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Bladder cancer (BC) is one of the most prevalent cancers around the world and, if not treated well, has high morbidity and mortality. Many studies have indicated that there may be various roles for the aryl hydrocarbon receptor (AHR) in the immune system. The aim of this study was to determine the frequency of Foxp3(+) regulatory T (Treg) and T helper 17 cells (Th17) in BC tissue in comparison with controls and determine the relationship between AHR, Foxp3(+) Treg and Th17 cells in BC. A total of 40 patients with BC were enrolled in this study. The control group was selected from non‐tumoural parts of bladder tissues from the patients who have undergone cystoscopy. The percentage of regulatory T cells (Foxp3(+)/CD4(+)) and Th17 (IL‐17(+)/CD4(+)), as well as AHR(+) cells in BC tissues and controls, were determined by immunohistochemistry. The results of this study showed that the number of Foxp3(+) Treg and Th17 is significantly higher in bladder tumour tissues in comparison with non‐tumoural tissues. Also, the percentage of AHR(+) lymphocytes and AHR(+) cells was increased significantly in bladder tumour tissues rather than non‐tumoural tissues. This study also found a relation between AHR and Foxp3(+)/CD4(+) T lymphocytes ratio cells in BC. The percentage of Foxp3(+) Tregs and AHR(+) cells were significantly correlated with the grade and stage of BC. An increase in the percentage of Foxp3(+) Treg and Th17 cells may play an important role in tumour immunity; and determining the relationship between AHR and differentiation of Th17/Foxp3(+)Treg in BC can lead to a potential cancer therapeutic possibility.

Programmed death‐ligand 1 (PD‐L1) blockade has revolutionized the prognosis of several cancers, but shows a weak effect on pancreatic cancer (PC) due to poor effective immune infiltration. Chemokine C‐C motif ligand 21 (CCL21), a chemokine promoting T cell immunity by recruiting and colocalizing dendritic cells (DCs) and T cells, serves as a potential antitumor agent in many cancers. However, its antitumor response and mechanism combined with PD‐L1 blockade in PC remain unclear. In our study, we found CCL21 played an important role in leukocyte chemotaxis, inflammatory response, and positive regulation of PI3K‐AKT signaling in PC using Metascape and gene set enrichment analysis. The CCL21 level was verified to be positively correlated with infiltration of CD8+ T cells by the CIBERSORT algorithm, but no significant difference in survival was observed in either The Cancer Genome Atlas or the International Cancer Genome Consortium cohort when stratified by CCL21 expression. Additionally, we found the growth rate of allograft tumors was reduced and T cell infiltration was increased, but tumor PD‐L1 abundance elevated simultaneously in the CCL21‐overexpressed tumors. Then, CCL21 was further verified to increase tumor PD‐L1 level through the AKT‐glycogen synthase kinase‐3β axis in human PC cells, which partly impaired the antitumor T cell immunity. Finally, the combination of CCL21 and PD‐L1 blockade showed superior synergistic tumor suppression in vitro and in vivo. Together, our findings suggested that CCL21 in combination with PD‐L1 blockade might be an efficient and promising option for the treatment of PC., Chemokine C‐C motif ligand 21 (CCL21) in the tumor microenvironment can promote T cell infiltration and enhance their antitumor immune response. However, CCL21 also stabilizes and upregulates programmed death‐ligand 1 (PD‐L1) expression on pancreatic cancer cells through the AKT‐glycogen synthase kinase‐3β signaling pathway at the same time, which could partly impair the local immune response. Thus, PD‐L1 blockade was used in combination with CCL21, which showed a powerful synergism and promising therapeutic efficacy.

