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Cancer Research 68, 8446, October 15, 2008. doi: 10.1158/0008-5472.CAN-08-1864
© 2008 American Association for Cancer Research
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Immunology

Balancing between Antitumor Efficacy and Autoimmune Pathology in T-Cell–Mediated Targeting of Carcinoembryonic Antigen

Rinke Bos1, Suzanne van Duikeren1, Hans Morreau3, Kees Franken1, Ton N.M. Schumacher4, John B. Haanen4, Sjoerd H. van der Burg2, Cornelis J.M. Melief1 and Rienk Offringa1

1 Department of Immunohematology and Blood Transfusion, Tumor Immunology Group, 2 Department of Clinical Oncology, and 3 Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands and 4 Division of Immunology, The Netherlands Cancer Institute, Amsterdam, the Netherlands

Requests for reprints: Rienk Offringa, Department of Immunohematology and Blood Transfusion, Tumor Immunology Group, E3-Q, Leiden University Medical Center, 2300 RC Leiden, the Netherlands. Phone: 31-71-5263845; Fax: 31-71-5216751; E-mail: r.offringa{at}lumc.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Carcinoembryonic antigen (CEA) is intensively studied as a potential target for immunotherapy of colorectal cancers. Although overexpressed by tumors, CEA is also expressed in normal tissues, raising questions about the feasibility and safety of CEA-targeted immunotherapy. We investigated these issues in transgenic mice in which the expression of human CEA in normal tissues closely resembles that in man. Our data show that the T-cell response against CEA in these mice is blunted by both thymic and peripheral tolerance. Consequently, effective tumor targeting is only achieved by adoptive transfer of T cells from nontolerant donors in combination with interventions that eliminate peripheral immune regulatory mechanisms. However, such treatments can result in severe intestinal autoimmune pathology associated with weight loss and mortality. Interestingly, preconditioning of recipient mice by depletion of T-regulatory cells results in immune-mediated tumor control in the absence of toxicity. In this setting, CEA-specific T-cell responses are lower than those induced by toxic regimens and accompanied by additional T-cell responses against non-self antigen. These findings illustrate the importance of testing adoptive immunotherapies targeting self antigens such as CEA in preclinical in vivo models and show that the choice of immune intervention regimen critically determines the balance between therapeutic efficacy and toxicity. [Cancer Res 2008;68(20):8446–55]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The feasibility of T-cell–mediated targeting of cancers through tumor-associated autoantigens is challenged by the notion that the T-cell repertoire against such antigens may be subject to central and/or peripheral tolerance. A variety of self antigens, including tissue-specific antigens, which are considered as targets for cancer immunotherapy, were found to be expressed in the thymus (1, 2). Furthermore, antigen expression by normal somatic tissues was shown to result in peripheral tolerance due to antigen encounter by T cells in the absence of costimulatory signals (3, 4). These tolerogenic mechanisms take out the high-affinity T cells and leave us with a low-affinity repertoire that has diminished efficacy against normal and diseased tissues expressing their cognate antigens (5, 6). Finally, tumor-induced tolerance can result from the chronic encounter of tumor-derived antigens (7). This coevolvement of tumor and antitumor immune response contributes to the further weakening of T-cell responses against autoantigens and can also cripple the efficacy of a T-cell repertoire targeting non-self tumor antigens (8). Awareness of these three levels of tolerance provokes the question whether the natural T-cell repertoire against tumor-associated autoantigens can be effectively exploited for cancer immunotherapy. Consequently, adoptive therapies involving the infusion of allogeneic or genetically engineered autologous T cells are gaining interest. Through the transfer of genes encoding T-cell receptors (TCR) that target tumor-associated autoantigens, the autologous T-cell repertoire can be replenished with specificities that have been eliminated due to tolerance (9, 10). A recent clinical study in melanoma patients showed the feasibility of this approach (11).

One of the tumor antigens for which reconstruction of the T-cell repertoire may be required is carcinoembryonic antigen (CEA). CEA was among the first tumor-associated autoantigens discovered, and over the past years, it has been intensively studied as a potential target for immunotherapy of epithelial cancers, particularly colorectal cancers (12). Although CEA is overexpressed in colorectal cancers, considerable levels of this antigen are present in normal intestinal epithelia. Due to its glycosylphosphatidylinositol linkage, it is readily shed into the circulation. These properties raise concerns with respect to the feasibility and safety of CEA-targeted immunotherapy of cancer. Nevertheless, a large number of preclinical and clinical studies suggest that CEA-specific immune intervention is not associated with significant adverse effects (reviewed in ref. 12). However, the risk for intestinal immune pathology has been clearly shown in clinical studies involving CTLA-4 blockade or allogeneic donor lymphocyte infusions (13, 14) as well as in preclinical studies involving adoptive transfer of autoreactive T cells (reviewed in ref. 15). This paradox prompted us to perform a detailed analysis of the feasibility and safety of CEA-targeted cancer immunotherapy in a preclinical mouse model in which human CEA, an antigen not conserved between mouse and man, is expressed as a self antigen.

