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Assessing Disease State in ACT treated TRAMP mice.

Expression of CCL24/eotaxin-2 in CAF supernatant-conditioned monocytes and in TDLNs.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Legends for Supplementary Table S1 and Supplementary Figures S1-S8.

Supplementary Figure 2

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.

Multivariate Cox regression analysis of the influence of the percentage of basophils in TDLNs on the survival of 35 stage IB-III pancreatic cancer patients after surgery.

LIMMA modeling of expression data.

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.

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

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.

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.

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.

CD4(+) T cells comprise a large fraction of tumor infiltrating lymphocytes and it is now established that they may exert an important role in tumor immune-surveillance. Several CD4(+) T cell subsets [i.e. T helper (Th)1, Th2, T regulatory (Treg), Th17, Th22 and follicular T helper (Tfh)] have been described and differentiation of each subset depends on both the antigen presenting cells responsible for its activation and the cytokine environment present at the site of priming. Tumor antigen-specific CD4(+) T cells with different functional activity have been found in the blood of cancer patients and different CD4(+) T cell subsets have been identified at the tumor site by the expression of specific transcription factors and the profile of secreted cytokines. Importantly, depending on the subset, CD4(+) T cells may exert antitumor versus pro-tumor functions. Here we review the studies that first identified the presence of tumor-specific CD4(+) T cells in cancer patients, the techniques used to identify the tumor antigens recognized, the role of the different CD4(+) T cell subsets in tumor immunity and in cancer prognosis and the development of therapeutic strategies aimed at activating efficient antitumor CD4(+) T cell effectors.

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.

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.

Infection by 'high-risk' human papillomaviruses (HPV) is associated with the development of neoplastic lesions. HPV-18 is responsible for a very aggressive form of cancer and poor survival. As for other HPV types, immune surveillance has probably a role in the control of the infection. However, very little is known on HPV-18 immunogenicity. CD4(+) T cells from 16 healthy donors were tested ex vivo for reactivity to synthetic peptides corresponding to 3 sequences on the HPV-18 E6 transforming protein predicted by bioinformatics as promiscuous HLA-DR ligands, and to the recombinant E6 protein. We found 3 donors with CD4(+) T cells that specifically proliferated in the presence of HPV-18 E6 antigens and produced IFN-gamma in the presence of the E6 protein. We then propagated CD4(+) T cell lines and clones from the responsive subjects to better characterize the recognized sequences. We show that E6(52-66) and E6(97-111) are indeed promiscuous and, most importantly, they contain naturally processed epitopes. Collectively, our data indicate that healthy donors may develop spontaneous CD4 immunity against HPV-18 E6 epitopes, thus strongly suggesting the potential for this protein to elicit in the host a natural productive immune response.

Dendritic cells (DCs) are involved in the initiation and regulation of innate and adaptive immune responses. Several molecular mechanisms regulate these diverse DC functions, and we have previously reported that mouse dendritic cells (mDCs) can produce interleukin-2 (IL-2) in vitro and in vivo, in response to microbial activation and T-cell-mediated stimuli. This property is shared by different DC subtypes, including Langerhans cells. Here we show that, on appropriate stimulation, human DCs, both plasmacytoid and myeloid subtypes, also express IL-2. Interestingly, the production of IL-2 by myeloid DCs is induced by T-cell-mediated stimuli and depends on the presence of IL-15. The key role of this cytokine in regulating IL-2 production was also confirmed in the mouse system. In particular, we could show that DCs from IL-15-deficient mice were strongly impaired in the ability to produce IL-2 after interactions with different microbial stimuli. Our results indicate that DC-produced IL-2 is tightly coregulated with the expression of IL-15.

Background: Profilins are conserved and ubiquitous plant and animal proteins. We wanted to discover whether the T-cell response to conserved epitopes on birch and grass profilins could account for cross-allergenicity in subjects allergic to these two pollens. Methods: Thirty-one patients allergic to grass and birch were recruited for the study. Grass and birch reactive T lymphocytes were studied by measuring proliferation to birch and grass allergen, respectively, followed by Vβ T-cell receptor family-specific polymerase chain reaction and heteroduplex analysis. T-cell clones were derived from patients with cross-proliferating T cells. Results: In 25 of 31 subjects the T-cell response to grass was quite distinct from that to birch. In contrast, in 6 of 31 individuals grass T cells cross-proliferated to birch and this was reproduced in 4 patients by birch profilin. CD4 Th2 cell clones were derived which promiscuously recognized homologously conserved regions on birch and grass profilins. Conclusion: We conclude that a functionally relevant T-cell response to conserved regions of panallergens underlie cross-allergenicity in a subset of allergic patients. These results suggest that a reciprocal modulation of the response to one sensitizing allergen can occur following natural exposure to or immunotherapy with another allergen. These results have relevance in the management of patients with multiple allergies.

