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.

Clinically-Relevant Rapamycin Treatment Regimens Enhance CD8+ Effector Memory T Cell Function In The Skin and Allow their Infiltration into Cutaneous Squamous Cell Carcinoma.

Patients receiving immunosuppressive drugs to prevent organ transplant rejection exhibit a greatly increased risk of developing cutaneous squamous cell carcinoma (SCC). However, not all immunosuppressive drugs confer the same risk. Randomised, controlled trials demonstrate that switching renal transplant recipients receiving calcineurin inhibitor-based therapies to mammalian target of rapamycin (mTOR) inhibitors results in a reduced incidence of de novo SSC formation, and can even result in the regression of pre-existing premalignant lesions. However, the contribution played by residual immune function in this setting is unclear. We examined the hypotheses that mTOR inhibitors promote the enhanced differentiation and function of CD8+ memory T cells in the skin. Here, we demonstrate that the long-term oral administration of rapamycin to achieve clinically-relevant whole blood drug target thresholds, creates a "low rapamycin dose" environment in the skin. While both rapamycin and the calcineurin inhibitor tacrolimus elongated the survival of OVA-expressing skin grafts, and inhibited short-term antigen-specific CD8+ T cell responses, rapamycin but not tacrolimus permitted the statistically significant infiltration of CD8+ effector memory T cells into UV-induced SCC lesions. Furthermore, rapamycin uniquely enhanced the number and function of CD8+ effector and central memory T cells in a model of long-term contact hypersensitivity provided that rapamycin was present during the antigen sensitization phase. Thus, our findings suggest that patients switched to mTOR inhibitor regimens likely experience enhanced CD8+ memory T cell function to new antigen-challenges in their skin, which could contribute to their lower risk of de novo SSC formation and regression of pre-existing premalignant lesions.

Cytokines, Chemokines, and Other Biomarkers of Response for Checkpoint Inhibitor Therapy in Skin Cancer.

Immunotherapy for skin malignancies has ushered in a new era for cancer treatments by demonstrating unprecedented durable responses in the setting of metastatic Melanoma. Consequently, checkpoint inhibitors are now the first-line treatment of metastatic melanoma and widely used as adjuvant therapy for stage III disease. With the observation that higher tumor mutational burden correlates with a better response, checkpoint inhibitors are tested in other skin cancer types of known high tumor mutational burden with promising results and recently became the first-ever FDA-approved treatment for metastatic Merkel cell carcinoma. The emerging new standards-of-care will necessitate more precise biomarkers and predictors for treatment response and immune-related adverse events. Measurable immune-related mediators are currently under investigation as factors that promote or block the response to cancer immunotherapy and may provide insights into the underlying immune response to the tumor. Cytokines and chemokines are such mediators and are crucial for facilitating the recruitment and activation of specific subsets of leukocytes within the microenvironment of skin cancers. The exact mechanisms of how these meditators, both immunological and non-immunological, operate in the tumor microenvironment is an area of active research, so to reliable biomarkers of responses to cancer immunotherapy. Here, we will review and summarize the expanding body of literature for immune-related biomarkers pertaining to Melanoma, Basal cell carcinoma, Squamous cell carcinoma, and Merkel cell carcinoma, highlighting clinically relevant checkpoint inhibitor therapy biomarker advancements.

CD4+CD8ß+ double-positive T cells in skin-draining lymph nodes respond to inflammatory signals from the skin.

CD4+CD8+ double-positive (DP), mature, peripheral T cells are readily detectable in a variety of species and tissues. Despite a common association with autoimmune and malignant skin disorders, however, little is understood about their role or function. Herein, we show that DP T cells are readily detectable in the blood, spleen, and peripheral lymph nodes of naïve C57BL/6 mice. DP T cells were also present in Jα18-/- and CD1d-/- mice, indicating that these cells are not NK-T cells. After skin administration of CASAC adjuvant, but not Quil A adjuvant, both total DP T cells and skin-infiltrating DP T cells increased in number. We explored the possibility that DP T cells could represent aggregates between CD4+ and CD8+ single-positive T cells and found strong evidence that a large proportion of apparent DP T cells were indeed aggregates. However, the existence of true CD4+CD8+ DP T cells was confirmed by Amnis ImageStream (Millipore Sigma, Billerica, MA, USA) imaging. Multiple rounds of FACS sorting separated true DP cells from aggregates and indicated that conventional analyses may lead to ∼10-fold overestimation of DP T cell numbers. The high degree of aggregate contamination and overestimation of DP abundance using conventional analysis techniques may explain discrepancies reported in the literature for DP T cell origin, phenotype, and function.