Studies with CEA-transgenic (CEA-tg) mice, in which the expression of CEA closely resembles that in man, have provided evidence that the immune system is indeed tolerized for CEA (12). Our own studies in CEA-tg mice showed that thymic expression, in medullary thymic epithelial cells, results in deletion of the major part of the CEA-specific T-cell repertoire and that CEA is similarly expressed in the human thymus (16). These findings suggested that therapeutic efficacy of the CEA-specific immune response against colorectal cancers will most likely require reconstruction of the CEA-specific T-cell repertoire. We have now tested this concept in a CEA-tg mouse model, in which the effect of a potent, adoptively transferred CEA-specific T-cell response was analyzed against transplantable tumors, autochthonous tumors, and normal intestinal epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Mice. Human CEA-tg mice were originally obtained from Dr. John Thompson (Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany). APC1638N mice were originally obtained from Dr. Ricardo Fodde (Josephine Nefkens Institute, Erasmus Medical Centre, Rotterdam, the Netherlands). The experiments were approved by the animal experimental commission (UDEC) of Leiden University.

Immunizations, adoptive transfer, tumor challenge, and analysis of T-cell responses. Immunizations, in vivo depletion of CD4+ or CD8+ T cells, tumor challenge experiments, and ex vivo evaluation of CEA-specific T-cell immunity were performed essentially as described by us previously (16, 17). For adoptive transfer experiments, spleen cells were isolated from donor mice that were immunized twice (unless otherwise indicated) with a DNA vaccine encoding CEA and the costimulatory molecule B7.1 (B7.1-CEA DNA). Splenocytes were isolated 2 wk after the last vaccination and depleted of erythrocytes. Recipient mice were reconstituted by i.v. injection of 5 x 107 spleen cells suspended in 200 µL PBS. Recipient mice received either 4.5 Gy total body irradiation (TBI) 1 d before reconstitution with spleen cells or 9.5 Gy TBI 5 d before reconstitution. TBI (9.5 Gy) was followed by bone marrow transplantation (BMT) 1 d later. Tumor challenge was performed by s.c. injection of 1.5 x 105 MC38-CEA cells 1 d after adoptive transfer. Mice treated with immunomodulatory agents received either 250 µg interleukin-10 receptor (IL-10R) blocking antibody that was injected weekly i.p., starting at the day of the tumor challenge, or 80 µg of CD25-specific antibodies (PC61) that were injected i.p. 6 d before adoptive transfer. Immunization of recipient mice with B7.1-CEA DNA started 1 d after adoptive transfer. IFN-{gamma} production by freshly isolated lymphocytes from spleen, lymph node, or intestine was measured by intracellular cytokine staining in the presence of previously defined CEA peptide T-cell epitopes (16, 17), CD8+ CTL epitope (CEA571-579), and CD4+ Th epitopes [mixtures of five dominant epitopes (C57BL/6) and two subdominant epitopes (CEA-tg)].

Histologic analysis. Tissues were snap frozen in isopentane and stored at –80°C. Cryosections (4 µm) were fixed in 4% paraformaldehyde and stained with H&E. To compare the histopathologic changes that occurred in the intestine of CEA-tg and wild-type mice, a scoring system was used based on a previously described system (18), with the following variables: (a) degree of inflammatory cell infiltrate in epithelia and stroma, giving a score ranging from 0 to 6; (b) mucin depletion, giving a score ranging from 0 to 3; (c) crypt elongation and hyperplasia, giving a score ranging from 0 to 3; and (d) crypt destruction, giving a score ranging from 0 to 3. The severity of the inflammatory changes in the intestine was based on the sum of the scores reported for each variable listed above. The higher the score, the greater the inflammatory changes. Cryosections (4 µm) were fixed for 10 min with ethanol-acetone at room temperature. Subsequently, sections were incubated with primary antibody Thy1.1 biotin (clone HIS51, BD PharMingen) followed by horseradish peroxidase (HRP)-labeled secondary antibody (StreptABCcomplex/HRP, DAKO). HRP activity was revealed by incubation in diaminobenzidine and counterstained with hematoxylin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Reconstitution of the CEA-specific T-cell repertoire through adoptive transfer. Our previous work indicated that wild-type C57BL/6 mice can raise strong CEA-specific T-cell responses on immunization with CEA-specific ALVAC or DNA-based vaccines (16, 17), whereas the breadth and magnitude of this response in human CEA-tg C57BL/6 mice is severely limited by central tolerance and, consequently, is incapable of rejecting CEA-positive tumors (16). A clinically relevant approach to induce potent CEA-specific immunity in vivo could involve gene transfer of a CEA-specific TCR into autologous lymphocytes followed by adoptive transfer of the genetically engineered T cells into tumor-bearing subjects (11, 19). To test, as a proof of concept, whether reconstitution of the CEA-specific T-cell repertoire could protect CEA-tg mice against the outgrowth of CEA-expressing tumors, we adoptively transferred the T-cell repertoire of CEA-immunized wild-type mice into CEA-tg mice that were subsequently challenged with a tumorigenic dose of MC38-CEA cells. In accordance with our earlier studies (17), two sequential immunizations of wild-type donor mice with a CEA-specific DNA vaccine induced strong CEA-specific CD4+ and CD8+ T-cell immunity, whereas this response is largely absent in CEA-tg mice (Fig. 1A ). As reported previously, the weak CEA-specific CD4+ T-cell responses in CEA-tg mice were only observed after at least three vaccinations and after in vitro restimulation of the splenocytes (16). Under these conditions, involving a variety of vaccination protocols, we never found CEA-tg mice to display a detectable CD8+ T-cell response against the epitope CEA571-579 nor against several other potential CEA peptide epitopes.5


