Using Context to Drive Tumor Antigen Cross-Presentation Towards Useful Antitumor Immunity

Review

Cranking the Immunologic Engine with Chemotherapy: Using Context to Drive Tumor Antigen Cross-Presentation towards Useful Antitumor Immunity

Robbert G. van der Most, Andrew Currie, Bruce W.S. Robinson, and Richard A. Lake

School of Medicine and Pharmacology and Western Australian Institute for Medical Research, University of Western Australia, Sir Charles Gairdner Hospital, Nedlands, Perth, Australia

Abstract notherapy but that the transition from cross-presentation to cross- This review shows how tumor antigen cross-presentation is priming needs help, either from CD4 T helper cells or via targeted affected by the major therapeutic modalities including immunotherapy. The combination of chemotherapy with its chemotherapy, radiotherapy, and surgery. We argue that this capacity to alter the rate of cross-presentation with immunotherapy process could affect the way that a tumor works as its own may therefore present an excellent opportunity to achieve this. cellular vaccine, and that it is differentially modulated by the choice of treatment. (Cancer Res 2006; 66(2): 601-4) Which Antigens Are Cross-Presented? The process of antigen cross-presentation was discovered in 12 It is estimated that tumors typically accumulate 10 or more 1976, when it was shown that immunization with lymphoid cells mutations (1). Even though many of these were already present in congenic for minor histocompatibility antigens (7) or with the tissue cells from which the tumor originated and only a small allogeneic or semisyngeneic SV40-transformed cells (8) resulted proportion occur in the open reading frames of genes expressed by in the generation of specific CTLs that were restricted by host MHC tumor cells, it is inevitable that any given cancer will express at class I molecules. Thus, antigen must have been transferred from least a few new antigenic determinants that could be recognized by the donor cells to the hosts APCs. Now, almost 30 years later, it is the immune system. Despite this, the immune system rarely stops evident that cross-presentation of tumor and self-antigens is a very established tumor growth. It is likely that the same tolerance efficient process, but it is still not entirely clear where the antigen mechanisms that prevent immune responses against normal tissue comes from. Controversy persists over the question of whether live, cells expressing random mutations also block antitumor responses. apoptotic, or necrotic cells are the best source of cross-presented Similar to self-antigens, tumor neo-antigens are constitutively and antigen. Also it is not clear how antigen transfer takes place and efficiently offered to the immune system through a process known what form of antigen is required. Answering these questions will be as cross-presentation (2, 3). The fact that tumor neo-antigens are important for the design of new immunotherapeutic strategies. likely to be highly individuated suggests that the most promising Particulate and cellular antigens are cross-presented much more approaches to immunotherapy will depend on our ability to turn a efficiently than soluble antigens (3), suggesting that antigen is tumor into its own cellular vaccine (4). captured by phagocytosis. Several different mechanisms have been proposed to explain antigen transfer from donor cells to APC. What Is Cross-Presentation? These mechanisms include phagocytosis of apoptotic bodies, Most cells, including many tumor cells, express MHC class I nibbling from live cells and transfer of antigen by molecular molecules and can present antigenic peptides to T cells. However, chaperones such as heat-shock proteins (Hsp). A related issue is only professional antigen-presenting cells (APC), such as dendritic whether the transferred antigen consists of mature proteins, cells (DC) and macrophages, have the capacity to prime immune peptides, or defective ribosomal products, which are known to be responses. These cells acquire antigen from donor cells such as the main source of antigenic peptides for the cells’ endogenous tumor cells, then present the captured antigens via their own MHC MHC class I processing pathway (9). Current evidence implicates class I molecules to CD8 T cells in a process known as antigen cross- mature proteins as the primary source of cross-presented antigen. presentation (3). However, the precise mechanisms are still First, treating donor cells with irreversible proteasome inhibitors controversial (5), and, importantly, the relevance of tumor antigen blocks direct antigen presentation but enhances cross-presentation cross-presentation for the induction of antitumor responses has (10). Second, epitopes located within cleaved leader sequences are been questioned (6). In this review, we will discuss the molecular not cross-presented, suggesting that short-lived peptides cannot basis for cross-presentation, which we judge to be a constitutive enter the cross-presentation pathway (11). On the other hand, data but limited process and describe the basis of the subsequent from Binder and Srivastava indicate that when cell lysates are used immune response; cross-priming, when a response is measurable as a source of antigen, transfer depends on Hsps (12). This suggests versus cross-tolerance, when it is not. We will argue that cross- that Hsp-chaperoned peptides can enter the cross-presentation presentation of tumor antigens is highly relevant for tumor immu- pathway, presumably via receptor-mediated endocytosis (12). The issue is further complicated by another study implicating mature proteins as the source of cross-presented antigen in cell lysates (13).

