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In this study, we investigated the role of the Wnt/β-catenin signaling pathway in antitumor immune responses. We report that the concentration of secreted Wnt3a was significantly higher in conditioned medium from tumor or nontumor tissues obtained from all hepatocellular carcinoma or colorectal cancer patients tested, than in serum of healthy donors or patients. In addition, both Wnt3a and β-catenin were overexpressed by tumor-infiltrating and nontumor-infiltrating CD4+ or CD8+ T cells. The majority of these T cells expressed a dysfunctional effector memory Eomes+T-bet−phenotype that we defined as partially exhausted, because they performed effector functions (in terms of interferon-γ and tumor necrosis factor-α production, as well as CD107a mobilization) despite their PD-1 expression. Wnt3a/β-catenin signaling in T naïve cells in vitro recapitulated the T-cell setting in vivo. Indeed, the differentiation of cultured T naïve cells was arrested, producing cells that resembled the EomeshighT-betlowβ-cateninhigh T cells with moderate effector functions that infiltrated tumor and nontumor areas. Wnt3a blockade improved the capacity of T naïve cells to differentiate into effector cells in vitro. However, Wnt3a blockade did not affect the function and phenotype of differentiated, partially exhausted, tumor-infiltrating T cells ex vivo. Taken together, our data suggest that Wnt3a blockade halts the capacity of Wnt/β-catenin signaling to inhibit the differentiation of T naïve cells, but it does not restore the dysfunction of differentiated T cells, in the tumor setting. Cancer Immunol Res; 6(8); 941–52. ©2018 AACR.

Table S1. Characteristics of all enrolled patients (HCC=35, CRC=16), not previous treated with chemotherapy Table S2. List of all antibodies for FC analysis Figure S1: CD4 and CD8 T cells expressed high levels of PD1 in TUM district and showed mostly an effector memory phenotype in CRC Figure S2: Distribution of Wnt3a in serum, NTUM- or TUM-derived CM, and expression of Wnt3a/β-catenin axis in NTUM and TUM-infiltrating CD4 and CD8 T cells from CRC patients Figure S3: Analysis of Frizzled receptor 1 expression among CD4 and CD8 T cells in periphery, nontumor and tumor samples of HCC patients Figure S4: β-catenin+ tumor-infiltrating CD4 T cells in presence of anti-Wnt3a do not down-regulated TNF-α prduction

In our study, we investigated the role of CD39 on tumor-infiltrating CD8

In our study, we investigated the role of CD39 on tumor-infiltrating CD8+ T lymphocytes (CD8+ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39+ CD8+ TILs. On the one hand, CD39+ CD8+ TILs (as compared to their CD39- counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39+ CD8+ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8+ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39+ CD8+ T-cell effector function ex vivo, and that CD39+ CD8+ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39+ CD8+ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8+ TILs.

Background Intrapancreatic accessory spleen (IPAS) is an uncommon finding of pancreatic mass. Differential diagnosis with pancreatic tumor, especially with non-functional neuroendocrine tumor (NF-NET), may be very hard and sometimes it entails unnecessary surgery. A combination of CT scan, MRI, and nuclear medicine can confirm the diagnosis of IPAS. 68-Ga-Dotatoc PET/CT is the gold standard in NET diagnosis and it can allow to distinguish between IPAS and NET. Case presentation A 69-year-old man was admitted to our hospital for an incidental nodule in the tail of the pancreas with focal uptake of 68-Ga-dotatate at PET/CT. NET was suspected and open distal splenopancreatectomy was performed. Pathologic examination revealed an IPAS. Conclusion This is the second IPAS case in which a positive 68Ga-Dotatoc uptake led to a false diagnosis of pancreatic NET. Here is a proposal of a literature review.