Figure 1
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  		Figure 1. Antitumor efficacy of therapeutic regimens involving adoptive transfer of CEA-reactive lymphocytes into CEA-tg and wild-type mice. A, C57BL/6 wild-type and CEA-tg mice were vaccinated twice at a 2-wk interval with 100 µg B7.1-CEA DNA. Two weeks after the last vaccination, splenocytes were isolated and analyzed for IFN-{gamma} production by intracellular cytokine staining as described in Materials and Methods. Representative examples are shown. The capacity of CEA-tg (B) and wild-type mice (C) to reject a lethal dose of 1.5 x 105 MC38-CEA cells was determined as follows. Mice were left nontreated (B: n = 37; C: n = 15), infused with 5 x 107 donor lymphocytes from CEA-immunized wild-type mice (B: n = 10; C: n = 6), irradiated with 4.5 Gy TBI (B: n = 10; C: n = 8), or infused with 5 x 107 donor lymphocytes from CEA-immunized wild-type mice in combination with 4.5 Gy TBI (B: n = 37; C: n = 26). Donor cells were injected i.v. 5 d after irradiation. To sustain the transferred immune response, recipient mice received weekly doses of CEA-specific DNA vaccine. Where indicated, additional immune modulation was performed by injecting CD25-specific antibodies (B: n = 35; C: not tested) or IL-10R blocking antibody (B: n = 19; C: n = 10) as further specified in Materials and Methods or by combining adoptive transfer with 9.5 Gy TBI (B: n = 17; C: n = 14). In the latter case (*), mice also received BMT 1 d after irradiation. Tumor challenge involved s.c. injection of 1.5 x 105 MC38-CEA cells 1 d after adoptive transfer. Tumor size was measured every 3 d and mice were sacrificed when tumor size exceeded 100 mm2. Depicted is the long-term survival percentage, as defined by a tumor size <10 mm2 at day 40 after tumor challenge. Data are cumulative over four experiments. D, tumor growth in CEA-tg mice (12 per group) as recorded in a representative experiment involving the indicated treatment regimens. Each ascending line represents one mouse. The horizontal lines represent one or more mice in which no tumor outgrowth was detected. Numbers below these lines indicate the fractions of tumor-free mice.

 
The response raised in wild-type mice was transferred to naive CEA-tg or wild-type recipient mice by infusion of 5 x 107 donor splenocytes. To sustain the transferred immune response, recipient mice received weekly doses of the CEA-specific DNA vaccine. Remarkably, this treatment failed to prevent, or even delay, tumor outgrowth in CEA-tg mice (Fig. 1B). In contrast, this regimen did efficiently protect wild-type mice against tumor outgrowth (Fig. 1C), indicating that the antitumor efficacy of the CEA-specific T-cell response in CEA-tg mice is not only restricted by central tolerance (16) but also by peripheral tolerance.

Elimination of regulatory mechanisms involved in peripheral tolerance. Others have shown that the antitumor efficacy of an adoptively transferred T-cell response can be significantly enhanced if the recipient mice are irradiated before lymphocyte infusion. This pretreatment was proposed to improve the performance of adoptively transferred T cells through elimination of immunoregulatory lymphocyte subsets and by creating a lymphoid environment that is more supportive of homeostatic T-cell proliferation (20). We therefore applied our adaptive transfer protocol to CEA-tg and wild-type mice that had received 4.5 Gy TBI. Pretreatment with 4.5 Gy TBI resulted in a marked improvement of the efficacy of the adoptive therapy in that 35% of the CEA-tg mice did not develop tumors (Fig. 1B), whereas tumor growth was delayed in most of the other mice (Fig. 1D). Irradiation did not improve treatment efficacy in wild-type recipient mice (Fig. 1C), indicating that this intervention alleviates the suppression of the adoptively transferred CEA-specific T-cell response in CEA-tg recipient mice. Furthermore, 4.5 Gy TBI alone had no effect on the tumor outgrowth in either CEA-tg or wild-type mice (Fig. 1B and C), showing that the adoptively transferred T-cell response was primarily responsible for the antitumor effects observed.

