Peritumoral administration of immunomodulatory antibodies as a triple combination suppresses skin tumor growth without systemic toxicity.

BACKGROUND: Skin cancers, particularly keratinocyte cancers, are the most commonly diagnosed tumors. Although surgery is often effective in early-stage disease, skin tumors are not always easily accessible, can reoccur and have the ability to metastasize. More recently, immunotherapies, including intravenously administered checkpoint inhibitors, have been shown to control some skin cancers, but with off-target toxicities when used in combination. Our study investigated whether peritumoral administration of an antibody combination targeting PD-1, 4-1BB (CD137) and VISTA might control skin tumors and lead to circulating antitumor immunity without off-target toxicity. METHODS: The efficacy of combination immunotherapy administered peritumorally or intravenously was tested using transplantable tumor models injected into mouse ears (primary tumors) or subcutaneously in flank skin (secondary tumors). Changes to the tumor microenvironment were tracked using flow cytometry while tumor-specific, CD8 T cells were identified through enzyme-linked immunospot (ELISPOT) assays. Off-target toxicity of the combination immunotherapy was assessed via serum alanine aminotransferase ELISA and histological analysis of liver sections. RESULTS: The data showed that local administration of antibody therapy eliminated syngeneic murine tumors transplanted in the ear skin at a lower dose than required intravenously, and without measured hepatic toxicity. Tumor elimination was dependent on CD8 T cells and was associated with an increased percentage of CD8 T cells expressing granzyme B, KLRG1 and Eomes, and a decreased population of CD4 T cells including CD4+FoxP3+ cells in the treated tumor microenvironment. Importantly, untreated, distal tumors regressed following antibody treatment of a primary tumor, and immune memory prevented growth of subcutaneous flank tumors administered 50 days after regression of a primary tumor. CONCLUSIONS: Together, these data suggest that peritumoral immunotherapy for skin tumors offers advantages over conventional intravenous delivery, allowing antibody dose sparing, improved safety and inducing long-term systemic memory. Future clinical trials of immunotherapy for primary skin cancer should focus on peritumoral delivery of combinations of immune checkpoint antibodies.

Metabolomics at the tumor microenvironment interface: Decoding cellular conversations.

Cancer heterogeneity remains a significant challenge for effective cancer treatments. Altered energetics is one of the hallmarks of cancer and influences tumor growth and drug resistance. Studies have shown that heterogeneity exists within the metabolic profile of tumors, and personalized-combination therapy with relevant metabolic interventions could improve patient response. Metabolomic studies are identifying novel biomarkers and therapeutic targets that have improved treatment response. The spatial location of elements in the tumor microenvironment are becoming increasingly important for understanding disease progression. The evolution of spatial metabolomics analysis now allows scientists to deeply understand how metabolite distribution contributes to cancer biology. Recently, these techniques have spatially resolved metabolite distribution to a subcellular level. It has been proposed that metabolite mapping could improve patient outcomes by improving precision medicine, enabling earlier diagnosis and intraoperatively identifying tumor margins. This review will discuss how altered metabolic pathways contribute to cancer progression and drug resistance and will explore the current capabilities of spatial metabolomics technologies and how these could be integrated into clinical practice to improve patient outcomes.

PD-1 and beyond to Activate T Cells in Cutaneous Squamous Cell Cancers: The Case for 4-1BB and VISTA Antibodies in Combination Therapy.

Non-melanoma skin cancers (NMSC) have a higher incidence than all other cancers combined with cutaneous squamous cell carcinoma (cSCC), capable of metastasis, representing approximately 20% of NMSCs. Given the accessibility of the skin, surgery is frequently employed to treat localized disease, although certain localities, the delineation of clear margins, frequency and recurrence of tumors can make these cancers inoperable in a subset of patients. Other treatment modalities, including cryotherapy, are commonly used for individual lesions, with varying success. Immunotherapy, particularly with checkpoint antibodies, is increasingly a promising therapeutic approach in many cancers, offering the potential advantage of immune memory for protection against lesion recurrence. This review addresses a role for PD-1, 4-1BB and VISTA checkpoint antibodies as monotherapies, or in combination as a therapeutic treatment for both early and late-stage cSCC.

