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.
Gamma delta T cells (gd T cells) possess innate-like properties and are proposed to bridge the ga... more Gamma delta T cells (gd T cells) possess innate-like properties and are proposed to bridge the gap between innate and adaptive immunity. In this study, we explored the role of gd T cells in cutaneous immunity using a skin transplantation model. Following engraftment of skin expressing cell-associated model antigen (Ag) (ovalbumin) in epithelial keratinocytes, skin-resident gd T cells enhanced graft rejection. Although the effector function of CD8 T cells was intact in the absence of gd T cells, cross-priming of CD8 T cell to graft-derived Ag was impaired in the absence of gd T cells. The reduced graft rejection and graft priming of gd T-cell-deficient mice was evident in both acutely inflamed and well-healed grafting models. Furthermore, expression of the CD40 activation marker on migrating dendritic cells was lower in TCRd À/À mice compared with wild-type mice, regardless of the presence or absence of inflammation associated with grafting. These results indicate that gd T cells enhance graft priming and consequently the likelihood of a successful immune outcome in the context of skin graft rejection, suggesting that gd T cells may be an important component of immunity to epithelial cancers or infection.
Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear t... more Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16) E7 oncoprotein under a keratin 14 promoter (K14E7 mice) display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb) led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes) to premalignant skin.
IFN-g has a central role in the defense against infections and cancer. More recently, however, IF... more IFN-g has a central role in the defense against infections and cancer. More recently, however, IFN-g has also been reported to have immunosuppressive effects in models of autoimmune disease, melanoma, and premalignant skin disease. Although IL-12 and IL-18 are critical inducers of IFN-g during infection, the mechanisms that induce IFN-g in an immunosuppressive context are unknown. Previously, we identified a key role for IFN-g in mediating the suppression of antigen-specific immune responses in a transgenic mouse model of human papillomavirus (HPV)-associated epidermal hyperplasia, driven by the expression of the HPV16 E7 oncoprotein from a keratin 14 promoter (K14E7). We now demonstrate elevated production of IFN-g, IL-18, and IL-12 by K14E7 transgenic skin compared with nontransgenic skin. IFN-g in K14E7 transgenic skin was produced predominantly by CD8 þ and CD4 þ T cells, which were present in greater numbers in K14E7 transgenic skin. Production of IFN-g in K14E7 skin required IL-18 but not IL-12. Our findings show that IL-18 contributes to inducing IFN-g in an immunosuppressive cutaneous environment caused by viral oncogene-driven hyperplasia.
Human papillomavirus (HPV) 16 E7 (E7) protein expression in skin promotes epithelial hyperprolife... more Human papillomavirus (HPV) 16 E7 (E7) protein expression in skin promotes epithelial hyperproliferation and transformation to malignancy. Grafts of murine skin expressing E7 protein as a transgene in keratinocytes are not rejected from immunocompetent recipients, whereas grafts expressing ovalbumin (OVA), with or without coexpression of E7 protein, are promptly rejected, demonstrating that E7-associated non-antigen-specific local immunosuppression is not a major determinant of lack of rejection of E7 transgenic skin. To determine whether failure of rejection of E7 skin grafts is due to failure to attract E7-specific effector T cells, E7-and OVA-specific effector CD8 + T cells, activated in vitro, were transferred to animals bearing E7 transgenic skin grafts. Three days after T cell transfer, E7-specific T cells were present in significantly greater numbers than OVAspecific T cells in the grafted skin on animals bearing recently placed or healed E7 grafts, without graft rejection, and also in the ear skin of E7 transgenic animals, without obvious pathology. E7 and OVA-specific T cells were present in lesser numbers in healed E7 grafts than in recently placed grafts and in lesser numbers in recently placed E7 transgenic epidermal grafts without E7-associated hyperproliferation, derived from E7 transgenic mice with a mutated retinoblastoma gene. These data demonstrate that effector T cells are to some extent attracted to E7 transgenic skin specifically by E7 expression, but in large measure non-specifically by the epithelial proliferation associated with E7 expression, and by the local inflammation produced by grafting. Failure of E7 graft rejection was observed despite trafficking of E7-specific effector T cells to E7-expressing epithelium, a finding of consequence for immunotherapy of HPV 16 E7-associated human cancers.
High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 p... more High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRb L/ L mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRb L/ L skin grafts. Here, we further show that K14.E7xRb L/ L skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b + antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRb L/ L grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRb L/ L grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRb L/ L keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro. We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression.
