Tumor Supernatants Research Articles

Twist and miR-34a Are Involved in the Generation of Tumor-Educated Myeloid-Derived Suppressor Cells

Tumors can induce the generation and accumulation of immunosuppressive cells such as myeloid-derived suppressor cells in the tumor microenvironment, contributing to tumor immunological escapes. Many studies have demonstrated that multiple factors could induce myeloid precursor cells into myeloid-derived suppressor cells, not dendritic cells. In our study, we found that tumor supernatants could induce the generation of myeloid-derived suppressor cells by disturbing the development of dendritic cells. Twist and miR-34a may regulate the effect of tumor cells inducing myeloid-derived suppressor cells via TGF-β and/or IL-10.

Effect of hypoxic stress on induction and plasticity of tumor‐induced immune cells in uveal melanoma

AbstractPurpose Highly malignant uveal melanoma contain increased numbers of lymphocytes and macrophages. We wondered whether hypoxia plays a role in the development of this inflammation. We analysed whether hypoxia induces uveal melanoma cells to release pro‐inflammatory cytokines, and whether tumor supernatant (TSN) affects monocyte migration and differentiation.Methods The expression of pro‐inflammatory genes in freshly cultured uveal melanoma was studied in an in vitro 24 hour hypoxic culture system using qPCR. Cell lines were cultured under normoxic and hypoxic conditions. Chemotaxis was tested using a transwell system with purified monocytes and TSN. Differentiation was tested by adding TSN to a monocyte‐DC culture. CCL2, IL‐6 and PGE2 levels in TSN were determined by ELISA.Results Exposure of freshly‐cultured uveal melanoma cells to hypoxia led to an increased expression of the pro‐inflammatory cytokines PLGF, TGFß, END1, ICAM1 and a lower expression of AIMP1 (EMAP2), CCL2 (MCP‐1), and IL1b. TSN from melanoma cells lines, cultured in normoxic as well as hypoxic conditions, was able to attract monocytes. Migration was independent of tumor‐produced CCL2. Uveal melanoma supernatant inhibits monocyte differentiation.Conclusion Under hypoxic conditions, immune response genes are differentially expressed in cultured primary uveal melanoma cells. TSN from uveal melanoma cell lines is capable of affecting the chemotactic response of monocytes in vitro, irrespectively of the hypoxic or normoxic conditions. Our data suggest that the induction of immune cells is not dependent on intratumoral oxygen level, and the uveal melanoma TSN does not skew macrophage polarization phenotype.

Induction of M2-macrophages by tumour cells and tumour growth promotion by M2-macrophages: A quid pro quo in pancreatic cancer

IntroductionMore effective therapies are required to improve survival of pancreatic cancer. Possible immunologic targets include tumour associated macrophages (TAMs), generally consisting of M1- and M2-macrophages. We have analysed the impact of TAMS on pancreatic cancer in a syngeneic orthotopic murine model. Methods6606PDA murine pancreatic cancer cells were orthotopically injected into C57BL6 mice. Tumour growth was monitored using MRI. Macrophages were depleted by clodronate liposomes. Tumours including microvessel density were evaluated using immunohistochemistry, immunofluorescence and/or cytometric beads assays. Naïve macrophages were generated employing peritoneal macrophages. In vitro experiments included culturing of macrophages in tumour supernatants as well as tumour cells cultured in macrophage supernatants using arginase as well as Griess assays. ResultsClodronate treatment depleted macrophages by 80% in livers (p = 0.0051) and by 60% in pancreatic tumours (p = 0.0169). MRI revealed tumour growth inhibition from 221.8 mm3 to 92.3 mm3 (p = 0.0216). Micro vessel densities were decreased by 44% (p = 0.0315). Yet, MCP-1-, IL-4- and IL-10-levels within pancreatic tumours were unchanged. 6606PDA culture supernatants led to a shift from naïve macrophages towards an M2-phenotype after a 36 h treatment (p < 0.0001), reducing M1-macrophages at the same time (p < 0.037). In vivo, M2-macrophages represented 85% of all TAMs (p < 0.0001). Finally, culture supernatants of M2-macrophages induced tumour growth in vitro by 63.2% (p = 0.0034). ConclusionsThis quid pro quo of tumour cells and M2-macrophages could serve as a new target for future immunotherapies that interrupt tumour promoting activities of TAMs and change the iNOS-arginase balance towards their tumoricidal capacities.

