[Frontiers in Bioscience 7, d853-871, April 1, 2002]

IMMUNOTHERAPY OF HUMAN PAPILLOMAVIRUS-ASSOCIATED MALIGNANCIES AND THE CHALLENGES POSED BY T-CELL TOLERANCE

Ryan A. Wilcox and Lieping Chen

Department of Immunology, Mayo Clinic, Rochester, MN 55905

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Immunotherapeutic strategies: dendritic cell and tumor cell based vaccines
4. T-cell tolerance, immunologic ignorance and incompetence
4.1. Tolerance
4.2. Immunologic ignorance
4.3. Immunologic incompetence
4.4. HPV transgenic models
4.5. Tolerance, immunologic ignorance and incompetence in cervical cancer
4.6. Tolerance and immunotherapy
5. T-cell costimulation in immunotherapy
5.1. 4-1BB (CD137) immune regulation
6.Conclusion
7. Acknowledgement

8. References

1. ABSTRACT

Human papillomaviruses are associated with a broad range of carcinomas, including cervical cancer. Although the delivery of immunogenic tumor-associated antigens represents a promising approach in the treatment of these malignancies, the imposition of T cell tolerance poses a significant challenge in this endeavor. The purpose of this review is to discuss T cell tolerance and the role of T cell costimulation in the immunotherapy of HPV-associated malignancies.

2. INTRODUCTION

The neoplastic transformation of a cell, by most accounts, may be attributed to a multistep process involving many rounds of genetic mutation followed by natural selection within the host. Therefore, any physical, chemical, or biological conditions, which increase the rate of cellular proliferation or prevent a damaged cell from undergoing apoptosis may promote the neoplastic transformation of the cell. Exposure to chemical carcinogens and UV irradiation have long been associated with human malignancies; however, the last decade has witnessed a growing awareness of the link between infectious agents and human cancers (1), leading some to estimate that a significant portion of human cancers worldwide may be associated with viral infections (2, 3), including hepatitis B virus (hepatocellular carcinoma), Epstein Barr virus (Burkitt's lymphoma and nasopharyngeal carcinoma), hepatitis C virus (immunoblastic lymphoma), human T cell leukemia virus (adult T-cell leukemias and lymphomas), HHV-8 (Kaposi's sarcoma, Castleman's disease, and body cavity lymphomas), HHV-6 (non-Hodgkin's lymphoma) and human papillomaviruses.

Human papillomaviruses (family papovaviridae) are epitheliotropic, non-lytic, double-stranded circular DNA viruses, producing hyperkeratotic lesions with little to no inflammation. Infectious virions are thought to be shed from the epithelial surface within desquamating keratinocytes. The HPV genome may be divided into both early (E) genes, encoding proteins involved in DNA replication, transcription and cellular transformation, and late (L) genes, which encode the viral capsid proteins, L1 and L2. HPV replication is exquisitely linked to the differentiation state of the keratinocyte. HPV particles are thought to infect basal keratinocytes coincident with local damage. Although viral gene expression is virtually undetectable in the basal layer, the early genes (including E1, E2, E5, E6, and E7) are transcribed as the keratinocyte differentiates and migrates from the basal layer, eventually to be exfoliated. An overwhelming body of evidence, too extensive to enumerate here, has implicated the E6 and E7 gene products as the major transforming proteins. This may be attributed to the ability of these viral oncogenes to associate with and functionally neutralize the tumor suppressors p53 and Rb (4-6).

Over 100 subtypes of HPV may exist in humans, with each belonging to either a low-, intermediate-, or high-risk subtype. An important distinguishing characteristic between these subtypes is the binding affinity of the E6 and E7 gene products for the tumor suppressors p53 and Rb, respectively (6-8). These early gene products of the low-risk HPVs, such as HPVs 1, 6 and 11, demonstrate no such binding, and are associated with the common wart, genital warts, and condylomata acuminata. In contrast, the transforming, high-risk HPVs, including HPV 16, 18, 31, 33, and 45 are associated with carcinoma development.

It is estimated that approximately 95% of cervical carcinomas may be associated with high-risk HPVs, most notably HPV 16 and 18 (9-12). Although both high- and low-risk subtypes may be associated with grade I cervical intraepithelial neoplasia (CIN), most CIN III (and many CIN II) lesions are associated with high-risk HPV types. Even though an accurate estimate of the incidence rate of HPV is not available, recent reports indicate the incidence of HPV has increased over the past few decades (10). Prevalence rates vary widely, depending upon the detection method utilized (with higher prevalence rates reported in those studies using PCR-based technologies) and the population examined, with current estimates ranging between 5% and 50% in women with a normal pap smear. Most infected women become HPV negative (i.e. HPV DNA undetectable) within two years; however, that subset of women who become persistently infected have an increased likelihood of developing CIN III lesions and cervical carcinoma (13-16).

Although HPV's causative role in cervical cancer is now well established, the association between HPV infection and a growing number of carcinomas in other tissues is less clear as the PCR-based technology used to detect a large variety of HPV types is extremely sensitive and prone to false positives, possibly attributable to incidental HPV contaminants. Despite these difficulties, faced years ago by those attempting to demonstrate the association between cervical cancer and HPV, a growing body of evidence supports the contention that HPV's role in epithelial cell transformation may not be limited to cervical cancer. For example, the association between HPV infection and a subset of head and neck squamous cell carcinomas (HNSCC) is now well established (17-20). Therefore, therapeutic vaccines being developed for use in cervical cancer patients may be useful in the treatment of some HNSCC patients.

