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This study analyzed magnetic resonance imaging (MRI) scans of Glioblastoma (GB) patients to develop an imaging-derived predictive model for assessing the extent of intratumoral CD3 T-cell infiltration. Pre-surgical T1-weighted post-contrast and T2-weighted Fluid-Attenuated-Inversion-Recovery (FLAIR) MRI scans, with corresponding mRNA expression of CD3D/E/G were obtained through The Cancer Genome Atlas (TCGA) for 79 GB patients. The tumor region was contoured and 86 image-derived features were extracted across the T1-post contrast and FLAIR images. Six imaging features-kurtosis, contrast, small zone size emphasis, low gray level zone size emphasis, high gray level zone size emphasis, small zone high gray level emphasis-were found associated with CD3 activity and used to build a predictive model for CD3 infiltration in an independent data set of 69 GB patients (using a 50-50 split for training and testing). For the training set, the image-based prediction model for CD3 infiltration achieved accuracy of 97.1% and area under the curve (AUC) of 0.993. For the test set, the model achieved accuracy of 76.5% and AUC of 0.847. This suggests a relationship between image-derived textural features and CD3 T-cell infiltration enabling the non-invasive inference of intratumoral CD3 T-cell infiltration in GB patients, with potential value for the radiological assessment of response to immune therapeutics.

Immunotherapy is a currently popular treatment strategy for cancer patients. Although recent developments in cancer immunotherapy have had significant clinical impact, only a subset of patients exhibits clinical response. Therefore, understanding the molecular mechanisms of immunotherapy resistance is necessary. The mechanisms of immune escape appear to consist of two distinct tumor characteristics: a decrease in effective immunocyte infiltration and function and the accumulation of immunosuppressive cells in the tumor microenvironment. Several host-derived factors may also contribute to immune escape. Moreover, inter-patient heterogeneity predominantly results from differences in somatic mutations between cancers, which has led to the hypothesis that differential activation of specific tumor-intrinsic pathways may explain the phenomenon of immune exclusion in a subset of cancers. Increasing evidence has also shown that tumor-intrinsic signaling plays a key role in regulating the immunosuppressive tumor microenvironment and tumor immune escape. Therefore, understanding the mechanisms underlying immune avoidance mediated by tumor-intrinsic signaling may help identify new therapeutic targets for expanding the efficacy of cancer immunotherapies.

Background Changes in glycogen metabolism is an essential feature among the various metabolic adaptations used by cancer cells to adjust to the conditions imposed by the tumor microenvironment. Our previous study showed that glycogen branching enzyme (GBE1) is downstream of the HIF1 pathway in hypoxia-conditioned lung cancer cells. In the present study, we investigated whether GBE1 is involved in the immune regulation of the tumor microenvironment in lung adenocarcinoma (LUAD). Methods We used RNA-sequencing analysis and the multiplex assay to determine changes in GBE1 knockdown cells. The role of GBE1 in LUAD was evaluated both in vitro and in vivo. Results GBE1 knockdown increased the expression of chemokines CCL5 and CXCL10 in A549 cells. CD8 expression correlated positively with CCL5 and CXCL10 expression in LUAD. The supernatants from the GBE1 knockdown cells increased recruitment of CD8+ T lymphocytes. However, the neutralizing antibodies of CCL5 or CXCL10 significantly inhibited cell migration induced by shGBE1 cell supernatants. STING/IFN-I pathway mediated the effect of GBE1 knockdown for CCL5 and CXCL10 upregulation. Moreover, PD-L1 increased significantly in shGBE1 A549 cells compared to those in control cells. Additionally, in LUAD tumor tissues, a negative link between PD-L1 and GBE1 was observed. Lastly, blockade of GBE1 signaling combined with anti-PD-L1 antibody significantly inhibited tumor growth in vivo. Conclusions GBE1 blockade promotes the secretion of CCL5 and CXCL10 to recruit CD8+ T lymphocytes to the tumor microenvironment via the IFN-I/STING signaling pathway, accompanied by upregulation of PD-L1 in LUAD cells, suggesting that GBE1 could be a promising target for achieving tumor regression through cancer immunotherapy in LUAD. Electronic supplementary material The online version of this article (10.1186/s12943-019-1027-x) contains supplementary material, which is available to authorized users.

