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*
Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, NE 68198; and
Mayo Clinic, Scottsdale, AZ 85259
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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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C57BL/6 mice transgenic for human MUC1 (MUC1.Tg3) were developed to investigate anti-MUC1 tumor immunity in an animal that expresses MUC1 as a self Ag on nontransformed epithelia (8, 9, 10). MUC1.Tg mice challenged with MUC1-expressing tumors (B16.MUC1) developed progressively growing, MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at significantly slower rates. The degree of dissimilarity between MUC1 and its murine homologue, Muc-1, enables the immune system of wt mice to recognize MUC1 as a foreign protein and to eliminate MUC1-expressing tumors. In contrast, MUC1.Tg mice are immunologically tolerant to MUC1 and do not reject B16.MUC1 cells.
A limiting dilution CTL frequency assay was used to quantify the cellular cytotoxic responses to MUC1 or non-MUC1 Ags in wt or MUC1.Tg mice during the course of progressive B16.MUC1 tumor growth. To complement the in vitro results, an in vivo adoptive transfer system was used to evaluate the degree of MUC1 tumor immunity in wt or MUC1.Tg donor mice. Both experimental systems were used to analyze the time course in which MUC1-specific immune responses developed during the course of B16.MUC1 tumor progression.
The MUC1.Tg mice are a preclinical model to evaluate immunotherapies designed to overcome tolerance to MUC1 by immunization with MUC1 vaccine formulations or by adoptive transfer of MUC1-specific cells. Similar to cancer patients, MUC1-specific responses in MUC1.Tg mice have the potential to result in autoimmunity since these mice express MUC1 on epithelia of many organs. To investigate the relationship between MUC1-specific tumor immunity and autoimmunity, MUC1-specific immune cells primed in vivo were adoptively transferred to MUC1.Tg mice, and the degree of MUC1-specific tumor immunity and autoimmunity was investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female wild-type C57BL/6 (wt) mice, 6 wk of age, were purchased from National Cancer Institute (Frederick, MD). Age-matched female MUC1.Tg C57BL/6 (MUC1.Tg) mice were obtained from a breeding colony at University of Nebraska Medical Center (Omaha, NE). Mice were treated in accordance with IACUC Institutional Animal Care and Use Committee guidelines.
Cell lines
B16.MUC1 or B16.Neo are murine (C57BL/6) melanoma tumor lines transfected with a MUC1 cDNA or control expression vector, respectively (9, 11). Both B16.MUC1 and B16.Neo cell lines express MHC class I in vitro, as detected by flow-cytometric analysis (data not shown).
Limiting dilution CTL frequency assay
Anesthetized (Metofane; Pitman-Moore, Madelein, IL) MUC1.Tg or wt mice were injected s.c. between the scapulae with 2 x 104 viable B16.MUC1 tumor cells. Splenocytes and brachial LN cells were mechanically separated to a single cell suspension, and RBCs were lysed by hypotonic shock. Effector lymphocytes were derived from draining LN, and splenocytes obtained from the same mouse were used as APCs. Limiting dilutions (6, 6 x 101, 6 x 102, 6 x 103, or 6 x 104) of brachial LN cells were incubated with 4 x 103 irradiated (6 x 103 rad) B16.MUC1 cells and 3 x 104 irradiated splenocytes (2 x 103 rad) in flat-bottom 96-well plates (Becton Dickinson, Franklin Lanes, NJ) with 250 µl DMEM high glucose (Life Technologies, Grand Island, NY) supplemented with 20% heat-inactivated FBS (Biowittaker, Walkersville, MD), 10% T-stim (Collaborative Biomedical Products, Bedford, MD), nonessential and essential amino acids (Mediatech, Hernden, VA), vitamins (Life Technologies), 4 mM glutamine (Life Technologies), 625 µM HEPES (Sigma, St. Louis, MO), 50 µM ß-mercaptoethanol (Sigma), 100 µg/ml gentamicin (Life Technologies), and 1 