Adaptive Antitumor Responses

The adaptive antitumor response is typically initiated by DCs, which capture dying tumor cells, process the antigenic cargo for MHC class I and II presentation, migrate to draining lymph nodes, and stimulate antigen specific T and B lymphocytes. In the tumor microenvironment, DCs may be activated by "danger signals" released from stressed or necrotic tumor cells, thereby triggering a maturation program that includes expression of multiple costimula-tory molecules and cytokines that result in effector T cell responses (Matzinger, 2002) (see Chapter 4). Alternatively, the production of immunosuppressive cytokines, such as TGF-p, IL-10, and VEGF, in the microenvironment may inhibit DC function, instead yielding abortive T cell effector responses and the augmented regulatory T cell function (Gabrilovich, 2004).

Productive antitumor CD4+ T cell responses promote potent and long-lasting CD8 + T cell responses and contribute to further DC maturation (Hung et al., 1998). These functions are accomplished in part through the secretion of a broad array of cytokines, including IFN-y, IL-4, IL-5, IL-6, IL-10, and IL-13. CD4+ T cell CD40 ligand expression may trigger CD40 signaling on DCs, resulting in enhanced IL-12 production and the differentiation of IFN-y secreting Th1 cells and cytotoxic T lymphocytes.

CD4 + T cells also stimulate B cell production of antitumor antibodies. These may include immunoglobulins that block growth or survival pathways dependent on cell surface receptors, such as Her-2/Neu. Antibodies may also target innate immune components to the tumor microenvironment, where specific cytolytic mechanisms may be unleashed. These include complement fixation, with resultant membrane disruption, and antibody-dependent cellular cyto-toxicity mediated by NK cells, macrophages, and granulocytes. Antibodies might also opsonize tumor cells for DC Fc receptor-mediated cross-presentation of tumor antigens, leading to the induction of additional CD4+ and CD8+ T cell responses (Dhodapkar et al., 2002). Studies have illuminated differences in the affinities of

IgG subclasses to bind the array of activating and inhibitory Fcy receptors expressed on DCs, which influences the balance of effector versus tolerizing function (Nimmerjahn and Ravetch, 2005). In this regard, IgG2a and IgG2b display higher affinity than IgG1 and IgG3 for the activating Fcy IV receptor and are more potent for antibody-dependent cellular cytotoxicity. Together, these properties resulted in superior suppression of B16 melanoma growth in vivo. These findings should advance the development of therapeutic monoclonal antibodies and provide a framework for clarifying the roles of endogenously produced antitumor antibodies.

A. Adaptive Immunity in Immunosurveillance

The importance of adaptive immunity for tumor immunosurveillance was first established through studies of mice harboring targeted mutations of the recombinase-activating gene 2 (RAG-2), which is required for immunoglobulin and T cell receptor gene rearrangement (Shankaran et al., 2001). Since the assembly of B and T lymphocyte antigen receptors requires RAG-mediated double-stranded DNA breaks to initiate V(D)J immunoglobulin gene recombination, RAG-2-deficient mice lack all B lymphocytes, ap- and yS-T cells, and NKT cells. These mice manifested an increased susceptibility to chemical carcinogen-induced tumors, and the fibrosarcomas arising in these animals were frequently rejected upon transplant to wild-type animals. Subsequent studies of mice deficient in ap- or yS-T cells alone revealed a similar enhanced susceptibility to chemical carcinogens, highlighting the key roles of T lymphocytes in tumor protection. Consistent with these findings, the development of intratumoral T cell infiltrates in multiple cancers is correlated with the absence of early metastasis and prolonged disease-free survival. The adaptive immune system, however, may also promote carcinogenesis within the background of chronic inflammation. In transgenic models of hepatitis B-induced liver cancer, smoldering CD4+ and CD8 + T cell responses are required for progression of hepatocellular carcinoma (Nakamoto et al., 1998). Similarly, CD4 + T cells activated by normal cutaneous bacterial flora promote the evolution of squamous cell carcinoma in a human papilloma virus transgenic model (Daniel et al., 2003).