The potential of neuroendocrine neoplasms (NEN) to respond to checkpoint inhibitors is largely unknown and full of great expectations. Immunohistochemical (IHC) studies of programmed cell death ligand 1 (PD-L1) expression in the tumor microenvironment and its implications in predicting the response to checkpoint inhibition is a very active subject. Currently, the combined analysis of PD-L1 expression and tumor-associated immune cell (TAIC) infiltration is considered the best predictive marker of therapeutic response. Here we investigated the expression of PD-L1 on tumor cells (TC) and tumor-infiltrating immune cells (IC) by IHC in 68 NEN samples with a high proliferation rate (Ki-67 >20%) from 57 patients and in 22 samples we correlated it with TAIC density by assessing intratumoral infiltration of CD3+, CD8+, and CD68+ cells. Furthermore, the tumor microenvironment was evaluated according to the classification of Teng et al. We detected PD-L1 expression in 31.6% of NEN G3. Its expression usually was weak and more IC than TC expressed PD-L1. The proportion of tumors positive for PD-L1 was comparable in NEN from different sites of origin but varied depending on tumor differentiation and disease extension. No positive IHC staining was found in 3 well-differentiated neuroendocrine tumors (NETs) with a proliferation rate above 20% (NET G3). When analyzing TAIC, we rarely (18.2%) detected intratumoral CD8+ cells, whereas infiltration by CD3+ and CD68+ cells was more common (45.5 and 59.1%, respectively). By combining CD3+ cells and PD-L1 status, we identified the immune ignorant phenotype of tumor microenvironment as being the most common phenotype, supporting the concept of a preferably combined immunotherapeutic approach in neuroendocrine carcinoma (NEC).

Elucidating how tumor-intrinsic pathways regulate T cell infiltration in tumors is crucial for developing new therapeutic strategies for immune checkpoint blockade therapy. Here, we review recent progress on how these pathways orchestrate immune status in tumors and discuss the potential interventions for reprogramming the tumor microenvironment for cancer treatment.

In adult cancer, immune signatures such as the T cell-inflamed gene expression profile (GEP) have been developed to predict which patients are likely to respond to immune checkpoint inhibitors (ICIs) beyond high tumor mutation burden (TMB) and PD-L1 expression. The GEP infers T cell infiltration and activation in the tumor microenvironment (TME) from transcriptomic data. However, it is not known whether tools such as GEP are applicable in pediatric cancer, as the TME in childhood cancers is largely unexplored and response to ICIs are rare. We have undertaken an integrated analysis of the pediatric TME using RNA-sequencing (RNA-seq) and immunohistochemistry (IHC). Our goal is to identify patients with T cell-inflamed or “hot” tumors who may benefit from ICIs. Through Australia's ZERO childhood cancer precision medicine program we performed RNA-seq on 347 high-risk pediatric cancers (estimated Citation Format: Chelsea Mayoh, Rachael L. Terry, Marie Wong, Loretta M. Lau, Dong Anh Khuong-Quang, Marion K. Mateos, Vanessa Tyrrell, Michelle Haber, David S. Ziegler, Mark J. Cowley, Joseph A. Trapani, Paul J. Neeson, Paul G. Ekert. The unexplored immune landscape of high-risk pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3044.

T cells in the tumor microenvironment, or tumor-infiltrating lymphocytes (TILs), have been recognized to play a critical role in tumor immunity. TILs are heterogeneously defined, and different T cell subsets have various effects. The properties of some T cell markers, such as CD3, CD4, CD8, and forkhead box P3 (FOXP3), are poor prognostic factors in several types of cancers. The effects of sex steroid hormones on T cell function in inflammation are known, and estrogen and androgen regulate the secretion of cytokines to enhance and attenuate, respectively, the immune response. While intratumoral estrogen and androgen synthesis plays an important role in the progression of cancer cells in hormone-dependent cancers, the relationship between intratumoral hormone levels and TIL subtypes is unclear. In this study, we examined the correlation between intratumoral hormone concentrations and expression scores of TIL markers in endometrial cancer. We employed frozen and formalin-fixed paraffin-embedded (FFPE) tissues of 19 endometrial cancer tissues (G1, n=6; G2, n=5; G3, n=6; Serous, n=2). Hormone concentrations were measured by LC-MC/MS from the frozen sample, and immunohistochemistry of T cell markers was performed using FFPE tissues. The five T cell markers examined in this study were CD3 (total T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), FOXP3 (regulatory T cells), and thymocyte selection-associated HMG box (TOX) (exhausted T cells). The number of intratumoral CD3 in an arbitrary two-field (magnification, ×200) was counted and used as the CD3 score. Similarly, the number of other markers was counted, and the ratio to CD3 was used as the score for each marker. A correlation was found between TOX and CD8 using correlation between markers. The median scores for CD3 and CD8 were significantly higher in type 1 (G1 and G2) than in type 2 (G3 and serous) (CD3, p=0.031; CD8, P=0.031). The CD4 score had a positive correlation with estrogen (estrone, r2=0.591; estradiol, r2=0.501) and androgen (dihydrotestosterone, r2=0.327) concentrations. There was a poor negative correlation between cortisol concentration and CD8 score (r2=0.203). Estrogen and androgen at the tumor in situ were considered to affect TILs, especially CD4 positive T cells. It is also suggested that cortisol acts on immunosuppression, even locally in the tumor. This study showed the potential of a combination of immunotherapy and hormone therapy in endometrial cancer. Citation Format: Yasuhiro Miki, Fuka Onuma, Kiyoshi Takagi, Erina Iwabuchi, Takashi Suzuki, Hironobu Sasano, Kiyoshi Ito. Correlation between T cell infiltration subtypes and intratumoral hormone levels in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 726.

