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Tumor biopsy is the gold standard for the assessment of clinical biomarkers for treatment. However, tumors change dynamically in response to therapy, and there remains a need for a more representative biomarker that can be assayed over the course of treatment. Circulating tumor cells (CTCs) may provide clinically important and comprehensive tumoral information that is predictive of treatment response and outcome. Blood samples were processed for CTCs from 56 patients using the ClearCell FX system. Captured cells were phenotyped for CTC clusters and markers for immunotherapy (PD‐L1) CTC chromosomal architecture (ALK, EGFR). CTCs were isolated in 11/23 (47.8%) of head and neck cancer (HNC) patients and 17/33 (51.5%) of non‐small‐cell lung cancer (NSCLC) patients. CTCs were determined to be PD‐L1‐positive in 6/11 (54.4%) HNC and 11/17 (64.7%) NSCLC cases, respectively. 3D chromosomal DNA FISH for ALK and EGFR molecular targets showed better resolution than in 2D when imaging CTCs. HNC CTC‐positive patients had shorter progression‐free survival (PFS) (hazard ratio[HR]: 4.946; 95% confidence internal[CI]:1.571‐15.57; P = 0.0063), and PD‐L1‐positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011‐26.33; P = 0.0485). In the advanced stage NSCLC patient cohort, PFS was not found to be associated with CTCs prior to therapy ([HR]:2.246; 95% [CI]:0.9565‐5.273; P = 0.0632), nor the presence of PD‐L1 expression ([HR]:1.646; 95% [CI]:0.5128‐5.283; P = 0.4023). This study demonstrated that CTCs are predictive of poorer outcomes in HNC and provides distinct and separate utility for CTCs in HNC and NSCLC, which may be more representative of the disease burden and overall survival than the parameters used to measure them. 3D DNA FISH of circulating tumor cells demonstrates a higher resolution for genomic aberrations compared to 2D imaging modalities.

Invariant natural killer T (iNKT) cells are a unique innate T lymphocyte population that possess cytolytic properties and profound immunoregulatory activities. iNKT cells play an important role in the immune surveillance of blood cancers. They predominantly recognize glycolipid antigens presented on CD1d, but their activation and cytolytic activities are not confined to CD1d expressing cells. iNKT cell stimulation and subsequent production of immunomodulatory cytokines serve to enhance the overall antitumor immune response. Crucially, the activation of iNKT cells in cancer often precedes the activation and priming of other immune effector cells, such as NK cells and T cells, thereby influencing the generation and outcome of the antitumor immune response. Blood cancers can evade or dampen iNKT cell responses by downregulating expression of recognition receptors or by actively suppressing or diverting iNKT cell functions. This review will discuss literature on iNKT cell activity and associated dysregulation in blood cancers as well as highlight some of the strategies designed to harness and enhance iNKT cell functions against blood cancers.

It has previously been shown that expression of human papillomavirus type 16 (HPV) E7 in epidermis causes hyperplasia and chronic inflammation, characteristics of pre-malignant lesions. Importantly, E7-expressing epidermis is strongly immune suppressed and is not rejected when transplanted onto immune competent mice. Professional antigen presenting cells are considered essential for initiation of the adaptive immune response that results in graft rejection. Langerhans cells (LC) are the only antigen presenting cells located in normal epidermis and altered phenotype and function of these cells may contribute to the immune suppressive microenvironment. Here, we show that LC are atypically activated as a direct result of E7 expression in the epidermis, and independent of the presence of lymphocytes. The number of LC was significantly increased and the LC are functionally impaired, both in migration and in antigen uptake. However when the LC were extracted from K14E7 skin and matured in vitro they were functionally competent to present and cross-present antigen, and to activate T cells. The ability of the LC to present and cross-present antigen following maturation supports retention of full functional capacity when removed from the hyperplastic skin microenvironment. As such, opportunities are afforded for the development of therapies to restore normal LC function in hyperplastic skin.

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.

\"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 mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRb skin grafts. Here, we further show that K14.E7xRb 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 grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRb grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRb keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis . 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.

IFN-γ has a central role in the defense against infections and cancer. More recently, however, IFN-γ 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-γ during infection, the mechanisms that induce IFN-γ in an immunosuppressive context are unknown. Previously, we identified a key role for IFN-γ 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-γ, IL-18, and IL-12 by K14E7 transgenic skin compared with nontransgenic skin. IFN-γ in K14E7 transgenic skin was produced predominantly by CD8+ and CD4+ T cells, which were present in greater numbers in K14E7 transgenic skin. Production of IFN-γ in K14E7 skin required IL-18 but not IL-12. Our findings show that IL-18 contributes to inducing IFN-γ in an immunosuppressive cutaneous environment caused by viral oncogene–driven hyperplasia.

Immunomodulatory therapies can effectively control haematological malignancies by promoting antitumour immunity. Previously, we reported transient growth of poorly immunogenic murine non‐Hodgkin B‐cell lymphomas (B‐NHL) by targeting natural killer T (NKT) cells with a therapeutic vaccine approach. Therapeutic efficacy was highly dependent on the ability of the vaccine to provoke rapid interferon‐gamma (IFNγ) production from NKT and NK cells. By manipulating the capacity of either host or lymphoma cells to signal through the IFNγ receptor (IFNγR), we investigated whether the therapeutic effect conferred by vaccine‐induced IFNγ is a result of immune cell activation, lymphoma IFNγ sensitivity or a combination of both. We demonstrated that antitumour immunity elicited by vaccination requires IFNγ signalling within host cells but not tumour cells. IFNγR‐deficient mice failed to mount an effective antitumour immune response following vaccination despite elevated IFNγ levels. With successive exposure to vaccination, lymphomas acquired an increasingly therapy‐resistant phenotype and displayed a reduction in major histocompatibility complex I and CD1d surface expression, which is independent of tumour intrinsic IFNγ signalling. Our results suggest that immunotherapy‐induced IFNγ production mainly exerts its therapeutic effect via signalling through host cells, rather than directly to tumour cells in B‐NHL. This signifies that intact IFNγ signalling within patients’ immune compartment rather than tumour cell sensitivity to IFNγ is more critical for successful treatment. Finally, tumour IFNγ signalling alone does not drive acquired tumour resistance to vaccination, implying that additional immunoediting pathways are responsible for tumour immune escape.

The natural killer T (NKT) cell ligand, alpha‐galactosylceramide (α‐GalCer), represents a potential adjuvant to boost immunotherapeutic vaccination strategies against poorly immunogenic cancers. The objective of this study was to assess the therapeutic potential of an α‐GalCer‐loaded tumor‐cell vaccine against solid tumors in mice and to enhance the effectiveness of this approach by removing immune suppression associated with the activity of Foxp3+ regulatory T cells (Tregs). In the B16F10 melanoma model, we show that single vaccination with irradiated, α‐GalCer‐loaded tumor cells resulted in suppression of established subcutaneous (s.c.) B16F10 tumor growth, which was mediated by NKT cell‐dependent IFN‐γ production and enhanced in the absence of IL‐17 A. Selective depletion of Foxp3+ Tregs in transgenic DEpletion of REGulatory T cells (DEREG) mice led to significant inhibition of B16F10 tumor growth and enhanced survival of mice receiving vaccination. Short‐term elimination of Foxp3+ Tregs (<7 days) was sufficient to boost vaccine‐induced immunity. Enhanced antitumor activity with combination therapy was associated with an increase in systemic NK cell and effector CD8+ T‐cell activation and IFN‐γ production, as well as infiltration of effector CD8+ T cells into the tumor. Overall, these findings demonstrate that transient depletion of Foxp3+ Tregs constitutes a highly effective strategy to improve the therapeutic efficacy of anticancer vaccination with NKT cell adjuvants.

We have shown that the expression of human papillomavirus type 16 E7 (HPV16.E7) protein within epithelial cells results in local immune suppression and a weak and ineffective immune response to E7 similar to that occuring in HPV-associated premalignancy and cancers. However, a robust acute inflammatory stimulus can overcome this to enable immune elimination of HPV16.E7-transformed epithelial cells. 2,4-Dinitrochlorobenzene (DNCB) can elicit acute inflammation and it has been shown to initiate the regression of HPV-associated genital warts. Although the clinical use of DNCB is discouraged owing to its mutagenic potential, understanding how DNCB-induced acute inflammation alters local HPV16.E7-mediated immune suppression might lead to better treatments. Here, we show that topical DNCB application to skin expressing HPV16.E7 as a transgene induces a hyperinflammatory response, which is not seen in nontransgenic control animals. The E7-associated inflammatory response is characterized by enhanced expression of Th2 cytokines and increased infiltration of CD11b+Gr1intF4/80+Ly6ChiLy6Glow myeloid cells, producing arginase-1. Inhibition of arginase with an arginase-specific inhibitor, N(omega)-hydroxy-nor-L-arginine, ameliorates the DNCB-induced inflammatory response. Our results demonstrate that HPV16.E7 protein enhances DNCB-associated production of arginase-1 by myeloid cells and consequent inflammatory cellular infiltration of skin.

Gamma delta T cells (γδ 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 γδ 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 γδ T cells enhanced graft rejection. Although the effector function of CD8 T cells was intact in the absence of γδ T cells, cross-priming of CD8 T cell to graft-derived Ag was impaired in the absence of γδ T cells. The reduced graft rejection and graft priming of γδ 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 TCRδ−/− mice compared with wild-type mice, regardless of the presence or absence of inflammation associated with grafting. These results indicate that γδ T cells enhance graft priming and consequently the likelihood of a successful immune outcome in the context of skin graft rejection, suggesting that γδ T cells may be an important component of immunity to epithelial cancers or infection.