CD4+ follicular helper T cell infiltration predicts breast cancer survival

CD4⁺ T cells are critical regulators of immune responses, but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce an image of CD4⁺ T cells infiltrating breast tumors using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. We performed comprehensive molecular profiling of infiltrating CD4⁺ T cells isolated from untreated invasive primary tumors and found that the infiltrating T cell subpopulations included follicular helper T (Tfh) cells, which have not previously been found in solid tumors, as well as Th1, Th2, and Th17 effector memory cells and Tregs. T cell signaling pathway alterations included a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely paralleled lymphoid infiltration levels and could be reproduced in activated donor CD4⁺ T cells treated with primary tumor supernatant. A comparison of extensively versus minimally infiltrated tumors showed that CXCL13-producing CD4⁺ Tfh cells distinguish extensive immune infiltrates, principally located in tertiary lymphoid structure germinal centers. An 8-gene Tfh signature, signifying organized antitumor immunity, robustly predicted survival or preoperative response to chemotherapy. Our identification of CD4⁺ Tfh cells in breast cancer suggests that they are an important immune element whose presence in the tumor is a prognostic factor.

Cross Talk between Follicular Th Cells and Tumor Cells in Human Follicular Lymphoma Promotes Immune Evasion in the Tumor Microenvironment

The microenvironment of human follicular lymphoma (FL), an incurable B cell non-Hodgkin's lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular Th cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17 but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17, but not CCL22, in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4-producing T cells rather than IFN-γ-producing T cells, and Abs to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4-producing T cells, which, in turn, may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk among TFH, tumor cells, and Tregs in FL, and offer potential targets for development of therapeutic strategies to overcome immune evasion.

Identification of Multiple Novel Protein Biomarkers Shed by Human Serous Ovarian Tumors into the Blood of Immunocompromised Mice and Verified in Patient Sera

The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.

Targeting macrophages rescues age‐related immune deficiencies in C57BL/6J geriatric mice

Changes to innate cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), during aging in healthy or tumor-bearing hosts are not well understood. We compared macrophage subpopulations and MDSCs from healthy young (6-8 weeks) C57BL/6J mice to those from healthy geriatric (24-28 months) mice. Spleens, lymph nodes, and bone marrow of geriatric hosts contained significantly more M2 macrophages and MDSCs than their younger counterparts. Peritoneal macrophages from geriatric, but not young, mice co-expressed CD40 and CX3CR1 that are usually mutually exclusively expressed by M1 or M2 macrophages. Nonetheless, macrophages from geriatric mice responded to M1 or M2 stimuli similarly to macrophages from young mice, although they secreted higher levels of TGF-β in response to IL-4. We mimicked conditions that may occur within tumors by exposing macrophages from young vs. geriatric mice to mesothelioma or lung carcinoma tumor cell-derived supernatants. While both supernatants skewed macrophages toward the M2-phenotype regardless of age, only geriatric-derived macrophages produced IL-4, suggesting a more immunosuppressive tumor microenvironment will be established in the elderly. Both geriatric- and young-derived macrophages induced allogeneic T-cell proliferation, regardless of the stimuli used, including tumor supernatant. However, only macrophages from young mice induced T-cell IFN-γ production. We examined the potential of an IL-2/agonist anti-CD40 antibody immunotherapy that eradicates large tumors in young hosts to activate macrophages from geriatric mice. IL-2-/CD40-activated macrophages rescued T-cell production of IFN-γ in geriatric mice. Therefore, targeting macrophages with IL-2/anti-CD40 antibody may improve innate and T-cell immunity in aging hosts.

Osteopontin splice variants expressed by breast tumors regulate monocyte activation via MCP-1 and TGF-β1