mM sodium pyruvate (Sigma). Cocultures were incubated at 37°C and 5% CO2 for 7 days and then split equally into triplicate microtiter plates and incubated with 4 x 104 freshly irradiated B16.MUC1 cells and 3 x 104 freshly irradiated APCs obtained from naive wt mice. Four days later, cytotoxicity was determined in each microculture by using a standard 51Cr release assay with MUC1-expressing EL-4 cells (EL4-MUC1) (a kind gift of Dr. Olivera Finn) and control EL-4 cells as targets. Briefly, target cells were labeled with 250 µCi of 51Cr (ICN, Costa Mesa, CA) at 37°C for 75 min and washed. Microcultures in one set of plates were incubated with 104 EL4-MUC1 targets, and microcultures in the second set of plates were incubated with 104 control EL-4 targets. The microcultures in the third set of plates were reserved to expand reactive clones identified in the lytic assay. The plates were spun at 103 rpm for 2 min and incubated for 4 h at 37°C and 5% CO2. Following the incubation, 100 µl of culture supernatant was collected (Skatron SCS system; Skatron, Sterling, VA) and radioactivity was quantified in a gamma counter (LKB Wallac, 1272 CliniGamma, Gaithersburg, MD). Positive wells were defined as those target cell supernatants with radioactivity 3 SD above the mean spontaneous target cell release. MUC1-specific wells were identified by positive reactivity with the EL4-MUC1 targets and negative reactivity in wells containing the EL4 control targets. Plotting the frequency of negative wells on a logarithmic scale as a function of increasing responder cell concentration (linear scale) enabled the derivation of the MUC1 or non-MUC1 CTL frequency, as previously described (12). Two-tailed t test was used for statistical analysis.
Cellular adoptive transfer and tumor challenge assay
Splenocytes and LN cells were harvested from donor mice primed with viable B16.MUC1 tumor cells, as indicated above. The lymphoid tissue was manually and mechanically processed to yield a single cell suspension, and in some cases the red cells were lysed by hypotonic shock. Harvested immune cells were treated as individual aliquots yielding approximately 120–220 x 106 leukocytes. Donor mice were paired with recipient mice such that aliquots equivalent to one-half of the total splenocyte and LN cell harvest from each donor animal were injected i.p. into two nonirradiated recipient mice (matched set). One day later, control or matched sets of recipient mice were injected between the scapulae s.c. with either 2 x 104 B16.MUC1 or B16.Neo tumor cells, and tumor growth was evaluated over time. The matched set design meant that equivalent cell fractions were always evaluated in control and experimental groups. Kaplan-Meier survival curves are shown for some tumor challenge studies. Survival is defined as a tumor less than 1 cm in diameter; animals were euthanized when tumors reached 1.2 cm in diameter. The log-rank test was used for statistical analysis.
Purified populations of CD4+ or CD8+ lymphocytes were obtained from freshly harvested splenocytes and LN cells from wt mice and incubated in HBSS (Life Technologies). Adherent cells were depleted by incubation of this suspension for 1.5 h at 37°C in 10-cm culture dishes (Falcon, Franklin Lakes, NJ). Nonadherent cells were removed by gentle aspiration, washed, and further purified into CD4+ or CD8+ T cells using negative selection columns, according to manufacturer’s specifications (Biotex Laboratories, Edmonton, Canada). Cells purified in this manner were >90% positive for the selected T cell phenotype, and were <2% positive for CD19 or the reciprocal T cell phenotype (data not shown). Approximately 15–22 x 106 purified CD4+ or CD8+ T cells were obtained from each donor mouse. Adoptively transferred or control wt mice were challenged with 2 x 104 B16.MUC1 tumor cells. Tumor size was quantified as described previously (9), and the two-tailed t test was used for statistical analysis.