To further restrain peripheral immune regulatory mechanisms in the CEA-tg host, we modified the treatment in three different ways. In addition to 4.5 Gy TBI, we suppressed the action of the key regulatory cytokine IL-10 (21) by injecting IL-10R blocking antibodies (22). Alternatively, we combined 4.5 Gy TBI with injection of CD25-specific antibodies (PC61) that are known to deplete CD25+ cells, particularly CD4+CD25high regulatory T cells (23). In a third regimen, we increased irradiation to a myeloablative dose of 9.5 Gy TBI because this is known to more rigorously deplete regulatory cells from the periphery and enhance homeostatic proliferation of adoptively transferred T cells (20). To preserve viability of the irradiated mice, their hematopoietic system was reconstituted through BMT with syngeneic bone marrow cells. We found these three modified treatments to further improve the antitumor efficacy of the adoptively transferred lymphocytes in CEA-tg mice (Fig. 1B). In particular, the modalities involving 9.5 or 4.5 Gy TBI in combination with CD25 depletion were highly effective in that the majority of the mice were capable of clearing their tumors (Fig. 1D). In all three modalities, tumor rejection depended on the adoptive transfer of donor lymphocytes (data not shown).

Taken together, our data show that the failure of the CEA-specific T-cell response in CEA-tg mice is a result of both central and peripheral tolerance and that these hurdles can be overcome by reconstitution of the CEA-specific T-cell repertoire and suppression of immune regulatory mechanisms, respectively.

Association between effective antitumor treatment and autoimmune colitis. Notably, two of the aforementioned modalities, although resulting in improved antitumor efficacy, were accompanied by symptoms that are typical for experimental colitis (24–26), including severe weight loss, which occasionally resulted in death (Fig. 2A ). Weight loss became apparent around 1 week after adoptive transfer. Surviving mice started to regain weight 2 weeks later, whereas their full recovery took 6 to 8 weeks. Treatment involving the combination of 4.5 Gy TBI and IL-10R blocking antibody resulted in severe weight loss in 50% of the cases, whereas this occurred in 100% of the mice if treatment involved 9.5 Gy TBI. These symptoms were not observed in any of the wild-type recipients receiving these treatments (Fig. 2A; data not shown), suggesting that they were caused by the immune response against the CEA-positive intestinal epithelia in CEA-tg mice. In accordance with this notion, weight loss was accompanied by significant colon shortening and thickening (data not shown). Histologic examination of the intestine showed loss of goblet cells, crypt elongation, crypt abscesses, and strong infiltration in colon and small intestine (Fig. 2B). Pathology was more pronounced in the colon than in the small intestine (Fig. 2B), in correspondence with the higher CEA expression levels in the colon (data not shown; ref. 27). Furthermore, the histopathologic changes and increased lymphocyte infiltration, although also observed in wild-type recipients, were much more severe in CEA-tg recipients (Fig. 2B and C). To provide further evidence that the adoptively transferred CEA-specific T-cell response was involved in colitis, Thy1.1+ donor lymphocytes were adoptively transferred into Thy1.2+ CEA-tg recipient mice. Histopathologic analyses showed massive infiltration of the intestinal epithelium by Thy1.1+ cells (Fig. 2D). Finally, adoptive transfer of lymphocytes from CEA-tg donor mice, which display a greatly reduced CEA-specific T-cell response (Fig. 1A; ref. 16), resulted neither in tumor clearance nor in colitis (Fig. 3A and B ), indicating that the potent CEA-specific T-cell response in wild-type donor cells was responsible for both effects.


Figure 2
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  		Figure 2. CEA-specific immunotherapy is accompanied by intestinal autoimmune pathology. A, weight changes of wild-type ({square}) and CEA-tg ({Delta}) mice after infusion with donor lymphocytes from CEA-immunized wild-type mice in combination with 9.5 Gy TBI/BMT and tumor challenge as described in the legend to Fig. 1. Points, mean percentage of the original weight over time for five surviving mice per group; bars, SD. Treatment resulted in death of some of the mice at this stage. B, H&E staining of cryosections from the colon and small intestine of wild-type and CEA-tg mice that were treated by 9.5 Gy TBI and BMT in combination with adoptive transfer of lymphocytes from CEA-immunized (CEA) or mock-immunized (control) wild-type mice. C, histopathologic scoring (see Materials and Methods) of the colon from wild-type and CEA-tg recipients 1 and 5 wk after adoptive transfer of lymphocytes from CEA-immunized wild-type mice. D, immunohistochemical detection of donor-type Thy1.1+ T cells in a cryosection of the colon of a CEA-tg mouse at day 7 after adoptive transfer.

 

Figure 3
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  		Figure 3. Dissection of the tumoricidal and pathologic components of the CEA-specific T-cell response. The importance of the source (wild-type versus CEA-tg mice) and T-cell subsets (CD4+ versus CD8+) for antitumor efficacy and intestinal pathology was evaluated in CEA-tg mice that were submitted to three different treatment regimens. A, 9.5 Gy TBI and BMT in combination with adoptive transfer of lymphocytes from CEA-immunized donor mice. B, 4.5 Gy TBI in combination with adoptive transfer of lymphocytes from CEA-immunized donor mice and administration of anti-IL-10R antibodies. C, 4.5 Gy TBI in combination with adoptive transfer of lymphocytes from CEA-immunized donor mice and administration of anti-CD25 antibodies. Where indicated, adoptively transferred T cells were depleted of CD4+ or CD8+ T cells (WT AT CD4– and WT AT CD8–, respectively) or derived from CEA-tg donor mice (CEA-tg AT). Survival is defined as described in the legend to Fig. 1. Where indicated, treatment resulted in intestinal pathology (marked with "colitis" and incidence).

