Peripheral mechanisms of CD8+ T cell tolerance in H-2b K14E7 transgenic mice

The human papillomavirus (HPV) early protein 7 (E7) represents one of the major transforming proteins in HPV-associated epithelial malignances and as such is considered a potential target for T cell mediated activity against HPVassociated epithelial tumours. Nevertheless, patients with HPV-associated cervical carcinomas are reported to generate ineffectual E7-specific cellular responses to either endogenous or vaccine administrated E7. Previous studies have reported that HPV16 E7 expression in peripheral keratinocytes of H-2q transgenic mice (K14E7 transgenic mice) facilitates global downregulation of CD8+ T cell responses, suggesting that the E7 oncoprotein has the ability to actively tolerize components of the immune system. The aim of this study was to confirm the existence of peripheral CD8+ T cell tolerance in the newly generated H-2b K14E7 mouse line and to utilize this new mouse line to understand the underlying mechanism(s) behind CD8+ T cell tolerance in K14E7 transgenic animals. Downregulation of CD8+ T cell function were verified in H-2b K14E7 mice. Furthermore, immunologically H-2b K14E7 animals were shown to behave similarly to previously described K14E7 transgenic mice lines. To demonstrate peripheral CD8+ T cell tolerance in H-2b K14E7 mice, the function of adoptively transferred E7-specific and Ovalbumin (OVA)-specific CD8+ T cells were assessed in the periphery of these animals, with the hypothesis that these adoptively transferred CD8+ T cells would be rendered unresponsive due to inhibitory influences within the H-2b K14E7 peripheral environment. Short-term transfers of E7-specific and OVA-specific CD8+ T cells was shown to significantly enhance E7-specific and OVA-specific CD8+ T cell responsiveness respectively, in H-2b K14E7 mice. However, over time antigen-specific CD8+ T cell responsiveness in H-2b K14E7 transfer recipients declined, which at least in the E7-specific CD8+ T cell transfer situation correlated with a progressive reduction in transferred E7-specific CD8+ T cell numbers in the spleen of H-2b K14E7 mice. Further in vivo tracking experiments revealed that transferred E7- specific CD8+ T cells were not being deleted in the periphery of H-2b K14E7 animals over time but were preferentially accumulating in the skin. Consistent with these findings, accumulation of E7-specific CD8+ T cells was also evident in the skin of native H-2b K14E7 animals and furthermore these CD8+ T cells were found to be functionally E7 unresponsive. Interestingly, elevated numbers of CD4+ Foxp3+ cells were detected in the skin of H-2b K14E7 mice, with H-2b K14E7 skin-derived CD4+ CD25+ cells shown capable of suppressing E7- specific interferon-5 (IFN-5) production by in vitro activated CD8+ T cells. Increased percentages of Foxp3+ cells were also evident within the CD4+ CD25+ population found in the spleen and skin-draining lymph nodes of H-2b K14E7 mice. Moreover, elevated numbers of Foxp3+ cells within H-2b K14E7 skindraining lymph node CD4+ CD25+ cells correlated with increased suppressive ability. Depletion of CD4+ or CD25+ cells in H-2b K14E7 mice resulted in enhanced E7-specific and OVA-specific CD8+ T cell responsiveness following specific peptide immunization, thus confirming a role for CD4+ CD25+ cells in regulating CD8+ T cell tolerance in these transgenic animals. In addition, significant suppression of E7-specific IFN-F production by activated CD8+ T cells in C57BL/6 mice was achieved by adoptive transfer of H-2b K14E7 CD4+ cells. Dendritic cells (DCs) harvested from naive H-2b K14E7 skin-draining lymph node were shown to express lower levels of costimulatory molecules when compared to non-transgenic DCs. Moreover, incubation of E7-specific CD8+ T cells with E7 pulsed H-2b K14E7 skin-draining lymph node DCs resulted in reduced IFN-F production following T cell activation. In conclusion, this study highlights a role for CD4+ CD25+ cells and tolerogenic DCs in maintaining peripheral CD8+ T cell tolerance in H-2b K14E7 mice. These results provide some insight into how HPV16 E7 may influence the immune system of patients with cervical carcinomas and other HPV-associated epithelial cancers and may have implications for immunotherapies against such malignancies