Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV‐mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL‐sensitive E7‐expressing cell lines but are not susceptible to CTL‐mediated lysis in vitro . E7 transgenic and non‐transgenic KC are susceptible to conventional mechanisms of CTL‐mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL‐4), despite large differences in H‐2D b expression at the cell surface. Furthermore, exposure of KC to IFN‐γ increased H‐2D b expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN‐γ. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.
The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer.
Abstract Interferon gamma (IFN γ ) is a key moderator of cell-mediated immunity with diverse, mainly pro-inflammatory actions on immunocytes and target tissue. Recent studies have shown it may enhance anti-tumor and antiviral effects of CD8 T cells. Here we investigate the mechanisms by which IFN γ mediates CD8 T-cell cytotoxic function. We show that in vivo , antigen-specific CD8 T cells that produce INF γ are necessary to effect rejection of skin grafts expressing OVA as a transgene in keratinocytes. The ability of CD8 T cells to produce IFN γ enhanced their ability to migrate to the site of antigen-presenting skin cells. By in vivo imaging, we show that CTL motility, particularly speed, during graft rejection was enhanced by locally available IFN γ . We then used a reductionist two-cell model of CTL effectors and keratinocyte targets to investigate the effects of locally available (paracrine) and CTL-producing (autocrine) IFN γ on the motility behavior and killing ability of the CTL. Using live-cell imaging by prolonged time-lapse microscopy of primary effector CD8 T cells and antigen-expressing primary keratinocyte targets, we show that CD8 T-cell cytotoxic function and motility is enhanced by locally available IFN γ . Conversely, deprivation of either autocrine or paracrine IFN γ , or blockade of IFN γ signaling to CTL markedly reduced their cytotoxic function, their kinematics, and effector cell survival. We conclude that in vitro and in vivo, autocrine production of IFN γ by CTL enhances their motility and promotes killing of primary target keratinocytes. The absolute need for local IFN γ to enable cytotoxic CD8 T-cell function is of significance for immunotherapy for chronic viral infection and for cancer.
Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.
Some recently obtained data from our laboratory on the molecular characterization of Echinococcus and Taenia solium are described and are complimented by relevant new information obtained by other groups. Progress made in the development of satisfactory immunodiagnostic assays and in the production of recombinant molecules, suitable for application in serology of hydatid disease and cysticercosis, is highlighted. Results arising from the application of polymerase chain reaction and direct sequencing, using primers homologous to evolutionarily conserved sequences, in phylogenetic studies and for distinguishing individual taeniid species are also discussed.
Most of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.
Chemokines and their receptors play an important role in the recruitment, activation and differentiation of immune cells. The chemokine receptor, CXCR3, and its ligands, CXCL9, CXCL10, and CXCL11 are key immune chemoattractants during interferon-induced inflammatory responses. Inflammation of the skin resulting from infections or autoimmune disease drives expression of CXCL9/10/11 and the subsequent recruitment of effector, CXCR3+ T cells from the circulation. The relative contributions of the different CXCR3 chemokines and the three variant isoforms of CXCR3 (CXCR3A, CXCR3B, CXCR3alt) to the inflammatory process in human skin requires further investigation. In skin cancers, the CXCR3 receptor can play a dual role whereby expression on tumour cells can lead to cancer metastasis to systemic sites while receptor expression on immune cells can frequently promote anti-tumour immune responses. This review will discuss the biology of CXCR3 and its associated ligands with particular emphasis on the skin during inflammation and carcinogenesis.
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