Objectives. To compare immunohistochemical scoring with clinical scoring and radiology for the as... more Objectives. To compare immunohistochemical scoring with clinical scoring and radiology for the assessment of rheumatoid arthritis (RA) disease activity, synovial tissue (ST) biopsied arthroscopically was assessed from 18 patients before and after commencement of disease-modifying anti-rheumatic drug (DMARD) therapy. Methods. Lymphocytes, macrophages, differentiated dendritic cells (DC), vascularity, tumour necrosis factor (TNF) a and interleukin-1b levels were scored. Clinical status was scored using the American College of Rheumatology (ACR) core set and serial radiographs were scored using the Larsen and Sharp methods. Histopathological evidence of activity included infiltration by lymphocytes, DC, macrophages, tissue vascularity, and expression of lining and sublining TNFa. These indices co-varied across the set of ST biopsies and were combined as a synovial activity score for each biopsy. Results. The change in synovial activity with treatment correlated with the ACR clinical response and with decreased radiological progression by the Larsen score. The ACR response to DMARD therapy, the change in synovial activity score and the slowing of radiological progression were each greatest in patients with high initial synovial vascularity. Conclusions. The data demonstrate an association between clinical, radiological and synovial immunopathological responses to anti-rheumatic treatment in RA. High ST vascularity may predict favourable clinical and radiological responses to treatment.
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.
Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia th... more Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL-23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skingrafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against the graft. IL-17 was produced by CD3 + T cells, predominantly CD4 + T cells in human, and CD4 + and gd T cells in mouse hyperplastic lesions. IL-23 and IL-1b, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer.
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.
Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven... more 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.
Vaccines prophylactic against infection with human papillomavirus (HPV) are based on alum adjuvan... more Vaccines prophylactic against infection with human papillomavirus (HPV) are based on alum adjuvanted virus-like particles. Two such vaccines have recently been shown to prevent persistent HPV infection and associated cervical cancer precursor lesions. The genotype-specific neutralising antibody directed at conformational epitopes of the L1 major capsid protein is likely to mediate protection. Vaccines therapeutic for persisting HPV infection can eliminate transplantable tumors in animal models, but are of limited efficacy in mice grafted with skin that expresses HPV antigens or in humans. This paradox has been partially resolved by data clarifying the immunoregulatory role of skin cytokines (e.g. transforming growth factor-b and interleukin-10) and the consequences of antigen presentation by subsets of skinassociated antigen-presenting cells.
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.
Further, blockade of either IL-17A or arginase activity alleviates DNCB-induced hyperinflammation... more Further, blockade of either IL-17A or arginase activity alleviates DNCB-induced hyperinflammation through reduced recruitment of neutrophils, as a consequence of decreased CXCL1 and CXCL5 chemokine production. Thus, our findings suggest that increased IL-17A expression by macrophages in E7-expressing skin exposed to DNCB promotes arginase-1 induction and contributes directly to the observed hyperinflammation.
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.
Gamma delta T cells (gd T cells) possess innate-like properties and are proposed to bridge the ga... more Gamma delta T cells (gd T cells) possess innate-like properties and are proposed to bridge the gap between innate and adaptive immunity. In this study, we explored the role of gd T cells in cutaneous immunity using a skin transplantation model. Following engraftment of skin expressing cell-associated model antigen (Ag) (ovalbumin) in epithelial keratinocytes, skin-resident gd T cells enhanced graft rejection. Although the effector function of CD8 T cells was intact in the absence of gd T cells, cross-priming of CD8 T cell to graft-derived Ag was impaired in the absence of gd T cells. The reduced graft rejection and graft priming of gd T-cell-deficient mice was evident in both acutely inflamed and well-healed grafting models. Furthermore, expression of the CD40 activation marker on migrating dendritic cells was lower in TCRd À/À mice compared with wild-type mice, regardless of the presence or absence of inflammation associated with grafting. These results indicate that gd T cells enhance graft priming and consequently the likelihood of a successful immune outcome in the context of skin graft rejection, suggesting that gd T cells may be an important component of immunity to epithelial cancers or infection.
Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear t... more Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16) E7 oncoprotein under a keratin 14 promoter (K14E7 mice) display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb) led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes) to premalignant skin.
IFN-g has a central role in the defense against infections and cancer. More recently, however, IF... more IFN-g has a central role in the defense against infections and cancer. More recently, however, IFN-g has also been reported to have immunosuppressive effects in models of autoimmune disease, melanoma, and premalignant skin disease. Although IL-12 and IL-18 are critical inducers of IFN-g during infection, the mechanisms that induce IFN-g in an immunosuppressive context are unknown. Previously, we identified a key role for IFN-g in mediating the suppression of antigen-specific immune responses in a transgenic mouse model of human papillomavirus (HPV)-associated epidermal hyperplasia, driven by the expression of the HPV16 E7 oncoprotein from a keratin 14 promoter (K14E7). We now demonstrate elevated production of IFN-g, IL-18, and IL-12 by K14E7 transgenic skin compared with nontransgenic skin. IFN-g in K14E7 transgenic skin was produced predominantly by CD8 þ and CD4 þ T cells, which were present in greater numbers in K14E7 transgenic skin. Production of IFN-g in K14E7 skin required IL-18 but not IL-12. Our findings show that IL-18 contributes to inducing IFN-g in an immunosuppressive cutaneous environment caused by viral oncogene-driven hyperplasia.
