Tumor resident memory CD8 T cells and concomitant tumor immunity develop independently of CD4 help.

Tissue resident memory (Trm) CD8 T cells infiltrating tumors represent an enriched population of tumor antigen-specific T cells, and their presence is associated with improved outcomes in patients. Using genetically engineered mouse pancreatic tumor models we demonstrate that tumor implantation generates a Trm niche that is dependent on direct antigen presentation by cancer cells. However, we observe that initial CCR7-mediated localization of CD8 T cells to tumor draining lymph nodes is required to subsequently generate CD103+ CD8 T cells in tumors. We observe that the formation of CD103+ CD8 T cells in tumors is dependent on CD40L but independent of CD4 T cells, and using mixed chimeras we show that CD8 T cells can provide their own CD40L to permit CD103+ CD8 T cell differentiation. Finally, we show that CD40L is required to provide systemic protection against secondary tumors. These data suggest that CD103+ CD8 T cell formation in tumors can occur independent of the two-factor authentication provided by CD4 T cells and highlight CD103+ CD8 T cells as a distinct differentiation decision from CD4-dependent central memory.

Ex vivo analysis of radiation effects on tumor infiltrating immune cells using tumor explants.

The response to radiation therapy incorporates both the direct impacts of radiation on cancer cells as well as the immune consequences that can help or hinder control of residual disease. Understanding the response of an individual patient's cancer to radiation, and the impact of radiation on the immune cell subsets present in the tumor prior to radiation therapy, can help identify potential predictors of outcome. Here, we describe a methodological approach to using an explant tumor model to characterize and study the immune cell subsets in murine tumors following exposure to ex vivo radiation treatment. The broader tumor environment incorporates distinct microenvironments consisting of tumor stroma and cancer cell nests, with limited interchange between these zones. Ex vivo analysis of tumor explants ensures that these environments remain intact and allows patient-specific response assessments with a broader range of treatment conditions to find optimal conditions and immunotherapy combinations. While this protocol describes the treatment of murine tumors, with minor variations the same protocol can be used to study and characterize various immune populations following radiation therapy in human tumors.

ICOS is upregulated on T cells following radiation and agonism combined with radiation results in enhanced tumor control.

Multiple preclinical studies have shown improved outcomes when radiation therapy is combined with immune modulating antibodies. However, to date, many of these promising results have failed to translate to successful clinical studies. This led us to explore additional checkpoint and co-stimulatory pathways that may be regulated by radiation therapy. Here, we demonstrate that radiation increases the expression of inducible T cell co-stimulator (ICOS) on both CD4 and CD8 T cells in the blood following treatment. Moreover, when we combined a novel ICOS agonist antibody with radiation we observed durable cures across multiple tumor models and mouse strains. Depletion studies revealed that CD8 T cells were ultimately required for treatment efficacy, but CD4 T cells and NK cells also partially contributed to tumor control. Phenotypic analysis showed that the combination therapy diminished the increased infiltration of regulatory T cells into the tumor that typically occurs following radiation alone. Finally, we demonstrate in a poorly immunogenic pancreatic tumor model which is resistant to combined radiation and anti-PD1 checkpoint blockade that the addition of this novel ICOS agonist antibody to the treatment regimen results in tumor control. These findings identify ICOS as part of a T cell pathway that is modulated by radiation and targeting this pathway with a novel ICOS antibody results in durable tumor control in preclinical models.

Fluorescent tracking identifies key migratory dendritic cells in the lymph node after radiotherapy.

Radiation therapy generates extensive cancer cell death capable of promoting tumor-specific immunity. Within the tumor, conventional dendritic cells (cDCs) are known to carry tumor-associated antigens to the draining lymph node (TdLN) where they initiate T-cell priming. How radiation influences cDC migration is poorly understood. Here, we show that immunological efficacy of radiation therapy is dependent on cDC migration in radioimmunogenic tumors. Using photoconvertible mice, we demonstrate that radiation impairs cDC migration to the TdLN in poorly radioimmunogenic tumors. Comparative transcriptional analysis revealed that cDCs in radioimmunogenic tumors express genes associated with activation of endogenous adjuvant signaling pathways when compared with poorly radioimmunogenic tumors. Moreover, an exogenous adjuvant combined with radiation increased the number of migrating cDCs in these poorly radioimmunogenic tumors. Taken together, our data demonstrate that cDC migration play a critical role in the response to radiation therapy.

Listeria monocytogenes-infected human monocytic derived dendritic cells activate Vγ9Vδ2 T cells independently of HMBPP production.

Gamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP.

A platform for locoregional T-cell immunotherapy to control HNSCC recurrence following tumor resection.

Surgical resection of head and neck squamous-cell carcinoma (HNSCC) is associated with high rates of local and distant recurrence, partially mitigated by adjuvant therapy. A pre-existing immune response in the patient's tumor is associated with better outcomes following treatment with conventional therapies, but improved options are needed for patients with poor anti-tumor immunity. We hypothesized that local delivery of tumor antigen-specific T-cells into the resection cavity following surgery would direct T-cells to residual antigens in the margins and draining lymphatics and present a platform for T-cell-targeted immunotherapy. We loaded T-cells into a biomaterial that conformed to the resection cavity and demonstrated that it could release T-cells that retained their functional activity in-vitro, and in a HNSCC model in-vivo. Locally delivered T-cells loaded in a biomaterial were equivalent in control of established tumors to intravenous adoptive T-cell transfer, and resulted in the systemic circulation of tumor antigen-specific T-cells as well as local accumulation in the tumor. We demonstrate that adjuvant therapy with anti-PD1 following surgical resection was ineffective unless combined with local delivery of T-cells. These data demonstrate that local delivery of tumor-specific T-cells is an efficient option to convert tumors that are unresponsive to checkpoint inhibitors to permit tumor cures.

Targeting MerTK Enhances Adaptive Immune Responses After Radiation Therapy.

PURPOSE: The role of MerTK, a member of the Tyro3-Axl-MerTK family of receptor tyrosine kinase, in the immune response to radiation therapy (RT) is unclear. We investigated immune-mediated tumor control after RT in murine models of colorectal and pancreatic adenocarcinoma using MerTK wild-type and knock-out hosts and whether inhibition of MerTK signaling with warfarin could replicate MerTK knock-out phenotypes. METHODS AND MATERIALS: Wild-type and MerTK-/- BALB/c mice were grafted in the flanks with CT26 tumors and treated with computed tomography guided RT. The role of macrophages and CD8 T cells in the response to radiation were demonstrated with cell depletion studies. The role of MerTK in priming immune responses after RT alone and with agonist antibodies to the T cell costimulatory molecule OX40 was evaluated in a Panc02-SIY model antigen system. The effect of warfarin therapy on the in-field and abscopal response to RT was demonstrated in murine models of colorectal adenocarcinoma. The association between warfarin and progression-free survival for patients treated with SABR for early-stage non-small cell lung cancer was evaluated in a multi-institutional retrospective study. RESULTS: MerTK-/- hosts had better tumor control after RT compared with wild-type mice in a macrophage and CD8 T cell-dependent manner. MerTK-/- mice showed increased counts of tumor antigen-specific CD8 T cells in the peripheral blood after tumor-directed RT alone and in combination with agonist anti-OX40. Warfarin therapy phenocopied MerTK-/- for single-flank tumors treated with RT and improved abscopal responses for RT combined with anti-CTLA4. Patients on warfarin therapy when treated with SABR for non-small cell lung cancer had higher progression-free survival rates compared with non-warfarin users. CONCLUSIONS: MerTK inhibits adaptive immune responses after SABR. Because warfarin inhibits MerTK signaling and phenocopies genetic deletion of MerTK in mice, warfarin therapy may have beneficial effects in combination with SABR and immune therapy in patients with cancer.

Dendritic Cell Maturation Defines Immunological Responsiveness of Tumors to Radiation Therapy.

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated Ags. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor Ags, and migrate to the tumor draining lymph node to initiate cross-priming of tumor-reactive cytotoxic CD8+ T cells. In this study, we report that radiation therapy promotes the activation of intratumoral cDC1s in radioimmunogenic murine tumors, and this process fails to occur in poorly radioimmunogenic murine tumors. In poorly radioimmunogenic tumors, the adjuvant polyinosinic-polycytidylic acid overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1 and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and polyinosinic-polycytidylic acid significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from lymph nodes for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly radioimmunogenic tumors, and coadministration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.

Evaluation of Explant Responses to STING Ligands: Personalized Immunosurgical Therapy for Head and Neck Squamous Cell Carcinoma.

