Tumour immunosurveillance : the spontaneous recognition and elimination of tumours by T cells.

In the 1950s, it was shown that the immune system could recognize and respond to tumor-associated antigens^ref. However, reports in the 1970s that nude mice do not demonstrate an increase in tumor incidence were considered by some to contradict the hypothesis of tumor immunosurveillance^ref. The hypothesis was rejuvenated by Robert Schreiber (St.Louis, USA) who reported an increase in the incidence of chemically induced tumors, and wider spectrum of tumors deficient in the tumor suppressor p53, in mice defective in responsiveness to IFN-g^{ref1, ref2, ref3}. Those studies also could explain results obtained using nude mice that, though deficient in lymphocytes, have IFN-g-producing innate immune cells, such as natural killer (NK) cells. Papers from Mark Smyth (East Melbourne, Australia) have provided further evidence for immune surveillance and have shown that perforin-mediated killing is an important mechanism in tumor immunity^{ref1, ref2, ref3}. Perforin functions as a tumor suppressor in mice that have a variety of molecular genetic defects, including v-abl transgenic mice and mice deficient in mlh1 (a DNA-mismatch repair gene) and p53, that, when crossed with perforin-deficient mice, produce offspring that have increased plasmacytomas and lymphomas^ref. An important issue is whether these mouse models relate to human diseases. Notably, some patients with lymphoma may have mutations in the gene encoding perforin^ref. Metastatic cancers is a systemic disease that is expected to be monitored by  ...

• killer cells (e.g. wild-type mice reject transplants of MHC class-I-defient spontaneous B-cell lymphomas by NK cells and gd T cells^ref)
• recognition of markers of abnormal self
• Va14-Ja18 TcR NKT lymphocytes recognize the tumour-associated disialoganglioside GD₃
• expression of NKG2D ligands confers an effective barrier to tumour formation as they are targeted by NK cells and gd T lymphocytes : Upregulation of the inducible gene products MICA (human) and Rae-1 (mouse) may promote tumor surveillance and autoimmunity by engaging the activating receptor NKG2D on NK cells and T cells. Nevertheless, sustained expression of MICA by tumors can also elicit NKG2D downregulation, perhaps indicating 'immunoevasion'. Investigating this paradox, we report here that constitutive Rae-1e transgene expression in normal epithelium elicited local and systemic NKG2D downregulation, generalized but reversible defects in NK cell-mediated cytotoxicity and mild CD8+ T cell defects. The extent of NKG2D downregulation correlated well with the incidence and progression of cutaneous carcinogenesis, emphasizing the utility of NKG2D as a marker of tumor resistance. Thus, NKG2D engagement is a natural mediator of immunosurveillance, which can be compromised by locally sustained ligand expression but potentially restored by innate immune activation^ref. If other stem cells or tissues upregulate expression of NKG2D ligands after transplantation, NKG2D may contribute to allogeneic bone marrow graft rejection in immunocompetent hosts^ref.
• vitamin D₃ upregulated protein 1 (VDUP1) is a stress-response gene that is upregulated by 1,25(OH)₂D₃ in tumor cells : it drives differentiation of HSCs towards NK cells^ref
• recognition of missing markers of normal self
• down-regulation of MHC class I expression : NK cells inhibitory receptors are no longer activated
• T_h1-cell immunity that promotes CTL response : the importance of perforin  in tumour surveiilance is clear from studies of perforin-deficient mice, which spontaneously develop B-cell lymphomas. Granzymes A and B are not essential for CTL- or NK-cell-mediated rejection of spontaneous or experimental tumours.
• MHC class I processing pathway :
• cross-presentation of tumor antigens : heat shock proteins (HSPs) family members released in the interstitium from necrotic cancer cells carry donor Ags for endocytosis and cross-presentation by APCs
• TAP-independent pathways are useful for TAP-deficient cells, e.g. in those tumors where there is a down-regulation of TAP (see below)
• extralymphatic tumor cells that lack conventional second signals efficiently induce CD8⁺ T cells if they reach draining secondary lymphoid organs and if helper T cells are available^ref.
• when autologous dendritic cells that had taken up apoptotic B lymphoma cells and induced to a fully mature state were used to stimulate nonadherent PBMCs from healthy donors, they induced strong cytotoxicity against B lymphoma cells in a HLA-A0201-restricted manner. The cells failed to induce cytotoxicity, however, when purified T cells were used as effector cells. Depletion of CD56⁺ cells, but not CD14⁺ or CD19⁺ cells, abrogated the cytotoxicity of nonadherent cells, showing that the help was provided by natural killer cells. Further, when natural killer cells were present in the cultures, a strong and persistent production of IL-18, but not IL-12 and IL-15, was observed. Blocking IL-18 significantly reduced the cytotoxicity of nonadherent cells against B lymphoma cells^ref
• IFNs contribute to tumor suppression by inducing p53 expression and boosting its response to stress signals, leading to an enhancement of cancer cell apoptosis
• TNF and TNFR superfamilies : anti-tumor mechanisms could be classified into direct actions onto tumor cells and indirect effects through immune or non-immune components of tumor-bearing host^ref

• signs of a T_h1 immune response within colorectal cancers are associated with the absence of pathological evidence of early metastatic invasion (venous emboli and lymphatic and perineural invasion) and with prolonged survival^ref

Many primary tumours originate at mucosal sites in which they are first encountered by the mucosal immune system : most experiments carried out with animal models available at present, and expecially with transplantable tumour that grow in subcutaneous sites, do not shed light on this subject.

There may be fundamental differences between the development of tumours injected into a mouse or induced by chemical carcinogens compared to naturally occurring spontaneous tumors in humans. For example, the act of injection itself and the presence of dead tumour cells as well as tissue-culture contaminants, such as fetal bovine serum in the inoculum, may induce local inflammatory reactions. In tumours induced by painting chemical carcinogens onto the skin of highly susceptible mice, Rae-1b and H-60 expression may be up-regulated, resulting in their rejection by gd T lymphocytes. In addition, whereas carcinogenesis in humans may be a slow process that occurs over several years, mouse experiments use high doses of carcinogens, which cause transformation in just weeks or months. At these doses, carcinogens may cause massive and rapid mutagenesis, resulting in greatly increased number of neoantigens that may alert the immune system.