Diet can alter the gut microbiota and shift its production of metabolites, which affect systemic immune function. In Nature Immunology, Mariño et al. (2017) explore diet-gut microbiome interactions in type 1 diabetes and identify mechanisms by which short-chain fatty acids prevent T cell destruction of pancreatic β-cells.

BACKGROUND Severe local and systemic immune suppression in glioblastoma (GBM) contributes to the failure of single-agent immunotherapies in clinical trials. In this study, we evaluated the efficacy of locally delivered combination immunotherapy in a poorly immunogenic murine GBM model. METHODS Immunomodulators used in these studies included: IL-15 and IL-7 (T cell activation), LIGHT (T cell tumor infiltration), FLT3L (dendritic cell maturation/proliferation), a surface T cell engager (T cell killing of tumor cells), and a bispecific PD-L1/T cell engager (T cell killing targeted to PD-L1-expressing cells). We first assessed T cell-mediated cytotoxicity in vitro against SB28, a poorly immunogenic murine GBM cell line, after expressing these immunomodulators in combination. Next, tumor growth inhibition in vivo was evaluated in syngeneic C57BL/6 mice, initially by establishment of intracranial tumors with pre-transduced SB28 cells, and subsequently by delivering these immunomodulators to pre-established naïve SB28 tumors using neural stem cells (NSCs) and retroviral replicating vectors (RRV). RESULTS SB28 cells transduced with immunomodulators activated dose-dependent T cell-mediated cytotoxicity in vitro. Mice with pre-transduced intracranial SB28 gliomas showed significantly longer survival (minimum survival: 60 days, long-term survival in 57% of mice) vs. control mice (median survival: 20 days) (p< 0.001), and significantly increased tumor infiltration of CD4+ and CD8+ T cells. NSC- and RRV-mediated immunomodulator delivery to pre-established SB28 gliomas also resulted in significantly increased survival of treated mice vs. controls (median survival: 31 days vs. 22 days, p< 0.001). Immunomodulator-treated tumors again showed significantly increased infiltration of CD4+ and CD8+ T cells, along with decreased CD11b+ cell infiltration. CONCLUSIONS A novel combination therapy for GBM immunotherapy activated T cell killing of SB28 GBM cells in vitro and achieved a significant survival benefit in vivo, associated with anti-tumor alterations to the GBM tumor microenvironment. Further studies to optimize the efficiency of combinatorial immunomodulator delivery are currently underway.

