Interference with Functions Survival of Effector T Lymphocytes

In addition to defective APC functions described in cancer patients, which interfere with the induction of antitumor responses, other functional aberrations result in effector T cell paralysis or their untimely elimination [56, 190, 195]. In this respect, the presence of death ligands and receptors, including the Fas/FasL system, in human lymphocytes has been of special interest, because of their role in immune homeostasis. Activated T cells are especially sensitive to receptor-mediated apop-tosis through increased sensitivity to FasL [177, 194], leading to the elimination of CD95/Fas+ activated T cells, a process known as activation-induced cell death (AICD). This mechanism is used for downregulation of the immune response, when it is no longer necessary, to control unlimited lymphocyte expansion and to maintain homeostasis. TAA-specific T cells may be especially sensitive to apopto-sis because most TAA are self-antigens, which are subject to control by peripheral tolerance [59]. Thus, suppressed tumor-specific immunity may reflect tolerance to self [59]. The underlying mechanisms are still poorly defined and difficult to study in vivo; however, several pathways used by tumors to counteract immune surveillance have been described, including the role played by regulatory T cells (Treg) and myeloid suppressor cells (MSC). Suppression of T-cell Responses by Treg

Treg are currently considered to be responsible for maintaining peripheral tolerance [141, 148, 165, 166, 179, 207], including transplantation tolerance and the prevention of autoimmune diseases [141, 148]. Treg have a beneficial role in preventing autoimmunity but are the most potent opponents of antitumor immune cells in cancer and play an important role in suppressing TA-specific immunity [141, 147]. Studies indicate that CD4+CD25bright FoxP3+T cells are present in blood or lymph nodes of subjects with cancer and accumulate at tumor sites [31, 146, 147, 206]. To date, at least three types of CD4+ Treg have been described in humans: (i) naturally occurring CD4+CD25brightFoxp3+T cells (nTreg), which arise in the thymus and can suppress responses of both CD4+CD25- and CD8+CD25- T cells in a contact-dependent, cytokine-independent, Ag-nonspecific manner [81, 110, 183]; (ii) CD4+CD25negative Foxp3low cells known as Type-1 regulatory (Tr1) T cells, which arise in the periphery upon encountering Ag in a tolerogenic environment via an IL-10-dependent process [79, 80]; and (iii) Th3 suppressor cells [39], which are dependent on IL-4 for functional differentiation. The nTreg are a heterogenous population endowed with regulatory functions and differentially expressing CTLA-4, glucocorticoid-induced tumor necrosis factor receptor (GITR) and CD62L [35, 159]. Tr1 cells can be generated in vitro by activating naive CD4+T cells by CD2 co-stimulation, IL-10 or IL-4 plus IL-10 [50, 64, 182]. The Tr1 cells appear to be modulated by IL-10 and TGF-beta and may play a prominent role in cancer. Tr1

cells are induced in the tumor microenvironment, and tumor-derived factors such as, e.g. PGE2, have been shown to promote this process [18]. We have recently established an in vitro model simulating the tumor microenvironment, in which COX-2+ , PGE2-producing carcinoma co-cultured with CD4+CD25- T cells, autologous DC and low doses of IL-2, IL-15 and IL-10 induces CD4+CD25-CD122+C D132+Foxp3+IL-10+TGF0+Tr1 cells [19]. Apart from CD4+ cells, CD8+ regulatory T cells have been recently reported. CD8+ CD28- T cells can be generated in vitro after stimulation of human PBMC with either allogeneic or xenogeneic APC. CD8+ CD28- T cells induce Ag-specific tolerance by increasing the expression of inhibitory receptor ILT3 and ILT4 on APC rather than by IL-10 production [26]. Today, the nature of human Treg is only partially defined. The phenotype, functions (Ag specificity, stability, trafficking or survival), lineage, differentiation and the relationship between the various Treg subsets are under investigation. No single specific marker is sufficient for distinguishing Treg subpopulations. Given the expansion of these populations in the circulation and tumor tissues of cancer patients [6, 84, 142, 158, 205], it is important to perform Treg phenotypic and functional evaluations to better define their role in the regulation of tumor-specific responses. From a practical point of view, it is important to distinguish Treg from activated CD4+CD25+ T cells which mediate helper functions and are sensitive to AICD. In contrast, Treg appear to be resistant to apoptosis [159]. Recent reports suggest that human Treg preferentially expand and survive in the presence of Rapamycin [16, 159], providing a novel approach to the large-scale culture of these cells for possible therapeutic use. Suppression of T-cell Responses by Myeloid Suppressor Cells