Although a model explains why established tumors grow and do not undergo immune-mediated rejection is that tumors function as immunologically normal tissue, as evidence exists that in some models and under some conditions the immune system can play an active role in suppressing the growth of very early tumours (immune attack), in this paradigm, when tumour do successfully grow, they are thought of as having escaped from this immunosurveillance due to natural selection of tumour immune "escape" or "evasion" phenotypes as viewed in a Darwinian light (cancer immunoediting). The central engine for generating immunoresistant tumour cells variants is the genomic instability and dysregulation that is characteristic of the transformed genome. Tumor-induced immunosuppression^ref generates ...

• defects in CTLs activation
• the stromal barrier that forms around tumours keeps the immune system in the dark about cancerous cells
• inhibition of maturation and function of DCs :
• recognition of tumor antigens by DCs under noninflammatory conditions will not lead to DC activation and maturation.
• up-regulation of PD-L1 / B7-H1 in tumor cells^ref
• STAT3 is costitutively activated in many tumour cells, inhibiting the production of pro-inflammatory mediators including TNF-a, IL-6, CCL5 / RANTES and CXCL10 : blocking STAT3 signalling with specific inhibitors leads to DC activation (mediated by induction of STAT3 in the DCs themselves) and could have direct anti-tumour effects. Further transformed cells directly block DC maturation by secreting inhibitory facotrs that are induced by Stat3 activity, diverse depending on the tumour type : VEGF from B16 tumours, IL-10 from Src-transformed fibroblasts, ...^ref
• a new model of tumour angiogenesis in which DEFB29 recruits CD11c⁺ immature DCs that have the potential to adopt endothelial characteristics (endothelial-cell markers, such as CD34 and VE-cadherin) in the presence of VEGF via VEGFR2, without proliferation (ruling out the involvement of a stem-cell population), increasing tumour growth through vasculogenesis. This model is also applicable to human ovarian tumours, which express endogenous b-defensins. By promoting the endothelial specialization of immature DCs, the tumour might not only ensure a sufficient blood supply but also prevent these DCs from initiating an immune response against tumour antigens. In this case, therapeutic targeting of this pathway could both reduce tumour growth and promote immune attack. Interestingly, tumour-infiltrating CD11c+ cells could present antigen to T cells when removed from the tumour milieu in vitro, showing that these cells can function as endothelial-like cells or DCs depending on the environment^ref
• B16 melanoma cells actively modulate the interaction between DCs derived from bone marrow precursors and NK/LAK cells propagated from the spleen of C57BL/6 mice. DCs increased in a dose-dependent manner the ability of NK/LAK cells to kill melanoma cells and to produce cytokines. This activatory cross-talk entailed the production of IL-18 by DCs and of IFN-g by NK/LAK cells. Melanoma cells were not a passive target of NK activity; they regulated the outcome of the interaction between DCs and NK/LAK cells, inhibiting the in vitro production of cytokines as effectively as the genetic deletion of IL-18 or IFN-g^ref
• loss or down-regulation of MHC class I antigens in tumor cells : anyway loss of H-2 expression in TAP1^-/- or LMP-2^-/- mice does not increase the onset or incidence of a variety of spontaneous tumors. On the other hand, some recurring tumors lose cell-surface MHC class I both in mice with pre-existing immunity induced by immunization with B7-1-transfected tumor cells and in humans after partial responses to T cell-based immunotherapy
• total loss of HLA class I expression is not uncommon in many tumors, including melanoma, colorectal carcinoma and prostate adenocarcinoma. In breast carcinoma, the frequency of total HLA class I loss is > 50%. The mechanisms than underlie total loss of HLA class I include
• mutations in one copy of the b₂-microglobulin gene in association with LOH involving the second allele : the loss of b₂-microglobulin has been observedin patients experiencing objective partial responses after T-cell based immunotherapy, although a causal relationship in clinical scenarios is difficult to demonstrate unequivocally
• defects in MHC genes and in the antigen processing and transport pathway
• down-regulation of the proteasome multi-catalytic complex subunits LMP-2 and LMP-7 and of peptide transporters TAP-1 and TAP-2 have been reported in tumor histologies that include small cell lung carcinoma (SCLC), non-small cell lung cancer (NSCLC), prostate adenocarcinoma and renal cell carcinoma (RCC). In these situations, HLA class I can often be up-regulated by treatment with IFN-g.
• selective loss of HLA class I haplotype, locus or allele. HLA haplotype loss can be due to LOH on chromosome 6. Several mechanisms are involved in locus down-regulation, which is more frequent with HLA-B than HLA-A antigens. In melanomas, c-Myc oncogene overexpression correlates with selective HLA-B locus down-regulation. Loss of transcription factor binding to locus-specific regulatory elements can induce HLA-B locus down-regulation in colorectal carcinoma cells. In melanoma, the gene products of the HLA-C locus are often expressed poorly or not at all. The defects underlying HLA class I allele-specific loss include mutations in the genes encoding HLA class I heavy chain.
• in leukaemic cells, downregulation of HLA-B expression is detected in most of the samples, but is restricted to HLA-Bw6 alleles, which - in contrast to HLA-Bw4 alleles - are not thought to be involved in inhibiting the attack by NK cells, so that selective HLA-Bw6 donregulation could prevent attack by CTLs while avoiding NK-cell killing^ref
Explanations for why tumor cells that have lost HLA class I are not destroyed by NK cells :
• loss or downregulation of NKG2D ligands expression (see below)
• lack of stimulatory cytokines such as IL-2, IL-12, IL-15 or type I IFNs in tumors
• lack of expression of costimulatory molecules CD80 / B7-1, CD86 / B7-2 / B7.2, CD40 and CD70 by tumors may also hinder optimal NK cell activation via CD28 and CD27 costimulatory pathways
• immunosuppressant cytokines such as TGF-b₁ or MIF produced by tumor cells
• direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells^ref.
• lack of appropriate costimulation
• lack of expression of costimulatory molecules by tumor cells may lead to T cell anergy and suboptimal activation of NK cells. In an experimental setting, insertion of genes encoding CD80 / B7-1, CD86 / B7-2 / B7.2 or both into tumors generally increases the immunogenicity of those tumors but does not necessarily lead to regression
• constitutive activation of STAT3, a common oncogenic signaling pathway, suppresses tumor expression of proinflammatory mediators^ref