Antigen-encoding bone marrow terminates islet-directed memory CD8+ T-cell responses to alleviate islet transplant rejection.

Islet-specific memory T cells arise early in type 1 diabetes (T1D), persist for long periods, perpetuate disease and are rapidly reactivated by islet transplantation. As memory T cells are poorly controlled by 'conventional' therapies, memory T-cell mediated attack is a substantial challenge in islet transplantation and this will extend to application of personalized approaches using stem-cell derived replacement ß cells. New approaches are required to limit memory autoimmune attack of transplanted islets or replacement ß cells. Here we show that transfer of bone marrow encoding cognate antigen directed to dendritic cells, under mild, immune-preserving conditions inactivates established memory CD8+ T-cell populations and generates a long-lived, antigen-specific tolerogenic environment. Consequently, CD8+ memory T cell-mediated targeting of islet-expressed antigens is prevented and islet graft rejection alleviated. The immunological mechanisms of protection are mediated through deletion and induction of unresponsiveness in targeted memory T-cell populations. The data demonstrate that hematopoietic stem cell-mediated gene therapy effectively terminates antigen-specific memory T-cell responses and this can alleviate destruction of antigen-expressing islets. This addresses a key challenge facing islet transplantation and importantly, the clinical application of personalized ß-cell replacement therapies using patient-derived stem cells.

Comparative immune phenotypic analysis of cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in immune-competent individuals: proportional representation of CD8+ T-cells but not FoxP3+ Regulatory T-cells is associated with disease stage.

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3+ T-cells than photo-damaged skin, however, the abundance of CD3-CD56+ Natural Killer (NK) cells, CD11c+HLA-DR+ conventional Dendritic Cells (cDC), BDCA-2+HLA-DR+ plasmacytoid DC (pDC), FoxP3+ Regulatory T-cells (T-reg), Vα24+Vß11+ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4+ T-cells were associated with SCC, yet CD8+ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8+ T-cells than IEC. We propose that differences in CD8+ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4+ T-cells to CD8+ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.

IL-17 suppresses immune effector functions in human papillomavirus-associated epithelial hyperplasia.

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 skin-grafting 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 γδ T cells in mouse hyperplastic lesions. IL-23 and IL-1ß, 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.

Tolerance induction with gene-modified stem cells and immune-preserving conditioning in primed mice: restricting antigen to differentiated antigen-presenting cells permits efficacy.

Bone marrow (BM) or hematopoietic stem cell (HSC) transplantation is used as curative therapy for hematologic malignancies. Incorporation of gene therapy to drive tolerogenic expression of antigens is a promising strategy to overcome the limited long-term efficacy of autologous HSC transplantation for autoimmune diseases. HSC engraftment and tolerance induction is readily achieved after myeloablative or immune-depleting conditioning regardless of the cellular compartment in which antigen is expressed. It is unclear whether the efficiency of engraftment and tolerance induction is influenced by targeting antigen to specific cellular compartments. This is particularly important when using clinically feasible low-intensity conditioning aimed at preserving infectious immunity in individuals where immunologic memory exists to the autoantigen to be expressed. Here we demonstrate that, under immune-preserving conditions, confining expression of a transgenically expressed antigen to dendritic cells permits stable, long-term engraftment of genetically modified BM even when recipients are immune to the expressed antigen. In contrast, broader expression within the hematopoietic compartment leads to graft rejection and therapeutic failure because of antigen expression in HSCs. These findings are relevant to the clinical application of genetically engineered HSCs and provide evidence that careful selection of promoters for HSC-mediated gene therapy is important, particularly where tolerance is sought under immune-preserving conditions.