In this study, we investigated the role of the Wnt/β-catenin signaling pathway in antitumor immune responses. We report that the concentration of secreted Wnt3a was significantly higher in conditioned medium from tumor or nontumor tissues obtained from all hepatocellular carcinoma or colorectal cancer patients tested, than in serum of healthy donors or patients. In addition, both Wnt3a and β-catenin were overexpressed by tumor-infiltrating and nontumor-infiltrating CD4+ or CD8+ T cells. The majority of these T cells expressed a dysfunctional effector memory Eomes+T-bet−phenotype that we defined as partially exhausted, because they performed effector functions (in terms of interferon-γ and tumor necrosis factor-α production, as well as CD107a mobilization) despite their PD-1 expression. Wnt3a/β-catenin signaling in T naïve cells in vitro recapitulated the T-cell setting in vivo. Indeed, the differentiation of cultured T naïve cells was arrested, producing cells that resembled the EomeshighT-betlowβ-cateninhigh T cells with moderate effector functions that infiltrated tumor and nontumor areas. Wnt3a blockade improved the capacity of T naïve cells to differentiate into effector cells in vitro. However, Wnt3a blockade did not affect the function and phenotype of differentiated, partially exhausted, tumor-infiltrating T cells ex vivo. Taken together, our data suggest that Wnt3a blockade halts the capacity of Wnt/β-catenin signaling to inhibit the differentiation of T naïve cells, but it does not restore the dysfunction of differentiated T cells, in the tumor setting. Cancer Immunol Res; 6(8); 941–52. ©2018 AACR.

Tregs can contribute to tumor progression by suppressing antitumor immunity. Exceptionally, in human colorectal cancer (CRC), Tregs are thought to exert beneficial roles in controlling pro-tumor chronic inflammation. The goal of our study was to characterize CRC-infiltrating Tregs at multiple levels, by phenotypical, molecular and functional evaluation of Tregs from the tumor site, compared to non-tumoral mucosa and peripheral blood of CRC patients. The frequency of Tregs was higher in mucosa than in blood, and further significantly increased in tumor. Ex vivo, those Tregs suppressed the proliferation of tumor-infiltrating CD8(+) and CD4(+) T cells. A differential compartmentalization was detected between Helios(high) and Helios(low) Treg subsets (thymus-derived versus peripherally induced): while Helios(low) Tregs were enriched in both sites, only Helios(high) Tregs accumulated significantly and specifically in tumors, displayed a highly demethylated TSDR region and contained high proportions of cells expressing CD39 and OX40, markers of activation and suppression. Besides the suppression of T cells, Tregs may contribute to CRC progression also through releasing IL-17, or differentiating into Tfr cells that potentially antagonize a protective Tfh response, events that were both detected in tumor-associated Tregs. Overall, our data indicate that Treg accumulation may contribute through multiple mechanisms to CRC establishment and progression.

Pancreatic cancer is one of the most common causes of cancer-related death in Western countries. In particular, pancreatic ductal adenocarcinoma represents more than 80% of all pancreatic neoplasms diagnosed.1 At present, surgical resection represents the best curative treatment for pancreatic cancer, with approximately 15% of patients considered suitable.2 The outcome of pancreatic tumor resection has been associated with several histological factors,3–7 including tumor size, histopathological grading,8 and cleared margin of resection.9 In the last few years, a great debate arose on the best surgical procedure for tumor eradication to obtain long-term results with significant survival benefit.10 In particular, the duodenopancreatectomy (DP) associated with extended lymphadenectomy and mesopancreas en bloc resection is deemed necessary for an adequate retropancreatic clearance of the tumor.11 The mesopancreas consists in a perineural lymphatic layer located dorsally to the pancreas, extending from the posterior surface of the pancreatic head to behind the mesenteric vessels (superior mesenteric vein and superior mesenteric artery [SMA]).12 Other authors have defined the mesopancreas as the soft tissue between the pancreatic parenchyma and the SMA that contains vascular, lymphatic, and nervous structures.11 Overall, the retropancreatic area can be considered as not a single entity with well-defined boundaries but an anatomical site of embryological fusion of peritoneal layers that are continuous and connected through the contained vessels, LNs, and nerves to the para-aortic area.13 The lack of anatomical boundaries of the mesopancreas renders difficult to obtain a microscopically negative retropancreatic resection margin and to control the neoplastic spread along the lymphovascular and neural structures; thus, it is highly recommended to perform an extended dissection of the retropancreatic and para-aortic areas to achieve an almost complete posterior clearance.11,13 This total mesopancreas excision should decrease the recidive rate (R1) and improve survival.12,14–17 Duodenopancreatectomy associated with extended lymphadenectomy and mesopancreas exeresis, by removing a sizeable portion of lymphatic tissue, most probably affects immune system status significantly. The role that the immune system exerts in recognizing and eliminating continually arising transformed cells is well established. This host protection process, named immunosurveillance, is aimed at maintaining physiological tissue homeostasis and controlling the initiation, growth, and progression of tumor.18 Both innate and adaptive effector populations such as natural killer (NK) cells, dendritic cells, and CD4+ and CD8+ T-cells contribute to immunosurveillance; in particular, NK cells, phenotypically defined by the absence of CD3 and the expression of CD56 surface antigen (CD3-CD56+), are a heterogeneous lymphocyte population composed of CD3-CD56dim and CD3-CD56bright subsets, which participates to antitumor responses by exhibiting cytotoxic function (CD3-CD56dim NK cells) and secreting a number of cytokines (CD3-CD56bright NK cells). Natural killer cells are resident in the secondary lymphoid tissue, such as lymph nodes and tonsils, where they are almost totally CD3-CD56bright, which circulate in the peripheral blood (PB) and rapidly accumulate in the parenchyma of several organs during inflammation, tumor growth, and invasion. Circulating NK cells consist for about 90% of CD3-CD56dim subset, whose “natural” cytotoxicity is regulated by a balance of signals deriving from inhibitory and activating receptors that recognize ligands either normally expressed or stress-induced on target cells. Malignant transformation often induces alteration in the expression pattern of the ligands of NK receptors, which in turn results in NK cell loss of inhibition and induction of activation, being both events essential to achieve NK cell-mediated cytotoxicity.19 Natural killer cell subset distribution, trafficking, and functions, including proliferation, differentiation, cytotoxicity, and cytokine production, are also controlled by a great variety of cytokines, such as IL-2, IL-15, IL-18, IL-21, and chemokines, whose specific receptors are differentially expressed on CD3-CD56dim and CD3-CD56bright subsets.20 It is becoming increasingly clear that the establishment of an immunosuppressive local and systemic environment represents an important mechanism for tumor capability to circumvent both innate and adaptive immune defenses.21 Indeed, several immunosuppressive cell populations (such as regulatory T-cells and myeloid-derived suppressor cells) and cytokines (such as IL-10, TGF-β, and the tumor-associated IL-18 that exerts an indirect negative regulation on NK and effector T-cell cytotoxic activity) have been found in tumor lesions and in the PB of pancreatic cancer patients.22–26 In this complex context, the study of the impact of surgical tumor mass eradication on patient immune status is gaining particular importance. Surgery can induce a rapid and transient inflammatory status and also modify, both locally and systemically, immune cell distribution and/or activation and release of soluble factors27; this results in a long-lasting alteration of patient’s immune system and, ultimately, of immunosurveillance.28,29 In this study, we analyzed the dynamics of the main PB lymphocyte subsets (CD4+ and CD8+ T-cells, B-cells, and CD56dim and CD56bright NK cell subsets) of the NK cell cytotoxic activity and of neutrophil count as an important systemic inflammation marker in a cohort of pancreatic cancer patients before and at different time points after DP with extended lymphadenectomy and mesopancreas resection. The effect of tumor stage on presurgery immune status and on postsurgery phenotypic and functional modifications was also evaluated.