Introduction: Epithelial ovarian cancers are highly aggressive and often diagnosed at late stages. To date, a limited number of these patients benefit from immunotherapies, and recurrent ovarian cancer remains an area of unmet medical need. Novel immunotherapies that generate and drive activated T cells into tumors, such as DPX-Survivac, represent a promising approach. To better understand the underlying mechanisms of action of this novel immunotherapy and how it may correlate with clinical outcomes, we used an orthotopic ovarian cancer model and a dual approach using molecular imaging to track immune cells in vivo followed by biological assays. Methods: Humanized transgenic mice (HLA-A2.1-/HLA-DR1; H-2 class-I/II knockout) were implanted with 104 syngeneic mouse ovarian surface epithelial cells in the left ovarian bursa and were either untreated or treated with DPX-Survivac, intermittent low dose cyclophosphamide and anti-PD-1. The CD8+ cytotoxic T cells (CTLs) and myeloid cells (MCs) were isolated from disease matched mice, cultured in vitro, loaded with superparamagnetic iron oxide (SPIO) nanoparticles, and injected i.v. in recipient mice. The next day, recipient mice were injected with 500µCi of 18F-fluorodeoxyglucose and imaged by magnetic resonance (MRI) and positron emission tomography (PET). Anatomical images and tumor volumes were collected with a balance steady-state free precession sequence, cells were semi-quantified using R2* maps from a multi-echo single point imaging sequence (TurboSPI) and tumor metabolism was assessed by simultaneous acquisition of PET. Mice were imaged days 41, 49, and 56 post-implant. CTLs and MCs were characterized by flow cytometry (FC) before injection into recipient mice. Following imaging completion, tumors and lymph nodes (LN) were assayed for tumor infiltrating lymphocytes. Ascites were analyzed for tumor cell to lymphocyte content by FC. Results: PET/MRI images showed that the combination immunotherapy significantly decreased primary tumor burden and improved survival rates. The recruitment of isolated CTLs and MCs was detected in both tumors and LNs. CTL recruitment to tumors was increased in mice receiving combination immunotherapy while MCs recruitment was decreased. CTLs were recruited at a higher rate to the DPX-Survivac draining LN than tumor draining LN and MCs were equally recruited to both. The expression of checkpoint inhibitor molecules on CTL did not appear to vary with treatment. CTLs from treated mice expressed higher rates of Ki67 (a proliferative marker) than CTLs from untreated mice. The treatment increased the lymphocyte:tumor cell ratio within the tumors of treated animals and included a population of CD8/CD4 double positive (DP) T cells. Differences in tumor infiltrating lymphocyte phenotype correlated with tumor burden. Conclusions: DPX-Survivac combination immunotherapy induces the recruitment of CD8+ T cells into tumors, resulting in tumor control in an orthotopic ovarian cancel model. Citation Format: Marie-Laurence Tremblay, Caitrin Sobey-Skelton, Hailey Wyatt, Victoria Gonzalez, Andrea Nuschke, Christa Davis, Alecia Mackay, Kim Bobbit, Andrea West, Barbara Vanderhyden, Genevieve Weir, Alexandra Merkx-Jacques, Kimberly Brewer, Marianne Stanford. T cell infiltration into tumors induced by DPX-Survivac combination immunotherapy demonstrated by PET/MRI imaging in an orthotopic ovarian cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 384.

TPS3160 Background: Immunotherapy with the anti-PD-(L)1 checkpoint inhibitors (CPIs) has been largely ineffective in so-called non-immunogenic “cold tumors”. Facilitating T cell infiltration is necessary to invoke an immune response which may be augmented or complemented by the activity of CPIs like durvalumab. Selective T cell Redirection Proteins (STRs) are fusion proteins that consist of genetically engineered Superantigen (Sag) linked to Fragment antigen binding (Fab) moieties directed to tumor-associated antigens. Nap is a first in class STR compound, recognizing the tumor-associated oncofetal antigen 5T4, whereas the SAg moiety selectively engages the T cell receptor β variable (TRBV) 7-9. Nap has been shown to induce specific T cells expansion, activation and infiltration into the tumor in pre-clinical and clinical studies. Pre-clinical data demonstrated that the combination of STR with CPI may lead to long term durable responses not possible in most patients receiving single agent CPI therapy and suggests that combining CPIs with STR may be a promising therapeutic strategy for patients with solid tumors. Methods: Patients will be treated with the combination of Nap and durvalumab using a flat dose of durvalumab (1120 mg) and the 3+3 design for Nap dose escalation (2, 5, 10, 15 and 20 mcg/kg). The MTD of Nap for the combination treatment will be established based on DLTs occurring during the first treatment cycle. The dose escalation part will be followed by MTD expansion cohort in which 10–15 patients will be treated with MTD of Nap and 1120 mg durvalumab (Clinical trial registry number NCT03983954). Major eligibility criteria include patients with pretreated advanced or metastatic, 5T4 expressing solid tumors, including patients previously progressed on CPI therapy. As of January 2020, enrollment to dose levels 2, 5 and 10 mcg/kg has been completed without DLT, enrollment to dose level 15 mcg/kg will start on February 2020. Clinical trial information: NCT03983954 .