Immunotherapy, represented by immune checkpoint inhibitors (ICIs), has greatly improved the clinical efficacy of malignant tumor therapy. ICI-mediated antitumor responses depend on the infiltration of T cells capable of recognizing and killing tumor cells. ICIs are not effective in "cold tumors", which are characterized by the lack of T-cell infiltration. To realize the full potential of immunotherapy and solve this obstacle, it is essential to understand the drivers of T-cell infiltration into tumors. We present a critical review of our understanding of the mechanisms underlying "cold tumors", including impaired T-cell priming and deficient T-cell homing to tumor beds. "Hot tumors" with significant T-cell infiltration are associated with better ICI efficacy. In this review, we summarize multiple strategies that promote the transformation of "cold tumors" into "hot tumors" and discuss the mechanisms by which these strategies lead to increased T-cell infiltration. Finally, we discuss the application of nanomaterials to tumor immunotherapy and provide an outlook on the future of this emerging field. The combination of nanomedicines and immunotherapy enhances cross-presentation of tumor antigens and promotes T-cell priming and infiltration. A deeper understanding of these mechanisms opens new possibilities for the development of multiple T cell-based combination therapies to improve ICI effectiveness.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous group of cancer with dismal prognosis. B7 families can be promising targets for cancer immunotherapy in TNBC. Previously we have found increased expression of B7-H3 and B7-H4 mRNA and protein in breast cancer including TNBC. In an effort to discover therapeutic targets, TNBC has been further stratified into molecular subtypes. However, little is known about the clinical impact and value of B7-H3 and B7-H4 in TNBC subtypes. The purpose of this study was to evaluate the clinicopathologic characteristics of the B7-H3 and B7-H4 mRNA and protein expression according to the TNBC subset. Materials and methods: A tissue microarray was constructed from 186 patients with TNBC. B7-H3 and B7-H4 mRNA and protein expression were assessed by the RNAscope in situ hybridization (ISH) and immunohistochemistry. Immunohistochemistry for the TNBC molecular subtype-surrogate markers [cytokeratin 5/6 (CK5/6), CK14, epidermal growth factor receptor (EGFR), and androgen receptor (AR)], CD3, and CD8 was also performed. TNBC subtypes were classified into three subtypes: basal-like (BL), luminal AR (LAR), and unclassifiable type (UN). Results: Based on the immunohistochemical results, 186 TNBCs were classified into BL (n=120, 64.5%), LAR (n=10, 10.8%), and UN (n=46, 24.7%) subtypes. BL and UN subtypes were associated with younger age than the LAR subtype. B7-H3 mRNA and protein expressions were expressed in the tumor and stromal cells of the TNBCs. On the contrary, B7-H4 mRNA and protein expressions were only observed in the tumor cells. High tumor mRNA and protein expression of B7-H3 and B7-H4 were found in 49 of 175 (28.0%) and 121 of 176 (68.8) cases, and 92 of 174 (52.9%) and 66 of 176 (37.5%) cases, respectively. High stromal B7-H3 mRNA and protein expression were observed in 22 of 175 (12.6%) and 43 of 176 (24.4%) cases. High stromal B7-H4 mRNA and protein expression were not observed. B7-H3 and B7-H4 protein expression were closely correlated with their mRNA expression according to the tumor compartment. Tumor B7-H4 mRNA expression was associated with younger age at the initial diagnosis and molecular TNBC subtypes. Expression of B7-H3 mRNA and protein in the tumor cells negatively correlated with CD3+ and CD8+ T cell infiltration density in the tumor and/or stromal compartment of the TNBCs and its subtypes. High stromal B7-H3 mRNA expression was associated with poor disease-free and overall survival in the TNBCs and overall survival in the UN subtype. Stromal B7-H3 mRNA expression was independently associated with overall survival in the TNBCs and disease-free survival in the BL subtype. Conclusions: As prognostic factors, our results point to the importance of the expression of B7-H3 mRNA by the stromal cells in the TNBCs and the BL subtype. The inverse relationship between B7-H3 expression and CD3+ and CD8+ T lymphocyte infiltration may represent a promising target in the immunotherapy of the TNBCs, irrespective of its molecular subtypes. Citation Format: Ji Shin Lee, Nah Ihm Kim, Min Ho Park. B7-H3 and B7-H4 expression in triple-negative breast cancer subtypes detected by RNA in situ hybridization and immunohistochemistry: Association with clinicopathological features and T-cell infiltration [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-35.

Breast cancer is the most common form of cancer in women. Despite significant therapeutic advances in recent years, breast cancer also still causes the greatest number of cancer-related deaths in women, the vast majority of which (> 90%) are caused by metastases. However, very few mouse mammary cancer models exist that faithfully recapitulate the multistep metastatic process in human patients. Here we assessed the suitability of a syngrafting protocol for a Myc-driven mammary tumor model (WAP-Myc) to study autochthonous metastasis. A moderate but robust spontaneous lung metastasis rate of around 25% was attained. In addition, increased T cell infiltration was observed in metastatic tumors compared to donor and syngrafted primary tumors. Thus, the WAP-Myc syngrafting protocol is a suitable tool to study the mechanisms of metastasis in MYC-driven breast cancer.