Requests for reprints: Richard A. Lake, School of Medicine and Pharmacology and Irrespective of the mechanism, it is evident that tumor antigens Western Australian Institute for Medical Research, University of Western Australia, 4th are efficiently cross-presented to the immune system (Fig. 1). The Floor, G Block, Sir Charles Gairdner Hospital, Nedlands, Perth 6009, Australia. Phone: question, therefore, is why the immune system fails to respond in 61-8-9346-3127; Fax: 61-8-9346-2816; E-mail: [email protected]. I2006 American Association for Cancer Research. an adequate fashion because tumors usually induce only relatively doi:10.1158/0008-5472.CAN-05-2967 weak immune responses. www.aacrjournals.org 601 Cancer Res 2006; 66: (2). January 15, 2006

What Does the Immune System Do with Cross- This is important for tumor immunity because tumors often Presented Antigens? masquerade as normal tissue. In an elegant series of experiments, Sherman and coworkers showed that when the influenza virus HA Cross-presented antigens do not necessarily induce an immune gene is transgenically expressed in the islets of Langerhans, it is response (3). Cross-presentation is involved in the maintenance constitutively cross-presented in the draining lymph nodes. In the of tolerance to self-antigens (cross-tolerance) as well as in the in- absence of any other signals, HA-specific CD8 T cells proliferate duction of immune responses (cross-priming). In both situations, but fail to acquire effector functions and are eventually deleted a subset of DCs that, at least in the mouse, expresses CD8 is most (16). In other words, tolerance is maintained. Adding the likely responsible for cross-presentation (3). We will now deal with proinflammatory cytokine IL-12 and a costimulatory signal in the question of how such APC ‘‘decide’’ whether the outcome of the form of an agonistic anti-CD40 antibody, triggered acquisition cross-presentation is tolerogenic or immunogenic. of effector functions by HA-specific CD8 T cells and led to islet Recent data indicate that the difference between tolerance and infiltration of these T cells (16). immunity depends on the presence or absence of inflammatory Taken together, the data suggest that cross-presented antigens signals associated with viral or bacterial pathogens, as well as on are likely to promote tolerance, or partial activation, in the absence costimulatory signals. Pathogen-associated molecular patterns of inflammatory signals or costimulation. In contrast, cross- (PAMP), such as lipopolysaccharide, double-stranded RNA, and priming occurs only when antigen is cross-presented in the unmethylated CpG motifs, are recognized by specific receptors, context of microbial infection or costimulation, or in the presence including the Toll-like receptors. Through their receptors, PAMPs of cognate CD4 T cell help. activate DCs and promote immune responses (14). In addition, soluble mediators, including the type I IFNs stimulate immune responses to cross-presented antigens (15). Why Does the Immune System Fail to Attack The necessity to activate DCs by external stimuli such as PAMPs, Tumors? costimulatory signals, and IFN-a/h suggests that the default action The two key features that differentiate tumors from somatic of such DCs is to maintain tolerance to cross-presented antigens. self in this regard are (a) antigenicity and (b) rate of growth. So it

Figure 1. Tumors constitutively present antigens via DCs (1). Chemotherapy increases the rate at which these antigens are cross-presented, but in the absence of any further signals, there is only weak T cell priming. APC-directed immunotherapy (2) promotes efficient cross-priming and can drive the activation and expansion of antigen-specific T cells (3).