In vivo CD4+/CD8+ lymphocyte depletion
The rat anti-mouse hybridoma clones 53-6.72 and GK1.5, and the rat anti-human hybridoma clone SFR3-DR5 were purchased from American Type Culture Collection (Manassas, VA) and cultured in vitro according to product sheet specifications. Hybridoma culture supernatants were filtered (0.45 µm) and purified on a protein G-Sepharose Fast Flow System (Pharmacia Biotech, Uppsala, Sweden). Binding conditions were in the presence of 20 mM Na2HPO4, pH 7, and elution was accomplished using 100 mM glycine, pH 2.7. Eluted solutions were immediately neutralized using 1 M Tris, pH 9, at a 1:25 ratio. Rat Ig was quantified by solid-phase ELISA, as described elsewhere (9). Four treatments at -6, -4, -2, and 7 days (tumor challenge occurred on day 0) of 0.5 mg of control Ab (SFR3-DR5), depleting rat anti-mouse CD4 (clone GK1.5), CD8 (clone 53.6-72), or both were administered i.p. to wt mice. The lymphocyte-depleted condition of representative mice was confirmed by flow cytometry at day 0. Splenocytes and LN cells obtained from representative depleted mice were stained with anti-mouse CD4, anti-mouse CD8, anti-mouse CD3, or anti-mouse CD19 Abs labeled with FITC. Compared with mice administered control Ab less than 2%, the depleted lymphocytes were detected in the CD4+-, CD8+-, or CD4+/CD8+-depleted groups (data not shown). Following initial Ab administration, administered control Ab wt, depleted wt, unmanipulated wt, or MUC1.Tg mice were challenged with 2 x 104 B16.MUC1 tumor cells, and tumor growth was evaluated over time.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MUC1.Tg or wt mice develop similar numbers of MUC1-specific CTLs following B16.MUC1 tumor challenge
A modified limiting dilution CTL frequency assay was used to
quantify in vitro MUC1-specific CTLs or CTLs reactive with Ags other
than MUC1 (referred to as non-MUC1-reactive CTLs) in MUC1.Tg or wt mice
challenged s.c. with 2 x 104 viable B16.MUC1 tumor
cells. No MUC1-specific and few (3.3/106) non-MUC1-reactive
CTLs were detected in naive wt mice (Fig. 1
, A and B).
Wild-type mice were challenged with 2 x 104 B16.MUC1
tumor cells, and CTL frequencies were quantified over the course of
progressive tumor growth (42 days). The frequency of MUC1-specific or
non-MUC1-reactive CTLs at different time points in these mice was
between 0–26/106 and 14.6–123/106,
respectively (Fig. 1, A and B).
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A). Both MUC1-specific (0–12.9/106) and
non-MUC1-reactive CTLs (4–55/106) were detected in naive
MUC1.Tg mice (Fig. 1, A and B). MUC1.Tg mice were
challenged with 2 x 104 viable B16.MUC1 cells, and
over the course of 28 days the frequency of MUC1-specific or
non-MUC1-reactive CTLs was between 12.9–38/106 and
14.6–36/106, respectively (Fig. 1, A and
B). Results of these CTL frequency studies indicated that the MUC1-specific or non-MUC1-reactive CTL responses detected in vitro were similar between the wt and MUC1.Tg mice. In contrast, results obtained from in vivo challenge with B16.MUC1 cells showed that MUC1.Tg mice developed progressively growing MUC1-positive tumors, while wt mice rejected MUC1-positive tumors and developed MUC1-negative tumors at a significantly slower rate (9). The tumor growth data provided in vivo evidence for a MUC1-specific tumor immune response in wt mice that was absent or ineffective in MUC1.Tg mice.
Cellular adoptive transfer indicated that wt but not MUC1.Tg mice develop MUC1-specific tumor immunity
The in vitro assays were not effective in detecting immune cells that mediated the MUC1-specific tumor immune response. A cellular adoptive transfer system was designed to evaluate MUC1-specific and non-MUC1-reactive tumor immunity in vivo. Splenocytes and LN cells from wt or MUC1.Tg mice previously challenged with 2 x 104 viable B16.MUC1 tumor cells (donor mice) were adoptively transferred to nonirradiated wt mice (recipient mice). One day later, recipient or unmanipulated control wt mice were challenged with 2 x 104 viable B16.MUC1 or B16.Neo tumor cells, and tumor growth was evaluated over time. Evidence of immunity to MUC1 is provided by an increase in recipient survival compared with control mice following challenge with B16.MUC1 tumor cells.