• most human cancer cells express PD-L1 / B7-H1, impairing of T cell function and survival. Patients harboring high intratumoral expression levels of B7-H1, contributed by renal cell carcinoma (RCC) cells alone, lymphocytes alone, or tumor and/or lymphocytes combined, exhibit aggressive tumors and are 4.5 times more likely to die from their cancer than patients exhibiting low levels of B7-H1 expression (RR 4.53)^ref
• other undefined soluble factors or cell-surface molecules expressed by tumour cells
• LIGHT stimulates tumour rejection via recruitment and costimulation of naive CTLs inside tumours^ref
• using an experimental system utilizing the adoptive transfer of transgenic T cells into naive recipients, the population of Gr-1⁺ immature dendritic cells (iDC) from tumor-bearing mice was able to induce CD8⁺ T cell tolerance. These iDC accumulate in large numbers in spleens, lymph nodes, and tumor tissues of tumor-bearing mice and are comprised of precursors of myeloid cells. Neither iDC from control mice nor progeny of tumor-derived iDC, including tumor-derived CD11c⁺ DCs, were able to render T cells nonresponsive. iDC are able to take up soluble protein in vivo, process it, and present antigenic epitopes on their surface and induce Ag-specific T cell anergy. Thus, this is a first demonstration that in tumor-bearing mice CD8⁺ T cell tolerance is induced primarily by iDC that may have direct implications for cancer immunotherapy^ref.
• immunosuppressive cytokines that tumours produce
• VEGF (secreted by gastric carcinoma, lung, head and neck, and breast cancers) inhibits DC differntiation and maturation through suppression of NF-kB in HSCs.
• IL-10 inhibits differentiation of DCs from stem cell precursors and compromises maturation and the functional status of DCs. It inhibits antigen presentation, IL-12 production and induction of T_h1 responses in vivo. IL-10 also enhances spontaneous DC apoptosis as well as susceptibility to autologous NK cell lysis. IL-10 may protect tumor cells from CTLs by down-regulation of HLA classes I and II and ICAM-1. The loss of HLA class I expression could also be due to IL-10-mediated down-regulation of TAP1 and TAP2 proteins in tumor cells. PGE₂ is expressed as a result of enhanced expression of COX-2 in multiple human tumours : PGE₂ increases the production of IL-10 by lymphocytes and macrophages and inhibits IL-12 production by macrophages.
• TGF-b₁ may also be released by cells dying apoptotically. It inhibits the activation, proliferation, and activity of lymphocytes in vivo.
• MIF
Tumors may not necessarily produce these cytokines as escape mechanisms : the hypothesized immunosuppressive functions may be mere side-effects of the angiogenic and growth factor functions of these cytokines.
• recently, a number of endogenous molecules have been reported to be ligands of TLRs. It has been suggested that the release of these putative endogenous ligands of TLRs during cancer progression may cause chronic inflammation leading to the recruitment of myeloid suppressor cells and down-regulation of T-cell and natural killer (NK) cell receptor z chain resulting in T and NK cell dysfunction. However, the reported putative endogenous TLR ligands may have been contaminated with PAMPs^ref.
• loss of tumor antigens and immunodominance : decreased expression of melanoma-melanocyte differentiation antigens (MDAs) is associated with disease progression. In one study, cells were MART-1⁺ in 100% of stage I lesions but only 75% of stage IV lesions. Decreased antigen expression has also been found in residual tumors after peptide vaccination. With a gp100 peptide (209-2M) vaccine, there was a decrease in gp100 expression in tumors after treatment (47% versus 32%), whereas expression of MART1 was unchanged (54%). In addition, the amount of tumor antigen expressed may also be important for recognition. Again, these are correlative studies and fully controlled human studies can be difficult or impossible to come by. It seems likely, however, that as T cell-based tumor immunotherapy becomes stronger, escape mechanisms such as antigen loss are likely to become more prominent. The exact mechanisms that control the down-regulation of tumor antigens are not known in most cases; however, the propagation of such antigen loss variants may be facilitated by epitope immunodominance. The theory of immunodominance, as it relates to tumor escape, predicts that one of the ways that antigen-loss variants within a tumor are shielded from immune pressure is that the parental tumor cells that carry the immunodominant epitope serve as a red flag for immune attack, thereby diverting attention fromm the tumor variants. Once the parental cells are eliminated, a new hierarchy is established among the variant subpopulations, and formerly immunorecessive epitopes become dominant. A tumor variant that has lost the restricting HLA class I allele while retaining the immunodominant antigen could cross-present this antigen to CD8⁺ CTLs by DCs and maintain an immunodominant response to a "phantom" target at the expense of more appropriate and effective responses to other antigens.
• defective death receptor signaling render tumor cells resistant to death receptor-mediated apoptosis
• defective CD178 / FasL signaling
• expression of cFLIP
• down-regulation or loss of CD95 / Fas / Apo-1 expression due to missense mutations and loss of the gene have been identified in multiple myeloma, non-Hodgkin's lymphoma (NHL) and melanoma
• mutations of the genes in the proximal pathways downstream of Fas signalling (including FADD and caspase-10) have also been found in metastatic lesions of non-small cell lung cancer (NSCLC) and lymphoma
• defective TRAIL signaling
• loss of TRAIL-Rs
• loss of caspase-8 by chromosomal loss or mutation
• no signalling from the death-inducing signalling complex (DISC) due to FADD mutation, X-linked inhibitor of apoptosis protein (XIAP) inhibition of caspase-3 and low second mitochondria-derived activator of caspase (SMAC) / direct IAP binding protein with low pI (Diablo) release
• low expression of death receptors by post-transcriptional regulation
• activation induced cell death (AICD) of activated T lymphocytes due to expression of death receptor ligands by tumor cells
• lung carcinoma, melanoma, colorectal carcinoma and hepatocellular carcinoma express functional CD178 / FasL, which induces activation induced cell death (AICD) of CD95 / Fas / Apo-1⁺-susceptible activated antitumor T lymphocytes (suicide) and between T lymphocytes (fratricide).  can regulte the proinflammatory effects of FasL
• TRAIL
• programmed death 1 (PD-1)
AICD in human melanoma epitope-specific primary CTL involves selective mitochondrio-nuclear translocation of AIF without cytochrome c release, caspase-3 and caspase-8 activation, and results from large-scale DNA fragmentation. The JNK inhibitor, SP600125, blocks the mitochondrio-nuclear translocation of AIF and prevents AICD in these CTL. These findings suggest that the AICD in human melanoma epitope specific primary CTL is mediated by mitochondrial AIF release and JNK is involved in regulation of this death process^ref.
• expanded CD4⁺CD25⁺ T_reg lymphocytes in response to the increased presentation of autoantigens during tumour growth. Their depletion plus injection of an anti-CTLA-4 antibody enhanced reactivity to a known TAA.
• tumor tissue and ascites from patients with ovarian cancer contain high levels of cells with all the hallmarks of regulatory T cells. These cells migrate into the tumor microenvironment in a process mediated by the chemokine CCL22, and are capable of suppressing antitumor responses^ref