Non-melanoma skin cancers (NMSC) are the most prevalent malignancies in the caucasion population worldwide. HPV has been implicated in the cutaneous malignancies associated with the rare and inherited disorder, epidermodysplasia verruciformis (EV). Recent studies have demonstrated that a significant number of NMSC in both immunocompetent and immunosuppressed individuals may be associated with various HPV types (21-23). Several groups have demonstrated that HPV gene products expressed under control of a keratinocyte-specific promoter in transgenic mice lead to the development of skin cancers in older mice (24), or in transgenic mice exposed to chemical carcinogens (25) or UV-irradiation (Wilcox et. al., unpublished data). The use of such transgenic models may not only help to elucidate the molecular events required for the malignant transformation of the HPV infected cell, but they may also improve our understanding of the immune response generated at each stage in this multi-step process (26).

The association of HPV infection and transitional cell carcinoma (TCC) of the bladder has been reported (27-31), although not all attempts to demonstrate the presence of HPV in neoplastic tissue have been successful (32-34). Cases of rapidly progressive multifocal TCC of the bladder have been reported in immunosuppressed transplant recipients, suggesting HPV's role in TCC of the bladder in immunocompromised patients (35, 36). One report has implicated HPV16 in a number of squamous cell carcinomas of the lung on the island of Okinawa (37, 38). A few studies would seem to support this finding (39-41), although others have failed to implicate HPV in lung cancer (42). Perhaps even more controversial is the recently proposed association between HPV and squamous cell carcinoma of the esophagus (43).

Data implicating a relatively few HPV subtypes in the neoplastic transformation of epithelial cells at a wide range of anatomic sites, not limited to the uterine cervix, implies that the vaccine strategies currently employed in cervical cancer patients may be beneficial in a number of other malignancies. The goal of this review is to summarize the current status of therapeutic HPV vaccine strategies, emphasizing the potential challenges posed by T cell tolerance and immunologic ignorance, and the role of T cell costimulation in the setting of tumor immunotherapy.

3. IMMUNOTHERAPEUTIC STRATEGIES: DENDRITIC CELL AND TUMOR CELL BASED VACCINES

Preclinical and clinical studies have shown the utility of prophylactic and therapeutic vaccines in the setting of HPV-associated disease (44). Whereas the goal of any prophylactic vaccine, which is beyond the scope of this review, is to prevent HPV infection by the induction of an appropriate humoral and cell-mediated immune response capable of producing neutralizing antibodies, the goal of a therapeutic vaccine is the eradication of premalignant and malignant HPV-associated lesions.

Several lines of evidence support the notion that the induction of a potent cell-mediated immune response may be capable of controlling HPV-associated malignancies. First, immunosuppressed patients have an increased incidence of both premalignant and malignant lesions of the cervix (45-50). Furthermore, spontaneous regression of HPV-associated lesions has been observed and is associated with a cell-mediated immune response (14). Tumor infiltrating lymphocytes may be demonstrated in cervical lesions by immunhistochemistry and HPV-specific T cells have been demonstrated, not only in peripheral blood, but in tumor draining lymph nodes and the tumors of CIN and cervical cancer patients (51). Not surprisingly, the downregulation of HLA class I molecules during the progression of dysplastic lesions to neoplastic lesions suggests that immune evasion may be an important contributing factor in disease progression (52, 53). Collectively, this data provides a strong rationale for the addition of T-cell based immunotherapies to our arsenal in the fight against HPV-associated malignancies.

As both the E6 and E7 gene products are required for the maintenance of the transformed phenotype, these viral proteins are attractive targets for immunotherapy. To date, many class I restricted peptides have been identified and have been shown to be immunogenic in both mouse models and phase I/II human trials (54-64). Studies performed in mice have demonstrated that peptide immunization may confer protection against a subsequent challenge with an HPV-transformed tumor. However, the same approach, although capable of generating a detectable CTL response, was incapable of treating established tumors. Van Elsas et. al. recently demonstrated that immunotherapy of a murine melanoma was dependent upon a CTL response, whereas tumor rejection was observed following a prophylactic vaccine in the absence of CD8+ T cells (65). These studies warrant a degree of caution when attempting to extrapolate data obtained from prophylactic vaccine studies to a therapeutic setting. The present challenge then, is to develop immunotherapeutic strategies capable of treating established tumors.

It may be argued that dendritic cells (DCs) are not only the most potent professional APC, undoubtedly due to their unique ability to process and present antigen, express costimulatory and adhesion molecules, and secrete proinflammatory cytokines; but they also play a pivotal role in the initiation, regulation, and maintenance of a cell-mediated anti-tumor immune response (66). Although DCs represent less than 1% of peripheral blood leukocytes, sufficient numbers of DCs may be obtained for use in immunotherapy protocols following the ex vivo expansion of DCs in IL-4 and GM-CSF from monocytes or monocyte (CD34+) precursors. Phenotypically immature DCs, although less efficient APCs, efficiently phagocytose apoptotic and necrotic cells and macropinocytose soluble proteins. Upon maturation, DCs become more potent APCs, capable of presenting antigenic peptides to CD4+ T cells and exogenous antigenic peptides to CD8+ T cells via "cross-priming." The upregulation of various costimulatory molecules and an increased ability to secrete inflammatory cytokines are hallmarks of a phenotypically mature DC. Further understanding of basic DC biology may prove useful in the determination of the "maturation state" which is optimal for any given DC-based immunotherapy. Furthermore, the ancestry of DCs used in immunotherapy, whether lymphoid or myeloid, or DC1 or DC2, may be important considerations (67-69). For example, Mohamadzadeh et. al recently demonstrated that IL-15 and GM-CSF could generate DCs similar to Langerhans cells from peripheral blood. This is an exciting finding, as these cells were more potent stimulators of a CTL response when compared to DCs generated in IL-4 and GM-CSF (70).