Adoptively transferred cells from naive wt mice showed no evidence of
tumor immunity in wt recipients (data not shown). Splenocytes and LN
cells from wt donors challenged with B16.MUC1 tumor cells for different
periods of times (15–42 days) were evaluated for immunity to B16.MUC1
or B16.Neo tumors in the adoptive transfer system. Wild-type donors
developed strong immunity to B16.MUC1 by day 28 after tumor challenge;
survival of wt recipients was increased dramatically compared with
control mice (p < 0.05) (Fig. 2A). Immune cells with this
phenotype are hereafter referred to as tumor immune cells, and were
used in adoptive transfer studies, unless otherwise indicated.
Approximately 60% of these wt recipients challenged with B16.MUC1
tumor cells were tumor free for as long as 60 days. Survival of wt
recipients challenged with B16.Neo tumor cells was increased compared
with control wt mice, although this was not statistically significant
(p > 0.30) (Fig. 2B). The adoptive
transfer of immune cells that eliminate B16.MUC1 tumor cells, but not
B16.Neo tumor cells, indicates that the immunogenicity of the B16.MUC1
tumor is largely due to the expression of MUC1. In some experiments,
the CTL frequencies of wt recipients of tumor immune cells or control
wt mice were quantified at 21 days after B16.MUC1 tumor challenge.
Compared with control mice, the recipient mice had increased MUC1- and
non-MUC1-reactive CTL frequencies detected in vitro, although the
difference was small and not statistically significant (data not
shown).
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The in vitro results shown in Fig. 1 suggested that MUC1-specific CTLs
exist in MUC1.Tg mice, yet the progressive growth of B16.MUC1 tumors in
the MUC1.Tg mice indicated that the CTLs detected in vitro were unable
to kill tumors expressing MUC1 in vivo. The wt and MUC1.Tg mice are
syngeneic, with the exception of the MUC1 gene, which usually is not
expressed on cells of hemopoetic origin (7). Therefore, it was possible
to use cellular adoptive transfer into recipient wt mice to evaluate
MUC1.Tg tumor immunity in vivo. Spleen and LN cells adoptively
transferred from naive MUC1.Tg donors or MUC1.Tg donors challenged with
B16.MUC1 tumor cells for 0 to 28 days did not increase the survival of
wt recipients challenged with B16.MUC1 or B16.Neo tumor cells compared
with control mice (p > 0.50) (data not shown
and Fig. 3). Collectively, these results
demonstrate that wt mice develop effective MUC1-specific cellular
responses that eliminate B16.MUC1 tumor cells in vivo; in contrast,
MUC1.Tg mice are functionally tolerant to MUC1 since they are unable to
eliminate B16.MUC1 tumors in vivo.
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Tumor immune cells from wt donors completely eliminated B16.MUC1
tumor cells in approximately 60% of wt recipients (Fig. 2). In vivo
Ab-mediated T cell depletion was used to determine phenotype of the
effector cell responsible for providing MUC1 tumor immunity in wt mice.
CD4+, CD8+, or both T cell subsets were
eliminated in vivo in wt mice by treatment with depleting Abs. Groups
of depleted wt, control wt, or MUC1.Tg mice were challenged with 2
x 104 B16.MUC1 tumor cells, and tumor growth was evaluated
over time. The results showed that CD4+ but not
CD8+ lymphocytes were required for MUC1-specific tumor
immunity (Fig. 4).
CD4-/CD8- wt, CD4- wt, and
MUC1.Tg mice developed progressively growing tumors at similar rates.
In contrast, CD8- wt or untreated wt mice developed tumors
that grew at significantly slower rates (p <
0.05) (Fig. 4). The observation that MUC1.Tg mice developed tumors at
rates similar to the CD4-/CD8- wt mice is
consistent with the hypothesis that MUC1 is the dominant Ag
responsible for in vivo tumor rejection and that non-MUC1 Ags are not
sufficient for tumor rejection in this system.