Human papillomavirus (HPV) 16 E7 (E7) protein expression in skin promotes epithelial hyperprolife... more Human papillomavirus (HPV) 16 E7 (E7) protein expression in skin promotes epithelial hyperproliferation and transformation to malignancy. Grafts of murine skin expressing E7 protein as a transgene in keratinocytes are not rejected from immunocompetent recipients, whereas grafts expressing ovalbumin (OVA), with or without coexpression of E7 protein, are promptly rejected, demonstrating that E7-associated non-antigen-specific local immunosuppression is not a major determinant of lack of rejection of E7 transgenic skin. To determine whether failure of rejection of E7 skin grafts is due to failure to attract E7-specific effector T cells, E7-and OVA-specific effector CD8 + T cells, activated in vitro, were transferred to animals bearing E7 transgenic skin grafts. Three days after T cell transfer, E7-specific T cells were present in significantly greater numbers than OVAspecific T cells in the grafted skin on animals bearing recently placed or healed E7 grafts, without graft rejection, and also in the ear skin of E7 transgenic animals, without obvious pathology. E7 and OVA-specific T cells were present in lesser numbers in healed E7 grafts than in recently placed grafts and in lesser numbers in recently placed E7 transgenic epidermal grafts without E7-associated hyperproliferation, derived from E7 transgenic mice with a mutated retinoblastoma gene. These data demonstrate that effector T cells are to some extent attracted to E7 transgenic skin specifically by E7 expression, but in large measure non-specifically by the epithelial proliferation associated with E7 expression, and by the local inflammation produced by grafting. Failure of E7 graft rejection was observed despite trafficking of E7-specific effector T cells to E7-expressing epithelium, a finding of consequence for immunotherapy of HPV 16 E7-associated human cancers.
High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 p... more High-risk" human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRb L/ L mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRb L/ L skin grafts. Here, we further show that K14.E7xRb L/ L skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b + antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRb L/ L grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRb L/ L grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRb L/ L keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro. We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression.
Objectives. To compare immunohistochemical scoring with clinical scoring and radiology for the as... more Objectives. To compare immunohistochemical scoring with clinical scoring and radiology for the assessment of rheumatoid arthritis (RA) disease activity, synovial tissue (ST) biopsied arthroscopically was assessed from 18 patients before and after commencement of disease-modifying anti-rheumatic drug (DMARD) therapy. Methods. Lymphocytes, macrophages, differentiated dendritic cells (DC), vascularity, tumour necrosis factor (TNF) a and interleukin-1b levels were scored. Clinical status was scored using the American College of Rheumatology (ACR) core set and serial radiographs were scored using the Larsen and Sharp methods. Histopathological evidence of activity included infiltration by lymphocytes, DC, macrophages, tissue vascularity, and expression of lining and sublining TNFa. These indices co-varied across the set of ST biopsies and were combined as a synovial activity score for each biopsy. Results. The change in synovial activity with treatment correlated with the ACR clinical response and with decreased radiological progression by the Larsen score. The ACR response to DMARD therapy, the change in synovial activity score and the slowing of radiological progression were each greatest in patients with high initial synovial vascularity. Conclusions. The data demonstrate an association between clinical, radiological and synovial immunopathological responses to anti-rheumatic treatment in RA. High ST vascularity may predict favourable clinical and radiological responses to treatment.
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.
Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia th... more Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL-23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skingrafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against the graft. IL-17 was produced by CD3 + T cells, predominantly CD4 + T cells in human, and CD4 + and gd T cells in mouse hyperplastic lesions. IL-23 and IL-1b, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer.
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.
Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven... more 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.
Vaccines prophylactic against infection with human papillomavirus (HPV) are based on alum adjuvan... more Vaccines prophylactic against infection with human papillomavirus (HPV) are based on alum adjuvanted virus-like particles. Two such vaccines have recently been shown to prevent persistent HPV infection and associated cervical cancer precursor lesions. The genotype-specific neutralising antibody directed at conformational epitopes of the L1 major capsid protein is likely to mediate protection. Vaccines therapeutic for persisting HPV infection can eliminate transplantable tumors in animal models, but are of limited efficacy in mice grafted with skin that expresses HPV antigens or in humans. This paradox has been partially resolved by data clarifying the immunoregulatory role of skin cytokines (e.g. transforming growth factor-b and interleukin-10) and the consequences of antigen presentation by subsets of skinassociated antigen-presenting cells.
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.
Further, blockade of either IL-17A or arginase activity alleviates DNCB-induced hyperinflammation... more Further, blockade of either IL-17A or arginase activity alleviates DNCB-induced hyperinflammation through reduced recruitment of neutrophils, as a consequence of decreased CXCL1 and CXCL5 chemokine production. Thus, our findings suggest that increased IL-17A expression by macrophages in E7-expressing skin exposed to DNCB promotes arginase-1 induction and contributes directly to the observed hyperinflammation.
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