Interference with the Induction of TAAspecific Responses

Tumors are known to interfere with the early stages of the immune response by targeting cellular and molecular mechanisms responsible for its initiation and subsequent progression. Molecular Signaling in the Tumor Microenvironment

Cells initiating immune responses that are driven by activating and co-stimulatory signals communicate with one another. However, normal signal transmission is disrupted or diverted in the tumor presence. Both murine and human tumors are known to interfere with different molecular pathways which mediate cellular cross talk [104, 129]. In addition to tumor cells, the tumor microenvironment contains stromal fibroblasts, infiltrating hematopoietic cells and blood vessel elements [193]. These normal cells were viewed as a scaffold necessary for tumor cell expansion (the stroma) or a manifestation of host antitumor activity (infiltrating leukocytes). Only recently molecular interactions between these cells and the tumor came to be recognized as regulatory elements of cell growth vs. cell death. Among them, the death receptor/ligand and NFkB activation pathways also play a key role in immune cell differentiation and functions [201]. However, the signaling immune cells encounter in the tumor milieu is distinct from that in normal tissues, and it does not favor the induction of TA-specific responses.

(a) Paucity and diversion of co-stimulatory signals

Immunohistology studies show that human tumors lack or down-regulate co-stimulatory molecules [75]. Although tumor cells are not "professional" APC, insufficient expression of molecules such as B7.1or B7.2 may result in tolerance, because the second signal necessary for T-cell activation is lacking [176]. If DC conditioned by the tumor have reduced levels of co-stimulatory molecules, their ability to fully activate T cells is diminished. Thus, tumors are inefficient in supporting T-cell differentiation and proliferation. In effect, DC in the tumor are locked in an immature state. Further, malignant cells express molecules such as programmed death ligands 1 and 2 (PD-L1 and PD-L2), which can interact with corresponding death receptors (PD-1 or PD-2) present on activated T cells and induce their apoptosis [118]. Also, PD-L1 and PD-L2 facilitate preferential development of CD25+CD152 (CTLA-4+) T suppressor cells and promote functional anergy [118]. Signaling processed via PD-L1 (B7-H1) has been shown to promote tumor progression in both murine and human SCID models [61], and its expression on tumors has been correlated with poor prognosis in humans [167]. CTLA-4, a homologue of CD28, is constitutively expressed on activated T cells and Treg. In the presence of DC cross-presenting TA and rich in B7.1 and B7.2 in the tumor microenvironment, activated T cells up-regulate CTLA-4. Because CTLA-4 molecule functions as a key negative regulator of CD28-dependent T-cell responses, such CTLA-4 upregulation on activated T cells results in the inhibition of Th1-type immunity and polarization to Th-2-type responses [164]. Evidence from animal models of tumor growth indicates that negative CTLA-4 signaling is in a large part responsible for functional paralysis of T cells in the tumor microenvironment [115].

(b) Death receptor/ligand signaling

Death ligands and their receptors, especially the Fas/FasL or TRAILR/TRAIL, are expressed on tumor cells and on activated immune cells [49, 200, reviewed in 201]. Immune cells expressing membrane-associated FasL and/or TRAIL are thus "armed" to eliminate death receptor-positive tumor cells and mediate immune surveillance [51, 100]. Death ligands, especially TRAIL, expressed on NK cells infiltrating tissues such as liver, contribute to protection from metastasis [51, 71, 101]. On the other hand, death ligands expressed on tissue cells maintain the immune privilege of the anterior chamber of the eye or testis, protecting them from immune interference [97, 162]. Death ligands present on the surface of tumor cells are used as means of achieving immune privilege. Best known as the "Fas-counterattack hypothesis," this concept is based on the demonstration that FasL+ colon cancer cells could induce Fas-mediated apoptosis of activated T cells in vitro [112, 113]. Expression of FasL in human tumors in situ has been confirmed by many studies [201, 209] and linked to Fas/FasL-mediated apoptosis of tumor-infiltrating lymphocytes (TIL) as well as to a poor prognosis for breast, ovarian, colon and liver cancers [9, 13, 28, 48, 89, 157]. While FasL+ tumor cells can counterattack immune cells, they have evolved tactics for protection from apoptosis by blocking signals delivered via Fas via intracellular inhibitors of apoptosis (IAPs) [203]. The overall results favor the tumor and interfere with antitumor immunity.

Surprisingly, FasL+ tumors are not protected from rejection in vivo. When implanted in experimental animals, FasL+ tumor cells are rapidly rejected due to a massive infiltration of the graft by granulocytes [103]. This suggests that FasL is a pro-inflammatory molecule, which promotes inflammatory cell infiltration into tissues [88, 93]. FasL has the ability to induce inflammatory gene expression in tissue-resident immune cells, especially macrophages, which leads to the release of TNF-a and IL-8, cytokines with potent pro-inflammatory activities [4, 22, 40, 181]. Pro-inflammatory cytokines, acting as autocrine or paracrine mediators, up-regulate death receptor (Fas, TRAILR) expression and death ligand (FasL, TRAIL) secretion from tumor/tissue cells, shifting signaling from the pro-apoptotic to pro-inflammatory mode. Death ligands can play diametrically opposing roles in tumor growth. On the one hand, their expression on the tumor confers immune privilege and promotes metastasis. However, they can also induce potent inflammatory responses leading to tumor eradication. The tumor takes advantage of this dual functionality of death ligands by consistently shifting the balance toward pro-tumorigenic effects. In the tumor microenvironment, immune cells are at a disadvantage, having been corrupted to produce factors favoring tumor growth.

(c) NFkB signaling

The developing tumor is a site of chronic inflammation [15]. It has been suggested that progression to malignancy is regulated at the level of NFkB signaling and pro-inflammatory cytokines [83, 122]. NFkB, a ubiquitous transcription factor, may be the link between cancer and inflammation [122]. NFkB, is regulated differently in normal vs. malignant tissues [83, 122]. In the former, NFkB regulates expression of various cytokines and the process of inflammation. In the latter, NFkB stimulates proliferation of tumor cells and inhibits their apop-tosis [122]. In most normal cells, NFkB complexes are present in the cytoplasm but remain inactive due to their interaction with inhibitor of NFkB (IkB). Upon cell activation, IkB kinases phosphorylate IkB, which is degraded and frees NFkB to translocate to the nucleus, where it regulates gene transcription. During inflammation, activation of NFkB is initiated by, e.g. binding of TNF-a to its receptor (TNFR1) expressed on inflammatory cells, and it induces regulated expression of cytokine genes, which control cell migration, proliferation and death. In chronic inflammation, the microenvironment favors overproduction of pro-inflammatory cytokines by infiltrating as well as local tissue cells. Such deregulated cytokine secretion is driven by the NFkB activation in tumor cells.

Many neoplastic cells show constitutive NFkB activation, which contributes to abnormal proliferation, resistance to apoptosis and disease progression [83, 120]. NFkB activation is already present in pre-malignant tissue cells, leading to establishment of the pro-inflammatory environment [120, 199]. As the tumor develops, tumor cells which depend on cytokines and growth factors for survival, either produce and secrete them (autocrine regulation) or reprogram leukocytes found in the tumor microenvironment to produce them (paracrine regulation). Responding to this cytokine storm, tumor and stromal cells produce a panoply of soluble factors with biologic effects ranging from enhancement of cell proliferation, matrix remodeling, vessel growth, inhibition of apoptosis or cellular differentiation to sustained release of pro-inflammatory mediators [15]. NFkB is also implicated in the promotion of metastasis by regulating cell adhesion and migration [83]. This microenvironment is assiduously maintained by the tumor, promotes tumor growth and invasiveness but is suppressive to immune cells and, in concert with tumor progression, assumes the features of chronic inflammation.

Tumors have the ability to usurp normal biologic process of inflammation to promote tumor progression. The prominent role of TNF-a in this process has been emphasized [102]. The NFkB pathway can either promote survival of malignant cells by inhibiting apoptosis or sustain the production of cytokines necessary for tumor growth and tissue restructuring, thus mediating opposite biologic effects. Signals available in the milieu, including FasL and/or TRAIL, undoubtedly contribute to this molecular diversion, promoting tumor escape. The tumor, manipulating molecular circuits to its advantage, "never heals," and the overall anti-apoptotic character of the milieu promotes tumor progression. Whether NFkB is a friend or foe of cancer cells is currently a central question of tumor biology [119]. It is becoming clear, however, that not only NFkB but other signaling molecules have dual functions, and the tumor can capitalize on this biologic redundancy. Dysfunction in TAA Cross-presentation by Dendritic Cells

The key role of DC in cross-presentation of TAA to T cells is well known [14, 156]. Dysfunction of DC cross-presenting TAA to T cells could lead to an inadequate or biased antitumor immune response. DC not only process and present TAA to T cells but are important sources of IL-1, IL-12, IL-15, IL-18, IL-23 and Interferons, among other cytokines and chemokines. They are also rich in co-stimulatory molecules necessary as second signals or in growth factors for T-cell differentiation, proliferation and memory development [46, 186]. Therefore, if DC are depleted or unable to perform normally, the induction of TAA-specific immunity is likely to be impaired. Indeed, inhibition of DC in the presence of tumors has been reported [1, 65]. Defective maturation of DC in the tumor microenvironment may be mediated by tumor-derived vascular endothelial growth factor (VEGF) [44]. Others report that tumor-derived exosomes interfere ex vivo with differentiation of human DC from peripheral blood monocytes [149]. Tumor-derived gangliosides interfere with expression of inducible proteasomal components of antigen processing machinery (APM) in DC [149, 168]. Expression of APM components in DC is likely to determine the functional potential of this cellular mechanism necessary for presentation of cell surface-bound HLA-peptide-02 microglobulin complexes to cognate T cells. Downregulation of APM component expression in DC co-incubated with apoptotic tumor cells (HNC, melanoma) has been observed, and it may contribute to poor induction of antitumor immune responses in cancer patients [189]. Apoptosis of DC in the Tumor Milieu

It has been recently shown that elimination of DC or DC precursors in the tumor microenvironment is an important element of tumor-induced immune suppression and may, in part, contribute to a deficient antitumor immune response in cancer patients [37, 150, 151]. The molecular pathways that may be involved include: (1) downregulation in DC of the anti-apoptotic Bcl-2 family proteins [38, 124]; (2) accumulation of ceramides which could interfere with PI3K-mediated survival signals [38, 123]; (3) production by the tumor of NO, which suppresses expression of cellular inhibitors of apoptosis proteins (cIAPs) [38] or cFLIP. Analysis of gene and protein expression in DC and DC precursors in the tumor microenvironment has demonstrated that expression of several intracellular signaling molecules is reproducibly altered in DC co-incubated with tumor cells, including IRF2, IL-2Ry, Mcl-1, and small Rho GTPases among others. It appears that both intrinsic and extrinsic apoptotic pathways are involved in tumor-induced apoptosis of DC, as determined by an increased resistance to apoptosis of DC genetically modified to overexpress XIAP, caspase-8, Bcl-xL or FLIP [123]. Finally, it has been shown that DC genetically engineered to overexpress Bcl-xL-induced strong antitumor immune responses and inhibited tumor growth in murine tumor models in vivo [123]. Taken together, these data suggest that protection of endogenous DC in cancer patients should significantly augment the generation of host antitumor immune responses and consequently inhibit tumor growth.

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