The generation of a robust CD8(+) cytotoxic T lymphocyte (CTL) response is a key feature of many ... more The generation of a robust CD8(+) cytotoxic T lymphocyte (CTL) response is a key feature of many immunotherapeutic strategies against epithelial tumors and virally infected epithelial tissue. However, surprisingly few studies have addressed whether primary epithelial cells, expressing defined endogenous antigens, are good targets for CTL-mediated lysis. Here, we show that primary keratinocytes (KCs), expressing endogenous ovalbumin (OVA) as a transgene, present measurable H-2K(b)/SIINFEKL complexes at the cell surface and are killed by OVA-specific CTL. Target cell lysis was comparable with a more traditional CTL target cell, EL4, and was enhanced by KC pretreatment with interferon-gamma. These results suggest that primary KCs will be susceptible to CTL-mediated apoptosis when endogenous KC antigens are targeted during immunotherapy.
Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ... more Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus‐specific immune response, and prolonged infection promotes anogenital cancer. To determine whether poor antigen presentation by HPV‐infected keratinocytes (KCs) contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7‐KCs) were compared for susceptibility to T‐cell‐mediated lysis directed to ovalbumin (OVA) processed for presentation by the KCs. Interferon (IFN)‐γ efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7‐KCs similarly presenting OVA. E7‐KCs also exhibited impaired IFN‐γ‐induced upregulation of transcription of major histocompatibility complex class I antigen processing and presentation‐associated genes, and of membrane SIINFEKL–H‐2Kb complexes. Thus, expression of HPV 16 E7 protein in KCs may inhibit enhancement by IFN‐γ of KC sensitivity to T‐cell lysis, by impairing antigen presentation.
Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechani... more Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non‐specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus‐induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced lo PV antigens, resting keratinocytes (KC) appear resistant to interferon‐γ‐enhanced mechanisms of cytotoxic T‐lymphocyte (CTL)‐mediated lysis, and expression of PV antigens by resting KC can tolerise PV‐specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV‐induced proliferative skin lesions for months to years, even in immunocompetent hosts.
We have previously demonstrated an immunosuppressive role for NKT cells that infiltrate hyperplas... more We have previously demonstrated an immunosuppressive role for NKT cells that infiltrate hyperplastic skin epithelium in transgenic mice expressing HPV16E7 oncoprotein under a keratin 14 promoter (K14E7 mice)(Mattarollo et al.(2010) J. Immunol. 184:1242-1250). However, depletion of NKT cells is not easily achieved in either mouse or human. We hypothesized that systemic depletion of lymphocytes might result in turnover of NKT cells (and other lymphocytes) within the precancerous skin without replacement from the circulation. Without these locally suppressive populations, the precancer might then be more susceptible to a transferred immune system. When K14E7 skin was grafted onto RAG1-/- mice, which lack lymphocytes, the grafted skin became depleted of T cells (including NKT cells) within 7 days and remained depleted. Depletion of skin lymphocytes could also be achieved using cyclophosphamide and fludarabine, although with less efficiency. Preliminary data suggests that K14E7 skin grafts on RAG1-/- mice become susceptible to graft rejection after transfer of a new immune system. Together the data suggest that systemic lymphodepletion results in a loss of local, suppressive lymphocytes at the precancerous site and that this might enhance the immune mediated clearance of skin precancers.
To examine the induction of immune response in the genital tract mucosa, subcutaneous and nasal r... more To examine the induction of immune response in the genital tract mucosa, subcutaneous and nasal routes of immunization with bovine papillomavirus type 1 (BPV1) virus-like particles (VLPs) were studied for their ability to induce systemic as well as mucosal immune responses. BPV1VLPs containing a HPV16E7 CTL epitope (BPVL1E7 VLP) were administered subcutaneously or intranasally to mice. After immunization, anti BPVL1E7 immunoglobulineG (IgG) antibodies, IgG1 and IgG2a in the serum and anti BPVL1E7 IgA antibodies in vaginal secretions were measured by ELISA assays. Specific IgG antibodies in serum were observed after immunization via subcutaneous and nasal routes. Both immunization routes gave a high ratio of IgG2a/IgG1 thus indicating a Th-1 type immune response. These data suggest that both systemic and mucosal immunization of BPVL1E7 VLP elicited a good Th1 type immune response which may be useful in protecting against local HPV infection.
