Search

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.

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...

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.

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.

The authors have requested that this preprint be removed from Research Square.

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.

The relationship between tumor immune responses and tumor neoantigens is one of the most fundamental and unsolved questions in tumor immunology, and is the key to understanding the inefficiency of immunotherapy observed in many cancer patients. However, the properties of neoantigens that can elicit immune responses remain unclear. This biological problem can be represented and solved under a multiple instance learning framework, which seeks to model multiple instances (neoantigens) within each bag (patient specimen) with the continuous response (T cell infiltration) observed for each bag. To this end, we develop a Bayesian multiple instance regression method, named BMIR, using a Gaussian distribution to address continuous responses and latent binary variables to model primary instances in bags. By means of such Bayesian modeling, BMIR can learn a function for predicting the bag-level responses and for identifying the primary instances within bags, as well as give access to Bayesian statistical inference, which are elusive in existing works. We demonstrate the superiority of BMIR over previously proposed optimization-based methods for multiple instance regression through simulation and real data analyses. Our method is implemented in R package entitled “BayesianMIR” and is available at https://github.com/inmybrain/BayesianMIR .

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.

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.

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 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.

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.

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.

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.

The establishment of immune heterogeneity in the tumor microenvironment (TME) is poorly understood, despite recent data that the success of immunotherapies is dictated by the immune environment of the tumor site. Pancreatic ductal adenocarcinoma (PDA) is characteristically devoid of CD8 T cells and resistant to therapeutic intervention. However, a small subset of patients (15%) have tumors highly infiltrated by CD8 T cells, correlating with improved overall survival. To better elucidate the determinants of immune heterogeneity in the PDA TME, we generated clones from spontaneous tumors harvested from KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mice, a genetically engineered mouse model of PDA. Using a panel of 17 tumor clones, we found the clones segregated in to two groups with differential immune cell infiltration upon implantation in congenic C57BL/6 mice. 7/17 tumor clones were categorized as ‘T cell high,' with an immune infiltrate comprising CD8 T cells, CD103+ dendritic cells (DCs), and a dearth of myeloid derived suppressor cells (MDSCs). In contrast, the remaining 10/17 tumor clones were categorized as ‘T cell low' lines, with TME dominated by myeloid cells and macrophages, especially granulocytic MDSCs. Hypothesizing that increased T cell infiltrate would render PDA sensitive to therapy, we treated two T cell high and two T cell low tumor clones with combination immunotherapy and chemotherapy. Mice bearing T cell high clones responded to therapy (7/7 and 4/7 mice cured) and formed protective memory responses against secondary tumor challenge, while none of the mice bearing T cell low tumors responded to treatment (0/7 and 0/7 mice cured). At baseline, T cell high tumors had similar proportions of functional CD8 T cells as in T cell low tumors (62.9% vs. 50.2% IFN-g+ in T cell high vs low, p=0.06). However, the proportion of activated CD44hiPD-1+ CD8 T cells was significantly increased in T cell high tumors (62.2% vs. 35.1% in T cell low clones, p Citation Format: Katelyn T. Byrne, Jinyang Li, Robert H. Vonderheide, Ben Z. Stanger. Tumor cell intrinsic factors dictate T cell infiltration and therapeutic response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1011.

The heterodimeric cytokine IL-23 plays a non-redundant function in the development of cell-mediated, organ- specific autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). To further characterize the mechanisms of action of IL-23 in autoimmune inflammation, we administered IL-23 systemically at different time points during both relapsing and chronic EAE. Surprisingly, we found suppression of disease in all treatment protocols. We ob- served a reduction in the number of activated macrophages and microglia in the CNS, while T cell infiltration was not significantly affected. Disease suppression correlated with reduced expansion of myelin-reactive T cells, loss of T-bet ex- pression, loss of lymphoid structures, and increased production of IL-6 and IL-4. Here we describe an unexpected func- tion of exogenous IL-23 in limiting the scope and extent of organ-specific autoimmunity.

e24160Background: The degree of tumor infiltration by CD8 T cells is associated with favorable outcomes to immunotherapy. Biopsies to assess T cell infiltration are invasive and may not capture het...

2556Background: Arginine deprivation with pegylated arginine deiminase (ADI-PEG 20) has been shown to upregulate tumor programmed death-ligand 1 (PD-L1) expression and T cell infiltration. Current ...

Meeting abstracts A subset of patients with cancer have evidence of a spontaneous anti-tumor immune response and T cell infiltration into tumor sites. This has prognostic significance and is associated with clinical response to immunotherapeutics in multiple cancers. It has been observed that high

Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5−/− recipients was investigated. Heart grafts from A/J (H-2a) donors were rejected by wild-type C57BL/6 (H-2b) recipients on day 8–10 posttransplant vs day 8–11 by CCR5−/− recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4+ and CD8+ T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-γ-, but not IL-4-producing cells in the CCR5−/− recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4+ than CD8+ T cells in the spleens of allograft wild-type recipients and on CD4+ T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5−/− recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5−/− recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.