Many DC-based immunotherapies have been attempted in mouse models, some of which have been applied in various clinical trials with a wide range of different malignancies. One approach is to load DCs in vitro with tumor antigens, whether in the form of antigenic peptides, recombinant protein, RNA or DNA encoding a known tumor antigen, tumor-derived RNA, tumor cell lysates, tumor-derived apoptotic bodies or exosomes, or CVLPs (71-84). Encouraging results have been obtained using HPV16 E7 peptide-pulsed DCs in mouse models. For example, peptide-pulsed DCs not only protect mice against subsequent challenge with an HVP16 E7-expressing tumor, but this approach also eradicated large established tumors (84-86).

Strategies, which do not require any prior knowledge of a patients MHC haplotype, or even identification of a relevant tumor antigen, would seem most attractive. One approach includes the use of autologous or allogeneic, irradiated, whole tumor cell vaccines transduced to express cytokines, such as GM-CSF or IL-12, or costimulatory molecules, like B7-1. The ability of tumor cell vaccines to induce a CTL response and confer protection against a tumor challenge has been well documented in the literature. These approaches have only recently been applied to HPV in preclinical studies (87, 88). Although this approach may have its advantages, especially in those malignancies in which tumor antigens have not yet been identified, this approach seems relatively less attractive in the setting of cervical cancer in which tumor antigens are well characterized. Furthermore, although tumor cell vaccines may be capable of conferring protection against a subsequent tumor challenge, these vaccines are considerably less effective in the therapeutic setting. With that in mind, the fusion of either autologous or allogeneic tumor cells with autologous DCs (89, 90), or loading of DCs with tumor cell derived products (e.g. heat shock proteins, exosomes, apoptotic bodies, RNA, or cell lysates) may not be unreasonable. Alternative approaches may include the delivery of DCs to tumors in vivo, or the mobilization of DCs using Flt-3 ligand or GM-CSF and IL-4 (91-96).

4. T-CELL TOLERANCE, IMMUNOLOGIC IGNORANCE AND INCOMPETENCE

The imposition of self-tolerance, both centrally and peripherally, may pose a significant barrier to the stimulation of a potent antitumor immune response capable of eradicating an established tumor or preventing tumor recurrence following conventional therapeutic modalities. (Although HPV clearly represents a "foreign" antigen, HPV infection is not usually associated with an inflammatory response. Furthermore, HPV is a non-lytic virus, and HPV-infected cells, once desquamated from the epithelial surface may be inaccesible to antigen presenting cells. This lack of "danger," and likely sequestration of antigen in desquamated epithelial cells may justify the comparison between a self-antigen expressed in the periphery and HPV antigens.) Therefore, the potential mechanisms responsible for the maintenance of self-tolerance and the methods that might be useful in breaking tolerance may be worth careful consideration in the cancer immunotherapy setting.

4.1. Tolerance

There are undoubtedly many factors which control the balance between immunity and tolerance in the periphery, many of which are poorly understood. Such mechanisms fall within a broad spectrum, ranging from T cell deletion (97-102), anergy or activation-induced nonresponsiveness (AINR) (103-107), to outright immunological ignorance or indifference (108-110), all of which may be operational in a given patient expressing multiple tumor associated antigens (111). T cell deletion has been observed in both autoimmune and tumor models (97-102) and is thought to be the result of a high antigen load, or in the case of a CTL response, a lack of CD4+ T cell help (100, 112, 113).

In contrast to deletional mechanisms, T cell anergy involves the induction of a functionally unresponsive state in antigen-specific T cells. T cell anergy has been classically defined in CD4+ T cells and may result from the presentation of antigen in the absence of the appropriate costimulatioin, leading to a defect in IL-2 secretion upon antigenic restimulation (114-116). Functional unresponsiveness in CD8+ T cells, recently described as activation-induced nonresponsiveness, is similar to the clonal anergy observed in CD4+ T cells, in that it involves an inability of CD8+ T cells to secrete IL-2 and proliferate upon reencounter with antigen (117). However, in contrast to clonal anergy, AINR is thought to occur following TCR engagement and costimulation (117-120). In addition, AINR in CD8+ T cells is independent of CTLA-4 stimulation (117, 120). AINR is reminiscent of the "split anergy" observed in CTL clones following activation in the absence of costimulation . Although the cells were unable to secrete IL-2 and proliferate upon restimulation, the ability to lyse target cells (121) and secrete IFN-f (wilcox et. al. unpublished data) remained intact. This dichotomy may be explained by the recent observation that AINR involves the defective upregulation of ERK and p38, MAP kinases essential for IL-2 production, whereas lytic function may be maintained due to the retention of the TCR-induced calcium flux (118). Anergy or AINR of CD4+ and CD8+ T cells, respectively, has been observed in mouse tumor models. Given the subtle differences in clonal anergy and AINR, the conditions needed to break the one may be innefective in reversing the other.

Systemic immune suppression may also play an important role in preventing an antitumor immune response. Many tumors, including cervical cancer, may result in systemic immunosuppression. Suppressor T cells may also play a role in the maintenance of self-tolerance. Sakaguchi et. al. made the important observation that depletion of CD4+CD25+ T cells from CD4+ cells prior to adoptive transfer into nu/nu recipients led to autoimmune disease (122, 123). Subsequent work has clearly identified this subpopulation of CD4+ T cells as important suppressors of self-reactive T cells in vivo and in vitro (124). The role of CD4+CD25+ suppressor cells in tumor immunity has been demonstrated by the enhanced anti-tumor immune response observed upon their depletion in vivo or upon their depletion in vitro prior to adoptive transfer of autologous T cells obtained from tumor-bearing mice (125).