Tregs may play an important role in modulating the dual roles of adaptive immunity in tumor protection and promotion (Dranoff, 2005). Substantial evidence in multiple tumor models indicates that Tregs present a major impediment to cytotoxic T cellmediated tumor rejection, particularly in the context of therapy-induced responses. Indeed, the presence of Tregs defined by the expression of the specific marker FoxP3 in ovarian cancer patients is tightly linked to inferior clinical outcomes (Curiel et al., 2004). On the other hand, Tregs function to maintain immune homeostasis, and their disruption leads to severe autoimmune disease and chronic inflammation. These functions may underlie the striking ability of Tregs to effectuate tumor destruction in murine models of inflammation-induced cancer (Erdman et al, 2005). Moreover, the presence of Tregs in the cellular infiltrates of Hodgkin's lymphomas has been linked to improved survival, perhaps reflecting a dependence of Reed-Sternberg cells (a unique characteristic of this disease) on particular components of the host response.

B lymphocytes similarly play dual roles in tumor immunosurveillance. While antibodies may promote tumor destruction through the mechanisms discussed earlier, in the HPV transgenic model of cutaneous squamous cell carcinoma, antibodies were required for disease development (de Visser et al, 2005). In this setting, antibodies promoted the recruitment of innate immune cells to the tumor microenvironment, where persistent inflammation promoted car-cinogenesis. Additional investigations are required to delineate the key mechanisms that determine if adaptive immune responses will inhibit or accelerate tumor development.

B. Targets of Antitumor T Cell Responses

The principal mechanism involved in the priming of antitumor T cells appears to be cross-presentation of tumor-associated antigens by professional antigen presenting cells, particularly DCs. This pathway allows processing of exogenously acquired tumor antigens into the MHC class I pathway, whereas proteins captured through endo-cytosis or autophagy are typically processed for MHC class II-restricted presentation. Tumor-associated antigens can be broadly divided into several categories, including cancer-testes antigens, which show restricted expression in adult germ cells but frequent upregulation in cancers, mutated proteins, differentiation antigens, and pathogen-encoded sequences, such as Epstein-Barr virus in some B cell lymphomas (Boon and van der Bruggen, 1996). Studies have unveiled an unexpected complexity to tumor antigen processing for CD8+ cyto-toxic T cells. Tumor infiltrating lymphocytes were specific for epitopes generated from posttranslational splicing of peptides derived from fibroblast growth factor 5 in renal cell carcinoma or gp100 in melanoma (Hanada et al, 2004; Vigneron et al, 2004).

Notwithstanding these striking examples of tumor-specific antigens, most of the gene products that stimulate endogenous responses in tumor-bearing hosts are non-mutated and expressed in some normal tissues. In these cases, the tumor-reactive T cells are of lower affinity because thymic deletion often purges the repertoire of high-affinity T cells with potential autoreactivity. Overexpressed self-antigens may also stimulate CD4+CD25+ regulatory T cells, recently demonstrated for the heat shock protein J-like 2 and the cancer-testis antigen LAGE-1 (Nishikawa et al, 2005; Wang et al., 2004). Together, these mechanisms result in intrinsic and extrinsic modes of T cell tolerance, which limit the overall potency of the antitumor T cell response. One possible way to avoid tumor antigen-specific tolerance is to identify recurrent mutations or novel epitopes arising from protein splicing, which could be incorporated into therapeutic strategies to augment antitumor T cell responses. However, a high priority is the elucidation of these tolerance mechanisms and the development of methods to bypass them (as described in more detail in Chapters 14-20).

C. Antitumor Effector Mechanisms 1. Cytokines

IFNs IFN-y plays a key role in tumor suppression (Dunn et al., 2004). NK, NKT, and yS-T cells are major sources of IFN-y early during tumor development, whereas CD4 + and CD8+ T cells may become additional sources as adaptive immunity evolves. IFN-y contributes to tumor protection in multiple ways, including inhibition of angiogenesis, the induction of phagocyte cytotoxicity, and the stimulation of DC IL-12 production, which in turn promote Th1 and cytotoxic T cell responses. Mice with targeted mutations of IFN-y or downstream signaling components established a major role for this cytokine in protection from chemically induced and spontaneous tumors. Moreover, IFN-y functions as the master regulator of tumor cell immunogenicity. Whereas methylcholanthrene-induced tumors that arose in RAG-2-deficientmice were efficiently rejected upon transplantation into wild-type mice, tumors that developed in mice doubly deficient for RAG-2 and the IFN-y receptor manifested robust growth after transplant into wild-type mice. The immunogenicity of these tumors was enhanced through the restoration of MHC class I presentation, identifying CD8+ T cells as a major component of IFN-y-stimulated tumor suppression.