 
The role of the adoptively transferred T-cell response in tumor control and colitis was further analyzed by using donor lymphocyte preparations depleted of either CD4+ or CD8+ T-cell subsets. Infusion of CD4-depleted donor lymphocytes resulted neither in tumor control nor in colitis (Fig. 3A and B). Infusion of CD8-depleted donor lymphocytes similarly failed to control tumor growth but did still cause colitis (Fig. 3A). These findings are in line with our previously published data in that protection of wild-type C57BL/6 mice by immunization with a CEA-specific DNA vaccine depended on both CD4+ and CD8+ T cells (17) as well as with work by others indicating a pivotal role for CD4+ T cells in inducing experimental colitis (28).

Kinetics of intraintestinal CEA-specific T-cell immunity. The effect of the adoptively transferred CEA-specific T-cell response was analyzed in more detail by isolation of Thy1.1+ donor-type cells from spleen, mesenteric lymph nodes (mLN), and colon lamina propria (LPL) of Thy1.2+ recipient mice. One week after adoptive transfer, at the time when mice began to display weight loss (Fig. 2A), high numbers of CEA-specific, IFN-{gamma}–producing CD4+ and CD8+ T cells could be found in CEA-tg mice, particularly in the intestinal epithelium and in the draining lymph nodes. This reactivity was only found in the Thy1.1+ donor-derived population (Fig. 4A ). Both the CEA-specific T-cell reactivity and the numbers of Thy1.1+ donor-type T cells gradually declined over time (Fig. 4B and C), a development that correlated with the decline of colitis-associated symptoms (Fig. 2A), supporting the direct involvement of the donor-type CEA-specific T cells in autoimmune colitis. The CEA-specific T-cell responses detected after adoptive transfer into wild-type mice were much weaker, and this reactivity was primarily found in spleen rather than the intestine or mLNs (Fig. 4A). Therefore, the vigorous CEA-specific T-cell responses detected in CEA-tg mice are primarily resulting from the encounter between these T cells and the CEA-positive intestinal epithelia and not from the response against the MC38-CEA tumor cells.


Figure 4
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  		Figure 4. Adoptive therapy results in potent systemic and intraintestinal CEA-specific CD4+ and CD8+ T-cell immunity. Wild-type and CEA-tg mice received 9.5 Gy TBI and BMT in combination with i.v. adoptive transfer of lymphocytes from CEA-immunized wild-type mice and s.c. tumor challenge (see legend to Fig. 1). Phenotype and CEA-specific reactivity of T cells were evaluated as indicated. A, CEA-specific IFN-{gamma} production, as measured by intracellular cytokine staining, by donor-derived (Thy1.1+) CD4+ and CD8+ T cells freshly isolated from spleen, mLNs, and LPL. Lymphocytes were isolated 7 d after adoptive transfer and incubated with D1 cells that were pulsed with the relevant CEA peptide epitopes (see Materials and Methods). B, CEA-specific IFN-{gamma} production by Thy1.1+CD4+ cells isolated from spleen, mLNs, and LPL. Cells were isolated 7, 21, and 35 d after adoptive transfer. Columns, mean percentage of IFN-{gamma}–producing Thy1.1+CD4+ T cells for groups of five mice; bars, SE. C, percentage of donor-type Thy1.1+ T cells recovered from mLNs of representative CEA-tg recipients 1, 3, and 5 wk after adoptive transfer.

 
CD25 depletion allows for tumor clearance in the absence of colitis. Intriguingly, if CEA-tg recipient mice were treated with 4.5 Gy TBI in combination with CD25-specific antibodies, antitumor efficacy of the adoptive transfer was not paralleled by any detectable signs of colitis (Fig. 1B; data not shown). This result was somewhat unexpected in view of reports showing that CD4+CD25+ T-regulatory cells have an important role in suppressing intestinal immune pathology in other mouse models (28). The dissociation between antitumor immunity and colitis in anti-CD25 antibody-treated mice implied that CEA-specific T-cell immunity could play a less prominent role in this setting compared with the other two modalities. We therefore compared the antitumor efficacy of adoptively transferred lymphocytes from CEA-vaccinated wild-type and CEA-tg donors. In contrast to what we found for the other two modalities, adoptive transfer of CEA-tg lymphocytes had a clear antitumor effect when applied to 4.5 Gy CD25-depleted recipient mice (Fig. 3C). Importantly, adoptive transfer of lymphocytes from CEA-vaccinated wild-type donors was still more effective (Fig. 5B and C ), indicating that CEA-specific T-cell responses did contribute to tumor eradication. Nevertheless, in this setting, the CEA-specific response seemed to be complemented by additional effector mechanisms of which the efficacy is enhanced by CD25 depletion. Because the tumor cell MC38 is known to express a CD8+ T-cell epitope derived from an endogenous retroviral gene product of murine leukemia virus (MuLV; ref. 29), we examined the CD8+ T-cell response against this epitope in mice that had successfully been treated through 4.5 Gy TBI and CD25 depletion and compared this with the response in control mice that had successfully been treated through 4.5 Gy TBI without CD25 depletion (~25% of these mice stay tumor-free; Fig. 1D). Indeed, the CD25-depleted mice showed a strong CTL response against this non-self MuLV epitope (Fig. 5D), whereas the majority (80%) of these mice were capable of rejecting a secondary challenge with the parental, CEA-negative MC38 tumor cells (data not shown). In contrast, MuLV-specific CTL responses were not observed if treatment did not involve CD25 depletion (Fig. 5D), whereas a secondary challenge with MC38 resulted in progressive tumor growth in all mice concerned (data not shown). Notably, we have previously shown that natural killer (NK) cell-mediated immunity is important for clearance of MC38-CEA tumors (17), whereas others have shown that treatment with anti-CD25 antibody can result in enhanced NK cell reactivity (30). Taken together, these data provide a plausible explanation for our finding that the antitumor effect in anti–CD25-treated recipients did not fully rely on a potent CEA-specific T-cell repertoire (Fig. 5C).