4.2. Immunologic ignorance

Immunologic ignorance may be particularly relevant when considering HPV. As previously mentioned, once epithelial cells are infected by HPV, viral antigens are likely sequestered from roaming APC's and the infection itself fails to elicit an inflammatory response. The coexistence of naïve, fully functional antigen-specific T cells and a peripheral antigen has been described as immunological ignorance. However, ignorance should be distinguished from the situation in which a T cell response is generated, either naturally or following immunotherapy, but the response is incapable of suppressing tumor growth, even though the tumor cells are susceptible targets. We will designate the latter situation in which T cells have been primed, but fail to control tumor outgroth as immunologic incompetence.

Ignorance was first demonstrated by Ohashi et. al. using double transgenic mice expressing both the LCMV-GP under control of the rat insulin promoter (RIP) and an LCMV-GP TCR transgene. The LCMV-GP specific T cells in these mice, while not deleted or anergized, ignored the viral antigen expressed on the islet b cells (108). This immunologic ignorance could be broken either by infection with the virus or by the local expression of B7-1 on islet cells (126). A similar study performed in mice expressing both B7-1 and TNFa in islet cells demonstrated the importance of both local inflammation and costimulation on the induction of autoimmunity (127). Similar experiments were performed in which a fragment of the SV40 large T antigen was expressed in the islets of mice which also expressed a TCR specific for an antigenic peptide of the large T antigen. In these mice, the antigen was ignored; however, the adoptive transfer of fully activated specific T cells into mice expressing the antigen did not result in autoimmunity (128). Similarly, ignorance has also been observed in transgenic mice expressing the E6 and E7 oncogenes of HPV16 under the control of a keratin-specific promoter (129). In these mice, the E6 and E7 gene products were expressed in all keratinized epithelial cells. Cultured keratinocytes from these mice could be lysed by E7 specific CTLs in vitro. Although immunization with an E7-derived peptide conferred protection against a subsequent challenge with a tumor expressing the E7 gene product, no skin pathology was noted in these transgenic mice. These studies demonstrate that although systemic ignorance may be broken upon immunization with the appropriate antigen , this does not guarantee that the CTL response generated will be sufficiently potent to mediate autoimmunity.

Immunologic ignorance has also been described in tumor models. For instance, Wick et. al. showed that Ag104 tumor cells transfected with Ld could grow in 2C mice, although Ld-expressing skin was rejected (110). Similarly, transgenic mice expressing a TCR specific for the nonmutated tumor antigen P1A cannot reject the P1A positive J558 plasmacytoma (130). While the evidence for immunological ignorance is clear, the mechanisms responsible for maintenance of the ignorant state are poorly understood.

One obvious factor which is likely important in the maintenance of immunological ignorance is the dose of antigen (131). This has been demonstrated in transgenic mice expressing either high (RIP-Ovahi) or low (RIP-Ovalo) levels of ovalbumin under the control of the rat insulin promoter (112). In these mice, the RIP-Ovahi deleted antigen-specific OT-1 cell in vivo. In contrast, Ova was completely ignored in the RIP-Ovalo mice. Importantly, both transgenic lines were susceptible to islet b cell destruction following the adoptive transfer of activated OT-1 cells (132). It was also noted that in chimeric RIP-Ovahi mice incapable of cross-presenting antigen, OT-1 cells were no longer deleted, but became ignorant. The latter observation highlights the potential influence of cross-priming in controlling the balance between tolerance and immunity.

Direct priming and cross-priming may both play redundant roles in tumor immunity (133, 134). However, not all tumors may be associated with cross-presentation of tumor antigens. For example, it has been shown that in various tumors expressing LCMV-GP, the LCMV-GP is not cross-presented to antigen specific T cells in vivo (135, 136). Given that all the tumors used in these studies were transfected with the same antigen, antigenic epitopes derived from this antigen may not be properly processed and presented by the immune proteasome. Therefore, to conclude from these studies that cross-priming is extremely rare may be an unjustified extrapolation. Despite this shortcoming, it was clearly demonstrated that the ability of a tumor to directly initiate a T cell response was associated with that tumor's ability to metastasize to secondary lymphoid organs, like the tumor draining lymph node. It was also noteworthy that tumors, which metastasize to the draining LN, may be encapsulated and thus remain inaccessible to naïve T cells. Therefore, a lack of cross-priming, whether or not that is determined by the dose of antigen or the anatomic site of the tumor (137), and/or a lack of direct-priming, presumably due to a tumors inability to gain access to naïve T cells in secondary lymphoid organs, or a tumor's ability to subvert these modes of antigen presentation, may explain immunologic ignorance.

Recent studies have also demonstrated the importance of secondary lymphoid organs in T cell activation following the cross-presentation of antigen. Bai et. al. demonstrated that following T cell activation, either by direct or indirect antigen presentation, P1A specific T cells underwent a phenotypic change, signified by the upregulation of HSA, at the tumor site. This change was also observed in MHC class I deficient tumors, suggesting the presence of cells, like macrophages or dendritic cells, capable of cross-presenting antigen to activated T cells. In contrast, T cell activation was not observed when naïve T cells were injected directly into the tumor site. Therefore, secondary lymphoid organs, but not the tumor microenvironment, may be permissive for the productive cross-presentation of tumor-associated antigens to naïve T cells. This limitation may not be restricted to tumors, as even allogeneic, vascularized organ transplants may be ignored in mice lacking secondary lymphoid organs (138). Although the mechanism responsible for this difference is unknown, several hypotheses may be offered.

First, the tumor microenvironment may not be sufficiently "dangerous" to sufficiently mature APCs involved in the cross-presentation of antigen (139, 140). Alternatively, the tumor may actively suppress APC maturation and T cell activation via the secretion of soluble factors like TGF-b or VEGF (141-144). A recent study has shown that in a collagen matrix, T cells interacted only transiently with antigen-presenting DCs (145). Therefore, collagen present in the periphery, including mucosal surfaces, may interfere with formation of the immunological synapse. In contrast, the absence or shielding of collagen by stromal cells in secondary lymphoid organs may favor an immunologically productive encounter between a naïve T cell and an antigen-presenting cell (146). Chemokine gradients present in vivo may also promote T cell ignorance in the tumor setting. Bromley et al. showed that "dominant chemokine gradients," including SLC may prevent immunological synapse formation (147). Therefore, in the absence of inflammation at the tumor site, a naïve T cell may exit the periphery, despite the presence of antigen, in response to an SLC gradient. In contrast, inflammatory chemokines like MIP-3b produced during an inflammatory response may favorably compete with any "dominant chemokine gradient," promoting immunological synapse formation and T cell activation in the periphery.

4.3. Immunologic incompetence

In contrast to immunological ignorance, incompetence is characterized by the activation of antigen-specific T cells; however, this T cell response is incapable of controlling tumor outgrowth. For example, Ag104 tumor cells transfected with Ld grew in 2C mice even though Ld-expressing skin grafts were rejected. In this model, incompetence could only be overcome if mice were preimmunized with Ld-expressing skin grafts and tumor cells expressed both B7-1 and CD48 (110). Furthermore, the P1A positive plasmacytoma J558 grow in transgenic mice expressing a TCR transgene specific for the P1A antigen. However, tumor cells expressing either B7-1 or B7H were rejected (130, 148, 149). Interestingly, in mice inoculated with both J558-neo and J558-B7-1, only the B7-1 expressing tumor cells were eliminated, further implicating a lack of local costimulation in the maintenance of immunological incompetence. Like ignorance, the mechanisms responsible for immunological incompetence are not yet fully understood.

The ability of activated T cells to gain access to the tumor is an important consideration. Transgenic mice expressing the SV40 large T antigen in islet b cells of the pancreas eventually leads to insulitis and the development of solid tumors (128). Interestingly, even when these mice were crossed with mice expressing a Tag-specific TCR transgene, the solid tumors were not infiltrated with T cells in the double transgenic mice, despite the presence of insulitis in adjacent tissue. Even the expression of B7-1 by islet b cells, although sufficient for the development of diabetes, did not prevent the outgrowth of solid tumors. Although these tumors maintained expression of the large T antigen and were not infiltrated by T cells, the tumors had lost expression of B7-1 (150). However, in this model, T cell extravasation into the tumor site was observed when ex vivo activated T cells were adoptively tranferred into mice which had previously received a high dose of whole body irradiation. Ionizing radiation has been shown to upregulate endothelial adhesion molecules which may be extremely important in the recruitment of effector T cells to the tumor site (151-153). These findings may be relevent given the observation that many tumors may regulate the expression of adhesion melecules by endothelial cells present within the tumor, thus preventing T cell extravasation at the tumor site (154, 155).

T cells capable of extravasating at the tumor site may not remain there long enough to completely eradicate a tumor. This has been observed in a mouse model in which the adoptive transfer of antigen-specific T cells was incapable of controlling the growth of lymphoma cells in the peritoneum. This was the result of T cell migration away from the tumor site into secondary lymphoid organs. After migration away from the tumor site, the T cells appeared to have undergone AINR, as they were capable of killing tumor targets in vitro, even though they were unable to proliferate upon restimulation (119).

The T cell repertoire, including the avidity of antigen specific T cells for their cognate antigen, may also be an important factor influencing immunological incompetence. This may be especially true in situations in which a tumor-associated antigen is presented in the thymus and central tolerance enforced (156). Alternatively, only T cells with a low avidity for a tumor-associated antigen may escape negative selection (157). Many studies have implicated costimulation at the tumor site as an important factor in immunological incompetence (110, 148, 149). Although costimulation itself may be an important factor in breaking immunological incompetence, it is also possible that the expression of costimulatory molecules on tumor transfectants may serve to increase the avidity of the T cell/tumor cell interaction.

4.4. HPV transgenic models

Whether or not HPV-associated malignancies induce tolerance is a difficult question given the problems associated with accurately measuring an antigen-specific T cell response in cancer patients. However, work performed in transgenic mice expressing HPV antigens in peripheral tissues, including skin , suggest that multiple modes of self-tolerance may be enforced. In these models, HPV16 E6 and/or E7 oncogenes are expressed under the control of promoters, such as the keratin 14 promoter, which restrict transgene expression to epithelial surfaces. As many of these mice also express the transgene in the thymus, both central and peripheral tolerance may be important (158). However, studies performed in radiation bone-marow chimeras have demonstrated that transgene expression in the periphery alone is sufficient to tolerize E7-specific CTLs (159). Studies performed in various lines of transgenic mice provide evidence for anergy (158, 160), ignorance (129, 161-163), and incompetence (129, 164). Interestingly, CTL tolerance could be maintained in transgenic mice in which antigen specific T helper cells were not tolerized or showed evidence of having been previously activated (165, 166). Therefore, the status of any B cell or CD4+ T cell response may not be a reliable indication of the immune status of tumor-specific CTLs.