e18016 Background: Cervical cancer is a Human papillomaviruses (HPV)-related disease. T cell infiltration is reported to be associated with a positive prognosis. We evaluated the prognostic value of the tumor-infiltrating dendritic cells (DC). Targeting this immune compartment might be clinically relevant. Methods: We characterized by immunohistochemistry the Dendritic cell lysosomal associated membrane glycoprotein (DC-Lamp) and CD8 T cell infiltration (high/low according to the median density) in patients with locally advanced cervical cancer (LACC) undergoing curative-intent concurrent chemoradiation followed by uterovaginal brachytherapy boost. They were treated in our institution between March 2004 and August 2011. The image acquisition was performed with a Zeiss Axio Scan Z1 microscope. CD8 T cells were detected using an algorithm created in Visiopharm software on manually selected regions of interest (ROI) and DC-Lamp was detected manually. We exported the number of cells and the surface of ROI to calculate the density. Results: A total of 91 patients were identified, with a median follow-up of 4.2 years (range: 0.1-10.3). Patient’s characteristics are listed in the table. DC-Lamphigh (n = 45) LACC patients showed a positive prognosis for overall survival (OS) by univariate (Odds Ratio (OR) = 0.20 [0.06 – 0.72], p = 0.013) and multivariate analyses (incorporating FIGO stage) (OR = 0.17 [0.05 – 0.61], p = 0.006). There was a positive correlation between DC-Lamp and CD8 (R = 0,25 et p = 0,095), which is significant in squamous cell carcinoma patients (R = 0.38, p = 0.039). There was a trend for improved risk stratification for the simultaneous assessment of DC-Lamp and CD8 levels, with DC-Lamphigh/CD8high patients having the best prognosis and DC-Lamplow/CD8low patients having the worst prognosis (p = 0.074). Conclusions: LACC patients with high intratumoral DC density have a favorable outcome. These results underscore the clinical potential of therapeutic strategies that target DC (e.g. Toll like receptor agonists) to render more patients responsive to immune checkpoint blockers. [Table: see text]

Immunotherapy resistance is associated with poor T cell infiltration into tumors. Tumor-cell-intrinsic oncogenic events that contribute to this defect include Wnt–β-catenin activation. PAK4 is now identified as an upstream modulator of this pathway, thus suggesting the potential of enhancing the efficacy of immunotherapy by targeting this druggable kinase.

BACKGROUND: Glioblastomas (GBMs) are considered immunologically cold tumors, making it difficult to apply immunotherapeutic strategies. Oncogenic pathways intrinsic to the tumor or the microenvironment can influence T-cell infiltration. WNT/β catenin is one such pathway implicated in modulating T-cell infiltration in other solid malignancies. In this study, we examine the influence of WNT signaling on the infiltration of T-cells in GBM. METHODS: Using the TCGA dataset, we analyzed the mRNA expression of both β-catenin dependent (canonical) and independent (non-canonical) WNT ligands and CTNNB1 (β-catenin) in GBM. The expressions of these components were correlated with a T-cell signature (CD3e, CD3d, CD3g, CD4, CD8a, and CD8b) and survival. Additionally, multiple GSC cell lines, derived from human GBM samples, were profiled for different WNT ligands and cells with the highest and lowest expression were selected for further experiments. In vitro, we also utilized a T-cell migration assay to measure the effect of WNT ligands (WNT3a and WNT5a) on T-cell migration towards a GL261 monolayer. RESULTS: In silico analysis of the TCGA dataset revealed downregulation of most WNTs in GBM and in 49 evaluated GSC cells lines. In cases with evidence of a T-cell signature, disease-free progression was 2-fold higher than those without evidence of a T-cell infiltration. Additionally, we observed a mutual exclusivity between the T-cell signature and β -catenin expression. WNT5a (non-canonical WNT ligand) had a significantly higher expression in the low-risk group (higher survival *p=1.42e-33) in GBM. Furthermore, in vitro, we observed a 2-fold increase in the migration ability of effector T-cells (CD4+ CD8+) towards the GL261 monolayer in presence of WNT5a. CONCLUSION: This data suggest that the WNT/β-catenin dependent pathway negatively regulates T-cell infiltration whereas the β-catenin independent pathway may have a promoting role.

Background Immunotherapy with immune checkpoint inhibitors has radically changed the management of a broad spectrum of tumors. In contrast, only very limited information is available about the efficacy of these therapies in non-melanoma skin cancers, especially in basal cell carcinoma. The latter malignancy is often associated with both an impairment of the host immune response and a high mutation burden, suggesting that immune checkpoint inhibitor-based immunotherapy may be effective in the treatment of this tumor. Case presentation A 78-year-old woman was diagnosed with a metastatic non-small-cell-lung-cancer. Following the lack of response to two lines of systemic chemotherapy, she was treated with the anti-PD-1 monoclonal antibody nivolumab, obtaining a prolonged stable disease. Under nivolumab treatment, the patient developed a basal cell carcinoma of the nose. The latter was surgically resected. Immunohistochemical staining of tumor tissue showed a PD-L1 expression

