Pro-tumor Tfh2 cells induce detrimental IgG4 production and PGE2-dependent IgE inhibition in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and it is characterized by predominant pro-tumor Th2-type inflammation. T follicular helper (Tfh) cells are relevant immunoregulators in cancer, and often correlate with better survival. How the Th2-skewed microenvironment in PDAC modulates the differentiation of Tfh cells and their immunoregulatory function is unknown. METHODS: We carried out high-dimensional flow cytometry and T-cell receptor- and RNA-sequencing, as well as bioinformatics, immunohistochemistry and in vitro mechanistic studies. FINDINGS: We identified Tfh1-, Tfh2-, and Tfh17-like cell clusters in the blood, tumors and tumor-draining lymph-nodes (TDLNs) of chemo-naïve PDAC patients and showed that high percentages of Tfh2 cells within the tumor tissue and TDLNs correlated with reduced patient survival. Moreover, only Tfh2 cells were highly activated and were reduced in frequency in patients who responded to neoadjuvant chemotherapy. RNA-sequencing analysis of immunoglobulin expression showed that tumor and TDLN samples expressed all immunoglobulin (IGH) isotypes apart from IGHE. Consistent with these findings, Tfh2 cells differentiated in vitro by tumor microenvironment-conditioned dendritic cells promoted the production of anti-inflammatory IgG4 antibodies by co-cultured B cells, dependent on IL-13. Moreover, unexpectedly, Tfh2 cells inhibited the secretion of pro-inflammatory IgE, dependent on prostaglandin E2. INTERPRETATION: Our results indicate that in PDAC, highly activated pro-tumor Tfh2 favor anti-inflammatory IgG4 production, while inhibit pro-inflammatory IgE. Thus, targeting the circuits that drive Tfh2 cells, in combination with chemotherapy, may re-establish beneficial anti-tumor Tfh-B cell interactions and facilitate more effective treatment. FUNDING: Research grants from the Italian Association for Cancer Research (AIRC) IG-19119 to MPP and the AIRC Special Program in Metastatic disease: the key unmet need in oncology, 5 per Mille no. 22737 to CB, MF, CD, MR and MPP; the ERA-NET EuroNanoMed III (a collaborative european grant financed by the Italian Ministry of Health, Italy) project PANIPAC (JTC2018/041) to MPP; the Fondazione Valsecchi to SC.

Immunomodulatory Effects of Endoscopic Ultrasound-Guided Thermal Ablation in Patients with Pancreatic Ductal Adenocarcinoma.

Immunological consequences of endoscopic ultrasound (EUS)-local thermal ablation (LTA) for pancreatic ductal adenocarcinoma (PDAC) have not been extensively assessed. We aimed to explore EUS-LTA effects on the systemic immune response in PDAC. Peripheral blood was collected from 10 treatment-naïve patients with borderline resectable and locally advanced PDAC, randomly allocated to Nab-paclitaxel plus Gemcitabine chemotherapy (CT-arm, n = 5) or EUS-LTA with HybridTherm Probe plus CT (HTP + CT-arm, n = 5). Twenty healthy donors were included as controls. Flow-cytometry and multiplex assays were used to profile immune cell subsets and measure serum cytokines/chemokines, respectively. At baseline, PDAC patients showed increased circulating monocytes and lower circulating lymphocytes and CD19+ B cells counts compared to healthy controls. After 4 months, CT induced decrease of B regulatory cells, CD4+ cytotoxic T cells and IL-1ß. The addition of EUS-HTP to CT selectively decreased the serum levels of APRIL/TNFSF13 as well as T regulatory cells, total, classic and inflammatory monocytes. Serum levels of APRIL/TNFSF13 and total, classic and inflammatory monocytes counts at baseline were associated with worse overall survival. EUS-HTP has the potential to selectively impact on immune cells and cytokines associated with poor outcomes in PDAC.

Thymic Stromal Lymphopoietin and Cancer: Th2-Dependent and -Independent Mechanisms.

The thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine originally cloned from a murine thymic stromal cell line, and subsequently a human homolog was identified using database search methods. Human TSLP is mostly expressed in epithelial cells, among which are keratinocytes as well as stromal cells such as fibroblasts and immune cells. Human TSLP was first described to activate myeloid dendritic cells, which prime naïve T helper cells to produce high concentrations of Th2 cytokines, thus representing a key cytokine in triggering dendritic cells-mediated allergic Th2 inflammation. TSLP and/or its receptor has been shown to be expressed in several tumor types, where TSLP expression is associated with functional activities that can be associated or not with the induction of a Th2-prone tumor microenvironment, i.e., Th2-dependent and Th2-independent mechanisms. These mechanisms involve tissue- and immune cell target-dependent tumor-promoting or tumor-suppressive functions in different or even the same tumor type. Here we report and discuss the Th2-dependent and Th2-independent roles of TSLP in cancer and possible therapeutic targeting.

B lymphocytes contribute to stromal reaction in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent stromal reaction that has been variably implicated in both tumor growth and tumor suppression. B-lymphocytes have been recently implicated in PDAC progression but their contribution to the characteristic stromal desmoplasia has never been assessed before. In the present work, we aimed to verify whether B-lymphocytes contribute to stromal cell activation in PDAC. CD19+ B-lymphocytes purified from peripheral blood of patients with PDAC were cultivated in the presence of human pancreatic fibroblasts and cancer-associated fibroblasts. Released pro-fibrotic soluble factors and collagen production were assessed by ELISA and Luminex assays. Quantitative RT-PCR was used to assess fibroblast activation in the presence of B cells. The expression of selected pro-fibrotic and inflammatory molecules was confirmed on PDAC tissue sections by multi-color immunofluorescence studies. We herein demonstrate that B-cells from PDAC patients (i) produce the pro-fibrotic molecule PDGF-B and stimulate collagen production by fibroblasts; (ii) express enzymes implicated in extracellular matrix remodeling including LOXL2; and (iii) produce the chemotactic factors CCL-4, CCL-5, and CCL-11. In addition we demonstrate that circulating plasmablasts are expanded in the peripheral blood of patients with PDAC, stimulate collagen production by fibroblasts, and infiltrate pancreatic lesions. Our results indicate that PDAC is characterized by perturbations of the B-cell compartment with expansion of B-lymphocyte subsets that directly contribute to the stromal reaction observed at disease site. These findings provide an additional rationale for modulating B-cell activity in patients with pancreatic cancer.

Dual Role of Inflammasome Adaptor ASC in Cancer.

Apoptosis-associated Speck-like protein containing a CARD (caspase activation and recruitment domain) (ASC), also called PYCARD/Target of Methylation-induced Silencing-1 (TMS1), was originally discovered as a protein that forms aggregates ("specks") in human leukemia cells treated with chemotherapeutic agents. Its expression was found to be silenced by methylation in many human tumors, preventing tumor cells from undergoing apoptosis and supporting its role as a tumor suppressor. Subsequently, ASC was also identified as a central adaptor molecule of the inflammasome complex, which mediates the secretion of inflammatory cytokines (i.e., IL-1ß and IL-18). Inflammatory cytokines have been shown to mediate tumor-promoting functions. Thus, in the context of cancer development and progression, ASC may exert opposing functions, i.e., be either tumor-suppressing by inducing tumor cell apoptosis, or tumor-promoting by favoring secretion of inflammatory cytokines (by tumor cells and/or tumor infiltrating myeloid cells) within the tumor microenvironment. Here, we report and discuss this dual role of ASC by also considering the final contribution of each of its two main functions in several cancer types, taking into consideration the correlation between ASC expression, clinical correlates, and patients' survival. ASC and inflammasome targeting strategies are being developed. However, before the use of such treatments in clinical practice, it is fundamental to better dissect the role of ASC in different tumors, in order to privilege or avoid their use in those tumors in which ASC exerts an anti-tumor or pro-tumor function, respectively.

The IL-1/IL-1 receptor axis and tumor cell released inflammasome adaptor ASC are key regulators of TSLP secretion by cancer associated fibroblasts in pancreatic cancer.