Most tumors secrete TGF-0 or induce TGF-0 secretion from immature myeloid cells (MSC) that tend to accumulate in the tumor microenvironment [145, 152]. Young et al. first reported accumulations of CD34+ cell-derived myeloid cells with immunosuppressive ability the peripheral blood of HNC patients [116]. These cells correspond to CD11b+ /Gr-1+ myeloid progenitor cells in mice [144]. In tumor-bearing mice, MSC accumulate in the spleen and peripheral circulation, reaching very high proportions and exerting potent immunosuppression, thus favoring tumor growth. MSC also control the availability of essential amino acids such as L-arginine and produce high levels of reactive oxygen species (ROS). MSC present in tumors constitutively express iNOS and arginase1, an enzyme involved in metabolism of L-arginine, which also synergizes with iNOS to increase superoxide and NO production, blunting lymphocyte responses [33]. MSC with the phenotype CD34+CD33+CD13+ and CD15- and suppressive functions were found to be increased in the peripheral blood of patients with various cancers [11]. Further, maturation defects in DC of patients with cancer have been described [10, 57] and are attributable, in part, to vascular endothelial growth factor (VEGF) production by human tumors [44, 45]. GM-CSF, which is also a frequently secreted product of tumor cells, recruits MSC and induces dose-dependent in vivo immune suppression and tumor promotion [144]. At the same time, GM-CSF is widely used as immune adjuvant in antitumor vaccines [36]. This dual role of GM-CSF (stimulatory and suppressive) suggests that GM-CSF and MSC are involved in maintaining immune homeostasis under normal physiologic conditions but in the tumor presence are subverted to promote its escape. Apoptosis of T cells in Patients with Cancer

It has been observed that when lymphocytes are co-incubated with autologous tumor cells, DNA fragmentation occurs in a proportion of activated T lymphocytes, presumably by the mechanism similar or identical to AICD. When TUNEL assays, which detect DNA fragmentation, were performed using human tumor biopsies and tumor-involved LN [130], it was not tumor cells, but TIL and DC that were TUNEL positive (Fig. 1.2). Control normal tissues or tumor-uninvolved tissues obtained from patients with cancer contained infrequent or no apoptotic lymphocytes [130]. This unexpected finding was subsequently confirmed, using tumor tissues isolated from a variety of patients with cancer [52, 66, 111, 129]. Further, TUNEL staining showed that CD8+ rather than CD4+ T cells were primarily undergoing apoptosis at the tumor site, suggesting that the fate of these two T-cell subsets in situ may differ due to their divergent sensitivity to apoptosis.

Apoptosis of immune cells is not limited to the tumor site. Apoptosis of circulating CD8+ T cells in subjects with cancer has been described in melanoma, breast, ovarian, and head and neck cancers [74, 139, 140, 190]. Studies involving TUNEL staining of TIL and Annexin V (ANX) binding to circulating T cells suggest that CD8+ rather than CD4+ T cells selectively undergo apoptosis at the tumor site and in the peripheral circulation of cancer patients [74, 190]. The proportion of


Fig. 1.2 Apoptosis of lymphocytes (A) or TADC (B) in the tumor microenvironment: TUNEL assay in sections of human oral CA (A) and caspase activity in prostate carcinoma in situ (B). TU = tumor; L = lymphocytes. The arrow points to a caspase+ (red) DC (blue = CD83+). (The photograph shown in B was generously contributed by Dr. Michael Shurin, University of Pittsburgh.)

CD8+Fas+ T cells that bind ANX is significantly increased in the patients' circulation relative to age-matched normal controls [56, 190]. Thus, the fate of CD8+ and CD4+ T-cell subsets may differ due to their divergent sensitivity to apoptosis [69]. Also, the effector subpopulations of CD8+ T cells (e.g. CD8+ CD45RO+ CD27-and CD8+ CD28-) appear to be preferentially targeted for apoptosis in cancer patients [169]. The CD8+ CCR7+ subset of effector cells, which is resistant to apoptosis, is replaced by apoptosis sensitive CD8+ CCR7- lymphocytes in patients with cancer [68 and Fig. 1.3]. Absolute numbers of circulating T-cell subsets are low in these patients [73]. Examination of the proliferative history of T-cells subsets using the T-cell receptor excision circle (TREC) PCR-based analysis confirms aberrant lymphocyte homeostasis characterized by a rapid turnover of T cells in cancer patients [73, 212]. Circulating VP-restricted CD8+ T cells are especially sensitive to apoptosis [7] and so are tetramer+ CD8+ T cells [5]. Tumor epitope-specific T cells (tetramer+) appear to preferentially bind ANX and are targeted for apoptosis [5]. Taken together, these findings suggest that a loss of effector T cell function through targeted apoptosis might compromise antitumor functions of the host immune system and contribute to tumor progression [73].