T cell infiltration in tumors has been investigated as a biomarker of response to checkpoint inhibitors. Neo-adjuvant studies in renal cell carcinoma (RCC) may provide a unique opportunity to compare T cell infiltration in a pretreatment renal mass biopsy to a posttreatment nephrectomy specimen, and thus evaluate the effects of immune checkpoint inhibitors. However, there are no data regarding the association of T cell infiltration in matched biopsy and nephrectomy samples without intervening treatment. Understanding this association will inform investigation of this potential biomarker in future studies. Matched biopsy and nephrectomy samples (without intervening systemic therapy) were identified from patients with nonmetastatic RCC. Selected tissue sections from biopsy and nephrectomy samples were reviewed and marked for intratumoral lymphocytes by a pathologist. Immunohistochemistry (IHC) was utilized to stain for T cell markers (CD3, CD4, and CD8). Intratumoral staining was then quantified in the tissue sections as counts per total tumor area surveyed. Spearman correlation (r) was used to measure associations. Thirty matched pairs were investigated. The median interval between biopsy and nephrectomy was 2.8 (0.2–87.7) months. Clear cell was the most common histology (29/30; 97%). There was a statistically significant positive correlation between the frequency of CD3 +and CD8+ T cells between matched biopsy and nephrectomy samples (r = 0.39; P = .036 and r = 0.38; P = .041, respectively). The frequencies of CD8+ T cells in matched biopsy and nephrectomy samples in RCC in the absence of intervening treatment have been characterized and show a positive correlation between matched biopsy and nephrectomy samples.

Tumour-infiltrating lymphocytes (TILs) are known to mediate potent anti-tumour activity. As T-cell-based therapies start to reach clinical practice, it becomes increasingly important to understand what characterizes a successful anti-tumour T-cell response and to exploit this knowledge for patient stratification. Next-generation sequencing of T-cell receptors (TCRs) promises to provide insights into the complexity of the tumour T-cell infiltrate that go beyond the phenotypic level. A recent study by Chen et al made use of this novel technology to demonstrate that the TIL repertoire of oesophageal squamous cell carcinoma patients is distinct from that of non-tumour sites and is characterized by significant intratumoural heterogeneity. This study illustrates the great potential of the method and addresses several technical and biological hurdles that need to be considered. Careful sampling, normalization, and error correction will be required to optimally use TCR sequencing to answer biological questions and define predictive biomarkers, e.g. for cancer immunotherapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic Tcell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ Tcells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response tocombination therapy did not appear to be associated with baseline CD8+ Tcell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.

e17542 Background: We sought to analyze the transcriptional landscape of HNSCC in an attempt to identify tumor-intrinsic oncogenic pathways that appear to mediate T-cell infiltration of tumor tissue. In this direction, we employ a methodology that integrates histopathology data of the tumor microenvironment with its corresponding transcriptome. Methods: 32 frozen HNSCCs were subjected to RNA-seq and corresponding FFPE were scored for plasma cells, tertiary lymphoid structures and CD8a+ TILs (center, invasive margin). RNA-seq data were analyzed to identify differentially expressed genes (DEGs) between tumors scored by immunohistochemistry (IHC) as CD8a high and CD8a low. Gene ontology analysis (GO) was performed based on DEGs > 1.5 fold expression change between CD8a high and CD8a low groups. Candidate genes were investigated by hierarchical clustering in TCGA RNA-seq data and further validated by IHC and quantitative RT-PCR in our cohort. Results: 32 HNSCCs were either scored as CD8a high or CD8a low based on IHC detection of CD8a+ cells in invasive margin of tumors. Comparative analysis of mRNA expression data between CD8a high and CD8a low groups in our cohort revealed that Muc1/16 overexpression and glycosylation was highly enriched in T cell infiltrated group of tumors. This finding was further validated using antibodies that detect glycosylated epitopes for both mucins. Analysis of TCGA RNA-seq data indicated that Muc1/16 overexpressing tumors share signatures of early keratinocyte differentiation and stem cell identity and co-express high levels of enzymes that promote Muc1/16 glycosylation. Interestingly, loss of CDH1 and acquisition of epithelial mesenchymal transition (EMT) markers in the cluster of Muc1/16 overexpressing tumors is strongly correlated with elevated CD8a, IDO1, CD274 and CXCL10 mRNA levels (P < 0.0001). Conclusions: Muc1/16 overexpressing tumors represent a very immunogenic HNSCC cluster. Previous studies have shown that mucins 1 and 16 in cancer cells expose glycosylated-specific epitopes that are recognized by T cells as cancer antigens. To this end, MUC1/16 expression may serve as predictive biomarkers for response to immunotherapy and MUC-targeted immunotherapy may function as an attractive partner to checkpoint inhibitors in HNSCC.

118 Background: Preclinical studies suggest that MMP9 inhibition relieves immune suppression and promotes T cell infiltration to potentiate checkpoint blockade. To test this hypothesis, patient tumor samples obtained in a phase 2, open-label, randomized study (NCT02864381) of previously treated advanced gastric cancer comparing the MMP9-specific inhibitor andecaliximab (ADX) plus nivolumab (N) vs N alone were evaluated for T cell biomarkers. Methods: CD8 and PD-L1 (28-8 DAKO) were assessed by immunohistochemistry. IFNg, Teffector (Teff), and activated CD8+ T cell (ActT) gene signatures (GS) were assessed by RNASeq in archival baseline (BL) and biopsies obtained between weeks 5 and 9 (on-treatment; OT). Results: For both N and ADX/N, intratumoral CD8+ cells were significantly increased in OT biopsies relative to BL. The CD8+ OT increase was significantly greater for the ADX/N treatment relative to N in the PD-L1+ subgroup. In the ADX/N group only, IFNg, Teff, and ActT GS were significantly higher in OT biopsies relative to BL. OT change from BL of ≥ 300% vs

Objective. The aim of this study was to identify serum markers that are modulated by an investigational anti-IFN-α mAb, sifalimumab, in adult DM or PM patients. Methods. In a phase 1b clinical trial, sera were collected from a total of 48 DM or PM adult patients receiving either placebo for 3 months or sifalimumab for 6 months. Samples were tested for 128 selected proteins using a multiplex luminex immunoassay. Muscle biopsies from selected patients were stained for T cell infiltration using an anti-CD3 antibody. Results. A robust overexpression of multiple serum proteins in DM or PM patients was observed, particularly in patients with an elevated baseline type I IFN gene signature in the blood or muscle. Neutralization of the type I IFN gene signature by sifalimumab resulted in coordinated suppression of T cell-related proteins such as soluble IL-2RA, TNF receptor 2 (TNFR2) and IL-18. Muscle biopsies from two patients with the highest serum protein suppression were selected and found to have a pronounced reduction of muscle T cell infiltration. Down-regulation of IL-2RA correlated with favourable manual muscle test 8 (MMT-8) alterations in sifalimumab-dosed patients. Conclusion. A reduced level of multiple T cell-associated proteins after sifalimumab but not placebo administration suggests a suppressive effect of blocking type I IFN signalling on T cell activation and chemoattraction that may lead to a reduction of T cell infiltration in the muscle of myositis patients. Further, soluble IL-2RA changes from baseline may serve as a responsive and/or predictive marker for type I IFN-targeted therapy in adult DM or PM patients.

Macrophage and T cell infiltration into metabolic tissues contributes to obesity-associated inflammation and insulin resistance (IR). C-C chemokine receptor 5 (CCR5), expressed on macrophages and T cells, plays a critical role in the recruitment and activation of proinflammatory M1 and TH1 immune cells to tissues and is elevated in adipose tissue (AT) and liver of obese humans and mice. Thus, we hypothesized that deficiency of CCR5 would protect against diet-induced inflammation and IR. CCR5-deficient (CCR5−/−) mice and C57BL/6 (WT) controls were fed 10% low-fat (LF) or 60% high-fat (HF) diets for 16 wk. HF feeding increased adiposity, blood glucose, and plasma insulin levels equally in both genotypes. Opposing our hypothesis, HF-fed CCR5−/− mice were significantly more glucose intolerant than WT mice. In AT, there was a significant reduction in the M1-associated gene CD11c, whereas M2 associated genes were not different between genotypes. In addition, HF feeding caused a twofold increase in CD4+ T cells in the AT of CCR5−/− compared with WT mice. In liver and muscle, no differences in immune cell infiltration or inflammatory cytokine expression were detected. However, in AT and muscle, there was a mild reduction in insulin-induced phosphorylation of AKT and IRβ in CCR5−/− compared with WT mice. These findings suggest that whereas CCR5 plays a minor role in regulating immune cell infiltration and inflammation in metabolic tissues, deficiency of CCR5 impairs systemic glucose tolerance as well as AT and muscle insulin signaling.

To study whether in-vivo recruitment of dendritic cells in response to antigen administration in the skin is altered during HIV-1 infection.Skin punch biopsies were collected from HIV-1-positive as well as seronegative individuals at 48 h after intradermal injection of inactivated antigens of mumps virus, Candida albicans, or purified protein derivate (PPD) from Mycobacterium tuberculosis.Cryosections were analyzed by in-situ staining and computerized imaging.Control skin biopsies showed that there was no difference in the number of skin-resident dendritic cells between seronegative and HIV-1-positive individuals. Antigen injection resulted in substantial infiltration of dendritic cells compared to the frequencies found in donor-matched control skin. In HIV-1-positive individuals, CD123(+)/CD303(+) plasmacytoid dendritic cells and CD11c myeloid dendritic cells, including the CD141(+) cross-presenting subset, were recruited at lower levels compared to healthy controls in response to PPD and mumps but not C. albicans. The level of dendritic cell recruitment correlated with the frequencies of T cells infiltrating the respective antigen sites. Ki67(+) cycling T cells at the injection sites were much more frequent in response to each of the antigens in the HIV-1-positive individuals, including those with AIDS, compared to healthy controls.Multiple dendritic cell subsets infiltrate the dermis in response to antigen exposure. There was no obvious depletion or deficiency in mobilization of dendritic cells in response to antigen skin tests during chronic HIV-1 infection. Instead, the levels of antigen-specific memory T cells that accumulate at the antigen site may determine the level of dendritic cell infiltration.