Background Immune system plays an important role in cancer evolution. However, cancer cells affect the immune system and use it for its growth and survival. Tumor infiltration lymphocytes and PDL-1 expression proved to play an important prognostic role in different malignancies. We aimed at evaluating this role in laryngeal cancer. Methodology We prospectively analyzed the pre-treatment paraffin blocks for laryngeal cancer patients, whether on the laryngoscopy or operative specimens, to identify the CD8-positive cells in the tumor epithelium or stroma and the PDL-1 expression on the tumor cells. Appropriate tissue was used as positive control; placenta for PDL-1 and tonsil for CD8. A total score formed of the sum of percentage and intensity of PDL-1. A final comprehensive rate was considered as low expression when combined percentage and intensity score from 0 to 4, and high expression when score from 5-7. According to the distribution, the CD8+T cell invasion was divided into strong infiltration (> 10/100 of epithelial cells; >20/100 stromal cell infiltration) and weak infiltration (< 10/100 epithelial cells; < 20/100 stromal cell infiltration). Results 40 patients were included in our study; 12 patients had early stage disease (I or II) and 28 with advanced stage (III or IV). PDL-1 expression was positive in 92.5%. Neither the PDL-1 nor CD-8 were found significantly correlated to OS in the whole population, however patients with advanced stage, median OS was significantly low for negative or low compared to high PDL-1 expression (11.7 vs 25.2 months (p = 0.036)), a trend of better OS in high CD8 Epithelial expression (25.1 versus 15.9, P = 0.514) and again trend of higher OS in high CD8 Stromal expression (25.3 versus 15.6 months, p = 0.375). Also there were significant correlation between the CD8 expression in the tumor epithelium and stroma with the PDL-1 expression with p-value of 0.001 and < 0.0001 respectively. Conclusion there is a positive correlation between tumor infiltration lymphocytes and PDL-1 expression in laryngeal squamous carcinoma and the higher PDL-1 expression in advanced stage laryngeal cancer is associated with better OS.

INTRODUCTION AND OBJECTIVE:Immunocompetent animal models that recapitulate human renal cell carcinoma (RCC) and its tumor-immune microenvironment are critical for the preclinical study of novel imm...

Background: Multiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A2A receptor (A2AR) is emerging as a potential pharmacological target to control EAE pathogenesis; however, the cellular basis for the A2AR-mediated protection remains undetermined. Methods: In EAE model, we assessed A2AR expression and leukocyte trafficking determinants in CP by immunohistochemistry and qPCR analyses. We determined the effect of the A2AR antagonist KW6002 treatment at days 8-12 or 8-14 post-immunization on T cell infiltration across CP and EAE pathology. We determined the critical role of the CP-A2AR on T cell infiltration and EAE pathology by focal knockdown the CP-A2AR with intracerebroventricular injection of CRE-TAT recombinase into the A2ARflox/flox mice. In cultured CP epithelium, we also evaluated the effect of overexpression of A2ARs or the A2AR agonist CGS21680 treatment on CP permeability and lymphocytes migration. Results: We found the specific upregulation of A2AR in CP in association with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8-12 or 8-14 post-immunization reduced T-cell trafficking across CP and attenuated EAE pathology. Importantly, focal CP-A2AR knockdown attenuated pathogenic infiltration of Th17+ cells across CP via inhibiting CCR6-CCL20 axis through NFκB / STAT3 pathway and protected against EAE pathology. Lastly, activation of A2AR in cultured epithelium by A2AR overexpression or CGS21680 treatment increased the permeability of CP epithelium and facilitated lymphocytes migration. Conclusion: These findings define the CP niche as one of the primary sites of A2AR action whereby A2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.

BackgroundAlthough immune checkpoint inhibitors (ICIs), especially programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis blockers, exhibit prominent antitumor effects against numerous malignancies, their benefit for patients with prostate cancer (PCa) has been somewhat marginal. This study aimed to assess the feasibility of B7-H3 or HHLA2 as alternative immunotherapeutic targets in PCa.MethodsImmunohistochemistry was performed to evaluate the expression pattern of PD-L1, B7-H3 and HHLA2 and the infiltration of CD8+ and Foxp3+ lymphocytes in 239 PCa tissues from two independent cohorts. The correlations between B7-H3 and HHLA2 and clinicopathological features, including the presence of CD8+ and Foxp3+ tumor-infiltrating lymphocytes (TILs), were explored.ResultsHHLA2 expression was much higher than PD-L1 expression but lower than B7-H3 expression in PCa tissues. High expression of both B7-H3 and HHLA2 was significantly associated with higher Gleason score and tumor stage, lymph node metastasis and dismal overall survival (OS) and cancer-specific survival (CSS). Moreover, a high B7 score, defined as high B7-H3 expression and/or high HHLA2 expression, was an independent prognostic predictor for PCa. Of note, a high B7 score was negatively correlated with CD8+ TILs. Importantly, a new immune classification, based on the B7 score and CD8+ TILs, successfully stratified OS and CSS in PCa.ConclusionsBoth B7-H3 and HHLA2 have a critical impact on the immunosuppressive microenvironment, and the B7 score could be used as an independent prognostic factor for PCa. The B7 score combined with CD8+ TILs could be used as a new immune classification to stratify the risk of death, especially cancer-related death, for patients with PCa. These findings may provide insights that could improve response to immune-related comprehensive therapy for PCa in the future.