Figure 5
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  		Figure 5. Antitumor response in anti-CD25 antibody-treated CEA-tg mice involves epitope spreading to a non-self target antigen. CEA-tg mice received 4.5 Gy TBI in combination with i.v. adoptive transfer of lymphocytes from CEA-immunized mice and s.c. tumor challenge (see legend to Fig. 1). Where indicated, donor lymphocytes were derived from wild-type (A and B) or CEA-tg (C) mice, and/or treatment was accompanied by administration of anti-CD25 antibodies 6 d before adoptive transfer (B and C). Figures depict tumor growth in individual mice (25 per group). Data are cumulative over three experiments. Each ascending line represents one mouse. The horizontal lines represent one or more mice in which no tumor outgrowth was detected. Numbers below these lines indicate the fractions of tumor-free mice. D, CEA-tg mice were challenged with MC38-CEA and treated by 4.5 Gy TBI in combination with infusion of lymphocytes from CEA-immunized wild-type mice. Where indicated, treatment was accompanied by administration of anti-CD25 antibodies 6 d before adoptive transfer (see legend to Fig. 1). Mice that successfully rejected MC38-CEA (n = 5 per group) were evaluated for their capacity to respond to the endogenous MuLV-encoded CTL epitope expressed by MC38 through secondary challenge with a tumorigenic dose (1.5 x 105) of parental, CEA-negative MC38 cells. After 4 wk, the majority (four of five) of mice initially treated with anti-CD25Ab were tumor-free, whereas all mice from the other group displayed palpable, progressively growing tumors. At this stage, mice were sacrificed and splenocytes were evaluated for CD8+ T-cell responses against the MuLV epitope by intracellular staining for IFN-{gamma}. Two representative examples of each group are shown.

 
Efficacy of adoptive transfer regimens against autochthonous intestinal tumors. In the case of most transplantable tumor models, including that used in our studies, the tumor develops at a site distinct of that of natural carcinogenesis. Furthermore, transplantable tumors, including MC38-CEA, are known to express additional foreign target antigens, such as the retroviral epitope discussed above. Consequently, a given immune response may display greater efficacy and selectivity toward transplantable tumors compared with their natural counterparts, particularly in cases where the target antigen is shared by tumor and normal surrounding tissue. In view of these considerations, we analyzed the effect of the previously described regimens in a model for spontaneous intestinal carcinogenesis. APC1638N mice (31) were bred with CEA-tg mice, resulting in APC1638N x CEA-tg that display the same CEA expression pattern as CEA-tg mice (27) and spontaneously develop CEA-overexpressing intestinal lesions (32). When left untreated, average number and size of the tumors were highly comparable between APC1638N and APC1638N x CEA-tg mice (data not shown). Therefore, comparison of these two mouse types, which develop CEA-negative and CEA-positive tumors, respectively, permits assessment of the efficacy of CEA-targeted immune interventions. We first tested adoptive transfer of lymphocytes from CEA-vaccinated wild-type donor mice in combination with pretreatment of recipient mice through 9.5 Gy TBI and BMT. This treatment was started at an average age of 9 months when tumors become detectable macroscopically. Approximately 1 week after adoptive transfer of CEA-specific lymphocytes, all APC1638N x CEA-tg mice showed severe weight loss to 65% of their original weight and the severity of the colitis eventually resulted in death of all the mice (10 of 10; data not shown). APC1638N single-tg mice that underwent the same treatment did not show these symptoms and all survived. These data indicate that the risk for antigen-specific autoimmunity is greatly increased in this model for spontaneous intestinal carcinogenesis compared with the transplantable tumor model. This is most likely due to the fact that tumor and normal intestinal tissue, which share the target antigen, are colocalized.