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). Although the
differences did not attain statistical significance, the results shown
in Fig. 5 are consistent with the results of the T cell depletion study
shown in Fig. 4, and further support hypothesis that MUC1-specific
CD4+ T cells protect against B16.MUC1 tumor growth.
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The MUC1.Tg mice provide a preclinical model to evaluate
strategies to overcome immunologic tolerance to MUC1. Cellular adoptive
transfer is one in vivo strategy to analyze the antitumor efficacy and
autoimmune consequences of developing MUC1-specific effector cells in
MUC1.Tg mice. We hypothesized that adoptive transfer of the
MUC1-specific immune response from the wt mice (Figs. 2 and 4) to
MUC1.Tg mice would increase the survival of recipient MUC1.Tg mice
challenged with B16.MUC1 tumor cells. Cells adoptively transferred from
naive wt mice did not increase the survival of MUC1.Tg recipients after
B16.MUC1 or B16.Neo tumor challenge (data not shown). Interestingly,
MUC1.Tg recipients that received tumor immune cells from wt donors
showed increased survival compared with control MUC1.Tg mice following
challenge with B16.MUC1 tumor cells (p < 0.05)
(Fig. 6A), indicating that it
was possible to transfer the anti-MUC1 tumor immune response into
these mice. There was no alteration in survival of MUC1.Tg recipients
challenged with B16.Neo tumors compared with controls
(p > 0.50) (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A limiting dilution CTL frequency assay was used to quantify, over time, MUC1-specific or non-MUC1-reactive CTLs during the course of B16.MUC1 tumor progression. Low frequencies of MUC1-specific or non-MUC1-reactive CTLs were detected in both naive wt and MUC1.Tg mice, which were similar to MUC1-specific CTL frequencies detected in naive mice reported by other groups (13). Quantitatively, MUC1.Tg or wt mice challenged with B16.MUC1 tumor cells had similar frequencies of MUC1-specific and non-MUC1-reactive CTLs.
Since the results of the CTL frequency study did not correlate with the observed in vivo rejection of tumors, a cellular adoptive transfer system was used to investigate further the relationship between the immune response against MUC1 and tumor protection in vivo. In this system, evidence of the transfer of tumor immunity is provided by an increase in the survival of recipient mice compared with control mice following tumor challenge. Adoptive transfer of immune cells is thought to increase the frequency of MUC1-specific or non-MUC1-reactive effector cells in recipient mice and provides direct evidence of tumor immunity in vivo.
The adoptive transfer system was used to investigate the time course of
the wt immune response to MUC1 and non-MUC1 tumor Ags following
B16.MUC1 tumor challenge. Naive wt donors were unable to provide
B16.MUC1 tumor protection. Wild-type mice challenged with B16.MUC1
tumor cells for 21 to 35 days developed tumor immunity that, upon
adoptive transfer, provided complete protection in approximately 60%
of wt recipients challenged with B16.MUC1 tumor cells. The 60%
survival is the result of the tumor dose that we selected for these
studies. A relatively high dose of this tumor was selected to clearly
detect the differences between the untransferred wt animal response to
B16.MUC1 cells and the response in the mice that received an adoptive
transfer. Modest increases in survival were observed in wt recipients
challenged with B16.Neo tumor cells, suggesting that reactivity to
non-MUC1 tumor Ags was of minimal importance to the tumor immune
response. Tumor immunity was not detectable by the adoptive transfer
system in wt mice bearing B16.MUC1 tumors for greater than 35 days,
suggesting that immunity to MUC1 and non-MUC1 tumor Ags had been
down-regulated by that time point. The down-regulation of the wt tumor
immune response at 35 days is most likely due to anergy (14, 15, 16),
clonal exhaustion (17), or the effects of inhibitory substances
produced by the tumor cells (18, 19), such as MUC1 (20). Coincident
with the down-regulation of the immune response, wt mice began to
develop tumors at a more progressive rate (Fig. 2).
Different results were obtained when the adoptive transfer system was
used to evaluate tumor immunity in the MUC1.Tg mice over the course of
B16.MUC1 tumor growth. Adoptive transfer of spleen and LN cells from
MUC1.Tg mice challenged with B16.MUC1 tumors for 0 to 28 days provided
no significant increase in wt recipient survival following challenge
with either B16.MUC1 or B16.Neo tumor cells. This result indicates that
MUC1.Tg mice are functionally tolerant to MUC1 in vivo. Wild-type
recipients compared with unmanipulated control wt mice reproducibly
developed more progressively growing tumors following challenge with
B16.Neo tumor cells (Fig. 3B). One interpretation of these
results is that MUC1.Tg mice develop immune suppression during tumor
progression that inhibits the development of non-MUC1 immunity in wt
mice.
The MUC1.Tg mice retain immune responsiveness to non-MUC1 tumor Ags (9, unpublished results), yet adoptive transfer of their primed immune cells did not result in increased wt recipient survival after challenge with B16.Neo tumor cells. This finding, in part, may be explained by concept of xenogenization in which strong immune responses to a foreign protein confer immunity to otherwise less immunogenic or nonimmunogenic proteins (21). The immune response of wt mice to the foreign Ag MUC1 and the resulting inflammatory response may have enhanced immune surveillance of non-MUC1 tumor Ags, thus allowing the development and transfer of non-MUC1 immunity and an increase in wt recipient survival after challenge with B16.Neo tumor cells.
The results obtained from the two experimental systems used to evaluate MUC1-specific responses in the MUC1.Tg mice were profoundly different. Results from the limiting dilution CTL frequency assay indicated that MUC1.Tg mice challenged with B16.MUC1 tumor cells produce MUC1-specific CTLs at frequencies similar to wt mice. However, the adoptive transfer system showed that wt but not the MUC1.Tg mice developed protective MUC1-specific immunity.
In vitro assays of cell-mediated cytotoxicity are sometimes difficult to interpret, in part because they are conducted under nonphysiologic conditions that include high concentrations of Ag and stimulatory cytokines. To kill tumor cells in vivo, lymphocytes must function within the tumor microenvironment, an environment that is likely to be distinct from the conditions encountered in vitro. The microenvironment of the tumor presents many challenges to effector lymphocytes, including changes in soluble Ag concentration with increased tumor development (22), antigenic heterogeneity of tumor cells (9, 23, 24), production of immunoregulatory cytokines by the tumor cells (18, 19), the immunosuppressive effect of tumor burden (25, 26), and physical barriers that preclude tumor cell-lymphocyte interaction (27, 28). Furthermore, it remains unclear whether CTL activity measured in vitro can provide tumor protection in vivo since these assays are at best only correlative. Indeed, the findings presented in this work and by others (29) demonstrate that mice transgenic for human MUC1 develop cell-mediated responses that are detectable in vitro; however, a more germane question is whether these responses eliminate tumors in vivo. The findings presented in this work raise questions regarding the use of the in vitro CTL lytic assay to quantify immunity against tumors. The results underscore the need for the development of other experimental systems to evaluate tumor immunity in vivo.
Results of in vivo, Ab-mediated, T cell depletion studies and the
adoptive transfer of purified T cell subsets showed that
CD4+ but not CD8+ lymphocytes were responsible
for providing MUC1-specific tumor immunity in wt mice. Failure to
detect immunity to B16.MUC1 tumor cells in the MUC1.Tg mice indicates
that tolerance to MUC1 exists in the CD4+ cell population.
Results of previous studies also suggest that MUC1.Tg CD4+
T lymphocytes were tolerant to MUC1: MUC1.Tg mice immunized with MUC1
peptides produced IgM, but only weak IgG Ab responses, which is
consistent with a lack of CD4+ Th cell function (9). It is
possible that tolerance to MUC1 expressed by the B16 transfectants is
maintained by an anergic state deficient in the production of cytokines
necessary for MUC1-specific CD8+CTL priming/activation in
vivo (30). While this is an attractive hypothesis, there are other
possibilities. CD4+ T cell effectors have been reported in
clinical and preclinical models to react with several tumor-associated
Ags (31, 32, 33, 34), including MUC1 (35). B16.MUC1 cells cultured in vitro
express MHC class I, but do not express MHC class II molecules within
the detectable limits of flow cytometry (data not shown). However, it
is not known whether B16.MUC1 cells up-regulate MHC class II molecules
in vivo as a response to inflammatory cytokines such as IFN-
.
MUC1-specific Abs have been shown to play little, if any, role in
protection against B16.MUC1 tumors; this lessens the likelihood that
CD4+ T cells mediate B16.MUC1 tumor immunity by
facilitating the production of high affinity MUC1-specific IgG Abs. The
finding that CD8--depleted mice rejected B16.MUC1 tumors
at rates similar to wt controls suggests that CD4+
lymphocytes may play a direct role in tumor rejection or function to
help a non-CD8+ immune cell population in the rejection
process.
MUC1-specific T cells have been detected in some cancer patients with adenocarcinoma of the breast or ovary (1, 2, 3). It is difficult to know whether this immune response detected in vitro destroys MUC1-bearing tumors in vivo. It is likely that these responses are ineffective, since these patients have a poor clinical prognosis. Immunologic tolerance to MUC1 prevents effective MUC1-specific immune responses in both cancer patients and the MUC1.Tg mice. In this respect, the MUC1.Tg mice are a reasonable preclinical model to evaluate immunization strategies designed to overcome tolerance to MUC1.
The ability of MUC1-specific CD4+ effector cells produced in wt mice to provide tumor immunity or provoke autoimmune reactions in normal epithelia-expressing MUC1 was evaluated by the adoptive transfer system. A statistically significant increase in survival was detected in MUC1.Tg recipients after challenge with B16.MUC1 tumor cells. There were no obvious or histologic signs of autoimmunity in recipient MUC1.Tg mice. The observed tumor protection and lack of detectable autoimmunity in the recipient MUC1.Tg mice are consistent with findings in other experimental models. For example, adoptive transfer of anti-Friend leukemia virus envelope or anti-p53 T cells into Friend leukemia virus envelop transgenic mice or p53+/+ mice, respectively, eliminated tumor cells expressing these self Ags with no detectable autoimmunity (36, 37, 38).
The lack of a detectable autoimmune response may be explained by several different hypotheses. One simple explanation is that levels of MUC1 protein expression in nontransformed cells may not be sufficient to stimulate immune reactivity. Alternatively, the glycosylation patterns may be altered in tumor-associated MUC1 compared with MUC1 expressed by nontransformed epithelia, obscuring immunodominant epitopes. It is not known whether the MUC1-specific CD4+ cells described in this work recognize MUC1 in the context of MHC class II molecules. However, the targeted stimulation or adoptive transfer of MUC1-specific CD4+ cells with MHC class II restriction may promote MUC1-specific tumor immunity with little autoimmunity, since epithelial tissues normally do not express MHC class II molecules.
Peripheral mechanisms of tolerance have been described, including lack of T cell costimulation (14, 15, 16, 39), activation-induced cell death (40), and suppressor T cells (41, 42). It remains possible that suppressor T cells are present in the MUC1.Tg mice and function to prevent autoreactive T cells from inducing autoimmunity. In vitro data indicate that both MUC1.Tg and wt mice have equal abilities to respond to MUC1; however, the antitumor responses in the MUC1.Tg mice may be suppressed by immunoregulatory mechanisms that mediate peripheral tolerance to MUC1. An alternative hypothesis is that regulatory cytokines or chemokines produced by normal epithelial cells inhibit T cell function.
In summary, MUC1.Tg mice develop MUC1-specific CTLs that are detectable in vitro; however, they remain functionally tolerant to MUC1 in vivo, as indicated by their inability to reject tumors expressing MUC1 and the failure of adoptively transferred cells to confer MUC1-specific immunity to wt mice. In contrast, wt mice challenged with B16.MUC1 tumor cells develop a MUC1-specific CD4+ effector population that is capable of rejecting tumor cells expressing MUC1 in vivo. Adoptive transfer of the wt tumor immune cells to MUC1.Tg recipients significantly increased the survival of MUC1.Tg recipients challenged with B16.MUC1 tumor cells without induction of autoimmunity. The MUC1.Tg mice provide a useful preclinical model for evaluating novel vaccine formulations and their translation into effective anti-MUC1 tumor responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael A. Hollingsworth, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6805. E-mail address:
3 Abbreviations used in this paper: MUC1.Tg, MUC1 transgenic; LN, lymph node; wt, wild type.
4 R. M. Tempero, G. J. Rowse, S. J. Gendler, and M. A. Hollingsworth. Passively transferred anti-MUC1 antibodies cause neither autoimmune disorders nor immunity against transplanted tumors in MUC1 transgenic mice. Int. J. Cancer, In press.
Received for publication April 27, 1998. Accepted for publication July 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1, 3)Gal antibodies in humans switch immune responses from cellular to humoral. Nat. Med. 4:315.[Medline]
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J. Li, W. Li, S. Liang, D. Cai, M. P. Kieny, L. Jacob, A. Linnenbach, J. W. Abramczuk, H. Bender, K. Sproesser, et al. Recombinant CD63/ME491/Neuroglandular/NKI/C-3 Antigen Inhibits Growth of Established Tumors in Transgenic Mice J. Immunol., September 15, 2003; 171(6): 2922 - 2929. [Abstract] [Full Text] [PDF]
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Y. Lu, Y.-q. Wei, L. Tian, X. Zhao, L. Yang, B. Hu, B. Kan, Y.-j. Wen, F. Liu, H.-x. Deng, et al. Immunogene Therapy of Tumors with Vaccine Based on Xenogeneic Epidermal Growth Factor Receptor J. Immunol., March 15, 2003; 170(6): 3162 - 3170. [Abstract] [Full Text] [PDF]
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M. M. Soares, V. Mehta, and O. J. Finn Three Different Vaccines Based on the 140-Amino Acid MUC1 Peptide with Seven Tandemly Repeated Tumor-Specific Epitopes Elicit Distinct Immune Effector Mechanisms in Wild-Type Versus MUC1-Transgenic Mice with Different Potential for Tumor Rejection J. Immunol., June 1, 2001; 166(11): 6555 - 6563. [Abstract] [Full Text] [PDF]
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K. Morikane, R. M. Tempero, C. L. Sivinski, S. Kitajima, S. J. Gendler, and M. A. Hollingsworth Influence of organ site and tumor cell type on MUC1-specific tumor immunity Int. Immunol., February 1, 2001; 13(2): 233 - 240. [Abstract] [Full Text] [PDF]
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S. Koido, M. Kashiwaba, D. Chen, S. Gendler, D. Kufe, and J. Gong Induction of Antitumor Immunity by Vaccination of Dendritic Cells Transfected with MUC1 RNA J. Immunol., November 15, 2000; 165(10): 5713 - 5719. [Abstract] [Full Text] [PDF]
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P. Mukherjee, A. R. Ginardi, C. S. Madsen, C. J. Sterner, M. C. Adriance, M. J. Tevethia, and S. J. Gendler Mice with Spontaneous Pancreatic Cancer Naturally Develop MUC-1-Specific CTLs That Eradicate Tumors When Adoptively Transferred J. Immunol., September 15, 2000; 165(6): 3451 - 3460. [Abstract] [Full Text] [PDF]
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M. Gerloni, S. Xiong, S. Mukerjee, S. P. Schoenberger, M. Croft, and M. Zanetti From the Cover: Functional cooperation between T helper cell determinants PNAS, November 21, 2000; 97(24): 13269 - 13274. [Abstract] [Full Text] [PDF]
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) mice were challenged with 2 x 104
B16.MUC1 cells. At the indicated time after B16.MUC1 challenge, a
limiting dilution assay was designed to determine the A,
MUC1-specific, or B, non-MUC1-reactive CTL frequency
(reactive cells/106). Symbols represent the determined CTL
frequency from individual mice. No statistically significant
differences in MUC1-specific or non-MUC1-reactive CTL frequency were
observed between wt or MUC1.Tg mice.
, n = 5), unmanipulated wt (
),
CD8- (
) wt, CD4-/CD8- (
, n = 4), or nonpurified
(