Cancer Res 2006; 66: (2). January 15, 2006 602 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. From Cross-Presentation to Cross-Priming is reasonable to ask how important is the process of antigen rarely adequate to stop tumor growth. Thus, the challenge for cross-presentation in the development of an immune response to therapeutic intervention is to change the context in which tumor tumors? The observation by Trinchieri et al. that SV40-trans- antigens are cross-presented into a more inflammatory one. This formed tumor cells induce SV40-specific CTL that are restricted could be achieved by activating Toll-like receptors with, for by the host’s MHC class I molecules showed for the first time that example, CpG-containing oligonucleotides or poly I:C, by using not only are tumor antigens cross-presented, but also that they cytokines such as IL-12 or IFN-a/h or by providing additional can induce specific responses (8). It is now clear that in the same costimulatory signals such as agonistic anti-CD40 antibodies. All of way as normal self-antigens, tumor-derived antigens are consti- these approaches have been tested with some success in animal tutively cross-presented in draining lymph nodes (17–20). Cross- models and, to some extent, in human clinical trials. presentation of tumor antigens has also been shown in cancer A different strategy is to use such immunotherapeutic patients who received a vaccine comprised of irradiated allogeneic strategies in combination with conventional therapeutic modal- tumor cells (21). The rest of this section will deal with the issue of ities, such as chemotherapy, radiotherapy, and surgery (4). This how cross-presented tumor antigens are perceived by the immune approach has two advantages. A first, practical, advantage is system and what regulates whether cross-presentation results in that it takes into account the clinical reality. Integration of tolerance or immune activation? immunotherapy into standard treatment regimens should The evidence indicates that the immunogenicity of cross- facilitate its implementation. Second, conventional treatment presented tumor antigens ranges from tolerogenic (20) to weakly could enhance the efficacy of subsequent immunotherapy. For immunogenic (19). Using HA-transfected mesothelioma cells, instance, many chemotherapies kill cells by apoptosis and Marzo et al. showed that the HA protein is cross-presented in apoptotic cells are a good source of cross-presented antigen the tumor-draining lymph nodes and triggers a weak, transient (3). Indeed, work from our laboratory revealed that the massive cytotoxic T cell response (19). Subsequent experiments revealed tumor cell apoptosis induced by chemotherapy leads to that weak in vivo CTL activity was sustained over 4 weeks of increased levels of cross-presentation (4, 22). This process could tumor growth (22). Thus, cross-presentation in this tumor model unmask additional tumor neo-antigens by increasing the amount seems to be a cross-priming rather than a cross-tolerizing event. of material available for cross-priming (Fig. 1). In these studies, Nevertheless, cross-priming does not prevent tumor progression. subsequent immunotherapy, using an agonistic anti-CD40 Sherman and coworkers reported slightly different results using antibody, cured 80% of tumor-bearing mice, when neither a tumor model in which transgenic mice expressed both the HA therapy was effective on its own (23). Although this combination protein and the SV40 large T antigen under the control of the rat of therapies was not effective for very large tumors, the same insulin promoter (20). These mice spontaneously develop 80% rate of cure was achieved when partially resected large pancreatic h-cell tumors. The authors then adoptively transferred tumors were then treated with the chemotherapy/immunotherapy HA-specific CD8 T cells into tumor-bearing mice and found that combination (24). These experiments suggest that the increased the acquisition of effector functions by these T cells depended on levels of tumor neo-antigen cross-presentation after chemother- the number of cells transferred. Adoptive transfer of low numbers apy could be exploited by immunotherapy. Importantly, these of HA-specific T cells resulted in proliferation without effector results contest the notion that cross-presentation of antigen function. Armed CD8 effector T cells were only observed when from apoptotic cells is a priori a tolerogenic event (25). We very high numbers of HA-specific precursor T cells were injected. hypothesize that the massive apoptosis induced by chemother- Because only small numbers of tumor-specific CD8 T cells would apy could release proinflammatory mediators such as Hsps and be present under normal conditions, it can be argued that tumor IL-6, which promote cross-priming. Our current understanding antigen cross-presentation is nominally tolerogenic in that of antigen cross-presentation predicts that any therapy that the tumor does not provide the inflammatory signals needed delivers higher levels of cross-presented tumor antigens to the to support differentiation of CD8 T cell precursors into effector draining lymph nodes could synergize with immunotherapy cells (20). (Fig. 1). As a case in point, it has recently been shown that In summary, tumor antigens are efficiently cross-presented but it tumor destruction by radiofrequency ablation creates a source is less predictable how CD8 T cells will respond to these antigens. of antigen for the induction of antitumor immunity by immu- The examples that we have discussed illustrate this (19, 20, 22). The notherapy (26). variable levels of cross-priming, in the face of quite robust cross- In conclusion, the therapeutic exploitation of cross-presented presentation, may also provide an explanation for Zinkernagel’s tumor neo-antigens to boost antitumor immune responses shows conclusion that tumor antigen cross-presentation is of little that a tumor could be its own cellular vaccine. This idea relevance for the induction of CD8 T cell responses against tumor represents a paradigm shift because the historical emphasis in antigens (6). Indeed, there are many examples in which tumors fail tumor immunotherapy has been on the identification of and to cross-prime, as originally noted by Bevan in 1976 using p815 vaccination with defined tumor-associated antigens (27). We mastocytoma cells (7), and as shown by Ochsenbein et al. for a propose that in order to transform a tumor into its own vaccine, wider range of tumors (6). However, lack of cross-priming does not we need to crank the immunologic engine by using chemother- exclude cross-presentation, it may simply indicate an opportunity apy to increase the level at which tumor antigens are cross- for therapeutic intervention (20). presented (Fig. 1). The spark that fires the motor could be supplied by proinflammatory adjuvants that are directed at the APC (4). Implications for Therapy Cross-presentation does not necessarily lead to cross-priming, and even when cross-presented tumor antigens do prime Acknowledgments antitumor immune responses, it seems that these responses are Received 8/19/2005; revised 9/22/2005; accepted 11/18/2005. www.aacrjournals.org 603 Cancer Res 2006; 66: (2). January 15, 2006

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cranking the Immunologic Engine with Chemotherapy: Using Context to Drive Tumor Antigen Cross-Presentation towards Useful Antitumor Immunity

Robbert G. van der Most, Andrew Currie, Bruce W.S. Robinson, et al.

Cancer Res 2006;66:601-604.

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