Evolution of Cancer Vaccines-Challenges, Achievements, and Future Directions.

The development of cancer vaccines has been intensively pursued over the past 50 years with modest success. However, recent advancements in the fields of genetics, molecular biology, biochemistry, and immunology have renewed interest in these immunotherapies and allowed the development of promising cancer vaccine candidates. Numerous clinical trials testing the response evoked by tumour antigens, differing in origin and nature, have shed light on the desirable target characteristics capable of inducing strong tumour-specific non-toxic responses with increased potential to bring clinical benefit to patients. Novel delivery methods, ranging from a patient's autologous dendritic cells to liposome nanoparticles, have exponentially increased the abundance and exposure of the antigenic payloads. Furthermore, growing knowledge of the mechanisms by which tumours evade the immune response has led to new approaches to reverse these roadblocks and to re-invigorate previously suppressed anti-tumour surveillance. The use of new drugs in combination with antigen-based therapies is highly targeted and may represent the future of cancer vaccines. In this review, we address the main antigens and delivery methods used to develop cancer vaccines, their clinical outcomes, and the new directions that the vaccine immunotherapy field is taking.

Investigating T Cell Immunity in Cancer: Achievements and Prospects.

T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells. However, as tumours become established they form their own suppressive microenvironments capable of shutting down T cell function, and allowing tumours to persist and grow. To further understand the tumour microenvironment, including the interplay between different immune cells and their role in anti-tumour immune responses, a number of studies from mouse models to clinical trials have been performed. In this review, we examine mechanisms utilized by tumour cells to reduce their visibility to CD8+ Cytotoxic T lymphocytes (CTL), as well as therapeutic strategies trialled to overcome these tumour-evasion mechanisms. Next, we summarize recent advances in approaches to enhance CAR T cell activity and persistence over the past 10 years, including bispecific CAR T cell design and early evidence of efficacy. Lastly, we examine mechanisms of T cell infiltration and tumour regression, and discuss the strengths and weaknesses of different strategies to investigate T cell function in murine tumour models.

Regulatory T Cells but Not IL-10 Impair Cell-Mediated Immunity in Human Papillomavirus E7+ Hyperplastic Epithelium.

High-risk human papillomavirus infection can induce cervical and other intraepithelial neoplasia and invasive cancers. A transgenic mouse expressing keratin 14 promotor-driven HPV16 E7 oncoprotein exhibits epithelial hyperplasia and mimics many features of human papillomavirus-related intraepithelial precancers. We have previously demonstrated that HPV16 E7-mediated epithelial hyperplasia suppresses T helper type 1 responses to intradermally delivered antigen and directs differentiation of CD4+ T cells towards a Foxp3+ regulatory phenotype (Treg). Here we establish that Foxp3+ Treg expansion from a transferred naive T-cell population is driven directly by the hyperplastic skin and is independent of pre-existing immune-modulated lymphocytes. However, depletion of endogenous CD25+ Tregs before priming of adoptively transferred T cells significantly improves antigen-specific CD8+ T-cell responses but not T helper type 1 responses. Deletion of IL-10 had no effect on Treg expansion, epidermal dendritic cell alteration, and suppression of induced T helper type 1 immunity in HPV16 E7-driven hyperplastic mice. Thus, HPV16 E7-mediated epithelial hyperplasia promotes expansion of peripheral Tregs in response to intradermal immunization that suppress antigen-specific CD8+ T-cell responses independently of IL-10, but depletion of these Tregs is not sufficient to restore T helper type 1 immunity.

Increased lipid metabolism impairs NK cell function and mediates adaptation to the lymphoma environment.

Natural killer (NK) cells play critical roles in protection against hematological malignancies but can acquire a dysfunctional state, which limits antitumor immunity. However, the underlying reasons for this impaired NK cell function remain to be uncovered. We found that NK cells in aggressive B-cell lymphoma underwent substantial transcriptional reprogramming associated with increased lipid metabolism, including elevated expression of the transcriptional regulator peroxisome activator receptor-γ (PPAR-γ). Exposure to fatty acids in the lymphoma environment potently suppressed NK cell effector response and cellular metabolism. NK cells from both diffuse large B-cell lymphoma patients and Eµ-myc B-cell lymphoma-bearing mice displayed reduced interferon-γ (IFN-γ) production. Activation of PPAR-γ partially restored mitochondrial membrane potential and IFN-γ production. Overall, our data indicate that increased lipid metabolism, while impairing their function, is a functional adaptation of NK cells to the fatty-acid rich lymphoma environment.

Cytokine/chemokine profiles in squamous cell carcinoma correlate with precancerous and cancerous disease stage.

Actinic Keratosis (AK), Intraepidermal Carcinoma (IEC), and Squamous Cell Carcinoma (SCC) are generally considered to be advancing stages of the same disease spectrum. However, while AK often regress spontaneously, and IEC often regress in response to immune-activating treatments, SCC typically do not regress. Therefore, it is vital to define whether fundamental immunological changes occur during progression to SCC. Here we show that proinflammatory cytokine expression, chemokine expression, and immune cell infiltration density change during progression to SCC. Our findings suggest a switch from predominantly proinflammatory cytokine production to chemokine production is a key feature of progression from precancer to cancer. Together, these observations propose a model that can underpin current research and open new avenues of exploration into the clinical significance of these profiles with respect to immunotherapeutic or other treatment outcomes.

HPV16 E7-Driven Epithelial Hyperplasia Promotes Impaired Antigen Presentation and Regulatory T-Cell Development.

Human papillomaviruses infect keratinocytes and can lead to hyperproliferative dysplasia and malignant transformation if not cleared by the immune system. Human papillomavirus has evolved an array of mechanisms to evade and manipulate the immune system to improve replication efficiency and promote persistent infection. We here demonstrate that hyperproliferative skin expressing the high-risk human papillomavirus 16 E7 oncogene as a transgene drives immunomodulation of dendritic cells (DCs), resulting in reduced capacity to take up antigen and prime effector CD4+ T cell responses. The phenotype of DCs in the E7-expressing hyperproliferative skin was not reversible by activation through intradermal immunization. Naïve CD4+ T cells primed by E7-driven hyperproliferative skin acquired FoxP3 expression and an anergic phenotype. DC and T help modulation was dependent on E7-retinoblastoma protein interaction-driven epithelial hyperproliferation, rather than on expression of E7. Inhibition of binding of E7 to retinoblastoma protein, and of consequent epithelial hyperplasia, was associated with normal skin DC phenotype, and T helper type 1 effector responses to immunization were restored. We conclude that human papillomavirus-induced epithelial hyperplasia modulates epithelial DCs and inhibits T helper type 1 immunity while polarizing T-cell differentiation to a regulatory or anergic phenotype.

NKT Cell-Driven Enhancement of Antitumor Immunity Induced by Clec9a-Targeted Tailorable Nanoemulsion.

Invariant natural killer T (iNKT) cells are a subset of lymphocytes with immune regulatory activity. Their ability to bridge the innate and adaptive immune systems has been studied using the glycolipid ligand α-galactosylceramide (αGC). To better harness the immune adjuvant properties of iNKT cells to enhance priming of antigen-specific CD8+ T cells, we encapsulated both αGC and antigen in a Clec9a-targeted nanoemulsion (TNE) to deliver these molecules to cross-presenting CD8+ dendritic cells (DC). We demonstrate that, even in the absence of exogenous glycolipid, iNKT cells supported the maturation of CD8α+ DCs to drive efficient cross-priming of antigen-specific CD8+ T cells upon delivery of Clec9a/OVA-TNE. The addition of αGC to the TNE (Clec9a/OVA/αGC) further enhanced activation of iNKT cells, NK cells, CD8α+ DCs, and polyfunctional CD8+ T cells. When tested therapeutically against HPVE7-expressing TC-1 tumors, long-term tumor suppression was achieved with a single administration of Clec9a/E7 peptide/αGC TNE. Antitumor activity was correlated with the recruitment of mature DCs, NK cells, and tumor-specific effector CD8+ T cells to the tumor-draining lymph node and tumor tissue. Thus, Clec9a-TNE codelivery of CD8+ T-cell epitopes with αGC induces alternative helper signals from activated iNKT cells, elicits innate (iNKT, NK) immunity, and enhances antitumor CD8+ T-cell responses for control of solid tumors.