• the AE17 murine mesothelioma model was used for this study. Both intra-tumoural T_reg cells and those in the periphery of tumour-bearing mice were identified by flow cytometry. The effect on tumour growth of intra-tumoural depletion of T_reg cells using the PC61 anti-CD25 mAb was then examined. CD4⁺ T_reg cells co-expressing both the CD25 cell surface marker and the transcription factor Foxp3 were identified within murine mesotheliomas. These intra-tumoural T_reg cells increase significantly as a percentage of total CD4⁺ T cells within the tumour as it grows. Depletion of intra-tumoural T_reg cells with anti-CD25 mAb injected directly into the tumours can cause significantly reduced tumour growth. Localised, intra-tumoural depletion of T_reg cells is a new, clinically relevant treatment option for established tumours^ref.
• intratumoral T_reg cells inhibit the proliferation and granule production of activated autologous infiltrating CD8⁺ T cells. Degranulation and subsequent cytotoxic activity of infiltrating CD8⁺ T cells exposed to lymphoma B cells is completely attenuated by the presence of intratumoral T_reg cells. Furthermore increased numbers of intratumoral T_reg cells correlates with the number of CD8⁺ T cells in biopsy specimens from patients with B-cell NHL, supporting the in vitro findings that intratumoral T_reg cells inhibit proliferation of infiltrating CD8⁺ T cells. Taken together, these data indicate that human lymphoma B cells are sensitive to autologous CTL-mediated cell death but are protected by the inhibitory function of intratumoral T_reg cells^ref.
• an increased frequency of T_reg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of T_reg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4⁺ T cell responses, as observed by IFN-g release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group^ref
• improper activation of macrophages and polymorphonuclear cells induces a state of oxidative stress in cancer patients that markedly suppresses the function of T cells.
• the presence of leukocytes in human tumors was first noted in neoplastic tissues by Rudolf Virchow in 1863 (Virchow R. Aetiologie der neoplastischen Geschwulste/Pathogenie der neoplastischen Geschwulste. In: Hirschwald VvA, ed. Die Krankhaften Geschwustle. Berlin, Germany;1863: 57-101; Virchow R. Reizung und Reizbarkeit. Arch Pathol Anat Klin Med. 1858;14: 1-63). He suggested that the "lymphoreticular infiltrate" reflected an important interaction between cancer cells and cells involved in chronic inflammation.
• tumor-associated macrophages (TAMs) have been the subject of investigation in a number of different tumors, including breast, prostate, ovarian, and cervical cancers, where a clear correlation with inferior survival has been seen with increased TAM content^ref. Curiously, in other tumors such as gastric and lung cancer, the results have been contradictory^ref. These disparate findings may be due to specific functional characteristics of macrophages in some tumors and may be context dependent. Nonetheless, in the majority of studies increased TAM density has been associated with both metastatic spread and diminished survival^ref. Lymphoma-associated macrophage (LAM) counts are correlated with poor prognosis in follicular lymphoma patients. Distinct role of macrophages in different tumor microenvironments^ref
• proliferation arrest of T lymphocytes when exposed to tryptophan shortage, which can be provoked by indoleamine 2,3-dioxygenase (IDO), an enzyme that is expressed in placenta and catalyzes tryptophan degradation. Most human tumors constitutively express IDO causing a lack of accumulation of specific T cells at the tumor site.
• a wide variety of carcinomas abundantly express MHC-class-I-chain related (MIC) molecules, which are recognized by many sets of immune system cells, but ....
• tumour cells often express insufficient levels of NKG2D ligands to stimulate tumour-cell rejection, either because expression of the ligands is not sufficiently upregulted early in the development of the tumour, or because tumour cells with lower levels of ligand expression are selected by the immune system in vivo as the tumour evolves
• MICA is overexpressed and shed by MMPs as a soluble form from the cell surface of many carcinoma cells into the circulation, causing systemic endocytosis and degradation of the activating receptor NKG2D, and hence its down-regulation in T lymphocytes (and possible other immune system cells).
However, the existence of tumours that evade NKG2D does not detract from the possibility that many other tumours are successfully eliminated by NKG2D-mediated immune activation, because such evasion mechanisms are unlikely to be universally effective. Notably, rejection of tumour cells that expressed NKG2D ligands confers resistance to subsequent tumour cells that lack NKG2D ligands, inducing long-lasting immunity at least in some cases, indicating the promise of NKG2D ligands as adjuvants in tumour vaccines. How NKG2D ligands expressed by tumour cells enhance priming of tumour antigen-specific CD8⁺ T lymphocytes remains to be established. Depletion of NK lymphocytes before vaccination failed to prevent T-cell priming, indicating that NK-cell-derived cytokines or the formation of tumour debris as a result of NK-cell-mediated tumour-cell lysis are not required for this process, although they might contribute to it. An alternative possibility is that NKG2D ligands expressed by the tumour cells directly costimulate tumour-antigen-specific CD8⁺ T lymphocytes. It is also possible that the activation of APCs that express NKG2D is increased by the ligand-expressing tumour cells, leading to more effective processing and/or presentation of tumour antigens.
• galectin-1 (Gal1) is expressed by many different tumour types (e.g. mouse melanoma cells) and increased expression correlates with tumour aggressiveness and metastasis. It induces apoptosis in T lymphocytes. Blocking Gal1 production by melanoma cells not only restores the ability of the host immune system to initiate a tumor-specific primary T_h1 response, it also provides resistance to subsequent challenge with Gal1-expressing tumour cells^ref.
• cancer immunosurveillance is known to control certain virus-associated tumours but its role in the control of spontaneous tumours is a matter of some controversy. If immunosurveillance is involved, any immunogenic tumours that do eventually grow are likely to be escape variants, selected for low immunogenicity by T cells. By generating a transgenic mouse model of sporadic cancer based on rare spontaneous activation of a dormant oncogene, it has been shown that sporadic immunogenic tumours do not escape their recognition but induce T-cell tolerance. In this model, tumours derive slowly from single cells and express a TSA, thus reflecting physiological tumour development as closely as possible. Whereas vaccinated mice remain tumour-free throughout their lifetime, naive mice always develop a progressively growing tumour. Despite specific recognition by T cells, the tumours do not lose their intrinsic immunogenicity and are rejected after transplantation in T-cell-competent recipients. Furthermore, in the primary host tumour-induced tolerance is associated with the expansion of non-functional T cells. Together, these data argue against immunosurveillance of spontaneous cancer^{ref1, ref2, ref3}
• expression of the heterodimeric cytokine IL-23, but not of its close relative IL-12, is increased in human tumours. Expression of these cytokines antagonistically regulates local inflammatory responses in the tumour microenvironment and infiltration of intra-epithelial lymphocytes. Whereas IL-12 promotes infiltration of cytotoxic T cells, IL-23 promotes inflammatory responses such as upregulation of the matrix metalloprotease MMP9, and increases angiogenesis but reduces CD8 T-cell infiltration. Genetic deletion or antibody-mediated elimination of IL-23 leads to increased infiltration of cytotoxic T cells into the transformed tissue, rendering a protective effect against chemically induced carcinogenesis. Finally, transplanted tumours are growth-restricted in hosts depleted for IL-23^-/-or in IL-23R^-/- mice. Although many strategies for immune therapy of cancer attempt to stimulate an immune response against solid tumours, infiltration of effector cells into the tumour tissue often appears to be a critical hurdle. IL-23 is an important molecular link between tumour-promoting pro-inflammatory processes and the failure of the adaptive immune surveillance to infiltrate tumours^ref.