Aim: Squamous epithelial cancers are immunogenic, as demonstrated by an increased risk of develop... more Aim: Squamous epithelial cancers are immunogenic, as demonstrated by an increased risk of development in immunosuppressed patients. Some however evade host immunity, and establishing the mechanism of immune evasion could assist with squamous cancer immunotherapy. Method: We have used a model of squamous epithelial pre malignancy, in which HPV16 E7 transgenic and hyperproliferative mouse skin is transplanted to naïve mice, and have explored local regulation of effector T cell function by E7 transgenic epithelial cells through manipulation of the graft donor immune system. Results: E7 transgenic skin is not rejected by naïve recipient mice, or by mice immunised with E7 protein and expressing E7 specific cytotoxic T cells. E7 expressing hyper proliferative epithelium demonstrates caspase-1 activation, production of IL-1β, IL-18, and CCL2, and attraction and activation of NKT cells and mast cells. Rejection of E7 transgenic skin grafts can be observed following depletion from the donor skin of bone marrow derived cells, of NKT cells or of mast cells, whereas adjacent E7 transgenic grafts, otherwise unmanipulated, are not rejected. E7 graft rejection can also be induced by suppression in the donor skin or recipient animal of NKT cell secreted IL-17 or IFN-γ, or by depletion of the soluble IL-1 receptor IL-1R1 or blockade of Arginase activity in the recipient. Inhibition of graft rejection is not overcome by antibody to PD-1, PD-L1, or PD-L2. Hyperproliferative cervix epithelium in patients with CIN3 expresses the factors identified as inhibitory to E7 transgenic skin graft rejection, whereas skin from E7 transgenic mice with a mutated Rb gene preventing E7 induced epithelial proliferation lacks the inhibitory factors. Conclusion: Epithelial hyper proliferation induces immunosuppressive mechanisms that locally prevent cytotoxic T cell function through mechanisms independent of checkpoint blockade. We hypothesise from our data that the local balance of production of IL-1β and Il-18 locally determines cytotoxic T cell effector function targeted at epithelial antigens. Citation Format: Stephen Mattarollo, Graham Leggatt, James Wells, Paul Lambert, Ian H. Frazer. Modeling checkpoint blockade inhibitor resistant immunoregulation induced by squamous epithelial cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A064.
The generation of a robust CD8(+) cytotoxic T lymphocyte (CTL) response is a key feature of many ... more The generation of a robust CD8(+) cytotoxic T lymphocyte (CTL) response is a key feature of many immunotherapeutic strategies against epithelial tumors and virally infected epithelial tissue. However, surprisingly few studies have addressed whether primary epithelial cells, expressing defined endogenous antigens, are good targets for CTL-mediated lysis. Here, we show that primary keratinocytes (KCs), expressing endogenous ovalbumin (OVA) as a transgene, present measurable H-2K(b)/SIINFEKL complexes at the cell surface and are killed by OVA-specific CTL. Target cell lysis was comparable with a more traditional CTL target cell, EL4, and was enhanced by KC pretreatment with interferon-gamma. These results suggest that primary KCs will be susceptible to CTL-mediated apoptosis when endogenous KC antigens are targeted during immunotherapy.
Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ... more Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus‐specific immune response, and prolonged infection promotes anogenital cancer. To determine whether poor antigen presentation by HPV‐infected keratinocytes (KCs) contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7‐KCs) were compared for susceptibility to T‐cell‐mediated lysis directed to ovalbumin (OVA) processed for presentation by the KCs. Interferon (IFN)‐γ efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7‐KCs similarly presenting OVA. E7‐KCs also exhibited impaired IFN‐γ‐induced upregulation of transcription of major histocompatibility complex class I antigen processing and presentation‐associated genes, and of membrane SIINFEKL–H‐2Kb complexes. Thus, expression of HPV 16 E7 protein in KCs may inhibit enhancement by IFN‐γ of KC sensitivity to T‐cell lysis, by impairing antigen presentation.
Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechani... more Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non‐specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus‐induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced lo PV antigens, resting keratinocytes (KC) appear resistant to interferon‐γ‐enhanced mechanisms of cytotoxic T‐lymphocyte (CTL)‐mediated lysis, and expression of PV antigens by resting KC can tolerise PV‐specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV‐induced proliferative skin lesions for months to years, even in immunocompetent hosts.