An Ex Vivo Human Tumor Assay Shows Distinct Patterns of EGFR Trafficking in Squamous Cell Carcinoma Correlating to Therapeutic Outcomes.

EGFR overexpression is associated with squamous cell carcinoma development. Altered endocytosis and polarization of receptor tyrosine kinases, including EGFR, affect migration and invasion in three-dimensional culture. These studies have been completed via genetic sequencing, cell line, or three-dimensional in vitro and in vivo murine models. Here, we describe an imaging method that allows ex vivo examination of ligand-induced endocytosis of EGFR in non-dissociated human tumors. We analyzed sets of tumor samples from advanced cutaneous squamous cell carcinoma and head and neck squamous cell carcinoma, actinic keratosis, intraepidermal carcinoma, and cutaneous squamous cell carcinoma. We show that EGFR endocytosis is dysregulated in advanced SCC and correlates with anti-EGFR monoclonal antibody therapy outcomes. In actinic keratosis, intraepidermal carcinoma, and well-differentiated cutaneous squamous cell carcinoma, different patterns of epidermal growth factor ligand uptake and binding were observed at the leading edge of different dysplastic lesions, suggesting that these differences in EGFR endocytosis might influence the metastatic potential of dysplastic squamous epithelium. These studies in live ex vivo human tumors confirm that endocytosis dysregulation is a physiological event in human tumors and has therapeutic implications.

Therapeutic vaccination with 4-1BB co-stimulation eradicates mouse acute myeloid leukemia.

Immunomodulatory therapies can effectively control haematological malignancies. Previously we reported the effectiveness of combination immunotherapies that centre on 4-1BB-targeted co-stimulation of CD8 + T cells, particularly when simultaneously harnessing the immune adjuvant properties of Natural Killer T (NKT) cells. The objective of this study was to assess the effectiveness of agonistic anti-4-1BB antibody-based combination therapy against two aggressive forms of acute myeloid leukemia (AML). Anti-4-1BB treatment alone resulted in transient suppression of established AML-ETO9a tumor growth in 50% of mice, however the majority of these mice subsequently succumbed to disease. Combining alpha-galactosylceramide (α-GalCer)-loaded tumor cell vaccination with anti-4-1BB antibody treatment increased the proportion of responding mice to 100%, and protection led to long-term, tumor-free survival, demonstrating complete eradication of AML. This finding was extended to established mixed lymphocytic leukemia (MLL)-AF9 tumors, whereby vaccine plus anti-4-1BB combination similarly resulted in 100% protection. The addition of anti-PD-1 to anti-4-1BB treatment, although improving survival outcomes compared to anti-4-1BB alone, was not as effective as NKT cell vaccination. The effectiveness of 4-1BB combination therapies was dependent on IFN-γ signaling within host cells, but not tumors. Vaccine plus anti-4-1BB therapy elicited potent generation of functional effector and memory CD8 + T cells in all tumor-associated organs. Therapy induced KLRG1+ effector CD8 T cells were the most effective at controlling disease. We show that combining NKT cell-targeting vaccination with anti-4-1BB provides excellent therapeutic responses against AML and MLL in mice, and these results will guide ongoing efforts in finding immunotherapeutic solutions against acute myeloid leukemias.

The Role of CXCR3 and Its Chemokine Ligands in Skin Disease and Cancer.

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 tumor cells can lead to cancer metastasis to systemic sites while receptor expression on immune cells can frequently promote anti-tumor immune responses. This review will discuss the biology of CXCR3 and its associated ligands with particular emphasis on the skin during inflammation and carcinogenesis.

Development of a polyclonal anti-dugong immunoglobulin G (IgG) antibody with evaluation of total plasma IgG in a living dugong (Dugong dugon) population.