Many studies have suggested that renal T cell infiltration contributes to the pathogenesis of salt-sensitive hypertension. To investigate this mechanism further, we determined T cell profiles in the kidney and lymphoid tissues as a function of blood pressure in the female Envigo Dahl salt-sensitive (SS) rat maintained on low-Na+(LS) diet. Mean arterial pressure and heart rate were measured by telemetry in SS rats from 1 mo old (juvenile) to 4 mo old. Normotensive salt-resistant (SR) rats were included as controls. Frequencies of T helper (CD4+) cells were greater in the kidney, lymph nodes, and spleen in 4-mo-old hypertensive SS rats compared with normotensive SR animals and SS juvenile rats, suggesting that renal T cell infiltration contributes to hypertension in the SS rat on a LS diet. At 1.5 mo, half of the SS rats were treated with vehicle (Veh), and the rest received hydralazine (HDZ; 25 mg·kg−1·day−1) for 11 wk. HDZ impeded the development of hypertension compared with Veh-treated control rats [mean arterial pressure: 157 ± 4 mmHg in the Veh-treated group ( n = 6) vs. 133 ± 3 mmHg in the HDZ-treated group ( n = 7), P < 0.001] without impacting T helper cell frequencies in the tissues, suggesting that HDZ can overcome mechanisms of hypertension driven by renal T cell infiltration under the LS diet. Renal frequencies of CD4+CD25+and CD4+CD25+FoxP3+regulatory T cells were significantly higher in 4-mo-old hypertensive rats compared with normotensive SR rats and SS juvenile rats, suggesting that these T cell subpopulations play a compensatory role in the development of hypertension. Greater understanding of these T cell populations could lead to new therapeutic targets for treating inflammatory diseases associated with hypertension.

Background and Aims Todays increasing effort are made to detect non-invasive and reliable methods for prediction of organ rejection. In the current study, we evaluated the degree of CD3, CD20, Th17 and Tregs infiltration in kidney biopsy of the patients with acute cellular rejection and the possible relation with graft outcome. Method From 70 biopsy proven ATCMR according to Banff criteria in whom kidney biopsy were done based on acute creatinine rise, 50 patients were selected. 20 patients excluded because of insufficient specimen or clinical data. All of recruited patients experienced their first episode of ATCMR. 21 patients had deceased donors and 29 had living ones. Paraffin sections were deparaffinized then dehydrated using alcohols. The tissues were incubated with Antibodies in two stages. First, they were incubated with antihuman IL-17 monoclonal antibody and polyclonal antibody against FOXP3. The results analyzed with SPSS version 20. P value less than 0.05 was considered as significant. Results FOXP3 mean cell count in stable graft function group was 7.88 but in impaired graft group was 8.02, which was not statistically significant (P-value :0. 96). Th17 mean cell count in stable group was 5.0 but in impaired group was 10.1, which was not significant too (P-value: 0.24). FOXP3/Th17 ratio was higher in stable group (1.4 versus 1.12) but without any statistically important value (P-value: 0.22) .CD3 count was higher in impaired group (29.8 versus 27.7) and CD20 count was more in stable group (8.8 versus 7.8) without any significant values (P-value: 0.7 and 0.62) respectively. FOXP3 positive cell count was higher in failure to response group (9.95 vs. 6.63) but with non-significant P-value of 0.1. Th17 count was higher also in this group (11.3 vs. 8.3) but with non-significant value .The FOXP3/Th17 ratio was higher in appropriate response group (1.19 vs. 1.15) with P-value 0.8 .CD3 count was higher in response group and CD20 in failure group vice versa (31.2 vs. 27.3 and 7.96 vs. 8.06) also with nonsignificant values Conclusion We observed that allografts with higher Th17 at biopsy had lower levels of GFR in the next 12 months. One conclusion can be that assessing the Th17 infiltration is more effective than FOXP3 Tregs in predicting the prognosis of renal transplant .