• A_2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. A_2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in approximately 60% of A_2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A₂ receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. Effects of A_2AR are T cell autonomous. The inhibition of antitumor T cells via their A_2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the "Hellstrom paradox"). The hypoxia-->adenosine-->A_2AR pathway should be targeted as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A_2AR-deficient mice suggest that A_2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia-->adenosine-->A_2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage^ref.
• tumor-expressed albumin, unlike normal serum albumin purified from blood, is capable of inhibiting T-cell activation, proliferation, and function in both in vitro and in vivo settings. Tumor-expressed albumin does not appear to affect antigen processing or presentation by professional antigen-presenting cells. The activity appears to lie in relatively small, lipid-like moieties that are presumably cargo for tumor-expressed albumin, and that activity can be removed from the albumin by lipid removal or treatment with lipase^ref
• lymphocyte apoptosis within the tumor environment : CD70, a cytokine overexpressed in RCC, promotes lymphocyte apoptosis through interaction with its receptor CD27 and with the intracellular receptor-binding protein SIVA. Apoptosis increased after cocultivating lymphocytes with the RCC cell lines A498 and CAKI2. The addition of recombinant soluble CD70 to both native lymphocytes and a T-cell cell line resulted in increased lymphocyte apoptosis as well. Furthermore, induced apoptosis could be partially blocked with anti-CD27 and anti-CD70 antibodies^ref.
• inhibition of NK lymphocytes : CD4⁺CD25⁺ T_reg lymphocytes that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular,T_reg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, T_reg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-b-dependent mechanism and independently of IL-10. Adoptively transferred T_reg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of T_reg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. There is at least one mechanism by which T_reg can suppress NK cell antitumor activity and highlight the effectiveness of combining T_reg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity^ref. TGF-b₁ associated with other blood cells may be involved in limiting NK cell activation : blocking endogenous TGF-b resulted in significant NK cell IFN-g production under suboptimal stimulation conditions^ref. NK cells can induce DC maturation, a differentiation process whereby DCs respond to a environmental stimulus and acquire the ability of eliciting adaptive immunity. Conversely, maturing DCs promote NK functions in vivo and in vitro. This interplay has important consequences on the immune response to pathogens and possibly to neoplastic cells. B16 melanoma cells actively modulate the interaction between DCs derived from bone marrow precursors and NK/LAK cells propagated from the spleen of C57BL/6 mice. DCs increased in a dose-dependent manner the ability of NK/LAK cells to kill melanoma cells and to produce cytokines. This activatory cross-talk entailed the production of IL-18 by DCs and of IFN-g by NK/LAK cells. Melanoma cells were not a passive target of NK activity; they regulated the outcome of the interaction between DCs and NK/LAK cells, inhibiting the in vitro production of cytokines as effectively as the genetic deletion of IL-18 or IFN-g. Interference with the NK/DC interaction possibly represents a mechanism used by growing tumors to evade the immune response^ref
• activation of NKT lymphocytes inhibit effective CTL-mediated tumor rejection by IL-13 via the IL-4R-STAT6 pathway. CD4+DX5+ NKT suppressor cells regulate the growth of UV-induced skin cancers and mediate antigen-specific immune suppression.
• apoptosis of inflammatory cells within 3-5 days upon binding of their CD95 / Fas / Apo-1 to CD178 / FasL expressed on certain tumor cells (tumour counter-attack)
• resistance to apoptosis by cancer cells : cancer cells enhance their ability to withstand an attack by cytotoxic immune effector cells via acquisition of specific genetic alterations that interfere with the shared mitochondrial death signaling pathway entrained by granzyme B, IFN-g, and Apo2L/TRAIL, 3 key mediators of immunologic cell-mediated cytotoxicity. The coexistence of specific mitochondrial signaling defects (either deletion of Bax, overexpression of Bcl-x_L, or deletion of Smac) with expression of XIAP decreases the sensitivity of cancer cells to IFN-g/Apo2L/TRAIL- or granzyme B-induced apoptosis, lymphocyte-mediated cytotoxicity in vitro, and adoptive cellular immunotherapy in vivo. Conversely, negating XIAP expression or function in tumor cells with defective mitochondrial signaling enables direct activation of caspase-3/-7 by granzyme B or Apo2L/TRAIL, and restores their susceptibility to immunologic cytotoxicity^ref
• resistance to attack by complement by cancer cells (a major limitation in the use of monoclonal antibodies as monotherapies) : expression of the complement-regulatory proteins CD55 / decay accelerating factor (DAF), CD46 / MCP and CD59 / MIRL are deregulated in cancer with tumors showing loss of one or more inhibitors and strong overexpression of others. However, tumor sensitivity to complement can be restored by co-administration of antibodies that bind to the functional domains of complement-regulatory proteins. Overexpression of complement-regulatory proteins on tumors also makes them potential targets for cancer vaccines. However, these vaccines have to be carefully designed to induce immune responses that recognize inhibitors overexpressed on tumors and that do not detect the levels expressed by normal cells^ref.
• inhibition of granzyme B / granzyme 2 activity by the serine protease inhibitor PI9 / SPI-6 expressed by some tumour cells
• antibody-mediated immunologic enhancement : both B and T cell immune responses in situ are fully compatible with progressive growth of small inocula either as ascites or as solid subcutaneous tumors of a number of carcinogen-induced, immunogenic, murine tumors in their syngeneic hosts. Tumor cell, after 2 weeks of growth in vivo, become resistant to the induced killer cells. The modulating agent bound to these resistant tumor cells appears to be anti-tumor antibody directed against epitopes are uniquely expressed on tumor cells (oncotopes). These observations are compatible with data first reported 35 years ago suggesting that anti-tumor antibody promoted or enhanced tumor growth^ref
• unknown pathways : CCL-2 / MCP-1, osteopontin, and midkine from oncogenic TC-1 cells producing E6 and E7 oncoproteins confer resistance to anti-tumor immunity induced via DNA vaccination anti-E7^ref
• defect in the lymphocyte-vessel wall interactions : the angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-a and the proangiogenic VEGF. A dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. Overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-alpha-dependent increase in adhesion. VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. The NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy^ref

