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Fully functional, tumor-specific, SLAMF7-expressing cytotoxic CD4 T cells directly mediate immunity against human cancer., CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.

Background CD4 T cells have been implicated in cancer immunity for their helper functions. However, their direct cytotoxic potential remains elusive in cancer patients. Here, we aimed at assessing the presence, rate and cytotoxic function of tumor-specific Th-CTX directly in cancer patients. Methods We capitalized on published single cell transcriptomic analyses of patient samples, integrated with the direct phenotypic and functional characterization of clonal, tumor-specific CD4 T cell populations, using peptide-MHC class II multimers and a novel high-throughput single-cell cytotoxicity assay in picowell arrays. The direct tumor cell killing by cytolytic tumor-specific CD4 T cells in the arrays was monitored in a high-throughput manner by combining multi-channel time-lapse microscopy with deep neural networks. Results By mining single-cell RNA-seq datasets of tumor infiltrating lymphocytes, we identified CD4 T cells displaying cytotoxic phenotypes in different human tumors. The cytolytic CD4 T cells formed a distinct cluster and expressed genes related to classical cytotoxic functions, largely resembling CD8 T cell gene profiles. Using the peptide MHC class II multimer technology, we confirmed directly ex vivo the presence of cytolytic tumor antigen-specific CD4 T cells, both in the circulation and in the tumors of patients. We performed an integrated phenotypic and functional characterization of cytolytic tumor-specific CD4 T cells, down to the single cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells with sub-nanoliter volumes and machine learning. We demonstrated a direct, contact-dependent, granzyme-dependent cytotoxic activity against tumor cells, with delayed kinetics compared to classical cytotoxic lymphocytes. Lastly, we discovered that this cytotoxic activity was at least in part dependent on the expression of SLAMF7, a homophylic receptor known to regulate NK cell activity. Conclusions Our work provides a deep characterization of human Th-CTX in cancer and supports their role in tumor immunity. Moreover, our results showing that agonistic engagement of SLAMF7 enhances the cytolytic capacity of tumor-specific CD4 T cells, suggests that targeting these cells might prove synergistic with the use of other immunotherapies in cancer patients.

Chromogranin A (CgA), a secretory protein released in the blood by the neuroendocrine system, consists of a mixture of full-length molecules and fragments endowed of vasoregulatory activity. The extent and the role of CgA fragmentation were investigated in patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC, n=172). Multivariate analysis showed that full-length CgA was associated with better progression free and overall survival, whereas CgA C-terminal fragmentation was associated with worse prognosis. In vitro studies showed that PDAC cells can promote the cleavage of CgA C-terminal region by activating plasminogen to plasmin. Limited digestion of full-length CgA with plasmin abolished its anti-angiogenic activity and generated pro-angiogenic molecules. The fragmentation of CgA C-terminal region was increased also in murine models of PDAC. In these models, the inhibition of CgA fragmentation with aprotinin, an inhibitor of plasmin and other serine proteases, or the blockade of pro-angiogenic fragments with specific antibodies inhibited the growth of PDAC implanted subcutaneously in mice. Finally, administration of full-length CgA to mice bearing orthotopic PDAC reduced tumor perfusion, as measured by contrast-enhanced ultrasound. These findings suggest that PDAC can promote the cleavage of circulating CgA C-terminal region to generate fragments that regulate the tumor vascular biology and that may represent new potential therapeutic targets.

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.

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-1β 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.

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.

Donor-derived allogeneic T cells evoke potent graft versus tumor (GVT) effects likely due to the simultaneous recognition of tumor-specific and host-restricted minor histocompatibility (H) antigens. Here we investigated whether such effects could be reproduced in autologous settings by TCR gene–engineered lymphocytes. We report that T cells redirected either to a broadly expressed Y-encoded minor H antigen or to a tumor-associated antigen, although poorly effective if individually transferred, when simultaneously administered enabled acute autochthonous tumor debulking and resulted in durable clinical remission. Y-redirected T cells proved hyporesponsive in peripheral lymphoid organs, whereas they retained effector function at the tumor site, where in synergy with tumor-redirected lymphocytes, they instructed TNFα expression, endothelial cell activation, and intratumoral T-cell infiltration. While neutralizing TNFα hindered GVT effects by the combined T-cell infusion, a single injection of picogram amounts of NGR-TNF, a tumor vessel–targeted TNFα derivative currently in phase III clinical trials, substituted for Y-redirected cells and enabled tumor debulking by tumor-redirected lymphocytes. Together, our results provide new mechanistic insights into allogeneic GVT, validate the importance of targeting the tumor and its associated stroma, and prove the potency of a novel combined approach suitable for immediate clinical implementation. Cancer Res; 77(3); 658–71. ©2016 AACR.

Immunomodulatory drugs (IMiDs) are effective therapeutics for multiple myeloma (MM), where in different clinical settings they exert their function both directly on MM cells and indirectly by modulating immune cell subsets, although with not completely defined mechanisms. Here we studied the role of IMiDs in the context of autologous hematopoietic stem cell transplantation on the T cell subset distribution in the bone marrow of newly diagnosed MM patients. We found that after transplantation pro-tumor Th17-Th1 and Th22 cells and their related cytokines were lower in patients treated with IMiDs during induction chemotherapy compared to untreated patients. Of note, lower levels of IL-17, IL-22, and related IL-6, TNF-α, IL-1β, and IL-23 in the bone marrow sera correlated with treatment with IMiDs and favorable clinical outcome. Collectively, our results suggest a novel anti-inflammatory role for IMiDs in MM.

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-1β 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-1β 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-1β 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. Electronic supplementary material The online version of this article (10.1186/s40425-019-0521-4) contains supplementary material, which is available to authorized users.

Purpose: Characterization of tumor antigen–specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an in vitro amplified T-cell library screening procedure. Patients and Methods: A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones. Results: In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents (Tetanus toxoid). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells. Conclusions: This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell–based therapies.

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.

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.

There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17-IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10- and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17-IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.

The carcinoembryonic Ag (CEA) is an attractive target for immunotherapy because of its expression profile and role in tumor progression. To verify the existence of spontaneous anti-CEA CD4+ T cells in lung cancer patients, we first identified CEA sequences forming naturally processed epitopes, and then used the identified epitopes to test their recognition by CD4+ T cells from the patients. We had previously identified CEA177–189/355–367 as an immunodominant epitope recognized by CD4+ T cells in association with several HLA-DR alleles. In this study, we identified four additional subdominant CEA sequences (CEA99–111, CEA425–437, CEA568–582, and CEA666–678), recognized in association with one or more HLA-DR alleles. Peptide-specific CD4+ T cells produced proinflammatory cytokines when challenged with the native protein and CEA-expressing tumor cells, thus demonstrating that the identified CEA sequences contain naturally processed epitopes. However, CEA is expressed in the thymus and belongs to the CD66 family that comprises highly homologous molecules expressed on hemopoietic cells, raising concerns about tolerance interfering with the in vivo development of anti-CEA immunity. We thus tested the spontaneous reactivity to the identified epitopes of peripheral blood CD4+ T lymphocytes from eight early-stage lung cancer patients bearing CEA-positive tumors. We found GM-CSF- and IFN-γ- producing CD4+ T cells in two patients. Our data indicate that CD4+ immune responses against CEA develop in neoplastic patients, suggesting that tolerance toward CEA or cross-reactive CD66 homologous molecules might be either not absolute or be overcome in the neoplastic disease.

Fas and Fas ligand (FasL) have been found both in lymphoid and in non-lymphoid malignancies, and are thought to play a role in the interplay between tumors and the immune system. Here we investigated Fas/FasL expression, function and intracellular signalling pathways in human melanomas. Of 5 melanoma cell lines, 3 expressed Fas at their surface, and all of them expressed FasL. FasL was functional, since it triggered Fas-induced apoptosis of human T lymphocytes clones. Conversely, cross-linking of Fas molecule with a specific monoclonal antibody failed to induce apoptosis in any of the melanomas tested, or ceramide intracellular accumulation or caspase-3 activation, pointing to an early alteration in the Fas-triggered signaling cascade. All melanomas retained the ability to undergo apoptosis induced by cytotoxic lymphocytes, which was mediated by the granule exocytosis mechanism. This suggests that melanoma cells evade immune-mediated Fas-triggered apoptosis via a selective blockade of the Fas apoptotic pathway. Cytotoxic lymphocytes, however, may circumvent tumor resistance to Fas-induced death via granzyme-mediated apoptosis, further supporting the development of immunotherapeutic strategies in the treatment of cancer.

BACKGROUND & AIMS: One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice. METHODS: Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors resulting from expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed by immunohistochemistry, in situ hybridization, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunoblot assays; fluorescence-activated cell sorting was performed to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin 6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a Janus kinase 2 (JAK2) inhibitor; tumor growth was monitored by 3-dimensional ultrasound. RESULTS: STAT3 was phosphorylated constitutively in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2 compared with tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation. CONCLUSIONS: In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3 signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice. Transcript profiling: ArrayExpress accession number: E-MTAB-3278. ZB 0 Z8 0 ZR 0 ZS 0

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.

Pancreatic Ductal AdenoCarcinoma (PDAC) is a very aggressive disease, characterized by desmoplasia and Th2-inflammation. Th2-inflammation correlates with poor prognosis and it is driven by the release of the Thymic Stromal LymphoPoietin (TSLP) from Cancer Associated Fibroblasts (CAFs) activated by tumor-derived pro-inflammatory cytokines. The aims of our study are: i) to verify which tumor-derived cytokines are responsible for CAFs activation, ii) to understand the stimuli/mechanisms of tumor-derived cytokine production in pancreatic cancer cells. Analyzing a series of 9 PDAC cell lines by both Real-time PCR and immunofluorescence, we found heterogeneous expression of IL-1α, IL-1β and TNF-α. Furthermore, ELISA tests revealed that some PDAC cell lines could either actively secrete or release during necrosis these pro-inflammatory cytokines. Next, we treated CAFs obtained from surgical patient samples with rhIL-1α, rhIL-1β, rhTNF-α and PDAC cell supernatants. Interestingly, we observed an induction in TSLP secretion that was significantly reduced by anti-IL1α and Anakinra (the recombinant form of IL-1Ra), suggesting a predominant role of the IL-1/IL-1R axis. It is known that IL-1α and TLRs ligands can activate NF-kB that in turn induces the synthesis of proIL-1β that is cleaved to matureIL-1β by caspase-1, upon inflammasome activation. In line with this, we performed Western Blot analysis and we observed constitutive activation of both NF-kB and inflammasome. We also demonstrated that IL-1α is able to increase IL-1β production in PDAC cells, with an autocrine loop. Furthermore, treating THP1 cells (a monocytic cell line that secrete IL-1β only upon stimulation) with PDAC cell supernatants we found that both IL-1-expressing and non-IL-1-expressing cells can release inflammasome-activating factors. Since pancreatic cancer is highly hypoxic, we cultured PDAC cell lines in hypoxic conditions and we demonstrated that prolonged severe hypoxia, in combination with starvation, induces an increase in both IL-1α and IL-1β secretion. In conclusion, our study demonstrates that PDAC cells can constitutively express IL-1α, IL-1β and TNF-α, thus inducing TSLP secretion from CAFs and eventually Th2-inflammation. PDAC cells secrete inflammasome-activating factors that sustain cytokine production with an autocrine loop, and that could induce cytokine release from infiltrating macrophages. Finally, the hypoxic environment of pancreatic cancer can further increase IL-1α and IL-1β secretion. Our findings support the development of clinical studies combining the use of Anakinra with conventional chemotherapy, in order to interfere with the IL-1/IL-1R axis and therefore Th2-inflammation, thus improving the prognosis of pancreatic cancer patients. Citation Format: Emanuela Brunetto, Lucia De Monte, Silvia Heltai, Maria Pia Protti. Mechanisms of IL-1 production and release in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 443. doi:10.1158/1538-7445.AM2015-443

Purpose: Renal cell carcinoma (RCC) is considered immunogenic; nonetheless, rare tumor-associated antigens have been identified or are expressed in RCC. Peptidome (i.e., the total content of natural peptides of whole cells) from other tumors, such as melanoma, has proved to be immunogenic. The aims of this study were to determine whether peptidome from RCC is immunogenic and whether it contains tumor peptides shared among allogenic RCCs.Experimental Design: Autologous dendritic cells pulsed with RCC peptidome were used to activate in vitro CD4+ T cells from healthy donors and a metastatic RCC patient. CD4+ T-cell polyclonal lines and clones were characterized for tumor cell recognition by proliferation assay, killing activity, and cytokine secretion.Results: CD4+ T-cell lines and clones recognized HLA-DR-matched allogenic RCC and, for the patient, the autologous tumor. RCC-reactive CD4+ T cells showed a heterogeneous Th1 or Th0/Th2 pattern of cytokine secretion. Moreover, RCC-reactive CD4+ T cells recognized also melanoma, colon carcinoma, cervical carcinoma, pancreas carcinoma, lung carcinoma, gastric carcinoma, and lymphoma cells but not autologous T-cell blasts.Conclusions: Our results show that (a) the RCC peptidome contain antigens recognized by CD4+ T cells and (b) shared among tumors of different histology and (c) it induces both Th1-type and Th2/Th0-type immune responses. These data support the use of the peptidome from allogenic RCC for specific immunotherapy in RCC and possibly in other neoplastic diseases. Moreover, the CD4+ T-cell clones generated here are useful tools for tumor antigen identification.

Heat shock proteins (hsp) are a family of proteins characteristically produced under stress conditions in normal cells. Overexpression of hsp 70 kDa protects tumoral cells from tumor necrosis factor cytotoxicity and is related to drug resistance. In this study we investigated whether hsp are abnormally expressed in melanoma cells in vivo. Antibodies directed against hsp 72 and hsp 90 were used to identify hsp on non-stressed neoplastic cells derived from surgical specimens of two primary and four metastatic melanomas. Hsp 72 and hsp 90 were always present at high level in the cytoplasm of melanoma cells. It is possible that the activation of hsp genes during neoplastic transformation confers a selective advantage to tumoral cells in vivo, allowing them to escape the immunological surveillance.