PurposeThe aim of this study was to characterize chondrosarcoma tumor infiltration by immune cells and the expression of immunologically relevant molecules. This information may contribute to our understanding of the role of immunological events in the pathogenesis of chondrosarcoma and to the rational design of immunotherapeutic strategies.Patients and MethodsA tissue microarray (TMA) containing 52 conventional and 24 dedifferentiated chondrosarcoma specimens was analyzed by immunohistochemical staining for the expression of parameters associated with tumor antigen-specific immune responses, namely, CD4+ and CD8+ tumor infiltrating lymphocytes (TILs) and the expression of HLA class I heavy chain, beta-2 microglobulin (β2m), HLA class II and immune checkpoint molecules, B7-H3 and PD-1/PD-L1. The results were correlated with histopathological characteristics and the clinical course of the disease.ResultsCD8+ TILs were present in 21% of the conventional and 90% of the dedifferentiated chondrosarcoma tumors tested. B7-H3 was expressed in 69% of the conventional and 96% of the dedifferentiated chondrosarcoma tumors tested. PD-1 and PD-L1 were expressed 53% and 33% respectively of the dedifferentiated tumors tested. PD-L1 expression was associated with shorter time to metastasis.ConclusionThe tumor infiltration by lymphocytes suggests that chondrosarcoma is immunogenic. Defects in HLA class I antigen and expression of the checkpoint molecules B7-H3 and PD-1/PD-L1 suggest that tumor cells utilize escape mechanisms to avoid immune recognition and destruction. This data implies that chondrosarcoma will benefit from strategies that enhance the immunogenicity of tumor antigens and/or counteract the escape mechanisms.

Simple Summary High T-cell infiltration has been associated with improved clinical outcomes in many human solid tumors. However, these cells are not autonomous in their effector function, depending on the interaction with other immune and non-immune cells, as well as soluble factors released into the tumor microenvironment (TME). Identification of the key elements underlying T-cell recruitment within tumors is of fundamental importance to improve the success of immunotherapy strategies. This review summarizes the most recent findings on dendritic cells (DC), a key cellular element that regulates the recruitment of functional tumor-specific CD8+ T cells, and current strategies that exploit this innate immune cell to improve the efficacy of therapeutic treatments. Abstract Tumor-infiltrating CD8+ T cells have been shown to play a crucial role in controlling tumor progression. However, the recruitment and activation of these immune cells at the tumor site are strictly dependent on several factors, including the presence of dendritic cells (DCs), the main orchestrators of the antitumor immune responses. Among the various DC subsets, the role of cDC1s has been demonstrated in several preclinical experimental mouse models. In addition, the high density of tumor-infiltrating cDC1s has been associated with improved survival in many cancer patients. The ability of cDC1s to modulate antitumor activity depends on their interaction with other immune populations, such as NK cells. This evidence has led to the development of new strategies aimed at increasing the abundance and activity of cDC1s in tumors, thus providing attractive new avenues to enhance antitumor immunity for both established and novel anticancer immunotherapies. In this review, we provide an overview of the various subsets of DCs, focusing in particular on the role of cDC1s, their ability to interact with other intratumoral immune cells, and their prognostic significance on solid tumors. Finally, we outline key therapeutic strategies that promote the immunogenic functions of DCs in cancer immunotherapy.

Simple Summary The ability of the immune system to kill tumour cells is a natural and extremely effective defence mechanism for fighting cancer. Cytotoxic-T-cells are a critical component of our immune system which function is to eliminate cancer cells. In some cancers, especially those with a rich tumour stroma, these cytotoxic-T-cells are unable to reach and kill the tumour cells. Cancer-associated fibroblasts are the most abundant cells in the tumour stroma and play a key role of the recruitment, infiltration and function of cytotoxic T-cells in the tumour, via several molecular mechanisms which we describe in this review. Abstract The tumour microenvironment (TME) is the complex environment in which various non-cancerous stromal cell populations co-exist, co-evolve and interact with tumour cells, having a profound impact on the progression of solid tumours. The TME is comprised of various extracellular matrix (ECM) proteins in addition to a variety of immune and stromal cells. These include tumour-associated macrophages, regulatory T cells (Tregs), myeloid-derived suppressor cells, as well as endothelial cells, pericytes and cancer-associated fibroblasts (CAFs). CAFs are the most abundant stromal cell population in many tumours and support cancer progression, metastasis and resistance to therapies through bidirectional signalling with both tumour cells and other cells within the TME. More recently, CAFs have been shown to also affect the anti-tumour immune response through direct and indirect interactions with immune cells. In this review, we specifically focus on the interactions between CAFs and cytotoxic CD8+ T cells, and on how these interactions affect T cell recruitment, infiltration and function in the tumour. We additionally provide insight into the therapeutic implications of targeting these interactions, particularly in the context of cancer immunotherapy.