Surgeons have unique in situ access to tumors enabling them to apply immunotherapies to resection margins as a means to prevent local recurrence. Here, we developed a surgical approach to deliver stimulator of interferon genes (STING) ligands to the site of a purposeful partial tumor resection using a gel-based biomaterial. In a range of head and neck squamous cell carcinoma (HNSCC) murine tumor models, we demonstrate that although control-treated tumors recur locally, tumors treated with STING-loaded biomaterials are cured. The mechanism of tumor control required activation of STING and induction of type I IFN in host cells, not cancer cells, and resulted in CD8 T-cell-mediated cure of residual cancer cells. In addition, we used a novel tumor explant assay to screen individual murine and human HNSCC tumor responses to therapies ex vivo We then utilized this information to personalize the biomaterial and immunotherapy applied to previously unresponsive tumors in mice. These data demonstrate that explant assays identify the diversity of tumor-specific responses to STING ligands and establish the utility of the explant assay to personalize immunotherapies according to the local response.Significance: Delivery of immunotherapy directly to resection sites via a gel-based biomaterial prevents locoregional recurrence of head and neck squamous cell carcinoma. Cancer Res; 78(21); 6308-19. ©2018 AACR.

Tumor cure by radiation therapy and checkpoint inhibitors depends on pre-existing immunity.

Radiation therapy is a source of tumor antigen release that has the potential to serve as an endogenous tumor vaccination event. In preclinical models radiation therapy synergizes with checkpoint inhibitors to cure tumors via CD8 T cell responses. To evaluate the immune response initiated by radiation therapy, we used a range of approaches to block the pre-existing immune response artifact initiated by tumor implantation. We demonstrate that blocking immune responses at tumor implantation blocks development of a tumor-resident antigen specific T cell population and prevents tumor cure by radiation therapy combined with checkpoint immunotherapy. These data demonstrate that this treatment combination relies on a pre-existing immune response to cure tumors, and may not be a solution for patients without pre-existing immunity.

Correction: STING expression and response to treatment with STING ligands in premalignant and malignant disease.

[This corrects the article DOI: 10.1371/journal.pone.0187532.].

Amplifying IFN-γ Signaling in Dendritic Cells by CD11c-Specific Loss of SOCS1 Increases Innate Immunity to Infection while Decreasing Adaptive Immunity.

Although prophylactic vaccines provide protective humoral immunity against infectious agents, vaccines that elicit potent CD8 T cell responses are valuable tools to shape and drive cellular immunity against cancer and intracellular infection. In particular, IFN-γ-polarized cytotoxic CD8 T cell immunity is considered optimal for protective immunity against intracellular Ags. Suppressor of cytokine signaling (SOCS)1 is a cross-functional negative regulator of TLR and cytokine receptor signaling via degradation of the receptor-signaling complex. We hypothesized that loss of SOCS1 in dendritic cells (DCs) would improve T cell responses by accentuating IFN-γ-directed immune responses. We tested this hypothesis using a recombinant Listeria monocytogenes vaccine platform that targets CD11c+ DCs in mice in which SOCS1 is selectively deleted in all CD11c+ cells. Unexpectedly, in mice lacking SOCS1 expression in CD11c+ cells, we observed a decrease in CD8+ T cell response to the L. monocytogenes vaccine. NK cell responses were also decreased in mice lacking SOCS1 expression in CD11c+ cells but did not explain the defect in CD8+ T cell immunity. We found that DCs lacking SOCS1 expression were functional in driving Ag-specific CD8+ T cell expansion in vitro but that this process was defective following infection in vivo. Instead, monocyte-derived innate TNF-α and inducible NO synthase-producing DCs dominated the antibacterial response. Thus, loss of SOCS1 in CD11c+ cells skewed the balance of immune response to infection by increasing innate responses while decreasing Ag-specific adaptive responses to infectious Ags.

STING expression and response to treatment with STING ligands in premalignant and malignant disease.

Human papilloma virus positive (HPV+) tumors represent a large proportion of anal, vulvar, vaginal, cervical and head and neck squamous carcinomas (HNSCC) and late stage invasive disease is thought to originate from a premalignant state. Cyclic dinucleotides that activate STimulator of INterferon Genes (STING) have been shown to cause rapid regression of a range of advanced tumors. We aimed to investigate STING ligands as a novel treatment for papilloma. We tested therapies in a spontaneous mouse model of papilloma of the face and anogenital region that histologically resembles human HPV-associated papilloma. We demonstrate that STING ligands cause rapid regression of papilloma, associated with T cell infiltration, and are significantly more effective than Imiquimod, a current immunotherapy for papilloma. In humans, we show that STING is expressed in the basal layer of normal skin and lost during keratinocyte differentiation. We found STING was expressed in all HPV-associated cervical and anal dysplasia and was strongly expressed in the cancer cells of HPV+ HNSCC but not in HPV-unrelated HNSCC. We found no strong association between STING expression and progressive disease in non-HPV oral dysplasia and oral pre-malignancies that are not HPV-related. These data demonstrate that STING is expressed in basal cells of the skin and is retained in HPV+ pre-malignancies and advanced cancers, but not in HPV-unrelated HNSCC. However, using a murine HNSCC model that does not express STING, we demonstrate that STING ligands are an effective therapy regardless of expression of STING by the cancer cells.

Mertk on tumor macrophages is a therapeutic target to prevent tumor recurrence following radiation therapy.

Radiation therapy provides a means to kill large numbers of cancer cells in a controlled location resulting in the release of tumor-specific antigens and endogenous adjuvants. However, by activating pathways involved in apoptotic cell recognition and phagocytosis, irradiated cancer cells engender suppressive phenotypes in macrophages. We demonstrate that the macrophage-specific phagocytic receptor, Mertk is upregulated in macrophages in the tumor following radiation therapy. Ligation of Mertk on macrophages results in anti-inflammatory cytokine responses via NF-kB p50 upregulation, which in turn limits tumor control following radiation therapy. We demonstrate that in immunogenic tumors, loss of Mertk is sufficient to permit tumor cure following radiation therapy. However, in poorly immunogenic tumors, TGFß inhibition is also required to result in tumor cure following radiation therapy. These data demonstrate that Mertk is a highly specific target whose absence permits tumor control in combination with radiation therapy.

The nuclear matrix shell proteome of human epidermis.

BACKGROUND: Proteomic approaches have identified cancer specific biomarker proteins in the nuclear matrix fraction of cancer cells. We wanted to determine whether a similar approach could be used to investigate melanoma biomarkers. OBJECTIVE: Since it was not clear that a nuclear matrix fraction could be isolated from the intact human epidermis, we first wanted to determine whether a nuclear matrix fraction could be isolated from the intact epidermis of human skin. If this was possible, we secondarily wanted to compare the proteome of cultured melanoma and carcinoma cells to that of the intact epidermis. METHODS: We applied two-dimensional electrophoresis (2DGE) and LC/MS/MS to identify proteins isolated in the nuclear matrix shell protein fraction isolated from the human epidermis and from cultured primary skin and cancer cells. RESULTS: A subcellular fractionation of intact epidermis succeeded in yielding a nuclear matrix shell which made up approximately 40% of total tissue protein. Only 5-10% of total cell protein was fractionated in the nuclear matrix shell of cultured skin cells. The nuclear matrix shell of the intact epidermis was distinguishable from cultured keratinocytes or HaCaT cells by expression of keratin 1. The nuclear matrix of the epidermis was distinguishable from melanocytes and melanoma cells by expression of vimentin in melanocyte-derived cells and by expression of desmoplakin in the intact epidermis. CONCLUSION: The nuclear matrix-intermediate filament system can be isolated from the intact human epidermis. A careful examination of the protein composition of this subcellular fraction from the epidermis and skin cancers may identify useful cancer specific biomarkers.

Transient dephosphorylation of p53 serine 376 as an early response to ionizing radiation.

In a previous paper we reported that the cytoplasmic sequestered p53 in cells of the SK-N-SH neuroblastoma cell line could be induced to translocate to the nucleus by exposure to ionizing radiation. We have extended these studies to determine the fate of p53 in HCT116 colorectal carcinoma cells where constitutive p53 protein resides in the nucleus. A continuous increase in the nuclear p53 protein was observed in irradiated cells beginning 1 h after irradiation that persisted for 8 h. Surprisingly, immunofluorescence microscopy revealed a transient, rapid and sensitive increase in a radiation-induced nuclear dephosphorylated p53 using antibody PAb421, which detects p53 when serine 376 is dephosphorylated. The PAb421 epitope was detectable after exposure to radiation doses as low as 0.5 cGy and was 10 to 20 times more sensitive compared to detection of p53 protein levels. The results are consistent with a radiation-induced, sensitive and rapid dephosphorylation of p53 at serine 376. The rapid increase in the nuclear PAb421 epitope was blocked by the protein serine phosphatase inhibitor calyculin A but was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that serine 376 was dephosphorylated by protein serine phosphatase 1 or 2A acting on pre-existing p53 protein. The data suggest that dephosphorylation of serine 376 on constitutive nuclear p53 is a sensitive and early signaling event in the response of cells to DNA damage induced by ionizing radiation.

Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation.

Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation.