T cell-mediated help against tumors

[Cell Cycle 7:19, 2974-2977; 1 October 2008]; ©2008 Landes Bioscience

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T cell-mediated help against tumors Thomas Wieder,1,† Heidi Braumüller,1,† Manfred Kneilling,1 Bernd Pichler2 and Martin Röcken1,* 1Department

of Dermatology; Eberhard Karls University; Tübingen, Germany; 2Laboratory for Preclinical Imaging and Imaging Technology of the Werner Siemens-Foundation; Department of Radiology; Eberhard Karls University; Tübingen, Germany

†These

authors contributed equally to this work.

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Key words: immune therapy, tumor dormancy, senescence, T helper cells, interferon γ, tumor vaccination, apoptosis

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cancer Willimsky et al.2 showed that tolerance to a tumor antigen occurs already at the premalignant stage, when the tumor is still dormant. Furthermore, the authors concluded from their results that tumor latency is unlikely to be caused by CTL, which indirectly strengthens the hypothesis that other immune cells, possibly CD4+ T helper (Th) cells, play a more central role in immunosurveillance than previously anticipated. Here we shortly review the role of CD4+ or Th cells in controlling tumor growth and discuss their future use in tumor therapy and prevention.

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Adoptive transfer of tumor antigen-specific T helper (Th) cells is a surprisingly potent anti-tumor therapy. Even in RIP1-Tag2 mice with a rapidly growing, aggressive endogenous β cell tumor Th can significantly extend life time and are more efficient than any other therapy studied. The therapeutic effect of Th cells seems to be independent of tumor cell destruction. It critically relies on three principles: (1) inhibition of tumor angiogenesis, (2) inhibition of β cell proliferation, and (3) induction of tumor dormancy. As tumor cell destruction by cytotoxic CD8+ T cells (CTL) largely failed in tumor therapy, induction of tumor dormancy by Th cell-mediated immune responses represents a novel therapeutic option that may be combined with other cytotoxic regimens, e.g., radio- and/or chemotherapy, as it is established for bone marrow transplantation. Importantly, Th cell efficacy strictly requires interferon γ (IFNγ) signaling, and in the absence of IFNγ, Th cells may even worsen tumor diseases. Therefore, using the immune system to control tumor dormancy represents a novel approach, especially as therapy of tumors resistant to conventional therapies. Yet, it is important to underline that Th cell-based antitumor effects critically depend on a functional cytokine network, especially appropriate IFNγ signaling.

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Th1 Cells, the Most Efficient T Cell Phenotype in Fighting Cancer?

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A number of clinical studies show that adoptive transfer of melanoma-specific CTL have little if any antitumor effects.3,4 Furthermore, strong tumor-specific CTL responses do not correlate with effective tumor protection, presumably by a mechanism that induces T cell tolerance.5 Nevertheless, indirect activation of CD8+ CTL still seems to be a promising approach for cancer treament. For example, Papewalis et al.6 showed that dendritic cell immunization with a tumor cell-specific polypeptide led to CTL-associated tumor regression in a transgenic mouse model developing medullary thyroid carcinoma. Serum calcitonin levels in calcitonin-vaccinated mice were lower than in the control group indicating that hormonproducing tumor cells were actively controlled by the immune response. One of the adverse reactions of the therapy is the killing of all antigen-bearing cells and subsequent hormon deficiency. Thus, the ideal tumor therapy would not induce destruction of all cells, but rather induce a stable state of tumor dormancy that prevents tumor escape. The frequent failure of CTLs to control cancer growth may be attributed to the development of T cell tolerance by induction of regulatory T cells, to antigen loss of the tumor, an important “tumor escape mechanism”, or to a direct counter strike of the attacked tumor cells (reviewed by Abrams).7 The insufficiency of CTL in tumor therapy was not entirely unexpected. Lessons from autoimmune diseases showed that IFNγ-producing Th (Th1) cells are by far more efficient and more aggressive in inducing organ-specific autoimmune disease than antigen-specific CTL.8 Based on these observations, Th1 cells were compared to IL-4 producing Th2 cells or CTL in cancer therapy in mice: even under conditions where CTL or Th2 cells fail to show any protection, TAA-specific Th1 cells

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Introduction

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Although fulminant clinical courses of tumor development have been described in the literature, especially in the case of pancreatic cancer,1 cancer development is often characterized by a long latency. This long term arrest of tumor growth is characterized by sometimes very long premalignant, clinically unapparent phases. Since most tumors evoke immune responses at some point of the disease, it seems likely that tumors are normally surveyed by specific cells of the immune system. However, different mechanisms, such as anergy induction, inactivation or deletion of tumor-specific cytotoxic T lymphocytes (CTL) or antigen loss of tumor cells, may accomplish that tumors eventually escape immunosurveillance and start to grow and to metastasize. In a mouse model of sporadic immunogenic *Correspondence to: Martin Röcken; Department of Dermatology; Eberhard Karls University; Liebermeisterstr. 25; 72076 Tübingen, Germany; Email: mrocken@med. uni-tuebingen.de Submitted: 08/15/08; Accepted: 08/15/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6798 2974

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provide solid protection against various transplanted or endogenous tumors.9,10 A case report from a patient with metastatic melanoma underlines this view: in a study with autologous CD4+ T-cell clones directed against the melanoma-associated antigen NY-ESO-1 one T cell clone provided sustained clinical remission; this T cell clone exhibited a Th1 phenotype.11

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Figure 1. Specific homing of Tag-specific Th1 cells into tumor-bearing pancreas. Pseudocolor fluorescence image of the pancreas three days after i.p. injection of Cy5-labelled Tag-Th1 cells into a tumor-bearing RIP1-Tag2 mouse. The fluorescence intensity reflects the number of infiltrating Tag-Th1 cells. Colored bar: the fluorescence intensity raises from blue to green, yellow and red.

Besiegement or Invasion?

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Other than tumor models using transplantation of monoclonal tumors, RIP1-Tag2 mice develop endogenously growing β cell tumors. All pancreatic β cells of RIP1-Tag2 mice express T antigen (Tag) from the Simian Virus 40 under the control of the rat insulin promotor (RIP). Tag inhibits the tumor suppressor p53 and retinoblastoma protein (Rb). RIP1-Tag2 mice therefore develop islet adenomas that progress into carcinomas12 leading to early death of the animals after 14–16 weeks.10,13 Apart from the time course, RIP1-Tag2 mice develop cancers that, like many human tumors, develop as a consequence of a defect in apoptosis. A combined chemotherapy plus anti-cathepsin protocol increased the 50% survival of the mice from 16 to 20 weeks.13 No other treatment approach achieved longer remissions, so far. Even CTL-mediated destruction of all pancreatic β cells, associated with severe increases in blood glucose, provides only limited tumor regression.14 In sharp contrast, treatment of RIP1-Tag2 mice with Tag-specific Th1 cells results in efficient tumor control, surprisingly without inducing any signs of diabetes. Even more surprisingly, when treatment was started at very early stages of malignant transformation, i.e., at 5 weeks of age, many mice lived healthy for more than half a year.10 Meanwhile, Th1 cell-based immune therapy seems to be by far the most efficient regimen in most tumor models analyzed. Detailed investigations in the Rip1-Tag2 mouse model show that tumor therapy with Tag-specific Th1 cells induces tumor dormancy, importantly without causing destruction of the target cells.

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RIP1-Tag2 Mice: Lessons from an Animal Model of Endogenous Multistage Carcinogenesis

Figure 2. Besiegement of tumors by Tag-specific Th1 cells. Autoradiographic image of the pancreas three days after i.p. injection of 64Cu-labelled Tag-Th1 cells into a tumor-bearing RIP1-Tag2 mouse. Note that the radioactive immune cells (T cells) do not invade the tumor but surround the diseased tissue.

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A major question will be to understand the biological principles underlying Th cell-mediated control of cancer. To address these questions, we started by following the migration of tumor associated antigen (TAA)-specific Tag-Th1 cells in vivo. We labelled Tag-Th1 cells with Cy5 fluorescent dye prior to adoptive transfer into RIP1-Tag2 mice. As shown in Figure 1, TAA-specific Tag-Th1 cells enrich in the diseased organ, i.e., in the pancreas of transgenic mice, and in the draining lymph node, whereas no TAA-specific Tag-Th1 cells home to the pancreas of nontransgenic C3H mice.10 Surprisingly, such Tag-Th1 cells surround the pancreatic β cell tumors, where they established lymph follicle-like structures. Active infiltration of the tumors, however, is a rare event (Fig. 2). Thus, it is very unlikely that Tag-Th1 cells destroy the tumor cells by direct T cell-cancer cell interactions, as it is postulated for CTL-mediated killing. The data currently available suggest that Th1 cells prevent tumor growth through cytokine signaling. Indeed, the antitumor effect mediated by Tag-Th1 cells is strictly dependent on two cytokine pathways: (1) on efficient IFNγ signaling and (2) on signaling of TNF through the tumor necrosis factor p55 receptor (TNFR1).10 Through the combined action of efficient IFNγ signaling and TNFR1 signaling Tag-Th1 cells induce the potent antiangiogenic www.landesbioscience.com

chemokines, interferon-inducible protein 10 (CXCL10) and monokine induced by IFNγ (CXCL9), and prevent the generation of new, αvβ3 intergrin-expressing endothelia. Thus, it is likely that Th1 cells prevent the angiogenic switch that is required for the progression of Tag-positive β cell adenomas into cancers. Even though the source of the antiangiogenic factors remains enigmatic, it is unlikely that the biologically relevant amounts of antiangiogenic chemokines are derived from the transfered Th1 cells. Other cells, such as the tumor cells themselves, macrophages or dendritic cells, or the endothelia are potential other sources.

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Th1 cells against cancer

striking findings.10 In RIP1-Tag2, several lines of evidence show that Tag-Th1 cells control tumor growth in the absence of significant killing of the target cells: (1) mice treated with TAA-Th1 cells do not develop diabetes, even though all islet cells express the same TAA. (2) The efficacy of Tag-Th1 therapy is independent of the physical presence of CD8+ CTLs. (3) Treatment with TAA-Th1 cells does not cause detectable signs of necrosis or apoptosis induction in vivo. This is reminiscent of the experimental treatment of mammary carcinomas in HER-2/neu transgenic mice with a combined allogeneic tumor cell vaccination plus systemic IL-12 administration, where the immune therapy did not only prevent tumor development but preserved normal functioning of the duct glands.21 Using transgenic models of endogenous tumor development, future experiments will focus on the mechanisms of tumor control by Th1 cells. It is currently believed that immune responses against endogenous tumors may delete all cells expressing appropriate TAA, while tumor cells that have lost immunogenic TAA may escape from immune surveillance and thus expand under improved conditions.22 Using MHC class I-lost mutants, we were able to demonstrate that this scene seems unlikely, as suppression of MHC class I rather promotes tumor cell recognition by natural killer cells.23 An alternative to the currently favored model of tumor escape is a concept, where specific immune cells may either induce tumor dormancy or promote growth and malignant transformation of tumors, depending on the cytokine milieu they provide. In line with this concept, Th1 cells induce tumor dormancy only when IFNγ and TNF are both present. In the case of immune responses, where either the IFNγ signaling or the TNF signaling are impaired, the same immune response promotes tumor progression and possibly even malignant transformation and antigen loss of the tumor cells, in mice10 and possibly also in humans.24 Thus, it is mandatory to uncover the mechanisms underlying both the induction of tumor-dormancy and/or the induction of tumor escape and tumor progression by specific immune cells.

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Besides preventing tumor angiogenesis, Tag-Th1 cells also significantly blunt tumor cell proliferation without increasing tumor cell apoptosis, through an yet unknown mechanism. Future work has to focus on microdissection techniques to isolate β cells, fibroblasts, endothelia, T cells or macrophages from RIP1-Tag2 tumors. Comparative analysis of these populations will shed light on the cellular sources of the antiangiogenic factors and the antiproliferative mechanisms. RNA array techniques will allow to investigate the differences in gene expression profile of silenced and proliferating insulinoma cells. In this respect, arrays of p53- and nuclear factor κb (NFκb)-dependent genes, or of apoptosis- and cell cycle-related genes will be of special interest. Moreover, it will be of interest, whether such mechanisms are unique to Th1 cell-mediated inhibition of RIP1-Tag2 tumors, or whether such rules also apply to other endogenous or even transplanted tumors. In line with this, Quin and Blakenstein15 found in a transplanted model tumor that inhibiting tumor growth with TAA-Th1 cells prevents tumor angiogenesis in a stricly IFNγ-dependent fashion; thus, Th1 cellmediated inhibition of tumor angiogenesis seems to reflect a general mechanism. As the current state of the literature suggests that tumor angiogenesis is crucial for tumor development in general,16,17 and as the antiangiogenic activity of Tag-Th1 cells strictly correlates with the pronounced tumor growth inhibition, future work should answer whether the antiangiogenic, IFNγ-mediated action of Tag-Th1 cells alone is capable of preventing tumor cell growth, as suggested by some authors,18 or whether TAA-Th1 cells can also exert a direct, IFNγ- and/or TNF-dependent, and biologically relevant antiproliferative effect on tumor cells, as suggested by others.10,19,20

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Figure 3. Possible mechanisms of tumor growth control by Tag-specific Th1 cells. CD4+ Th1 cells are primed in the draining lymphnode and then home into the tumor-bearing pancreas where they secrete IFNγ. Depending on the cytokine network, the same Th1 cells either induce tumor dormancy (A) or enhanced growth of tumor cells (B). For details, see text. DC, dendritic cell; IFNγ, interferon γ; IP-10, interferon-inducible protein 10; MIG, monokine induced by IFNγ; MΩ, macrophage; Tag, T antigen; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor p55 receptor.

Destruction, Growth Inhibition, or Reeducation? The high efficiency of TAA-Th1 cells in cancer treatment in the absence of tumor cell destruction is currently one of the most 2976

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Depending on the target antigen, Th1 cells either cause autoimmune diseases, e.g., psoriasis,25,26 or control severe diseases, such as infections with intracellular pathogens27 or tumors.10 Yet one struggling difference remains between autoimmune diseases and cancer: while Th1 cells may cause tissue destruction in the case of autoimmune encephalitis,8 Th1 cell-mediated prevention of tumor progression does not involve detectable signs of destruction.10 The factors, decisive for such strictly opposing effects of Th1 cells remain elusive. There are at least three possibilities: (1) the decision is taken by the target organ itself, (2) the microenvironment, e.g., fibroblasts, endothelial cells, macrophages etc., in tumors and healthy organs react differently to Th1 cells, or (3) the cytokine network in the different clinical settings becomes tuned either towards destruction or education.

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In the present perspective, we summarized recent work dealing with the antitumoral effect of Th1 cells. Based on our work, we propose the following working hypothesis (Fig. 3A): antigenpresenting dendritic cells in the pancreatic lymph node stimulate transfered Tag-Th1 cells that, subsequently, migrate to the diseased pancreatic islets, where they secrete high amounts of IFNγ. IFNγ then induces the potent antiangiogenic factors CXCL9 and CXCL10, presumably indirectly, by stimulating macrophages. Thus, active inhibition of tumor angiogenesis reduces the proliferation of developing insulinoma cells and keeps tumors in a dormant state. Yet, when the anti-angiogenic effect of IFN or TNF is impaired, the same immune response creates an inflammatory milieu that dangerously promotes tumor progression (Fig. 3B). These insights pave the way for a combination of cellular immune therapy with a chemotherapy regimen. In view of the current literature, the adoptive transfer of TAA-Th1 cells seems to be the most promising immune approach, best in combination with either radiation or chemotherapy, similar to minigrafts established for hematologic malignancies.

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9. Egeter O, Mocikat R, Ghoreschi K, Dieckmann A, Röcken M. Eradication of disseminated lymphomas with CpG-DNA activated T helper type 1 cells from nontransgenic mice. Cancer Res 2000; 60:1515-20. 10. Müller-Hermelink N, Braumüller H, Pichler B, Wieder T, Mailhammer R, Schaak K, et al. TNFR1- and IFNγ-signaling determine whether T Cells induce tumor dormancy or promote multistage carcinogenesis. Cancer Cell 2008; 13:507-18. 11. Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R, et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med 2008; 358:2698-703. 12. Casanovas O, Hicklin DJ, Bergers G, Hanahan D. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 2005; 8:299-309. 13. Bell-McGuinn KM, Garfall AL, Bogyo M, Hanahan D, Joyce JA. Inhibition of cysteine cathepsin protease activity enhances chemotherapy regimens by decreasing tumor growth and invasiveness in a mouse model of multistage cancer. Cancer Res 2007;67:7378-85. 14. Speiser DE, Miranda R, Zakarian A, Bachmann MF, McKall-Faienza K, Odermatt B, et al. Self antigens expressed by solid tumors do not efficiently stimulate naive or activated T cells: implications for immunotherapy. J Exp Med 1997; 186:645-53. 15. Qin Z, Blankenstein T. CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFNgamma receptor expression by nonhematopoietic cells. Immunity 2000; 12:677-86. 16. Naumov GN, Akslen LA, Folkman J. Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch. Cell Cycle 2006; 5:1779-87. 17. Sessa C, Guibal A, Del Conte G, Rüegg C. Medscape. Biomarkers of angiogenesis for the development of antiangiogenic therapies in oncology: tools or decorations? Nat Clin Pract Oncol 2008; 5:378-91. 18. Blankenstein T, Qin Z. The role of IFNgamma in tumor transplantation immunity and inhibition of chemical carcinogenesis. Curr Opin Immunol 2003; 15:148-54. 19. Lynch RA, Etchin J, Battle TE, Frank DA. A small-molecule enhancer of signal transducer and activator of transcription 1 transcriptional activity accentuates the antiproliferative effects of IFNgamma in human cancer cells. Cancer Res 2007; 67:1254-61. 20. Conti L, Regis G, Longo A, Bernabei P, Chiarle R, Giovarelli M, et al. In the absence of IGF-1 signaling, IFNgamma suppresses human malignant T-cell growth. Blood 2007; 109:2496-504. 21. Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E, Cavallo F, et al. Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice. J Exp Med 2001; 194:1195-205. 22. Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, et al. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 2001; 410:1107-11. 23. Mocikat R, Braumüller H, Gumy A, Egeter O, Ziegler H, Reusch U, et al. Natural killer cells activated by MHC class I(low) targets prime dendritic cells to induce protective CD8 T cell responses. Immunity 2003; 19:561-9. 24. Erfurt C, Sun Z, Haendle I, Schuler-Thurner B, Heirman C, Thielemans K, et al. Tumorreactive CD4+ T cell responses to the melanoma-associated chondroitin sulphate proteoglycan in melanoma patients and healthy individuals in the absence of autoimmunity. J Immunol 2007; 178:7703-9. 25. Ghoreschi K, Thomas P, Breit S, Dugas M, Mailhammer R, van Eden W, et al. Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease. Nat Med 2003; 9:40-6. 26. Ghoreschi K, Weigert C, Röcken M. Immunopathogenesis and role of T cells in psoriasis. Clin Dermatol 2007; 25:574-80. 27. Biedermann T, Zimmermann S, Himmelrich H, Gumy A, Egeter O, Sakrauski AK, et al. IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nat Immunol 2001; 2:1054-60.

Acknowledgements

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References

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The work of the authors is supported by the Deutsche Forschungsgemeinschaft (SFB 685), the Wilhelm Sander-Stiftung (2005.043.1), and the Deutsche Krebshilfe (107128). The authors want to thank N. Bauer for excellent technical assistance.

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