We have previously demonstrated an immunosuppressive role for NKT cells that infiltrate hyperplas... more We have previously demonstrated an immunosuppressive role for NKT cells that infiltrate hyperplastic skin epithelium in transgenic mice expressing HPV16E7 oncoprotein under a keratin 14 promoter (K14E7 mice)(Mattarollo et al.(2010) J. Immunol. 184:1242-1250). However, depletion of NKT cells is not easily achieved in either mouse or human. We hypothesized that systemic depletion of lymphocytes might result in turnover of NKT cells (and other lymphocytes) within the precancerous skin without replacement from the circulation. Without these locally suppressive populations, the precancer might then be more susceptible to a transferred immune system. When K14E7 skin was grafted onto RAG1-/- mice, which lack lymphocytes, the grafted skin became depleted of T cells (including NKT cells) within 7 days and remained depleted. Depletion of skin lymphocytes could also be achieved using cyclophosphamide and fludarabine, although with less efficiency. Preliminary data suggests that K14E7 skin grafts on RAG1-/- mice become susceptible to graft rejection after transfer of a new immune system. Together the data suggest that systemic lymphodepletion results in a loss of local, suppressive lymphocytes at the precancerous site and that this might enhance the immune mediated clearance of skin precancers.
To examine the induction of immune response in the genital tract mucosa, subcutaneous and nasal r... more To examine the induction of immune response in the genital tract mucosa, subcutaneous and nasal routes of immunization with bovine papillomavirus type 1 (BPV1) virus-like particles (VLPs) were studied for their ability to induce systemic as well as mucosal immune responses. BPV1VLPs containing a HPV16E7 CTL epitope (BPVL1E7 VLP) were administered subcutaneously or intranasally to mice. After immunization, anti BPVL1E7 immunoglobulineG (IgG) antibodies, IgG1 and IgG2a in the serum and anti BPVL1E7 IgA antibodies in vaginal secretions were measured by ELISA assays. Specific IgG antibodies in serum were observed after immunization via subcutaneous and nasal routes. Both immunization routes gave a high ratio of IgG2a/IgG1 thus indicating a Th-1 type immune response. These data suggest that both systemic and mucosal immunization of BPVL1E7 VLP elicited a good Th1 type immune response which may be useful in protecting against local HPV infection.
Aim: Squamous epithelial cancers are immunogenic, as demonstrated by an increased risk of develop... more Aim: Squamous epithelial cancers are immunogenic, as demonstrated by an increased risk of development in immunosuppressed patients. Some however evade host immunity, and establishing the mechanism of immune evasion could assist with squamous cancer immunotherapy. Method: We have used a model of squamous epithelial pre malignancy, in which HPV16 E7 transgenic and hyperproliferative mouse skin is transplanted to naïve mice, and have explored local regulation of effector T cell function by E7 transgenic epithelial cells through manipulation of the graft donor immune system. Results: E7 transgenic skin is not rejected by naïve recipient mice, or by mice immunised with E7 protein and expressing E7 specific cytotoxic T cells. E7 expressing hyper proliferative epithelium demonstrates caspase-1 activation, production of IL-1β, IL-18, and CCL2, and attraction and activation of NKT cells and mast cells. Rejection of E7 transgenic skin grafts can be observed following depletion from the donor skin of bone marrow derived cells, of NKT cells or of mast cells, whereas adjacent E7 transgenic grafts, otherwise unmanipulated, are not rejected. E7 graft rejection can also be induced by suppression in the donor skin or recipient animal of NKT cell secreted IL-17 or IFN-γ, or by depletion of the soluble IL-1 receptor IL-1R1 or blockade of Arginase activity in the recipient. Inhibition of graft rejection is not overcome by antibody to PD-1, PD-L1, or PD-L2. Hyperproliferative cervix epithelium in patients with CIN3 expresses the factors identified as inhibitory to E7 transgenic skin graft rejection, whereas skin from E7 transgenic mice with a mutated Rb gene preventing E7 induced epithelial proliferation lacks the inhibitory factors. Conclusion: Epithelial hyper proliferation induces immunosuppressive mechanisms that locally prevent cytotoxic T cell function through mechanisms independent of checkpoint blockade. We hypothesise from our data that the local balance of production of IL-1β and Il-18 locally determines cytotoxic T cell effector function targeted at epithelial antigens. Citation Format: Stephen Mattarollo, Graham Leggatt, James Wells, Paul Lambert, Ian H. Frazer. Modeling checkpoint blockade inhibitor resistant immunoregulation induced by squamous epithelial cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A064.
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Papers by Ian Frazer