• tumour can damage or disrupt surrounding tissue or trigger a stress response when they outstrip their oxygen and nutrient supplies. These processes can cause pH imbalance that results from metabolic disturbance, generation of ROS, up-regulation of stress protective factors - such as hsps - and death by necrosis or apoptosis
• as tumours grow progressively, dysregulated genetic and epigenetic events lead to the expression of large numbers of neoantigens (one study estimates that the average malignancy contained > 10,000 mutations)

• of low affinity for peptide-MHC complex, associated generally with the presence of undesirable residues at crucial anchor positions in the antigen
• specific for epitopes that are presented at low density

Cancer in immune-privileged sites

• in a mouse experimental model, the immune-privileged state of the eye permits the outgrowth of xenogeneic uveal melanoma cells, but does not protect these tumors against adoptive immunotherapy with highly potent anti-tumor CTL^ref

Cancer immunotherapy : a major goal of cancer immunotherapy has been to generate a large number of highly avid, tumor-specific T cells that can last in vivo for a long time and resist tolerization^ref. Existing methods focus on reshaping the normal T cell repertoire and fall into 2 categories:

• methods relying on massive expansion of the few antigen-specific T cells, which is usually a demanding task and not always successful.
• active immunotherapy, which involves activating the effectors in the host immune system to inhibit cancer cell growth and reject tumor (e.g., cancer vaccination)^{ref1, ref2}
• immune system enhancers
• whole tumour cell therapeutic vaccines
• tumour antigen therapeutic vaccines
• anti-cancer monoclonal antibodies
• passive immunotherapy, which directly provides the host with effectors to react against cancer (e.g., adoptive transfer of in vitro expanded or modified anti-tumor T cells)^{ref1, ref2, ref3, ref4, ref5, ref6}. Antigen-specific T cells expanded from an individual's T cell repertoire cannot be readily transferred to others because of the risk of severe graft-versus-host disease^ref
• donor lymphocyte infusions (DLIs)
• creating autologous T cells of a desired specificity with T cell receptor (TCR) gene transfer (instructive immunotherapy) :
• conventional TCR transgenic animals can be made via pronuclear injection of prerearranged TCR genes^ref. However, this method offers no opportunity for therapeutic application in humans
• retrovirus-mediated expression of TCR genes in peripheral T cells could endow them with the desired specificity^{ref1, ref2}. However, such T cells present certain challenges: they are activated mature cells that have already expressed an endogenous TCR of the unknown specificity; their effector function may be restricted by the conditions under which they are activated in vitro; and it is unclear how long they persist in vivo^ref.
• retrovirus-mediated expression of TCR genes in hematopoietic stem cells (HSCs) : HSCs are attractive targets for TCR gene transfer because of their roles as T cell progenitors, enormous regeneration capacity, and availability for modification in humans^ref. A method to impart the desired anti-tumor specificities (anti-chicken OVA) to the RAG1^–/– mouse T cell repertoire by delivering tumor-specific TCR genes into HSCs via MIG retrovirus^ref, followed by adaptive transfer to generate a continuous stream of anti-tumor T cells in host mice, has been described^ref. OVA is the well characterized target antigen for both a CD8 TCR, OT1 (recognizing OVAp_257–269, designated as OVAp1)^ref, and a CD4 TCR, OT2 (recognizing OVAp_329–337, designated as OVAp2)^ref, offering us the opportunity to study both the CD8 cytotoxic and CD4 helper arms of antigen-specific T cell immunity. Coexpression of the 2 TCR subunits from the same vector has proved critical because no antigen-specific T cells were detected when a 2-vector system reported previously to infect WT HSCs was used^ref : the coexpression was achieved by linking cDNAs encoding TCR a and b chains with an internal ribosome entry site and inserting this into the retroviral vector under control of the viral LTR promote. The vectors expressing OT1 or OT2 TCR were designated MOT1 or MOT2. In addition to using HSCs as the target for gene transfer, this approach has other features that facilitate clinical application: single-vector gene delivery, small genetic elements (TCR a and b cDNAs), and a convenient viral promoter to drive the expression of transgenes. The recipients were allowed to reconstitute their T cell compartments for 7 weeks before analysis. Some 3–7% of the total BM cells were transduced, including long-term HSCs that expressed c-kit and Sca-1^ref. The percentage of transduced HSCs remained constant to 8-month posttransfer and persisted through a secondary BM transfer, indicating that they maintained their stem cell features of longevity and self-renewal. Therefore, this genetic method is sufficiently robust to ensure the maintenance of a transduced HSC population for the lifetime of the host.  25% of the total peripheral CD8 T cells in C57BL/6J(B6) female mice/MOT1 expressed OT1 TCR and 8% of the total peripheral CD4 T cells in B6/MOT2 mice expressed OT2 TCR, with no leakage into the other T cell compartment. In numerous experiments, an average of 20% (ranging from 15% to 30%) of the total peripheral CD8 T cells in B6/MOT1 mice carrying the OT1 TCR specificity was achieved, and an average of 8% (ranging from 5% to 10%) of the total peripheral CD4 T cells carrying the OT2 TCR specificity. This high percentage of antigen-specific T cells remained constant for 8 months posttransfer. Therefore, retrovirus-mediated expression of TCR cDNAs in WT HSCs can stably generate a significant population of T cells with the desired features and specificity. In natural conditions, this percentage can be achieved only by clonal expansion of a few high-affinity T cell clones after strong immune stimulation^ref. The RAG1^–/– background offered a clean system for our intended study, because of the lack of endogenous TCR expression^ref. Surprisingly, TCR a and b chain expression patterns in the thymocytes of RAG1/MOT2 mice closely resembled the b chain expression in RAG1/OT2 Tg mice that is under control of the endogenous TCR promoter and enhancers^ref. The mature OT2 T cells recovered from the periphery of the 2 kinds of mice were comparable, although there was more heterogeneity of TCR expression in the OT2 T cells from RAG1/MOT2 mice. A similar magnitude of antigenic response was observed when the animals were immunized with OVAp2 antigen. T cells harvested from mice displayed the canonical naïve phenotype of CD8 T cells: CD25^–CD69^–CD62L^highCD44^low. Upon stimulation with OVA in vitro, these T cells responded vigorously as measured by proliferation and IFN-g production, with a magnitude comparable to the response of transgenic T cells obtained from conventional TCR transgenic mice^ref (but we have no comparison to functionality of normally developed OVA-specific T cells). The activated T cells exhibited the typical effector cytotoxic T cells phenotype: CD25⁺CD69⁺CD62L^lowCD44^high. The ability of these OT1 T cells to generate and maintain CD8 T cell memory by adoptive transfer of in vitro-activated OT1 cytotoxic T cell to host mice was then tested because continuous generation of naïve OT1 T cells makes it difficult to directly analyze memory formation in B6/MOT1 mice. Sixteen weeks after transfer, long-lived OT1 T cells were detected in the hosts, which displayed the memory phenotype (CD25^–CD69^–CD62L^highCD44^high). These cells showed a stronger and faster response to antigen stimulation when compared with naïve OT1 T cells. Furthermore, they proliferated upon cytokine stimulation, which is a unique feature of memory T cells^ref. The therapeutic potential of this method was tested in the E.G7 mouse tumor model^ref. OVA serves as the tumor antigen in this model, allowing us to study either anti-tumor cytotoxic T cell immunity in B6/MOT1 mice, anti-tumor helper T cell immunity in B6/MOT2 mice, or the combination of both arms in B6/MOT1+MOT2 mice (B6 mice receiving both MOT1- and MOT2-transduced WT HSCs)^ref. Suppression of syngenic tumor growth was studied by using the following protocol: B6/MOT1, B6/MOT2, and B6/MOT1+MOT2 mice were allowed to reconstitute their T cell compartment over 8–10 weeks after receiving retroviral transduced HSCs. E.G7 tumor cells were then injected s.c., and EL.4 tumor cells were used as a control^ref. To evaluate the effects of peptide immunization, half of the experimental groups were immunized by using DCs loaded with OVAp1 (DC/OVAp1) 4 days after tumor injection. Four mice were used in each treatment group, and the experiments were performed three times. Compared with the B6 control, significant tumor suppression was observed in B6/MOT1 mice, with complete suppression in 50% of the mice. No recurrence of tumors was observed as long as the experiments ran (up to 200 days). For the other 50%, tumor growth was suppressed up to 18 days but eventually progressed. E.G7 tumor cells recovered from these mice were still recognized by naïve OT1 T cells , excluding the possibility of "epitope escape" by OVA antigen mutation. The OT1 cells recovered from the tumor-bearing mice could no longer respond to OVAp1 antigen stimulation in vitro, as measured by IFN-g production. These anergic OT1 T cells displayed a surface phenotype that resembled naïve T cells but with higher CD44 expression, indicating that they had encountered tumor antigen. Interestingly, total T cells still responded to anti-CD3 stimulation, suggesting the existence of an active antigen-specific tumor tolerogenic mechanism^ref. Similar results were obtained for B6/MOT2 tumor-bearing mice, suggesting that tolerogenic mechanisms act on both anti-tumor cytotoxic and helper T cells. DC-based peptide immunization enhanced OT1 T cell function^ref and thoroughly suppressed tumor growth in all of the B6/MOT1 mice. Interestingly, significant tumor suppression was also observed in B6/MOT2 mice despite the fact that unlike OT1 T cells OT2 T cells cannot directly recognize E.G7 tumor cells because of their lack of MHC II expression. The possible mechanism in this case would be cross-presentation of tumor antigens by the host-derived antigen-presenting cells^ref. Even more strikingly, when B6/MOT2 mice were immunized with DC/OVAp1 complete suppression of tumor growth was observed in all of the mice (some tumors grew up to a barely detectable size but soon regressed). This series of experiments indicated that the CD4 anti-tumor T cells could provide essential help for the endogenous cytotoxic T cell to mount an anti-tumor response, regardless of whether they can recognize the tumor directly or not. As expected, a combinatorial tumor suppression effect was observed in B6/MOT1+MOT2 mice, as exhibited by a slower tumor growth in mice even when the tumor broke through. DC/OVAp1 immunization totally suppressed tumor growth in all of the mice. Importantly, peptide immunization did not work for B6 control mice, and in all experimental groups challenged with control tumor EL.4, the tumors grew up at a similar rate regardless of immunization, confirming that suppression of E.G7 tumor growth was tumor antigen-specific and mediated by the engineered anti-tumor CD8 and/or CD4 T cells. The idea of combining the 2 arms of anti-tumor T cell immunity was further extended with the aim of eradicating an established large vascularized solid tumor. Physiologically, tumors achieve growth by initially overcoming the host's anti-tumor immuno-surveillance and then constantly tolerizing the anti-tumor T cells in the host^ref. A protocol was devised to mimic these processes: mice in which E.G7 tumors grew despite the presence of anti-tumor CD8 (B6/MOT1) or both anti-tumor CD8 and CD4 T cells (B6/MOT1+MOT2) were immunized with one dose of DCs loaded with both OVAp1 and OVAp2 (denoted as DC/OVAp1+2), at the time when their tumors reached 30 mm² [18 days after tumor challenge, tumors extant for this duration and with this size are considered to be long-term established tumors^ref]. The tumors in B6/MOT1 mice receiving immunization were suppressed for 10 days but then grew, reaching 400 mm2 in 50 days. In contrast, tumors in B6/MOT1+MOT2 mice shrank after the immunization and were not longer detectable within 12 days; no tumor recurrence was observed for as long as the experiments ran (>200 days). These results demonstrated that established solid vascularized tumors could be eradicated by providing both the CD8 and CD4 arms of anti-tumor T cell immunity combined with appropriate peptide immunization to boost the responses of both arms. This method can be easily extended to generate T cells targeting multiple epitopes of the antigen, thus countering "epitope escape" by antigen mutations^ref. It might also be extended to engineering the BcR on B cells, allowing the cells to secrete antibodies of predefined specificity. Therefore, instructive immunotherapy has the potential to establish in vivo a lifelong "complete" adaptive immunity (including CD8 cytotoxic T cell, CD4 helper T cell, and B cell arms) to target any pathogens where antigens and the cognate TCR/B cell receptors are known^{ref1, ref2}. In addition, this method could be used for rapid and efficient analysis of such problems as the development and life history of antigen-specific T cells and in vivo anti-tumor T cell response and tolerance, providing a useful tool for basic T cell studies.