BACKGROUND: The thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients' survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined. METHODS: We performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets. RESULTS: We show in vitro that IL-1α and IL-1ß released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1ß secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients. CONCLUSIONS: Our findings indicate that tumor released IL-1α and IL-1ß and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer.

Natural Antibodies to Tumor-Associated Antigens.

Natural autoantibodies raised by humoral immune response to cancer can be exploited to identify potential tumor-associated antigens (TAAs), and might constitute new putative prognostic and/or diagnostic biomarkers. Here we describe how sera from tumor patients can be used to identify TAAs by screening antibody immunoreactivity against the cancer proteome resolved by two-dimensional gel electrophoresis.

Quantitative and Qualitative Analysis of Tumor-Associated CD4⁺ T Cells.

CD4(+) T cells comprise a significant portion of tumor-infiltrating lymphocytes. Different subsets of CD4(+) T cells exist and they exert different effector functions in tumor immunity depending on the cytokines produced going from antitumor to pro-tumor. Methods that use small aliquots of cells to identify ex vivo the frequency and functional orientation of tumor-specific CD4(+) T cells in the blood and visualization of the presence of different CD4(+) T cell subsets and their localization at the tumor site are valuable tools to determine their clinical impact in neoplastic diseases.

Basophil Recruitment into Tumor-Draining Lymph Nodes Correlates with Th2 Inflammation and Reduced Survival in Pancreatic Cancer Patients.

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell-derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792-803. ©2016 AACR.

Protein disulfide isomerase A3-specific Th1 effector cells infiltrate colon cancer tissue of patients with circulating anti-protein disulfide isomerase A3 autoantibodies.

To investigate novel colorectal cancer (CRC)-associated antigens that could be targets of humoral or cellular responses, we analyzed the reactivity of serum from a long-surviving CRC patient (for more than 100 months of follow-up) in clinical remission, by serologic proteome analysis. Two-dimensional Western blotting (2D-WB) and mass spectrometry analysis revealed a strong reactivity of this serum against protein disulfide isomerase A3 (PDIA3). Anti-PDIA3 antibodies are not a diagnostic marker of CRC, 2D-WB and Luminex analysis revealed that they were equally present in about 10% of sera from healthy subjects and CRC patients. Kaplan-Meier analysis of survival in CRC patient cohort, after 48 months of follow-up, showed a trend of higher survival in patients with increased levels of autoantibodies to PDIA3. Therefore, the interplay between the presence of these antibodies and T-cell response was investigated. Peripheral blood T cells from CRC patients with high immunoglobulin G (IgG) reactivity to PDIA3 also secreted interferon gamma (IFN-γ) when stimulated in vitro with recombinant PDIA3, whereas those from CRC with low IgG reactivity to PDIA3 did not. PDIA3-pulsed dendritic cells efficiently induced proliferation and IFN-γ production of autologous CD4(+) and CD8(+) T cells. Finally, ex vivo analysis of tumor-infiltrating T lymphocytes from CRC patients with autoantibodies to PDIA3 revealed that PDIA3-specific Th1 effector cells accumulated in tumor tissue. These data indicate that the presence of autoantibodies to PDIA3 favors the development of an efficient and specific T-cell response against PDIA3 in CRC patients. These results may be relevant for the design of novel immunotherapeutic strategies in CRC patients.

Immune infiltrates as predictive markers of survival in pancreatic cancer patients.

Pancreatic cancer is a devastating disease with dismal prognosis. The tumor microenvironment is composed by multiple cell types, molecular factors, and extracellular matrix forming a strong desmoplastic reaction, which is a hallmark of the disease. A complex cross-talk between tumor cells and the stroma exists with reciprocal influence that dictates tumor progression and ultimately the clinical outcome. In this context, tumor infiltrating immune cells through secretion of chemokine and cytokines exert an important regulatory role. Here we review the correlation between the immune infiltrates, evaluated on tumor samples of pancreatic cancer patients underwent surgical resection, and disease free and/or overall survival after surgery. Specifically, we focus on tumor infiltrating lymphocytes (TILs), mast cells (MCs) and macrophages that all contribute to a Th2-type inflammatory and immunosuppressive microenvironment. In these patients tumor immune infiltrates not only do not contribute to disease eradication but rather the features of Th2-type inflammation and immunosuppression is significantly associated with more rapid disease progression and reduced survival.