A requirement for type I interferons (IFN-a/P) in tumor suppression has also recently been defined (Dunn et al., 2005). Mice with targeted mutations of the type I

IFN receptors or wild-type animals administered neutralizing antibodies to type I IFNs both manifested enhanced susceptibility to chemical carcinogenesis or tumor transplantation. Protection in these systems involved host immunity and p53 tumor suppressor function in cancer cells (Takaoka et al., 2003). Thus, IFN-y and IFN-a/p mediate critical but distinct functions in tumor immunosurveillance.

IL-12 and IL-18 Upon activation, phagocytes secrete IL-12 and IL-18, which in turn stimulate innate and adaptive cells to produce IFN-y and thereby contribute to tumor suppression. Mice deficient in p40, a subunit shared by IL-12 and IL-23, manifest an increased susceptibility to chemical carcinogens. IL-12 enhances NK and NKT cell antitumor activities through NKG2D and pfp-dependent pathways (Smyth et al., 2005). In contrast, IL-18 augments NK cell cytotoxicity in a NKG2D-independent fashion that involves through Fas ligand-mediated killing. Thus, IL-12 and IL-18 elicit both overlapping and distinct pathways for tumor protection.

IL-2, IL-15, and IL-21 IL-2 potently activates the antitumor effector functions of both innate and adaptive cytotoxic cells. The systemic infusion of high doses of recombinant IL-2 or the adaptive transfer of NK cell and CD8 + T lymphocytes stimulated ex vivo with IL-2 can evoke durable tumor regressions in a small minority of patients with advanced melanoma and renal cell carcinoma (Rosenberg, 2001). IL-2-induced tumor immunity involves both NKG2D-dependent pathways and pfp-mediated killing. In addition, IL-2 is critical for immune homeostasis, as mice deficient in the cytokine or signaling components succumb to chronic inflammatory disease due to defects in the maintenance of FoxP3-expressing regulatory T cells. Thus, IL-2 may also contribute to tumor protection through the control of inflammation-driven carcinogenesis.

The closely related cytokine IL-15 is critical for NK cell and memory CD8+ T cell homeostasis. The ability of IL-15 to amplify proximal TCR signaling in memory CD8+ T cells overcomes tolerance in a TCR trans-genic model of tumor-induced anergy (Teague et al, 2006). Constitutive IL-15 expression, however, may contribute to tumor promotion, as IL-15 transgenic mice succumbed to growth-factor-induced NKT cell leukemias (Fehniger et al., 2001).

The IL-21 receptor shares the common y-chain subunit that is also employed by the IL-2 and IL-15 receptors. IL-21 promotes NK cell differentiation from hematopoietic progenitors and induces their functional maturation, manifested by increased cell size, granularity, proliferation, and expression of activating receptors (Parrish-Novak et al., 2000). The therapeutic administration of IL-21 enhances the rejection of B16 melanomas and MCA205 fibrosarcomas through NK cell activities that involve NKG2D and IFN-y. Collectively, IL-2, IL-15, and IL-21 play complementary roles in tumor surveillance.

IL-23 and IL-17 IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. While activated macrophages and DCs produce both IL-12 and IL-23, these cytokines trigger distinct downstream effector pathways. IL-12 promotes the development of IFN-y-secreting Th1 cells, whereas IL-23 supports the expansion and activation of Th17 cells, a recently discovered CD4+ T subset that is critical for tissue inflammation. Consistent with these differences, the production of IL-23 promotes tumor cell growth and invasion through upregulation of MMP9, COX-2, and angiogenesis, whereas IL-23 deficiency attenuates tumor formation through a reduction in inflammation (Langowski et al, 2006). IL-23 also restrains protective immunity through inhibiting the intra-tumoral localization of Th1 cells and cytotoxic CD8 + lymphocytes. In some systems, though, IL-23 might also evoke protective antitumor responses, in part mediated by activated granulocytes, which as discussed earlier can trigger tumor cytotoxicity. Further investigations are required to understand the multiple roles of IL-23 in tumor surveillance.

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