• Departments of Internal Medicine and Obstetrics and Gynecology, University of Arkansas, Little Rock, AR : Hermonat PL, Liu Y,
• Department of Microbiology and Immunology, Texas Tech University, Lubbock, TX; USA : Wan W, Tedesco A, Chiriva-Internati M
• Scientific Direction, Istituto Clinico Humanitas, Milan; : Grizzi F
• Foundation “M. Rodriguez”, Milan; Italy : Grizzi F
• Immunovirology and Biotherapy Unit and Experimental Oncology 1, CRO, IRCCS Aviano : Guidoboni M, Caggiari L, De Re V, Gasparotto G, Marzotto A, Martorelli D, Baiocchi M, Dolcetti R
• Human Tumor Immunobiology Unit, INT,Milano; Italy : Anichini A
• Dipartimento di Scienze Oncologiche e Chirurgiche, Università di Padova : Bobisse S, Tisato V, Mandruzzato S, Zoso A, Indraccolo S, Zanovello P, Rosato A
• H San Raffaele Scientific Institute, Milano; Italy : Casorati G
• Flow Cytometry and Cell Therapy Unit, Medical Oncology, University and IRCCS “S. Matteo”, Pavia, Italy : Ferrari S, Rovati B, Collovà E, Grasso D, Sagrada P, Danova M, Riccardi A

• Associations : Cancer Research Institute (CRI) : Academy of Cancer Immunology
• Journals : Cancer Immunity

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