Although the mechanisms regulating recognition and phagocytosis of apoptotic cells by scavenger cells are the subject of intense investigation, little is known about the fate of the antigens contained in apoptotic cells and the constraints defining their immunogenicity. We developed a model in C57BL/6 mice to evaluate whether phagocytosis of apoptotic tumor cells yielded antigens able to get access to the MHC class I pathway and activate a specific cytotoxic T lymphocyte response. Our results demonstrate that apoptotic tumor cells are antigenic in vitro and can be immunogenic in vivo. Their immunogenicity depends on the number of cells used for immunization and the antigen-presenting cells involved in processing and presentation of antigens contained in the dying cells. The demonstration of the immunogenicity of apoptotic cells may have direct implications both in autoimmunity and cancer. J. Leukoc. Biol. 66: 247–251; 1999.

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.

In this study we used TEPITOPE, a new epitope prediction software, to identify sequence segments on the MAGE-3 protein with promiscuous binding to histocompatibility leukocyte antigen (HLA)-DR molecules. Synthetic peptides corresponding to the identified sequences were synthesized and used to propagate CD4+ T cells from the blood of a healthy donor. CD4+ T cells strongly recognized MAGE-3281–295 and, to a lesser extent, MAGE-3141–155 and MAGE-3146–160. Moreover, CD4+ T cells proliferated in the presence of recombinant MAGE-3 after processing and presentation by autologous antigen presenting cells, demonstrating that the MAGE-3 epitopes recognized are naturally processed. CD4+ T cells, mostly of the T helper 1 type, showed specific lytic activity against HLA-DR11/MAGE-3–positive melanoma cells. Cold target inhibition experiments demonstrated indeed that the CD4+ T cells recognized MAGE-3281–295 in association with HLA-DR11 on melanoma cells. This is the first evidence that a tumor-specific shared antigen forms CD4+ T cell epitopes. Furthermore, we validated the use of algorithms for the prediction of promiscuous CD4+ T cell epitopes, thus opening the possibility of wide application to other tumor-associated antigens. These results have direct implications for cancer immunotherapy in the design of peptide-based vaccines with tumor-specific CD4+ T cell epitopes.

In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.

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.

Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8(+) and CD4(+) T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8(+) and NY-ESO-183-99 for CD4(+) T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7(+) patients generated strong CD4(+) T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8(+) and CD4(+) T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

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

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.

BACKGROUND: Knowledge of antigen-specific CD4(+) T cells frequencies is pivotal to the choice of the antigen to be used in anti-viral and anti-tumor vaccination procedures and for monitoring of immune responses. Methods that employ small cell numbers from patient samples, are easy to perform and do not require complex techniques/instrumentations and therefore standardization are desirable. METHODOLOGY/PRINCIPAL FINDINGS: Purified blood CD4(+) T cells from healthy donors were cultured with autologous antigen presenting cells in several replicate wells in equal numbers in the absence (un-stimulated wells) or in the presence of synthetic peptides corresponding to viral antigens promiscuous HLA-DR epitopes (antigen-stimulated wells). At day 7 of culture low dose IL-2 was added and at day 14 IFN-γ and IL-5 release in the supernatant was measured. A statistical analysis approach, based on Poisson distribution, was then implemented to calculate the frequency of viral-specific CD4(+) T cells. We first determined a patient-specific exceptionality threshold of cytokine release in the un-stimulated wells and then, based on this threshold, we counted the inactive/active wells within the antigen-stimulated wells. This number, along with the number of cells per well, allowed the point and interval estimates of frequencies. A ready-to-use Excel worksheet template with automatic calculations for frequencies estimate was developed and is provided as a supplemental file (Table S9). CONCLUSIONS/SIGNIFICANCE: We report a simple experimental procedure combining short term in vitro cell culture with statistical analysis to calculate the frequency of antigen-specific CD4(+) T cells. The detailed experimental procedure along with the Excel applicative are a valuable tool for monitoring immune responses in the clinical practice.

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of target cells. Optimal induction of CD8+ CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stimulatory molecules on antigen-presenting cells (APC). The antigen-processing defective mutant cell line RMA-S, when cultured at low temperature, expresses high amounts of MHC-I molecules that do not contain endogenously derived peptides. These "empty" MHC-I molecules can be stabilized by addition of MHC-binding peptides. RMA-S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA-S cells do not express detectable amounts of B7 co-stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA-S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co-stimulation for CD8+ T lymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when "sub-optimal" antigen peptides were used. Most importantly, RMA-S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8+ cells against B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy of MHC-I by single peptide epitopes in CTL priming.