One line of transgenic mice frequently develop SCC of the skin and lenticular tumors upon aging. In younger mice, E7 specific T cells appear to ignore the antigen, however, development of DTH responses to E7 was observed in older mice with skin pathology. Therefore, immunological ignorance may be prominent early in disease progression (i.e. low grade CIN), but an observable immune response may be generated later as the disease progresses, potentially inducing an inflammatory response or metastasizing to secondary lymphoid organs (161). It should be noted that the depletion of CD4+ T cells in these mice was associated with a reduced incidence of skin pathology, thus raising the possibility that any immune response may precede noticeable skin pathology (163). Alternatively, loss of CD4+CD25+ suppresor T cells in these mice may result in the generation of a more robust cell-mediated immune response capable of limiting skin disease. Further studies in these mice should continue to shed light on the mechanisms of tolerance in HPV-associated malignancies and the potential methods capable of preventing tolerance induction or reversing tolerance once established.

4.5. Tolerance, immunologic ignorance and incompetence in cervical cancer

As in some of the transgenic mouse models, both T helper cell and CTL responses are detectable in CIN and cervical cancer patients (51, 167-173). However, the relevance of these findings in determining the significance of tolerance in patients is confounded by several variables. First, these studies inevitably involve the in vitro culture of patient PBMC in the presence of IL-2, a procedure which may reverse T cell anergy. As already noted, the detection of a CD4+ T cell proliferative response may not be associated with a CTL response. Furthermore, T cell responses in patients with advanced disease may be recent occurences associated with a high tumor burden and antigen load, or even an ongoing infection (170). Therefore, the detection of a T cell response in a patient with advanced disease may reveal very little about the putative immune response earlier in the disease course. Finally, most of these studies use PBMCs as a source of T cells; however, the frequency of tumor-specific T cells may be significantly higher in the tumor-draining lymph node or the tumor site (174). Therefore, the absence of a detectable T-cell response in peripheral blood may be uninformative, and most certainly cannot be interpreted as evidence for immunological ignorance.

Despite these pitfalls, evidence exists to suggest that self-tolerance may be as important a consideration in human disease as it is in both mouse tumor models and in studies performed in HPV transgenic mice. As has been observed in a variety of tumor types, HPV-associated malignancies may be associated with systemic or local immunosuppression. A variety of studies have shown that HPV-derived proteins may be immunosuppressive. This may be attributed to their ability to abrogate signaling mediated by type I interferons (175), to inhibit IL-18 induced IFN-f production (176), or to inhibit the secretion of IFN-a by APCs (177). Furthermore, HPV16 E6 oncoprotein may stimulate the release of VEGF at the tumor site, thus promoting angiogenesis and inhibiting DC maturation (178, 179). In fact, VEGF expression is a negative prognostic indicator following radiation therapy for cervical cancer (180). The presence of DCs within the tumor site is a positive prognostic indicator in a variety of different malignancies (181), including HPV-associated malignancies (182-185). The lack of DC infiltration within preneoplastic and neoplastic lesions may prevent the cross-presentation of tumor-associated antigens, thus preventing the induction of a cell-mediated immune response, favoring immunological ignorance. In contrast, antigen presentation by tumor-associated APCs may result in the induction of tolerance (186-190). This may be attributed to the secretion of a variety of immunosuppressive cytokines by tumor cells, including IL-10, TGF-b, or VEGF. Interestingly, an association between smoking and cervical cancer has been recognized for many years (191). It is unclear whether that may be attributed to the association between smoking and immune suppression, including a reduction in the density of Langerhans cells within the cervix (192). Furthermore, a persistent HPV infection may be associated with local infiltration of B cells capable of presenting HPV-associated antigens to CTL in a tolerogenic fashion (193-195) or may result in the generation of a Th2/Tc2 deviated immune response (196-198).

Collectively, the data enumerated above provides strong circumstantial evidence that various modes of immune tolerance, including immunological ignorance, may predominate at various stages of disease progression in HPV-associated malignancies. Therefore, the design of strategies capable of preventing and reversing tolerance and immunologic ignorance may be important additions to the tumor immunologist's arsenal.

4.6. Tolerance and immunotherapy

Tolerance is also an important consideration in the immunotherapy setting. For example, many mouse and human studies have demonstrated the great potential of peptide-based immunotherapeutic strategies; however, caution may be warranted as studies have shown that some peptides administered in IFA, due to their pharmacokinetics, may actually induce tolerance, thus promoting tumor outgrowth (199, 200). Consequently, studies in HLA congenic mice may be useful, not only for identifying immunogenic peptides, but in screening these peptides for their ability to induce tolerance. An alternative approach may be the development of strategies which prevent tolerance, thus converting an otherwise tolerogenic peptide into an immunogenic vaccine. For example, tolerization may be avoided by the presentation of antigenic peptides by peptide-pulsed DCs.

Chimeric VLPs, including HPV 16 E7/L1 or L2 fusions elicit an E7-specific CTL response capable of protecting immunized mice against subsequent challenge with E7-expressing tumors (201, 202). In addition, CVLPs may prevent the induction of tolerance. Nieland et. al. recently demonstrated that immunization of mice with a peptide derived from the nonmutated tumor antigen P1A in IFA induced tolerance leading to the outgrowth of a regressor P815 cell line. However, immunization of mice with a CVLP containing the same P1A peptide induced a protective immune response (203). It is unclear whether or not this may be explained by the ability of CVLPs to upregulate costimulatory molecule expression and cytokine secretion in immature dendritic cells which have taken up CVLPs (71, 204).