The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1−/− donors were tested. Wild-type C57BL/6 (H-2b) heart allografts were rejected by A/J (H-2a) recipients on days 7–9, whereas B6.ICAM-1−/− allografts survived until days 18–23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4+ and CD8+ T cells in the ICAM-1−/− allografts were 20–30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-γ from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1−/− mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1−/− allografts on days 5–6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

// Shenying Jin 1, 2 , Bo Xu 1 , Lixia Yu 1 , Yao Fu 3 , Hongyan Wu 3 , Xiangshan Fan 3 , Jia Wei 1 and Baorui Liu 1 1 The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China 2 The Comprehensive Cancer Center of Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing 210008, China 3 Department of Pathology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China Correspondence to: Baorui Liu, email: baoruiliu@nju.edu.cn Jia Wei, email: weijia01627@hotmail.com Keywords: programmed cell death protein 1, programmed cell death ligand 1, CD3+ T cell, microsatellite instability, gastric signet-ring cell carcinoma Received: January 09, 2017 Accepted: February 24, 2017 Published: March 21, 2017 ABSTRACT Recent data supports a potentially significant role for immune checkpoint inhibitors in the treatment of gastric cancer. However, there are few data on the clinical implications of immunotherapy markers in gastric signet-ring cell carcinoma (SRCC). We evaluated the expression of programmed cell death protein-1 (PD-1), programmed cell death ligand 1(PD-L1), infiltration by CD3+ T cell, microsatellite instability (MSI), and Epstein-Barr Virus (EBV), and the relationship of each factor to survival in 89 advanced SRCC patients. All patients received 5-FU-based first-line chemotherapy. PD-L1 and PD-1 were expressed in 40.4% and 18.0% of the patients, respectively. There was a significant correlation between PD-L1 and PD-1 expression (r=0.363, p

3599 Background: Pre-operative CRT followed by total mesorectal excision (TME) is nowadays the standard of care for patient with LARC (cT3-T4N0 or cTxN+). Currently, pathologic complete response occurs in +/- 15% after CRT. Colorectal cancer T-cell infiltration is a strong prognostic factor for survival after primary tumor resection. Our aim was to determine whether T-cell infiltration in PTB could be predictive of tumor response and relapse after CRT + TME. Methods: Between 1999 and 2012, patients with LARC who underwent CRT + TME and with available clinical follow-up and PTB (with sufficient tumor cells density) were identified at the Cliniques universitaires St-Luc. The density of CD3 (T cells) and CD8 (cytotoxic) was quantified on immunostained PTB slides and analyzed with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Cumulative disease-free survival (DFS) was performed using the Kaplan-Meier estimator and compared by log-rank tests. Cox regression we used for uni- and multi-variate analysis. P value of less than 0.05 was considered statistically significant. Results: 154 patients (sex ratio M/F 1.8; mean age 65 years-old; upper (20%), mid (29%) and low rectum (51%), synchronous metastases (11%)) were analyzed. High CD3 and CD8 PTB densities were significantly associated with a higher pathological response (Dworak 3-4) and lower ypTNM stage after CRT +TME (p < 0,05). Higher CD3 and CD8 PTB densities were associated with higher patient DFS (CD3: HR = 2,30 (CI95%:1,15-4,59) p = 0,02; CD8: HR = 1,95 (CI95%: 1,01-3,75) p = 0,04). These results were confirmed in uni and multivariate analysis. CD3 and CD8 PTB densities added to pathological response (ypTNM/Dworak) but also clinical response (ycTNM) after CRT + TME increases significantly the accuracy prediction of tumor relapse. Conclusions: Pretherapeutic T-cell infiltration of LARC is predictive of tumor response and relapse after CRT +TME. This biomarker could be helpful for patient treatment decision. It must be validated in larger patient cohorts.

3053 Background: Activation of the Wnt-β-catenin signaling pathway is associated with poor T cell infiltration of tumors. We have previously demonstrated that paracrine Wnt5a-β-catenin signaling is a critical trigger of dendritic cell (DC) tolerization and regulatory T cell (Treg) differentiation in the melanoma microenvironment. In a transgenic BRAFV600EPTEN-/- model, the genetic silencing of melanoma-derived Wnt5a potently enhances infiltrating CD8+T cell effector function and promotes responses to anti-PD-1 Ab therapy. Ipafricept (IPA) is a recombinant Wnt decoy receptor and Vantictumab (VAN) is a Fzd receptor monoclonal Ab. Both molecules inhibit Wnt-β-catenin signaling and have been well-tolerated in ongoing phase I/Ib clinical trials. We explored the ability of IPA/VAN to reverse tumor-mediated immune tolerance and enhance the efficacy of anti-PD-1 Ab immunotherapy in a pre-clinical model that closely recapitulates human melanoma. Methods: Both IPA and VAN were utilized to investigate Wnt-β-catenin inhibition as a strategy for suppressing melanoma-induced DC indoleamine 2,3-dioxygenase (IDO) expression and Treg differentiation in vitro. These agents were further tested for their ability to enhance anti-tumor T cell responses and to augment the efficacy of anti-PD-1 Ab therapy in syngeneic and autochthonous models of BRAFV600EPTEN-/- melanoma. Results: IPA and VAN effectively inhibit Wnt5a and melanoma-induced DC IDO expression and Treg differentiation in vitro. Further studies demonstrate that IPA and VAN significantly augment anti-PD-1 Ab-mediated suppression of primary and metastatic tumor progression in both syngeneic and autochthonous BRAFV600EPTEN-/- melanoma models. These anti-tumor effects correlated with suppressed IDO enzymatic activity, enhanced tumor-infiltrating CD8+T cell/Treg ratios, and increased activation of TRP2 antigen-specific effector T cells. Conclusions: The pharmacological inhibition of paracrine Wnt-β-catenin signaling with IPA and VAN augment the anti-tumor efficacy of anti-PD-1 Ab therapy and represent a promising strategy for further phase I testing in melanoma and other solid tumors.