Species-specific antibodies (Ab) for the measurement of immunoglobulins (Ig) are valuable tools for determining the humoral immune status of threatened and endangered wildlife species such as dugongs. However, no studies have reported antibody reagents against dugong immunoglobulin. The object of this study was to develop an Ab with specificity for dugong IgG and apply this tool to survey total IgG levels in plasma samples from a live wild population of dugongs in southern Queensland, Australia. Dugong IgG was isolated from plasma by protein A/G column chromatography and a polyclonal antiserum was successfully raised against the dugong IgG through immunization of mice. The anti-dugong antiserum was reactive with dugong serum but not immunoglobulin from other species such as rats and humans. When tested against a panel of dugong plasma samples, relative IgG levels from dugongs (n = 116) showed biologically relevant relationships with pregnancy status and a principal component of Body Mass Index (BMI)/globulin/fecal glucocorticosteroid (chronic stress) levels combined, which together accounted for 9.2% of the variation in total Ig levels. Together these data suggest that dugongs show variation in total IgG and that this correlates with some physiological parameters of dugong health.

B cell lymphoma progression promotes the accumulation of circulating Ly6Clo monocytes with immunosuppressive activity.

Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ production in vitro, but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity.

Recruitment of Antigen Presenting Cells to Skin Draining Lymph Node From HPV16E7-Expressing Skin Requires E7-Rb Interaction.

"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.E7xRbΔL/ΔL mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRbΔL/ΔL skin grafts. Here, we further show that K14.E7xRbΔL/ΔL 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.E7xRbΔL/ΔL grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRbΔL/ΔL grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRbΔL/ΔL 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.

HPV16E7-Induced Hyperplasia Promotes CXCL9/10 Expression and Induces CXCR3+ T-Cell Migration to Skin.

Chemokines regulate tissue immunity by recruiting specific subsets of immune cells. Mice expressing the E7 protein of human papilloma virus 16 as a transgene from a keratin 14 promoter (K14.E7) show increased epidermal and dermal lymphocytic infiltrates, epidermal hyperplasia, and suppressed local immunity. Here, we show that CXCL9 and CXCL10 are overexpressed in non-hematopoietic cells in skin of K14.E7 mice when compared with non-transgenic animals, and recruit CXCR3+ lymphocytes to the hyperplastic skin. Overexpression of CXCL9 and CXCL10 is not observed in E7 transgenic mice with mutated Rb gene whose protein product cannot interact with E7 (K14.E7xRbΔL/ΔL) and in consequence lack hyperplastic epithelium. CXCR3+ T cells are preferentially recruited by CXCL9 and CXCL10 in supernatants of K14.E7 but not K14.E7xRbΔL/ΔL skin cultures in vitro. CXCR3 signalling promotes infiltration of a subset of effector T lymphocytes that enables donor lymphocyte deficient, E7-expressing skin graft rejection. Taken together, this suggests that recruitment of CXCR3+ T cells can be an important factor in the rejection of precancerous skin epithelium providing they can overcome local immunosuppressive mechanisms driven by skin-resident lymphocytes.

Modulation of antigen presenting cell functions during chronic HPV infection.

High-risk human papillomaviruses (HR-HPV) infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs) are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity.

Interferon-γ derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity.

Interferon gamma (IFNγ) 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 IFNγ mediates CD8 T-cell cytotoxic function. We show that in vivo, antigen-specific CD8 T cells that produce INFγ are necessary to effect rejection of skin grafts expressing OVA as a transgene in keratinocytes. The ability of CD8 T cells to produce IFNγ 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 IFNγ. 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) IFNγ 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 IFNγ. Conversely, deprivation of either autocrine or paracrine IFNγ, or blockade of IFNγ 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 IFNγ by CTL enhances their motility and promotes killing of primary target keratinocytes. The absolute need for local IFNγ to enable cytotoxic CD8 T-cell function is of significance for immunotherapy for chronic viral infection and for cancer.

HPV16-E7-Specific Activated CD8 T Cells in E7 Transgenic Skin and Skin Grafts.

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 OVA-specific 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.