Although DCs have been exploited in the immunotherapy setting with promising results, the observation that DCs, particularly lymphoid DCs, may play a role in the induction of self-tolerance raises the possibility that vaccine preparations including DCs may be further optimized by the removal of any tolerogenic cells (205-211). For that reason, Ruedl et. al. compared the ability of myeloid and lymphoid DCs to prime a CTL response in vivo (212). Although both DC subsets were capable of eliciting a protective anti-viral CTL response, the possibility that a DC based vaccine may induce tolerance cannot be excluded given the different DC lineages and maturation states. The method used to load DCs with tumor antigens may be an additional consideration. For instance, necrotic tumor cells have been shown to be more immunogenic than apoptotic cells (213, 214), an observation that may be attributed, at least in part, to the ability of necrotic cells to stimulate DC maturation (215-218). In addition, DCs loaded with apoptotic tumor cells may induce tolerance (206, 207). The notion that DC based approaches to tumor immunotherapy are immune to the challenges posed by T cell tolerance may be naïve.

5. T-CELL COSTIMULATION IN IMMUNOTHERAPY

T cells require both antigen-dependent and independent (i.e. costimulatory) signals to become fully activated. Therefore, the administration of a tumor-associated antigen may be insufficient to eradicate established tumors in the absence of appropriate costimulation. On the other hand, immunogenic tumors capable of priming antigen-specific T cells may regress after those T cells receive an appropriate costimulatory signal. This may be achieved in a number of ways, including the expression of an appropriate costimulatory ligand by a tumor or by the systemic administration of agonistic monoclonal antibodies against any one of a number of costimulatory molecules. Manipulation of the growing repertoire of T cell costimulatory molecules may enable tumor immunologists to modulate the antitumor immune response in such a way so as to overcome some of the barriers, including those mentioned above, which prevent the induction and maintenance of a potent antitumor cell-mediated immune response.

Although the manipulation of many costimulatory receptors, including CD40 (219-223), OX-40 (224, 225), and CTLA-4 (222, 226-230), have already been demonstrated to play a role in the enhancement of the antitumor T cell response and/or in the prevention and reversal of tolerance, the remainder of this review will emphasize the role of 4-1BB (CD137) costimulation in the tumor immunotherapy setting.

5.1. 4-1BB (CD137) immune regulation

4-1BB is a member of the tumor necrosis factor receptor superfamily (TNFRSF) that is expressed in an activation-dependent fashion on CD8+ and CD4+ T cells, intraepithelial lymphocytes (IELs), NK cells, eosinophils, and endothelial cells (231-238). In vitro studies have demonstrated that both agonistic anti-4-1BB mAb and 4-1BB ligand (4-1BBL) can costimulate proliferation and cytokine secretion in both CD4+ and CD8+ T cells (239-241). However, studies performed in vivo suggest that 4-1BB may play a more prominent role in the generation of a CTL response than in a T-helper cell response. In fact, administration of anti-4-1BB mAb in mice may induce anergy in CD4+ T cells (242) or may even promote activation-induced cell death (243, 244). In contrast, the importance of 4-1BB costimulation in a purely CD4+ T cell response has been demonstrated in a graft versus host disease model (245). Therefore, the importance of 4-1BB costimulation in a CD4+ T cell response is not yet entirely clear; however, the importance of 4-1BB/4-1BBL interactions in the generation of a fully competent CTL response has been confirmed in studies performed in 4-1BBL deficient mice (246-248). From the tumor immunologist's point of view, the ability of agonistic anti-4-1BB mAb or 4-1BBL to eradicate established tumors in mouse models is of great interest (137, 249-255).

Although anti-4-1BB mAb may eradicate immunogenic tumors, the same treatment is considerably less effective or completely ineffective in the treatment of poorly immunogenic tumors. We have recently described two tumors expressing the HPV-16 E6 and/or E7 oncogenes. Both tumors may be lysed in vitro by E7(49-57)-specific T cells. However, in vivo, only one of the tumors is immunogenic, as demonstrated by both tetramer analysis and in vitro 51Cr-release assays for CTL activity. Not surprisingly, administration of anti-4-1BB mAb in tumor-bearing mice was capable of eradicating this tumor. In contrast, E7-specific T cells were neither deleted nor tolerized in mice bearing the second, less immunogenic tumor. In fact, no evidence of a T cell response to this tumor was observed in tumor-bearing mice, suggesting that the tumor may be ignored. Administration of anti-4-1BB mAb in these mice was completely ineffective. As T-cell priming is required to upregulate 4-1BB expression on antigen-specific T cells, the inability of anti-4-1BB mAb to overcome immunologic ignorance to this tumor may not be surprising. Although vaccination of tumor-bearing mice with the E7(49-57) peptide in incomplete Freund's adjuvant was incapable of eradicating established tumors, even though a CTL response was detected following vaccination, administration of anti-4-1BB mAb following vaccination was capable of overcoming immunologic ignorance to an extent capable of eradicating established tumors (256). This novel approach, which we have dubbed COPP (Costimulatory anti-4-1BB mAb Plus Peptide), was also successfully applied in lung metastases models using both B16-F10 and TC-1 tumor cells. It should also be noted that anti-4-1BB mAb was also affective in treating tumors when combined with an irradiated tumor-cell vaccine. Therefore, use of anti-4-1BB mAb may not be limited to peptide-based vaccines, but may be a useful addition to antigen-based immunotherapies, including tumor-cell vaccines and DC-based approaches. Immunotherapy with antigenic peptides and anti-4-1BB mAb may be an effective form of therapy in HPV-associated malignancies, many of which may be immunologically ignored.