Recent studies of apoptosis in immune cells suggest that in T lymphocytes, sensitivity to Fas-mediated death is a regulated phenomenon, in which both IL-2 and antigenic stimulation play a crucial regulatory role [78, 177]. In AICD, which is an

Fig. 1.3 Flow cytometry for expression of CCR7 on circulating CD8+ T cells in normal donors and patients with head and neck cancer (HNC). On the left, the boxplots show that the frequency of CCR7+ CD8+ T cells in HNC patients is significantly lower than that in normal donors. On the right, the data from a representative patient and normal donor indicate that in the patient CCR7+ CD8+ T cells are replaced by CCR7-CD8+ cells, which are sensitive to apoptosis. (Reproduced from Kim et al., 2005. With permission.)

Fig. 1.3 Flow cytometry for expression of CCR7 on circulating CD8+ T cells in normal donors and patients with head and neck cancer (HNC). On the left, the boxplots show that the frequency of CCR7+ CD8+ T cells in HNC patients is significantly lower than that in normal donors. On the right, the data from a representative patient and normal donor indicate that in the patient CCR7+ CD8+ T cells are replaced by CCR7-CD8+ cells, which are sensitive to apoptosis. (Reproduced from Kim et al., 2005. With permission.)

essential part of any normal immune response, IL-2 is a potentiating cytokine. At appropriate concentrations and in the presence of a relevant antigen, it enhances the Fas/FasL pathway in activated T cells leading to expression of CD95 [128, 160, 177]. AICD is induced by repeated or chronic antigenic stimulation, and neither co-stimulatory molecules nor the Bcl-2 family members can rescue T cells from AICD. Furthermore, Th1 cells appear to be more sensitive to AICD than Th2 cells [78]. T cells in the tumor, LN or peripheral circulation of patients with cancer experience chronic or repeated antigenic stimulation with TAA, express CD95 on the cell surface [129, 130] and might be particularly sensitive to AICD. However, expression of IL-2 in the tumor has been shown to be low or absent both at the message and protein levels [86, 128]. TIL in situ do not appear to produce IL-2 or express IL-2R [86, 128]. Translation of IL-2 mRNA is defective in TIL isolated from human breast carcinomas [86]. Therefore, if IL-2 is required for the assembly or function of the Fas death complex in T lymphocytes, then AICD may not be the only mechanism responsible for the demise of these cells in the tumor microenvironment. Microvesicles (MV) Released by Tumors as Intercellular Harbingers of T-cell Apoptosis

Sera and body fluids of patients with melanoma, ovarian carcinoma or HNC contain membranous 50-100 nm microvesicles (MV) presumably originating from the tumor, which contain biologically active 42 kDa FasL and MHC class I molecules and mediate apoptosis of Fas+ lymphocytes at sites distant from the tumor [2, 12, 70, 163]. Upon isolation, these MV induce TCRZ degradation and DNA fragmentation in activated T cells [reviewed in 196]. MV are prominent in tumor cell super-natants, but activated normal cells also produce MV [196]. However, tumor-derived MV have a molecular profile that that distinguishes them from MV produced by normal cells such as DC [196, 204]. While DC-derived MV do not express FasL, are rich in co-stimulatory and HLA class II molecules can promote T-cell proliferation and have been used for immunization in mice and man [27, 161], tumor-derived MV are immunosuppressive. They express TAA, death ligands and HLA class I molecules but not co-stimulatory epitopes, and they promote apoptosis of activated CD8+ antitumor effector T cells [196]. MV can also interfere with mono-cyte differentiation to DC, diverting it from a stimulatory to suppressive pathway [173]. As a result, monocytes fail to up-regulate HLA class II molecules, produce TGF-P and acquire the ability to suppress lymphocyte proliferation [173]. In effect, tumor-derived MV can turn monocytes into CD14-negativeHLA-DRlow TGF-P+ MSC [173]. The characteristic molecular profile of tumor-derived MV in cancer patients' sera could have a prognostic value. For example, patients with advanced HNC, i.e. those with tumor-involved lymph nodes or systemic metastases, had significantly higher levels of biologically active FasL+ MV as well as T-cell targeted apoptosis than patients with early stage disease [70]. In aggregate, these data suggest that MV represent another mechanism used by tumors to subvert differentiation and antitumor activities of immune cells.

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