Because adoptive treatment involving IL-10R blockade induced intestinal autoimmune pathology in our transplantable tumor model (Figs. 1B and 3B), we did not further test this regimen in the APC1638N x CEA-tg mouse model. Importantly, treatment involving CD25 depletion was not associated with autoimmune pathology in the transplantable tumor model. Therefore, this modality was applied on APC1638N x CEA-tg and APC1638N single-tg mice. In accordance with our experience with this treatment (Fig. 3), the APC1638N x CEA-tg mice did not develop any signs of colitis. Treatment was started at an average age of 9 months and mice were sacrificed and examined 8 weeks later. Interestingly, the average number of tumors and the average surface area of the tumors per mouse after treatment were significantly lower in the group of APC1638N x CEA-tg mice compared with the group of APC1638N single-tg mice (Fig. 6A ). Treatment not including adoptive transfer of donor lymphocytes did not show significant effect on tumor development in APC1638N x CEA-tg mice (data not shown). The notion that the adoptively transferred CEA-specific immunity suppressed tumor development in the APC1638N x CEA-tg mice was further supported by in vitro analyses of T-cell responses. CEA-specific IFN-{gamma} production by T cells isolated from the mLNs was much stronger in the APC1638N x CEA-tg mice than in the APC1638N single-tg mice (Fig. 6A). These results again show that expansion of the infused CEA-specific T-cell populations was greatly enhanced by their reactivity against the CEA-expressing intestinal epithelium (compare with Fig. 4A). In accordance with these findings, considerable numbers of Thy1.1+ donor-type T cells were found to concentrate in the CEA-positive tumors of APC1638N x CEA-tg mice, whereas much lower numbers of infiltrating Thy1.1+ donor-type T cells were detected in the CEA-negative tumors of APC1638N single-tg mice (Fig. 6B). Similar low numbers of infiltrating Thy1.1+ T cells were observed in tumors of APC1638N x CEA-tg and APC1638N single-tg mice that received lymphocyte populations from control-vaccinated donor mice (Fig. 6C). Therefore, the increased infiltration of CEA-positive tumors after adoptive transfer of CEA-reactive T-cell populations is directly related to the presence and recognition of this target antigen.


Figure 6
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  		Figure 6. Development of intestinal tumors in APC1638N and APC1638N x CEA-tg mice after CEA-specific adoptive immunotherapy. A, cohorts of APC1638N and APC1638N x CEA-tg mice received 4.5 Gy TBI in combination with i.v. adoptive transfer of lymphocytes from CEA-immunized wild-type mice. Treatment was accompanied by administration of anti-CD25 antibodies 6 d before adoptive transfer. Treatment was started at an average age of 9 mo and mice were vaccinated every 2 wk with CEA-specific DNA vaccine. Intestines were analyzed 8 wk after onset of treatment for the number and size of tumors. mLNs were isolated and analyzed for CEA-specific CD4+ and CD8+ T cells by intracellular IFN-{gamma} staining as described in the legend to Fig. 1. B and C, cryosections of intestinal tumors, isolated 8 wk after the start of the treatment from APC1638N and APC1638N x CEA-tg mice, were stained for Thy1.1+ to detect donor-derived infiltrating T cells. Infused lymphocytes were derived from mice immunized with CEA-specific DNA vaccine (B) or with canarypox virus (ALVAC empty vector)–immunized mice (C). The latter immunization is known to elicit potent CD4+ and CD8+ T-cell responses against canarypox virus antigens (17). A representative example for each group is shown.

 
In conclusion, reconstitution of the CEA-specific T-cell repertoire in CEA-tg mice can suppress the development of autochthonous, CEA-expressing intestinal tumors, in the absence of autoimmune pathology to the normal intestinal epithelium, provided that this treatment involves moderate levels of CEA-specific T-cell immunity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In the present study, CEA transgenic mice were used as a preclinical model to investigate the effect of potent CEA-specific T-cell immunity on CEA-overexpressing tumors in the context of a host that expresses CEA as a self antigen in the intestine. Because the magnitude and breadth of the CEA-specific T-cell repertoire of these mice was blunted by central tolerance (Fig. 1; ref. 16), induction of effective CEA-specific T-cell immunity required adoptive transfer of lymphocytes from nontolerant donor mice. These T cells were only capable of mounting a tumoricidal response when peripheral immunoregulation was eliminated (Fig. 1), indicating that CEA-specific immunity in the CEA-expressing host is subject to tight control in both thymus and periphery. For several of the immune regimens tested in our study, antitumor efficacy of the CEA-specific T-cell response was associated with severe autoimmune pathology in the CEA-positive intestine (Fig. 2), illustrating the vital importance of aforementioned control over CEA-specific immunity as well as the risks involved in tampering with these mechanisms in the context of cancer immunotherapy. In contrast, preconditioning of mice with the anti-CD25 antibody PC61 before T-cell infusion resulted in antitumor efficacy in the absence of intestinal immune pathology (Figs. 1, 3, and 6). Taken together, our data indicate that the use of CEA-specific T-cell immunity for cancer immunotherapy involves considerable hurdles and risks but that careful management of this immune response may offer a window of opportunity for cancer targeting in the absence of life-threatening intestinal pathology.

