kb-transgenic gp100-specific CTL in vitro as
well as for adoptive immunotherapy with these CTL in vivo. We employed
the human uveal melanoma cell line OMM-1, which is positive for both
HLA-A*0201 and gp100 (17). In accordance with the
expression of these Ags, OMM-1 was shown to be an excellent target for
lysis by A2/Kb-transgenic
gp100154–162-specific CTL in vitro (Fig. 4). We subsequently performed adoptive
transfer experiments in A2/Kb mice that were
challenged with an intraocular dose of this human uveal melanoma cell
line. We have recently found that highly immunogenic adenovirus type 5
(Ad5)-transformed cells, which otherwise fail to grow in syngeneic
immunocompetent mice (29), do form tumors when inoculated
in the anterior chamber of the eye (28). This observation
supports the notion that the eye is an immune-privileged site and
suggests that this setting may also permit the outgrowth of xenogeneic
tumors in immunocompetent mice. Indeed, injection of OMM-1 in the
anterior chamber of the eye of A2/Kb-transgenic
mice resulted in outgrowth of these tumor cells (Figs. 5, A–C, and
6), whereas s.c. injection of up to
107 OMM-1 cells failed to induce tumors (not
shown). Tumor take was 100% when 3 x 105
OMM-1 cells were applied. Although in the majority of mice the OMM-1
tumor regressed spontaneously at around day 20 after tumor inoculation,
similar to what was previously reported for intraocular Ad5-transformed
tumors (28), this setting still allowed us to study the
prevention of ocular human uveal melanoma engraftment. Importantly,
adoptive transfer of the A2/Kb-transgenic,
gp100154–162-specific CTL in combination with
IL-2 prevented the outgrowth of OMM-1 in the eyes of these mice (Fig. 6, A–G). Treatment with a control CTL and IL-2
(Fig. 6, H–N) did not restrict the growth of
this tumor. Treatment with IL-2 alone did not differ significantly from
treatment with control CTL and IL-2 (not shown). The tumoricidal effect
of the gp100-specific CTL indicated that, despite the immune privilege
of the eye, the CTL were capable of homing to the tumor site and
exerting their Ag-specific effector function. The eradication of the
tumor by the CTL was not accompanied by any sign of immunopathological
damage to the eye (Fig. 5, D–F). To confirm the homing of
the adoptively transferred CTL toward the anterior chamber of the
eye, we analyzed ocular single-cell suspensions for the presence
of gp100154–162-specific CTL. Mice
challenged with OMM-1 in the anterior chamber of the left eye were
treated with 107
gp100154–162-specific CTL and IL-2. As shown in
Fig. 7, gp100154–162-specific CTL can be detected in the
tumor-bearing eye after 24 h, whereas no CTL are detectable in the
tumor-free eye of the same animal. This result directly shows that the
CTL indeed are capable of homing toward OMM-1 tumors in the anterior
chamber of the eye.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain permits
efficient interaction of HLA-restricted murine CTL with their target
through their CD8 receptor (13). However, a drawback may
be that the HLA-restricted CTL generated in
A2/Kb-transgenic mice, especially in mice that
have been immunized with considerable quantities of synthetic peptides,
do not exhibit very high affinity for their target epitope and
therefore depend for their reactivity on the interaction with CD8. The
consequence of this CD8 dependency would be that such CTL, although
capable of recognizing their target epitope in the context of the
A2/Kb molecule, would fail to respond to this
epitope in the context of the physiologically relevant HLA-A*0201
molecule (36). To prevent the induction of CTL of
insufficient affinity, we avoided immunization with synthetic peptides
and instead immunized with recombinant canarypox viruses (ALVAC)
encoding these epitopes. To ensure induction of HLA-A*0201-restricted