Sarcoidosis is a disease of involving the unusual accumulation on inflammatory cells which forms lumps called as granulomas. Though the pathogenesis remains undefined, it has been proposed that an initial CD4+ T cell infiltration is the spark in the inflammatory cascade that results in granulomatous formation in affected organs. In this case report we have described an unusual presentation of sarcoidosis detailing the mutable iterations of this disease.

In previous chronic servo control experiments we showed that increased renal perfusion pressure (RPP) amplifies renal T-cell infiltration in Dahl salt-sensitive (SS) rats. Renal T-cell infiltration was significantly higher in the right-hypertensive kidneys vs. the left servo-controlled kidneys of SS rats fed high salt (HS) for 7-days. There was no significant difference in T-cell infiltration in sham rats (both kidneys exposed to comparable RPPs). Here we use the chronic servo control system to determine whether the reversal of salt-sensitive hypertension reverses renal T-cell infiltration in SS rats. An aortic balloon occluder was placed around the aorta, between the renal arteries to electronically servo-control RPP to the left kidney. Arterial catheters were used to measure MAP above and below the occluder (n=5). Baseline RPPs were measured on a control salt diet (0.4% NaCl) for 3-days (average left RPP, 124±1mmHg) after which rats were switched to a HS diet (4.0% NaCl) for 14-days. During the first 7-days of HS both kidneys were exposed to uncontrolled hypertension. After 7-days a servo control system was used to bring the RPP to the left kidney (the reversal kidney) back to the average control level, where it was held for the next 7-days of HS (from 139±2mmHg to 125±2mmHg). It was found that reversal of hypertension prevented a further increase of renal T-cell infiltration but the number of infiltrated T-cells did not diminish. Moreover, the number of T-cells in the left-reversal kidney was significantly higher than in the continuously-controlled, left-servo kidney despite comparable final RPPs (129±2mmHg to the left-servo kidney vs. 125±2mmHg to the left-reversal kidney). T-cell infiltration in the left-reversal kidney was comparable to the left-sham kidneys, in which in the final RPP was 148±2mmHg. In ‘T-cells per kidney’- CD3+ (mature): Reversal=287x10 3 ±65x10 3 Servo=78x10 3 ±12x10 3 Sham=186x10 3 ±47x10 3 . CD3+CD4+ (helper): Reversal=95x10 3 ±27x10 3 Servo=32x10 3 ±5x10 3 Sham=86x10 3 ±26x10 3 . CD3+CD8+ (cytotoxic): Reversal=196x10 3 ±42x10 3 Servo=42x10 3 ±7x10 3 Sham=102,082x10 3 ±23x10 3 We conclude that reversal of blood pressure alone, for 1-week, while HS intake is continued, stops but does not reverse renal T-cell infiltration in SS rats.

The majority of cancer patients respond poorly to either vaccine or checkpoint blockade, and even to the combination of both. They are often resistant to high doses of radiation therapy as well. We examined prognostic markers of immune cell infiltration in pancreatic cancer. Patients with low CD8+ T cell infiltration and high PD-L1 expression (CD8+ TloPD-L1hi) experienced poor outcomes. We developed a mouse tumor fragment model with a trackable model antigen (SIYRYYGL or SIY) to mimic CD8+ TloPD-L1hi cancers. Tumors arising from fragments contained few T cells, even after vaccination. Fragment tumors responded poorly to PD-L1 blockade, SIY vaccination or radiation individually. By contrast, local ionizing radiation coupled with vaccination increased CD8+ T cell infiltration that was associated with upregulation of CXCL10 and CCL5 chemokines in the tumor, but demonstrated modest inhibition of tumor growth. The addition of an anti-PD-L1 antibody enhanced the effector function of tumor-infiltrating T cells, leading to significantly improved tumor regression and increased survival compared to vaccination and radiation. These results indicate that sequential combination of radiation, vaccination and checkpoint blockade converts non-T cell-inflamed cancers to T cell-inflamed cancers, and mediates regression of established pancreatic tumors with an initial CD8+ TloPD-L1hi phenotype. This study has opened a new strategy for shifting "cold" to hot tumors that will respond to immunotherapy.