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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.

Perineural spread of tumour cells along cranial nerves is a severe complication of primary cutaneous squamous cell carcinomas of the head and neck region. While surgical excision of the tumour is the treatment of choice, removal of all the tumour is often complicated by the neural location and recurrence is frequent. Non-invasive immune treatments such as checkpoint inhibitor blockade may be useful in this set of tumours although little is understood about the immune response to perineural spread of squamous cell carcinomas. Immunohistochemistry studies suggest that perineural tumour contains a lymphocyte infiltrate but it is difficult to quantitate the different proportions of immune cell subsets and expression of checkpoint molecules such as PD-1, Tim-3 and CTLA-4. Using flow cytometry of excised perineural tumour tissue, we show that a T cell infiltrate is prominent in addition to less frequent B cell, NK cell and NKT cell infiltrates. CD8 T cells are more frequent than other T cells in the tumour tissue. Amongst CD8 T cells, the frequency of Tim-3, CTLA-4 and PD-1 expressing cells was significantly greater in the tumour relative to the blood, a pattern that was repeated for Tim-3, CTLA-4 and PD-1 amongst non-CD8 T cells. Using immunohistochemistry, PD-1 and PD-L1-expression could be detected in close proximity amongst perineural tumour tissue. The data suggest that perineural SCC contains a mixture of immune cells with a predominant T cell infiltrate containing CD8 T cells. Elevated frequencies of tumour-associated Tim-3+, CTLA-4+ and PD-1+ CD8 T cells suggests that a subset of patients may benefit from local antibody blockade of these checkpoint inhibitors.

Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions.

Chronic infection of anogenital epithelium with human papillomavirus (HPV) promotes development of cancer. Many pathogens evoke immunosuppressive mechanisms to enable persistent infection. We have previously shown that grafted skin expressing HPV16 E7 oncoprotein from a keratin-14 promoter (K14E7) is not rejected by a syngeneic, immunocompetent host. In this study we show that indoleamine 2,3-dioxygenase (IDO) 1, an IFN-γ-inducible immunoregulatory molecule, is more highly expressed by langerin−ve dermal dendritic cells (DCs) from K14E7 skin than nontransgenic control skin. Furthermore, inhibiting IDO activity using 1-methyl-dl-tryptophan (1-D/L-MT) promotes K14E7 skin graft rejection. Increased IDO1 expression and activity in K14E7 skin requires IFN-γ and invariant natural killer T (iNKT) cells, both of which have been shown to negatively regulate T-cell effector function and suppress K14E7 graft rejection. Furthermore, DCs from K14E7 skin express higher levels of IFN-γ receptor (IFN-γR) than DCs from control skin. K14E7 transgenic skin recruits significantly higher numbers of DCs, independent of IFN-γ and IFN-γR expression. Consistent with these observations in a murine model, we found higher expression of IDO1 and IFN-γ but not IDO2 in the cervical epithelium of patients with HPV-associated cervical intraepithelial neoplasia (CIN) 2/3. Our data support a hypothesis that induction of IDO1 in HPV-infected skin contributes to evasion of host immunity.

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.

Objectives Langerhans cells (LCs) are epithelial antigen‐presenting cells (APC) contributing to immune surveillance. LCs depend on interleukin 34 (IL34) production by epithelial cells. This study aimed to uncover mechanisms of alteration of IL34 and LC function in squamous cell carcinoma (SCC). Methods Cancer cohort data were used to identify associations between SCC and IL34. ATAC‐seq of keratinocytes (KCs) and LCs from a murine model of epithelial hyperplasia, driven by HPV16 E7 oncoprotein (K14E7), was analysed. Transcriptomic data were used to validate findings. RNAscope, RT‐qPCR, ELISA and confocal imaging was used to analyse IL34 expression and LCs in a spatial context. Results IL34 mRNA is downregulated in human SCCs of the head and neck, the cervix, the lung and the oesophagus, and low IL34 expression is associated with poor survival. We demonstrate that KCs of K14E7 mice have reduced Il34 gene accessibility, mRNA and protein, as well as broad changes in promotor accessibility associated with cell adhesion and immune responses. Chromatin accessibility was substantially changed in LCs, including increased accessibility of the Csf1r gene, and changes in promotors associated with cytoskeleton arrangement and antigen processing and presentation. We discovered altered spatial LC dendrite organisation in hyperproliferative epithelium. Conclusion Squamous cell carcinoma of the cervix, head and neck, oesophagus and lung demonstrate downregulation of IL34, which is associated with poor survival, and with alterations in LC spatial organisation and function. These findings suggest that reduced IL34 expression in SCC may contribute to impaired local immunity through LC dysregulation. IL34 is critical for the development and maintenance of Langerhans cells in squamous epithelium. This study shows that low expression of IL34 determines poor survival in squamous cell carcinomas. We demonstrate that the downregulation of IL34 transcript and protein in hyperproliferative squamous epithelial cells is because of the reduction in chromatin accessibility at the enhancer region of the Il34 gene. This epigenetic alteration is associated with substantial chromatin changes at genes pivotal to the antigen processing and presentation machinery of Langerhans cells.

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.

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.

Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.