Background and Aims Renal transplantation is considered as the best replacement therapy for advanced ESRD patients. An allograft rejection happens as a result of post transplant immune reactions, which change the outcome of the organ transplantation. Today a major challenge in the field of transplantation is the identification of easy, reliable and non-invasive markers or methods that being able to predict the probability of organ rejection. One of the possible methods is looking for type of infiltrated cells in tissue obtaining by biopsy stained with specific cellular markers and assesses the infiltration of these cells in different types of rejection. Here the severity of CD3, CD20, Th17 and FOXP3 infiltration in patients with biopsy proven acute cellular rejection was evaluated based on IHC staining, whether these specific infiltrations can show an association with graft outcome or not. Method 50 patients with biopsy proven Acute T Cell Mediated Rejection (ATCMR) recruited. Previous clinical data and 1 year clinical follow up collected. The entire specimen assessed for infiltration of CD3, CD20, FOXP3 Tregs and Th17 with IHC. Patients divided into subgroups: stable graft function versus impaired graft function based on serum creatinine course in one year follow up after rejection therapy and appropriate response to treatment versus failure to response, based on allograft function throughout the course of admission. Results In impaired graft function arm, FOXP3 (7.88 vs. 8.02 with P-value 0.96) and Th17 cells were higher (5.01 vs. 10.2 with P-value 0.24) but with non-significant values. FOXP3/Th17 ratio was higher in stable group (1.4 vs. 1.12 with P-value 0.22). In failure to response to therapy group both FOXP3 (9.95 vs. 6.63 with P-value 0.1) and Th17 (11.3 vs. 8.3 with P-value 0.15) cells were higher. FOXP3/Th17 ratio was higher in proper response group (1.19 vs. 1.15 with P-value 0.8). No significant difference was obtained between CD3 and CD20 infiltration in these two groups. Conclusion Final results showed that Th17 has more important role in predicting the graft outcome and response to treatment and FOXP3 infiltration had a minor part. This may be in controversial with previous facts about the role of FOXP3 cells, which drive the allograft into stable condition.

Objective To assess the potential impact of mutant ECM amino acids (AA) on melanoma-related matrix metalloproteinase-7 (MMP7) activity. Design and methods We applied a novel scripted algorithm, based on the MEROPS database, to reveal mutant-dependent sensitivity changes across the cancer genome atlas, melanoma dataset. Results This approach revealed a strong bias in favor of mutant AA dependent protease sensitivity increases. Thus, melanoma specimens with relatively few mutations had only MMP7 mutant sensitive, ECM peptides. As mutations increased, melanoma specimens included mutant AA representing mostly increased sensitivity and a small but increasing number of mutant AA representing decreased MMP7 sensitivity. There was no detection of melanoma specimens with only decreases in MMP7 sensitivity. Furthermore, melanoma specimens with exclusively increased sensitivity and thereby only a few overall mutations represented reduced T-cell infiltrates and worse outcomes. Conclusions Overall, the results indicated that changes in MMP7 sensitivity, attributable to mutant AA, have the potential of identifying patients with distinct survival outcomes as well as patients with cancer specimen immune activity.

Pancreatic cancer is the third leading cause of cancer mortality in the United States, recently surpassing breast cancer. A key component of pancreatic cancer’s lethality is its acquired immune privilege, which is driven by an immunosuppressive microenvironment, poor T-cell infiltration, and a low mutational burden. Although immunotherapies such as checkpoint blockade or engineered T cells have yet to demonstrate efficacy, a growing body of evidence suggests that orthogonal combinations of these and other strategies could unlock immunotherapy in pancreatic cancer. In this review, we will discuss promising immunotherapies currently under investigation in pancreatic cancer and provide a roadmap for the development of prevention vaccines for this and other cancers.

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.