In contrast to our findings, previous preclinical and clinical studies by others have not provided any indication that CEA-specific targeting of cancer could be associated with immune attack of the CEA-positive intestine and have thereby supported the long-standing hypothesis that the difference in CEA levels between colorectal cancers and normal intestine would allow selective tumor targeting (12). However, the CEA-specific T-cell responses observed in vaccinated cancer patients were of low magnitude (reviewed in ref. 33 and references therein). Furthermore, CEA is one of many tissue-specific antigens that is expressed in the human thymus (1, 2, 16). It is therefore conceivable that the high-affinity CEA-specific T-cell repertoire in humans, like that in CEA-tg mice (16), is deleted and that the responses observed in vaccinated patients primarily reflect low-affinity T cells. Such T cells were shown to display poor immune efficacy against tumors and normal somatic tissues expressing their target antigen (5, 6). Accordingly, the patient studies concerned have not resulted in striking clinical responses that could be clearly attributed to vaccine-induced CEA-specific immunity (33). Similarly, a series of previously published studies with CEA-tg mice (the same strain that was used in our studies), which suggested antitumor efficacy of the endogenous CEA-reactive T-cell response in the absence of intestinal pathology, did not involve detailed analysis of the CEA-specific T-cell response (reviewed in refs. 34–37 and references therein). In fact, T-cell immunity against a retroviral epitope was recently found to contribute to rejection of transplantable CEA-positive tumors in these experiments (38), offering a plausible explanation for the fact that tumor rejection was not associated with intestinal damage. Strikingly, our present data show that T-cell immunity against this same epitope was involved in tumor rejection in the absence of intestinal pathology in mice that were administered anti-CD25 antibodies before adoptive immunotherapy (Figs. 1 and 3). Furthermore, our data indicate that the CEA-specific T-cell response in CEA-tg recipient mice that were conditioned by 4.5 Gy TBI in combination with anti-CD25 antibody was ~10-fold lower than in mice that were conditioned by 9.5 Gy TBI in combination with BMT (Figs. 4A and 6A).5 The cumulative data lead us to conclude that CEA-specific T-cell immunity can contribute to the selective elimination of CEA-positive tumors in the CEA-positive host provided that the CEA-specific T-cell responses are not too strong and are supplemented, through antigen spreading of the immune response, by immune responses against additional tumor-specific antigens. Importantly, such tumor-specific antigens not only are an artifact of transplantable tumor models but also occur in human cancers (39–42). Furthermore, our experiments in the autochthonous APC1638N x CEA-tg tumor model also show, albeit modest, antitumor efficacy of CEA-targeted adoptive immunotherapy when combined with prior administration of anti-CD25 antibodies in the absence of intestinal pathology (Fig. 6).

The CEA gene expression pattern in the CEA-tg mice used in our experiments closely resembles that in humans, but the levels are 3- to 10-fold higher than in healthy humans (27). In fact, experiments by others with another CEA-tg strain, expressing CEA at levels ~10-fold lower than in healthy humans (43), revealed antitumor efficacy of the vaccination-induced endogenous T-cell repertoire against CEA-overexpressing tumors in the absence of overt autoimmune pathology (reviewed in refs. 44, 45 and references therein). Whereas our CEA-tg model therefore may reflect a worst case scenario with respect to both immune tolerance and intestinal immune pathology, experiments with the second, low CEA expressing strain are likely to underestimate the complications of CEA-targeted immunotherapy. The risk for intestinal immune pathology in immune intervention strategies has been clearly shown in clinical studies involving CTLA-4 blockade or allogeneic donor lymphocyte infusions (13, 14), regimens that do not involve intentional targeting of intestine-specific antigens. Furthermore, immune intervention strategies against melanoma that boost immune responses against lineage-specific antigens expressed by melanocytes involve immune attack against normal melanocytes, causing autoimmune skin depigmentation (23, 46), whereas spontaneous immunity against ectopically expressed self antigens in lung cancer and gynecologic tumors, although effectively suppressing cancer development, can cause life-threatening paraneoplastic neurologic disorders (47). It is therefore conceivable that powerful therapeutic T-cell responses targeting tumor-associated autoantigens will not spare normal tissues expressing these antigens. For target antigens expressed by "dispensable" cell types and tissues, such as melanocytes or the prostate, the resulting damage could be considered an acceptable price for cure of cancer. However, our studies show that there is only a thin line between therapeutic efficacy and life-threatening autoimmune disease if antitumor immunity is targeted against autoantigens expressed by vital somatic tissues.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
C.J.M. Melief and R. Offringa: Commercial research support, Sanofi Pasteur. The other authors disclosed no potential conflicts of interest.


   Acknowledgments
 
Grant support: Dutch Cancer Society grant UL2000-2035 and Sanofi Pasteur Toronto.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Alain Vicari and Giorgio Trinchiery for providing the anti-IL-10R antibody; Sandra Bres for additional technical assistance; and Neil Berinstein, Thorbald van Hall, and Peter Kuppen for inspiring discussions.


   Footnotes
 
Note: Current address for R. Offringa: Genentech, Inc., South San Francisco, California. Current address for R. Bos: The Scripps Research Institute, La Jolla, California.

5 Unpublished data. Back

Received 5/16/08. Revised 7/11/08. Accepted 7/28/08.


   References
Top
 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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