gp100-specific CTL instead of CTL directed against epitopes restricted
by the endogenous H-2Db and
Kb molecules, we immunized not only with
ALVAC-gp100, encoding the full-length human gp100 Ag, but also with
multiepitope ALVAC encoding synthetic polypeptides, which comprises a
string-of-beads arrangement of the three gp100 epitopes. In all cases,
CTL specific for the gp100154–162 peptide were
obtained, suggesting that this epitope is the immunodominant
HLA-A*0201-restricted gp100 epitope in A2/Kb
mice. This can most readily be explained by the fact that the
gp100154–162 and
gp100280–288 epitopes, in contrast to the
gp100209–217 epitope, are not conserved between
mice and humans (10), whereas the
gp100154–162 peptide exhibits the strongest
binding to HLA-A*0201 (37). Our adoptive immunotherapy experiments in A2/Kb mice challenged by intraocular injection with human uveal melanoma cells show that systemic (i.v.) administration of the A2/Kb-transgenic anti-gp100 CTL can completely control the outgrowth of these tumors. Moreover, we demonstrated that these CTL can be detected in eyes bearing an OMM-1 tumor in the anterior chamber. As noted before, mice receiving control treatment do exhibit spontaneous regression of established intraocular tumors (Ref. 28 , and this paper). Additional studies have indicated that this phenomenon largely depends on the activity of endogenous CD4+ T cells (L.R.H.M.S. and R.E.M.T., unpublished observations). Despite spontaneous regression, a sufficient time-window is available for evaluation of the tumoricidal efficacy of adoptively transferred CTL. In two previous papers, HLA-A*0201-restricted CTL isolated from HLA-transgenic mice were shown to partially control the outgrowth of human tumors in SCID mice (11, 12). Other studies with xenogeneic models in which in vivo efficacy of HLA-restricted anti-tumor CTL was demonstrated involved either peritumoral injection of effector cells (e.g., Refs. 38, 39, 40) or injection of a mixture of tumor and effector cells in a Winn-type assay (e.g., Refs. 41, 42, 43). In these latter experiments, homing of the effector cells to the tumor site is not a prerequisite. Notably, the requirement of homing involves not only the ability of the CTL to travel through the periphery of the host and track down their target, but also the capacity of a sufficient proportion of these cells to survive this journey as well as the power of this fraction to launch a ferocious tumoricidal attack. This implies that adoptive immunotherapy involving systemic injection of CTL into mice bearing xenogeneic tumors requires high-quality CTL.
In addition to the adoptive transfer experiments in A2/Kb-transgenic mice, we performed parallel experiments in C57BL/6 (A2/Kb-negative) nude mice. As mentioned above, immunodeficient mice such as nude or SCID mice are commonly used as hosts in xenogeneic tumor models. However, in the latter setting, our gp100-specific A2/Kb-transgenic CTL failed to eradicate the intraocular OMM-1 tumors (data not shown). This suggests that expression of the A2/Kb molecule in the host is essential for the in vivo efficacy of the A2/Kb-transgenic CTL. The mechanistic aspects of this phenomenon lie outside the scope of the present study. Importantly, we demonstrate that the in vivo efficacy of the A2/Kb-transgenic CTL against a xenogeneic tumor can be tested in an immunocompetent syngeneic host by engrafting the xenogeneic human tumor in the anterior chamber of the eye. Because we and others have shown that intraocular engraftment enables the in vivo growth of tumors that otherwise fail to grow (28, 44), this experimental setting will most likely be applicable for analyzing the efficacy of adoptive immunotherapy with HLA-transgenic T cells against a variety of human tumors.
Metastatic uveal melanoma constitutes a formidable therapeutic challenge. At first sight, these tumors seem an equally poor target for immunotherapeutic approaches. They arise in an immune-privileged site that can sustain the growth of foreign tissues and in which different mechanisms for suppression of immune responses are operational (19, 20, 45, 46). Furthermore, the microenvironment of the eye was shown to endow human uveal melanoma cells with lymphocyte-inhibitory properties (21). Another compelling observation is that lack of expression of class I HLA-Ags on human uveal melanoma was found to be correlated with a better, rather than poor, patient survival (34). This suggests that NK cells have a protective role in the development of metastatic disease, whereas, in contrast to what has been observed for skin melanoma (47, 48, 49), CTL-mediated immunity does not impose a selective pressure for loss of HLA-expression by these tumors. Paradoxically, work with animal models for intraocular tumors has demonstrated that adoptively transferred T cells can in principle be effective against such tumors (28, 50). In the latter study, we demonstrated that adoptive transfer of human Ad5-specific CTL resulted in rapid and complete eradication of intraocular tumors of Ad5-transformed cells. An explanation for this paradox could be that these murine studies made use of model tumors that had not originally developed in the eye and that therefore lacked certain features typical of uveal melanomas. Importantly, our present work in A2/Kb mice demonstrates that adoptive immunotherapy is effective not only against such model tumors but also against the intraocular engraftment of human uveal melanoma. This is in accordance with the fact that OMM-1 and many human uveal melanomas express class I HLA as well as a variety of melanoma Ags (16, 17, 18, 34), and that such cells are excellent targets for lysis by tumor-specific CTL in vitro (as shown by this study and previous studies (35)). Although we showed that adoptively transferred CTL eliminate the intraocular tumor in the absence of detectable damage to the eye, it should be noted that the CTL used so far were directed against foreign Ags. The gp100154–162 epitope is not conserved between mice and humans (10). Therefore, it is important to test the effect of CTL against autologous Ags, especially epitopes that are also expressed in the eye. An exquisite opportunity to address this issue is offered by the recent observation that potent anti-tumor CTL immunity against the gp100209–217 epitope, which is conserved between mice and humans, can be raised through immunization with recombinant virus encoding a variant of this peptide exibiting improved binding to HLA-A*0201 (10). Although in these experiments, involving vaccination of A2/Kb mice followed by evaluation of CTL immunity in vitro, no autoimmune vitiligo was observed, it is conceivable that adoptive transfer of large numbers of anti-gp100209–217 CTL would result in depigmentation in these mice, at least in the pigmented tissues of the eye where they exert their tumoricidal effects. The prime question will be whether such side effects would result in damage that would compromise the function of the eye.
Previous observations by our laboratory (28) have indicated the feasibility of specific vaccination as a therapy against intraocular tumors. Therefore, we evaluated whether vaccination of A2/Kb mice with ALVAC-gp100 vectors protected the mice from OMM-1 outgrowth. However, no significant effect of vaccination on outgrowth of OMM-1 could be observed when ALVAC-gp100-vaccinated mice were compared with control (parental ALVAC)-vaccinated animals (data not shown). This discrepancy between the efficacy of adoptive transfer and that of vaccination can readily be explained by the fact that the CTL clone has been selected for its capability to recognize the gp100154–162 epitope in the context of both the A2/Kb and the wild-type HLA-A*0201 molecules. In contrast, Ag-specific vaccination of A2/Kb mice is known primarily to elicit CTL capable of recognizing their epitope in the context of A2/Kb only (13, 51). This implies that gp100-specific vaccination induced only a limited number of CTL capable of reacting against the HLA-A*0201+, gp100+ OMM-1 cells and explains the failure of vaccination in this particular setting.
In conclusion, our study strongly argues in favor of considering T cell immunotherapy for the treatment of human uveal melanoma. In addition to adoptive immunotherapy, melanoma-specific vaccination is a valid option, because others have shown that vaccination can result in specific immunity capable of protecting mice against a subsequent intraocular tumor challenge (52). Although such therapy appears to be effective against primary uveal melanoma, the most important application would probably be to use this therapy in an adjuvant setting in combination with conventional treatment of the primary intraocular tumor. Especially in situations of minimal residual disease, which in the case of human uveal melanoma is associated with a high incidence of fatal metastases (15), T cell immunotherapy may offer a solution for a long-standing problem.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rienk Offringa, Department of Immunohematology and Bloodbank, Leiden University Medical Center, Building 1-E3-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
3 Abbreviations used in this paper: A2/Kb, HLA-A*0201/Kb; TIL, tumor-infiltrating lymphocyte; Ad5, adenovirus type 5.
Received for publication January 7, 2000. Accepted for publication September 11, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein. Cancer Res. 59:3134.2 and 3 domains in the recognition of self and nonself MHC molecules. J. Immunol. 156:2473.[Abstract]
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