Following COPP treatment, a significant increase in the number of E7-specific tumor-infiltrating lymphocytes (TILs) was observed at the site of regressing tumors. Although the mechanism involved is not yet known, many attractive hypotheses may be offered. First, 4-1BB stimulation may enhance the ability of T cells to migrate to the tumor site. Although the effect of 4-1BB stimulation on 4-1BB-expressing endothelial cells is unknown, the upregulation of adhesion molecules important in T cell trafficking is a possibility. Alternatively, 4-1BB costimulation has been shown to enhance the ability of T cells to adhere to components of the extracellular matrix in a b1-integrin-dependent fashion (257). Furthermore, 4-1BB stimulation may upregulate 4-1BB expression via a positive feedback loop (258, 259), and murine 4-1BB is capable of binding fibronectin (260). In both cases, any enhancement in a T cell's ability to adhere to the extracellular matrix may improve the cell's ability to traffic to a tumor or remain at the tumor site once there. Following peptide immunization, a significant increase in the clonal burst size was noted in the draining lymph nodes of mice following anti-4-1BB mAb treatment. Therefore, any increase in the number of TILs may be a mere reflection of this increase in clonal burst size. Finally, the ability of 4-1BB costimulation to alter the expression patterns of chemokines and their receptors on antigen-specific T cells has not been investigated, but may be a possibility worthy of consideration, as other costimulatory members of the TNFRSF have been shown to alter the expression of various chemokines and their receptors (261, 262).

Second, 4-1BB stimulation has been shown to prevent AICD (263-265). Therefore, the increased number of TILs observed after treatment may be explained by the prolonged survival of these T cells. Alternatively, 4-1BB stimulated T cells may escape AINR. AINR may be observed in the later phase of a CTL response that receives insufficient help from CD4+ T cells. T cells which escape AINR may be capable of proliferating upon encountering cognate antigen at the tumor site. In fact, we have demonstrated that the intravenous administration of high doses of antigenic peptide may induce AINR in antigen-specific CD8+ T cells. However, administation of anti-4-1BB mAb prevented the induction of AINR in this model. Furthermore, administration of peptide and anti-4-1BB mAb ten days following the induction of AINR was capable of reversing nonresponsiveness (Wilcox et. al., unpublished data). To our knowledge, anti-4-1BB mAb administration is the only therapy capable of reversing established AINR in vivo. The ability to reverse AINR may be extremely valuable in the immunotherapy setting. In summary, 4-1BB costimulation may promote the expansion, survival, and/or migration of antigen-specific T cells to sites of tumor growth, and may therefore represent an exciting new approach in the immunotherapy of HPV-associated malignancies.

Depletion studies in COPP-treated mice revealed that treatment was independent of CD4+ T cells, but was NK-cell dependent. Preliminary studies have revealed that 4-1BB-stimulated NK cells may play an important immunoregulatory role, raising the possibility that 4-1BB-stimulated NK cells may be able to compensate, at least in part, for a deficient CD4+ T cell response.

As previously described, many mechanisms may be invoked to explain immunological ignorance in the setting of HPV-associated malignancies. One potential mechanism, is the inefficient processing and presentation of HPV antigens by bone-marrow derived APCs, like the dendritic cell. Therefore, our recent observation that murine DCs express 4-1BB raised the possibility that 4-1BB stimulation may somehow alter the phenotype or function of the dendritic cell. With that in mind, we isolated splenic DCs from mice which had received anti-4-1BB mAb and were able to demonstate that these DCs were more potent stimulators of proliferation in both allogeneic and antigen-specific T cells (266). The mechanism involved in this apparent upregulation of the DCs ability to stimulate T cells following 4-1BB stimulation is currently under investigation and raises the possibility that the successful eradication of established tumors following anti-4-1BB mAb treatment may require 4-1BB stimulation of DCs. Although much remains to be learned, the ability of anti-4-1BB mAb treatment to, not only modulate the innate and adaptive immune responses, but also prevent and reverse AINR, are findings which, we believe, may prove useful in the treatment of HPV-associated malignancies.

6. CONCLUSION

With the identification of a vast number of tumor antigens has come great progress in the rational design of antigen delivery strategies capable of eliciting a detectable T cell response against HPV-associated antigens. Unfortunately, the goal of tumor immunotherapy is not the induction and subsequent detection of a tumor-specific T cell response, but tumor eradication and tumor-free survival. While the delivery of tumor antigens may induce a cell-mediated immune response, the regulation and maintenance of that response may be critical in tumor eradication. It remains to be determined whether or not more and better antigens alone are capable of achieving that goal. If not, immune regulation may be the next frontier in tumor immunotherapy.

7. ACKNOWLEDGEMENT

This work is supported by National Institutes of Health, Cancer Research Institute-New York, Natural Science Foundation of China, American Cancer Society, Amgen Inc., and Mayo Foundation. We would like to thank Julie Lau and Kathy Jensen for editing the manuscript and Drs. Koji Tamada and Andrei I. Chapoval for helpful discussion.

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Key Words: Papillomavirus, Cervical Cancer, Immunotherapy, CD137, Review

Send correspondence to: Dr. Lieping Chen, Professor of Immunology, Mayo Medical School and Consultant, Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, Tel: 507-538-0013, Fax: 507-284-1637, E-mail: chen.lieping@mayo.edu