Cross-talk within the tumor microenvironment mediates Th2-type inflammation in pancreatic cancer.

Th2-type inflammation has been proposed to facilitate tumor growth. In De Monte et al. (J Exp Med 208:469-478, 2011) we identify in pancreatic cancer a complex cytokine/chemokine cross-talk within the tumor microenvironment mediating Th2 immune-deviation and show that the ratio of Th2/Th1 tumor infiltrating lymphocytes is an independent predictive marker of patients survival.

Intratumor T helper type 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and reduced survival in pancreatic cancer.

Pancreatic cancer is a very aggressive disease characterized by a marked desmoplasia with a predominant Th2 (GATA-3+) over Th1 (T-bet+) lymphoid infiltrate. We found that the ratio of GATA-3+/T-bet+ tumor-infiltrating lymphoid cells is an independent predictive marker of patient survival. Patients surgically treated for stage IB/III disease with a ratio inferior to the median value had a statistically significant prolonged overall survival, implying an active role for Th2 responses in disease progression. Thymic stromal lymphopoietin (TSLP), which favors Th2 cell polarization through myeloid dendritic cell (DC) conditioning, was secreted by cancer-associated fibroblasts (CAFs) after activation with tumor-derived tumor necrosis factor α and interleukin 1ß. TSLP-containing supernatants from activated CAFs induced in vitro myeloid DCs to up-regulate the TSLP receptor (TSLPR), secrete Th2-attracting chemokines, and acquire TSLP-dependent Th2-polarizing capability in vitro. In vivo, Th2 chemoattractants were expressed in the tumor and in the stroma, and TSLPR-expressing DCs were present in the tumor stroma and in tumor-draining but not in nondraining lymph nodes. Collectively, this study identifies in pancreatic cancer a cross talk between tumor cells and CAFs, resulting in a TSLP-dependent induction of Th2-type inflammation which associates with reduced patient survival. Thus, blocking TSLP production by CAFs might help to improve prognosis in pancreatic cancer.

Serological immunoreactivity against colon cancer proteome varies upon disease progression.

Sera from colon carcinoma patients were used to identify tumor-associated antigens (TAAs) by screening tumor proteome resolved by 2D electrophoresis. A panel of six TAAs eliciting a serological immune response in colorectal cancer was identified, showing a modification in antigen recognition by B cells in patients as a function of colon cancer progression. The expression of these proteins was either confined or increased in tumor as compared to normal mucosa.

Differential expression of ceruloplasmin isoforms in the cerebrospinal fluid of amyotrophic lateral sclerosis patients.

Amyotrophic lateral sclerosis (ALS) a fatal degenerative disease that selectively affects motor neurons, likely results from a complex interplay among oxidative injury, excitotoxic stimulation, protein aggregation and genetic factors. Ceruloplasmin (Cp) protein is a ferroxidase that oxidizes toxic ferrous iron to its nontoxic ferric form, protecting the central nervous system (CNS) from iron deposition. Cp is thus considered as one of the main systems dedicated to the protection of the CNS from oxidative stress damage. We investigated Cp protein behaviour in the cerebrospinal fluid (CSF) of ALS patients of recent onset. An increased expression of Cp was observed in ALS (n = 16) compared to two control groups (healthy subjects, n = 11 and peripheral neuropathy patients, n = 10). 2-DE analysis revealed a differential expression of Cp isoforms in ALS patients compared to controls. ALS samples showed an increase in the relative abundance of more basic Cp forms, corresponding to the nonsialylated proteins. Despite the increase in protein expression, ferroxidase activity evaluated in the CSF of ALS patients was comparable to that of the controls, indicating a Cp functional impairment. Ceruloplasmin isoforms profile may be proposed as disease feature that could provide insight into the molecular mechanisms of ALS pathogenesis.

Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions.