Generalized substitution of isoencoding codons shortens the duration of papillomavirus L1 protein expression in transiently gene-transfected keratinocytes due to cell differentiation

Recently we reported that gene codon composition determines differentiation-dependent expression of the PV L1 genes in mouse primary keratinocytes (KCs) in vitro and in vivo (Zhao et al. 2005, Mol. Cell Biol. 25:8643–8655). Here, we investigated whether generalized substitution of isoencoding codons affects the duration of expression of PV L1 genes in mouse and human KCs in day 1 culture transiently transfected with native (Nat) and codon modified (Mod) L1 genes. Following transient transfection, KC continuously transcribed both Nat and Mod PV L1 genes for at least 12 days, with the levels of L1 mRNAs from the Mod L1 genes significantly higher than those from the Nat L1 genes. However, continuous L1 protein expression at day 9 post-transfection was observed for both mouse and human KCs transfected with the Nat L1 genes only. Further, aa-tRNAs prepared from D8 KC cultures enhanced translation of two PV Nat L1 DNAs in RRL lysate and PV Nat L1 mRNAs in D0 cell-free lysate, whereas aa-tRNAs from D0 KCs enhanced translation of PV Mod L1 mRNAs in D8 cell-free lysate. It appears that aa-tRNAs in less-differentiated and differentiated KCs differentially match the PV Nat and Mod L1 mRNAs to regulate their translations in vitro

Antigen-specific CD8 T cells can eliminate antigen-bearing keratinocytes with clonogenic potential via an IFN-γ-dependent mechanism

The immune system surveys the skin for keratinocytes (KCs) infected by viruses or with acquired genetic damage. The mechanism by which T cells mediate KC elimination is however undefined. In this study we show that antigen-specific CD8 T cells can eliminate antigen-bearing KCs in vivo and inhibit their clonogenic potential in vitro, independently of the effector molecules perforin and Fas-ligand (Fas-L). In contrast, IFN-gamma receptor expression on KCs and T cells producing IFN-gamma are each necessary and sufficient for in vitro inhibition of KC clonogenic potential. Thus, antigen-specific cytotoxic T lymphocytes (CTLs) may mediate destruction of epithelium expressing non-self antigen by eliminating KCs with potential for self-renewal through an IFN-gamma-dependent mechanism

Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers

Interferon-gamma enhances cytotoxic T lymphocyte recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide

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-gamma 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-gamma. 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

Bortezomib Improves Adoptive T-cell Therapy by Sensitizing Cancer Cells to FasL Cytotoxicity

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy

Bortezomib Improves Adoptive T-cell Therapy by Sensitizing Cancer Cells to FasL Cytotoxicity

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Since T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of co-stimulatory molecules and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T cell receptor CD3ζ and IL-2 receptor-α, while maintaining IFN-γ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(−/−) mice, bortezomib treatment after adoptive T cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T cell function in the context of cancer immunotherapy