Cancer Immunol Immunother (1998) 46: 75 ± 81
Ó Springer-Verlag 1998
SYMPOSIUM IN WRITING
Edith M. Lord ? John G. Frelinger
Tumor immunotherapy: cytokines and antigen presentation
Received: 10 October 1997 / Accepted: 12 January 1998
AbstractmIncreasing the ability of tumor-reactive T cells to mediate tumor regression in vivo has been a major goal of tumor immunologists. Progress toward this goal has been aided by the identification of tumor-associated antigens on both experimental mouse tumors and human tumors. However, the self-like nature and low immunogenicity of these antigens has made it clear that other measures to enhance the effectiveness of the T cells reactive to these antigens are essential if immunotherapy is to be clinically effective. An increased understanding of antigen processing and presentation is an important step in this process, as is the use of cytokines to increase immune responsiveness. Despite recent advances, there is still much to be learned before the specificity of the immune system is safely harnessed to halt malignant cell growth effectively. Key wordsmCytokines ? Antigen presentation Immunotherapy ? Cytolytic T lymphocytes
Introduction
Control of tumor growth presents a difficult problem for the host immune system. A major advance in recent years has been the clear identification of tumor-associated antigens. Tumors, in contrast to invading virus or bacteria, express few foreign antigens and are thus basically self. The immune system, which has evolved to resist foreign inThis article forms part of the Symposium in Writing on ªTumor and dentritic cells as cellular vaccines: confrontation and perspectivesº, published in this issue (vol 46, no 2) of the journal This work was supported in part by grant IM-493 from the American Cancer Society and grants CA28332 and CA70218 from the National Institutes of Health E.M. Lord ( ) ? J.G. Frelinger Cancer Center Immunology Program and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA Fax: +1 716 461 4019 E-mail:
[email protected]
vaders, appears to be much less well-equipped to deal with the slight differences between normal and malignant cells. Even in melanoma, one of the most immunogenic tumor types, many of the antigens that have been identified have been found to be self or only slightly modified self-antigens (reviewed in [44]). Thus, it is perhaps not surprising that stimulation of tumor-reactive lymphocytes has proven difficult. However, lack of immunogenicity of tumor-associated antigens alone is not solely responsible for the inability of the immune system to generate an effective antitumor response. Even in experimental tumor models, in which a foreign antigen is inserted to serve as a model ªtumor antigenº, the immunogenicity of the altered tumor cells is often increased only slightly, and the tumors continue to grow progressively (Fig. 1). Thus, even with an expressed foreign antigen, the immune system finds it difficult to eradicate a rapidly growing tumor. This strongly suggests that simply expressing a foreign protein is insufficient to engender a strong immune response, and other characteristics of tumors must be critical in limiting the generation of an effective antitumor response. We will focus on approaches to this problem that have utilized genetically engineered tumor cells, that secrete cytokines as reagents to alter the tumor cell microenvironment, and examine the subsequent immune response. As we will see, these factors can act at either the afferent or efferent arm of the immune response. In this review we will examine some of the issues that we believe play a significant role in generating an effective immune response within the unique microenvironment of a growing tumor. Of course, even within a growing tumor that has been genetically engineered to secrete a single cytokine, the in vivo cytokine milieu is complex. The tumor cells themselves may already secrete cytokines and growth factors, the identity of which depends upon the type of cell that has undergone transformation. For example, many melanoma cells secrete a variety of cytokines including interleukin-1 (IL-1b), IL-6 and IL-8 [28]. In addition, many tumor cells secrete transforming growth factor-b (TGFb), a pleiotrophic cytokine that can serve as a growth-stimulatory
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Fig. 1mLine 1 (Ð) or line 1 human-prostate-specific-antigen-expressing (hPSA) (- - -) cells (104) were injected intramuscularly into the hind limb of (A2/Kb ´ BALB/c) F1 mice and tumor growth was measured at regular intervals. Each line represents tumor growth in an individual animal
factor for many cell types [21]. Further, TGFb can have important down-regulatory effects on the immune system, including inhibition of the generation of cytotoxic T lymphocytes (CTL) [27]. Interestingly, we have found that, by engineering tumor cells to secrete IL-2, this inhibition by tumor-produced TGFb can be overcome in vivo [29]. These types of results illustrate that the combination or balance of cytokines expressed can determine the ultimate outcome: tumor growth or tumor rejection. Further, it has been appreciated that the infiltrating host cells can dramatically affect the cytokine milieu within the tumor. Indeed, this probably changes with time, and the chronic situation within a human tumor may be different from the acute short-term situation that we generally observe in fastgrowing rodent tumors.
Enhancement of CTL activation by cytokine secreting tumor cells
One method for enhancing the response to relatively weak antigens has been to provide a source of additional cytokines. Cytokines are designed to provide transient, shortrange signaling between cells. Thus, in studies of antitumor immunity, systems in which the cytokines are expressed locally within the microenvironment of the tumor provide high concentrations at the antigen site and avoid the potential side-effects that can result when cytokines are delivered by systemic administration. Experimentally, this has been achieved by transfecting tumor cell lines with genes for different cytokines. These genetically altered cell lines have been used in basically two ways. One has been to use irradiated cells as immunogens in which the cytokine serves as an adjuvant for the killed tumor cells. The effects of this immunization are then assessed by measuring the animals' response to subsequent challenge with parental cells. The second is to inject viable tumor cells allowing them to secrete cytokine within the growing tumor, thus
using these as a probe for how they alter subsequent events. A large number of these studies have been done in a variety of systems. Most informative have been the experiments in which multiple cytokines have been compared within a single tumor system. One example of using irradiated cytokine-transfected cells is the study by Dranoff et al. [11] in which B16-F10 melanoma cells were transfected with one of ten different cytokines. In this study, granulocyte/macrophage-colony-stimulating factor (GM-CSF) stood out as the cytokine providing the most protection against subsequent challenge with parental cells. In vivo depletion of either CD4+ or CD8+ cells abrogated the protection, although only minimal CTL activity was detectable in splenocytes from the immunized animals. Using the second experimental approach, Musiani et al. [31] have examined the cellular events that take place at the site of challenge with TSA mammary adenocarcinoma parental cells or the same cells engineered to produce one of eight different cytokines. They observed intense contacts between granulocytes, macrophages, fibroblasts and lymphocytes, which resulted in tumor rejection. Interestingly, they found a similar picture when the tumors were transfected with the suicide gene, cytosine deaminase, and the animals were treated with 5-fluorocytosine, which results in the selective destruction of the transfected tumor cells. They concluded that the process of tumor growth and regression was a very effective immunogen irrespective of cytokine production. In our experiments [30] we transfected the BALB/c lung carcinoma, line 1, with different cytokines, and examined the effect on tumor growth and the ability to generate nonspecific and specific CTL effector cells at the tumor site. Local production of IL-2 and IL-3 resulted, in both cases, in an increase in the number of specific CTL at the tumor site, while other cytokines increased only the nonspecific effector cells. Tumor regression in the absence of cytokine production was also examined in this system, in which cells were transfected with herpes thymidine kinase and the animals treated with ganciclovir. Tumor regression due to the drug treatment was accompanied by the appearance of nonspecific effector cells, but not specific CTL. Although the experiments are not directly comparable, since different tumor systems were used, specific CTL were apparently generated in the TSA system, in that the mice that had rejected the tumor exhibited a protective memory response that was at least partially due to CD8+ T cells. Thus the inherent immunogenicity of a tumor may be significant in generating specific immunity. It is obvious from these experiments, as well as many others, that both specific and nonspecific immunity are involved in the rejection of cytokine-transfected tumor cells. The effectiveness of these responses in causing tumor rejection may be more a function of the aggressiveness of the tumor cell line used than a measure of the magnitude of the immunity generated. Thus, the generation of effector cells or development of protective memory may be a more effective measure of effectiveness than rejection of the primary tumor. Development of quantitative measures to access accurately the type and extent of the
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immune response generated is essential in order to obtain information that will be translatable to clinical situations.
Direct compared to indirect antigen presentation
One of the original concepts behind the use of cytokinetransfected tumor cells was to provide the help that might normally be supplied by CD4+ T cells or other accessory cells. Indeed, the positive effects observed with IL-2 in a wide range of systems support the notion that help of the type normally supplied by CD4+ cells is limiting. The implication was that this cytokine was providing additional stimulation to CD8+ cells that were reacting to antigen/ MHC complexes presented directly by the tumor cells themselves. However, the stimulatory effects we observed with IL-3 and those observed by other investigators with GM-CSF [11] had a somewhat different implication, since these cytokines were primarily stimulators of myeloid progenitor cells rather than T cells. These results led to the revival of the idea of ªcross-primingº first described by Bevan [5] and Gooding and Edwards [14], in which antigens expressed by one set of cells are taken up by ªprofessionalº antigen-presenting cells (APC) and ªre-presentedº to T cells. Consideration of direct and indirect antigen presentation reveals fundamental differences in how we approach the development of tumor vaccines. If direct presentation is the process, then vaccines must provide not only tumor antigens, but also MHC molecules of the appropriate type as well as any other necessary costimulatory molecules. This approach has spurred the various attempts to make tumor cells better APC. Several investigators have transfected tumor cells with either B7-1 or B7-2 co-stimulatory molecules and shown that these cells are very effective immunogens (reviewed in [1]). However, expression of these molecules has not proven to be sufficient in all tumor models. Effectiveness appears to correlate with the immunogenicity of the tumors studied [10], and thus one wonders whether this approach, at least in its simplest form, will be effective for human tumors, which are generally not immunogenic. Although, in many of the tumors studied, evidence has been obtained indicating that CD8+ cells are involved in the rejection process [15], in other systems, CD4+ cells have been shown to play an important role [3, 22]. For example, in the SaI tumor, expression of both MHC class II and B7-1 results in optimal stimulation of antitumor immunity [3]. However, in other systems, with tumors of low immunogenicity, B7-1 is effective only in inducing natural-killer-cell-mediated cytotoxicity [17, 45]. Ample evidence also exists indicating that indirect presentation of tumor antigens by host APC occurs and may be an efficient means of eliciting specific antitumor immunity. Huang et al. [16] demonstrated, in a bone marrow chimera system, that GM-CSF-transfected tumor cells elicit antitumor responses resulting from antigen presentation by cells derived from the host bone marrow, and not by the tumor cells themselves. Similarly, we have
demonstrated that IL-3-transfected line 1 (H-2d) tumor cells elicit an increased population of host APC in F1 (H-2bxd) mice that can effectively present antigen and generate H-2brestricted CTL [36]. Our earlier work in this system demonstrated that this effect was dependent on CD4+ cells [35]. Interestingly, Heath and co-workers [4] recently demonstrated that CD4+ cells were required for crosspriming of CD8+ CTL to ovalbumin by host APC, which is consistent with our finding.
Generation of host antigen-presenting cells
The realization that a limiting feature in the generation of cell-mediated immunity to tumors is effective presentation of antigen to lymphocyte precursors has led to a variety of experiments designed to enhance this activity. Central to this approach has been the use of cytokines either in vivo or in vitro to increase the number and/or efficiency of these APC. Both dendritic cells and macrophages have been demonstrated to be very effective at processing and presenting tumor antigens. Perhaps most significant has been the realization that phagocytes can present exogenous antigens via their class I MHC antigens. Thus, such cells have the capability of stimulating not only class-II-restricted CD4+ T cells but also CD8+ CTL precursors (reviewed in [37]). This appears to be a property of cells that can ingest antigens by macropinocytosis and/or phagocytosis [19, 32, 34]. Particulate antigens, such as those complexed to 1- mmsized beads, appear to be handled particularly well by these cells, such that extremely small amounts of antigen can be presented efficiently to T cell precursors [19, 24, 34]. There is considerable controversy regarding the exact nature of the cells that perform this process in vivo, with macrophages and dendritic cells (DC) being the major contenders. The elegant studies of Rock and colleagues [19, 37] as well that of others [32] make it obvious that cells and cell lines with macrophage characteristics can very efficiently perform this function. Our own studies with IL-3 indicate that this cytokine, when expressed by gene-transfected tumor cells, can stimulate a population of host cells within the tumor that can efficiently present a tumorassociated antigen whether it is in a secreted or nonsecreted form [36]. The cell type responsible for this activity was visualized at the electron microscope level by its close association with an activated T cell hybridoma and also characterized by cell-surface phenotype. It appeared to have characteristics more similar to those of macrophages than those of classical DC. Macrophages have many of the characteristics expected of APC involved in antitumor immunity. They are highly phagocytic and thus capable of readily ingesting tumor fragments, they can perform macropinocytosis and they are present within tumors in abundance. However, their ability to traffic effectively to lymph nodes draining a tumor site and thus presenting antigen to a wide range of lymphocytes is not well-documented.
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Dendritic cells are widely regarded to be the most effective antigen-presenting cells and have been stated to be the only APC capable of presenting antigen to naive T cells (reviewed in [2] and [39]). Although thought to be somewhat less phagocytic than macrophages, they are clearly capable of ingesting large amounts of material by macropinocytosis and by mannose-receptor-mediated uptake [38]. Their presence within tumors has been reported [8, 18, 42], but it is not clear whether the markers used to identify DC are indeed absolutely specific for these cells. It is interesting that in one study examining tumors in rats, the DC isolated from the tumors were CD8+, and a population of mouse CD8+ DC has recently been described [40] as regulatory DC that can down-regulate T cell responses. They are, however, capable of trafficking to lymph nodes and are found within both T and B cell areas of these secondary lymphoid organs. DC are a relatively rare cell population and have been difficult to isolate from lymph nodes. However, with the use of cytokines, most notably GM-CSF in combination with IL-4 and/or TNFa, it is now possible to expand this cell population from both murine and human progenitor cells. This has created the possibility of obtaining a patient's DC from blood or bone marrow, exposing them to tumor antigens, and reinfusing them back into the patient. To date, this has only been attempted using peptide-pulsed DC, which does not address the ability of the DC to process tumor antigens. Thus, this technique might not be applicable for less-well-defined tumor antigens and the ability of such cultured cells to traffic effectively has not yet been demonstrated. This is also a
Fig. 2mSchematic representation of the possible pathways of tumor antigen presentation to host T lymphocytes. Presentation may occur within the tumor (A) or within draining lymph nodes (B). Tumor cells are shown as large, shaded cells.T T lymphocytes, APC antigenpresenting cells
potentially expensive and lengthy process. However, it would insure that the antigens were presented in the correct MHC context. The current controversy regarding the best type of APC (DC or macrophages) may become a moot point as we learn more about the ontogeny and differentiation of these cells. Although there may be very clear differences between these two cell types at certain stages of differentiation, at other stages it appears to be difficult, if not impossible, to distinguish between them. Indeed, both cell types appear to be able to differentiate from common CD14+ progenitor in humans [6, 41]. Further, they appear to be much more plastic in their differentiation than was originally thought, in that the microenvironment in which they find themselves and the cytokines available may markedly affect the differentiation pathway used (reviewed in [33]). What we call these cells would appear to be far less important than what they are capable of doing. An important and interesting question is where this antigen presentation actually occurs and whether there are ways to make this process more efficient. There are only limited data available that directly address this issue, and several scenarios are possible. These are shown schematically in Fig. 2. Presentation of antigen to T cells could occur
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primarily in the tumor (path A) or in the lymph nodes draining the tumor site (path B). At either site, antigen could be presented directly by the tumor cells themselves or indirectly by APC. Presentation within the tumor would require naive T cells to traffic into the tumor, a process that may be very inefficient. Direct presentation by the tumor cells themselves would also likely be inefficient, as most transformed cells fail to express the co-stimulatory molecules needed for efficient stimulation of naive T cells. Similarly, if presentation within the tumor is performed by infiltrating APC, they would also need to be expressing these co-stimulatory molecules. In one study, examining a regressing rat tumor, the inflammatory cells infiltrating the tumors failed to express B7 molecules [9]. The authors concluded that presentation of tumor antigen must be occurring at another tumor site. The macrophage-like cells we have identified to be present in increased amounts in IL-3-transfected tumors can indeed present the model tumor antigen (ovalbumin) to ovalbumin-specific T cells, and these APC express B7 [36]. Thus, indirect presentation within the tumor by host APC is certainly possible. Alternatively, if presentation is occurring within the lymph node, then tumor cells or antigen must be transported into the node (path B). One possibility is that APC infiltrate into the tumors and engulf either soluble antigens secreted from viable tumor cells or fragments of tumor cells resulting from tumor cell death within the tumor. These antigenbearing APC could then migrate to draining lymph nodes where they would encounter and stimulate the large numbers of naive CD8+ or possibly CD4+ T cells. If these cells are migrating from the tumors, then they should be capable of stimulating tumor-antigen-specific T cells in the absence of any added antigen, and they should be present within the lymph nodes prior to the presence of T cells within the tumors. Conclusive experiments testing this have not yet been reported. Since tumor cells themselves can also metastasize to the lymph nodes, direct presentation by tumor cells within the lymph nodes is also theoretically possible. However, since lymph node metastases are associated with poor prognosis, it is unlikely that this results in efficient T cell stimulation. We favor the idea that APC infiltrating into the tumor pick up and process tumor antigen, migrate to the draining lymph nodes and present antigen to the circulating T cells within the well-organized structure of the node. Although data supporting tumor antigen presentation within the lymph nodes are limited, experiments investigating autoimmunity and transplantation have provided evidence that localization of antigen within organized lymphoid tissue must occur for a response to be generated (reviewed in [46].
Infiltration and function of effector cells
Fundamental to tumor growth is the development of a system of blood vessels that is capable of supplying the rapidly dividing tumor with oxygen and other nutrients and removing waste products. This development of new blood
vessels or ªangiogenesisº is essential for tumor progression. However, unlike normal tissues, the development of vessels within tumors often does not follow an orderly progression. As a result, vessel formation within tumors is often somewhat disorganized and, as a consequence, there are often areas within the tumors that are not adequately vascularized. This disorganized vascularization of tumors creates a paradoxical situation. On the one hand, the limited vascular system probably slows the overall growth of the tumor as oxygen and essential nutrients limit tumor cell proliferation. On the other hand, it also creates microenvironmental conditions within the tumor that make standard therapies more difficult. If the cells in the poorly vascularized areas are without oxygen for long enough, they will die. However, at low levels of oxygen, the hypoxic cells become quiescent and noncycling. As a result of the low oxygen and their altered state, these cells are more resistant to standard tumor therapies such as radiation and chemotherapy. Cells within these poorly vascularized and hypoxic areas of tumors are also likely to be less susceptible to lysis by CTL simply because the T cells traffic less effectively to these areas [20], and the adverse conditions may affect T cell function [23]. The ultimate usefulness of cellular immunotherapy against solid tumors will probably require additional measures to make them more accessible to reactive host cells. Interestingly, cytokines and angiogenic factors produced by both the malignant cells and the infiltrating host cells appear to play an important role [13].
Immunotherapy
Central to the concept of understanding how tumor antigens are presented is determining how this process can be improved to generate tumor-specific cytotoxic cells more effectively. The recent giant strides in identifying antigens on human tumors [44] have made it feasible to use these peptides for vaccinations. However, it should be remembered that, in most cases, the individual has been exposed to the antigens on a growing tumor for some time, without mounting an effective immune response; there may even be an induced tolerance. Thus, if any positive effects are to be seen, the antigen will have to presented in a more immunogenic form. Despite this caveat, immunization with free peptides from the MAGE-3 antigen has induced regression in two cases of metastatic melanoma [26]. Peptides given in adjuvant have resulted in CTL generation in many cases [12, 25], but in other instances they have engendered tolerence [43], resulting in enhanced tumor growth. It would appear that a clear understanding of how these peptides are affecting the immune system is essential before immunizations of this type are used generally in the clinic. An alternative approach is to pulse APC with peptides or tumor fragments. This has been used successfully in animal tumor models with bone-marrow-derived APC. In one case [7], DC were prepared from bone marrow by culturing in GM-CSF, pulsed with ovalbumin peptide and used to immunize mice. The mice generated CD8+ anti-ovalbumin
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CTL and were protected against subsequent challenge with ovalbumin-expressing tumor cells. In another instance, bone marrow cells stimulated in vitro with IL-3 and pulsed with tumor cell fragments were able to transfer immunity [45]. Thus, the transfer of APC may be a very effective method of delivering antigen efficiently, even when a peptide has not been identified. As our experimental models improve and we understand more about tumor antigens and how they are viewed by the host immune system, our ability to translate these approaches to humans should also improve. However, no single animal model can perfectly replicate the human disease, and it is unlikely that late-stage disease will be curable by immunotherapy. Thus, it is important, as clinical trials are initiated, that they be designed to maximize our understanding of the efficacy of the vaccines or treatments used. This will ensure that progress towards understanding the interactions between host immune cells and malignant cells continues, even in the absence of cures.
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