Osteopontin (OPN), a multifunctional glycoprotein, has three transcripts that have distinct roles in tumors in vitro. Whether OPN transcripts have different functions in tumor processes in vivo is unclear. It has been reported that immune cell-derived OPN can promote tumor formation. We propose a hypothesis that tumor-derived OPN may facilitate tumor immune escape by affecting immune cell differentiation and function. In this study, we constructed lentiviral expression vectors of OPN transcripts and transfected them into the MCF-7 cell line. MCF-7 cells transfected with OPN transcripts were injected into the armpit of nude mice, and tumor growth was monitored. The results showed that all OPN transcripts promoted local tumor formation, but that there was no significant difference among transcripts. We also investigated the effect of the OPN expressed by tumor cells on monocyte differentiation by coculturing monocytes with tumor supernatant. We found OPN-c upregulated CD163 levels compared with OPN-a and OPN-b; however, none of the transcripts affected HLA-DR and CD206 levels. All OPN transcripts significantly inhibited TNF-α and enhanced IL-10 production by monocytes. Furthermore, we found that the overexpression of OPN transcripts significantly upregulated TGF-β1 and MCP-1 production by tumor cells. Using neutralizing antibody and recombinant cytokines, we found that OPN overexpressed by tumor cells regulates the production of TNF-α and IL-10 by monocytes partly via MCP-1 and TGF-β1, respectively. Collectively, our results show that OPN transcripts have no distinct role in breast cancer formation in vivo. We also demonstrate that OPN regulates the alternative activation of monocytes via TGF-β1 and MCP-1, which may represent an additional mechanism for tumor immune escape.

Selective recruitment and retainment of regulatory T cells in human colorectal cancer

BackgroundColorectal cancer (CRC) is the third most common malignancy in the UK, and lymphocytic infiltration is associated with improved survival. Analysis of lymphocytic infiltration yields a prognostic ability rivalling the TNM tumour staging system. Regulatory T cells (Treg) are known to be enriched in CRC, and it is postulated that they promote immunological tumour escape by suppression of effector T-cell responses. Little is known about the signals controlling entry of Treg into CRC. MethodsMatched CRC, distal colonic tissue, draining lymph node, and blood were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were isolated and phenotyped for chemokine receptors with flow cytometry. The presence of tissue chemokines was analysed with real-time PCR and western blotting. Standard chemotaxis and suppression assays were performed. Peripheral blood lymphocytes were co-cultured with tumour supernatant, and effects on lymphocyte phenotype were assessed. FindingsThe proportion of T cells with a Treg phenotype was significantly increased in CRC compared with that in colonic tissue (14·8%vs 5·1%, p<0·01). More than 95% of this cell population expressed the transcription factor FOXP3. CCR5 was found to be markedly upregulated on Treg compared with other T cells. The ligands for CCR5 were overexpressed in CRC compared with those in colonic tissue. CCL4 was found to localise to the tumour endothelium. Tumour Treg migrated in response to CCL4, and blockade of CCR5 inhibited migration. Co-culture experiments demonstrated that CCR5 was upregulated on peripheral blood lymphocytes, especially Treg, in mixed lymphocyte reactions. This effect could be augmented by the addition of tumour supernatant. InterpretationConditions exist to recruit CCR5+ Treg into human CRC. CCR5 upregulation is promoted by the tumour microenvironment, providing a retention signal. CCR5 inhibition may prove to be a novel immunotherapy for CRC by blocking the recruitment and egress of suppressive Treg, promoting an anti-tumour immune response. FundingUK Medical Research Council.

CD200 Upregulation in Vascular Endothelium Surrounding Cutaneous Squamous Cell Carcinoma

To characterize the presence of CD200 and CD200 receptor (CD200R) in the human cutaneous squamous cell carcinoma (SCC) microenvironment and to define a possible role for the CD200 axis in immune evasion by SCC. Gene expression in SCC vs normal skin was studied. Laser capture microdissection was used to determine differential expression of CD200 in normal skin vs actinic keratosis vs SCC in situ vs invasive SCC. Immunofluorescence microscopy was used to define expression of CD200R on macrophages, myeloid dendritic cells, natural killer cells, and T cells in SCC vs normal skin. The effects of SCC supernatant on induction of CD200 in human blood endothelial cells was also examined. Academic Medical Center with an established Section of Mohs and Dermatologic Surgery and an active Cutaneous Biology Research Program. Surgical discard tissue from deidentified patients and samples of normal skin from healthy volunteers were used in this study. Expression of CD200 on SCC-associated blood vessels; expression of CD200 receptor on SCC-associated macrophages and T cells; and induction of CD200 on endothelial cells by SCC supernatants. CD200 gene and message were upregulated in SCC stroma. Immunostaining revealed a higher number of CD200(+) cells in SCC stroma than in normal dermis (180.8 cells/mm(2) vs 24.6 cells/mm(2)) (P<.01). CD200 was further identified mainly on blood vessel endothelium in SCC. Tumor supernatant was able to induce CD200 expression on human dermal blood endothelial cells in culture. CD200R was identified on macrophages and dendritic cells in SCC microenvironment. CD200 expression on local blood vessels may promote tumor progression by suppressing CD200R myeloid cells during diapedesis. These data highlight a previously unrecognized mechanism of immune evasion by SCC and may provide guidance for the development of targeted therapy.

Abstract A73: The selective recruitment and retainment of regulatory T cells in human colorectal cancer.

Abstract Background: Colorectal cancer (CRC) is the third most common malignancy in the United States and United Kingdom and deadly in one third of patients. Tumor lymphocytic infiltration is associated with improved survival and analysis of some lymphocyte subsets has revealed that their prognostic ability rivals the TNM tumor staging system. Regulatory T cells (Tregs) are known to be enriched in CRC and it is postulated that they promote immunological tumor escape by suppression of effector T cell responses. Little is known however about the signals controlling entry of Tregs into CRC. Methods: Matched CRC, distal colonic tissue, draining lymph node and blood were obtained from patients undergoing resection of CRC having given informed consent. The study was approved by the Local Research and Ethics Committee. Tumor-infiltrating lymphocytes were isolated from fresh tissue and phenotyped for chemokine receptors and various other markers using multicolor flow cytometry. The presence of tissue chemokines was analyzed using real-time PCR and Western blotting. CD3+ cells were isolated from tumor tissue and placed in a transwell system to measure chemotaxis in response to various chemokines. Migrated and non-migrated T cells were phenotyped to establish differential migration of T cell subsets. CD4+CD127lowCD25+ T cells were isolated by fluorescent-activated cell sorting from tumor tissue and incubated with fluorescent-labeled responder cells in standard suppression assays. Peripheral blood lymphocytes were co-cultured with tumor supernatant and effects on lymphocyte phenotype and proliferation were analyzed by flow cytometry. Results: The proportion of T cells with a suppressive phenotype (Treg, CD4+CD127lowCD25+) was significantly increased in CRC compared to matched distal colon. More than 95% of this cell population expressed the transcription factor, foxp3. The chemokine receptor CCR5 was found to be markedly upregulated on Treg compared to other effector T cells and in CRC compared to distal colon. CCR4 and to a lesser extent, CCR6, were also upregulated on Treg compared to other T cells. The ligands for CCR5 (CCL3, CCL4 and CCL5) were overexpressed in CRC tissue compared to matched colon. CCL4 was found to localize to the tumor endothelium. The ligands for other chemokine receptors were not overexpressed in the tumor tissue compared to the colon and were therefore not investigated further. Tumor-resident Treg migrated in response to CCR5 ligands and blockade of CCR5 inhibited this migration. Co-culture experiments demonstrated that CCR5 was upregulated on peripheral blood lymphocytes, and especially Treg, in mixed lymphocyte reactions. This effect that could be augmented by the addition of tumor supernatant. Conclusion: Conditions exist to actively recruit CCR5+ Treg into colorectal cancer tissue in humans. CCR5 upregulation is also promoted by the tumor microenvironment that may provide a tumor-retention signal. CCR5 inhibition may prove to be a novel immunotherapy for colorectal cancer by blocking the selective recruitment and/or egress of suppressive Treg, thereby promoting an anti-tumor immune response. Citation Format: Stephen T. Ward, Elizabeth A. Hepburn, Ka-kit Li, Stuart M. Curbishley, Rahul K. Hejmadi, Tariq Ismail, Roy Bicknell, Antal Rot, David H. Adams. The selective recruitment and retainment of regulatory T cells in human colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A73.

Abstract B87: CXCL13-producing follicular helper CD4+ T cells infiltrating human breast cancer signal an organized immune response and predict a favorable clinical outcome.

Abstract CD4+ T cells are critical regulators of immune responses but their functional role in human breast cancer is relatively unknown. The goal of this study was to produce a comprehensive image of CD4+ T cells infiltrating breast tumors (TIL) using limited ex vivo manipulation to better understand the in vivo differences associated with patient prognosis. Toward this goal we employed gene expression arrays, qRT-PCR, flow cytometry and immunohistochemistry to produce molecular profiles of CD4+ T cells freshly isolated from untreated invasive primary breast tumors (n=70) compared with non-adjacent breast tissue and CD4+ T cells isolated from the lymph nodes and blood. Gene expression profiling of the TIL discovered a CD4+ follicular helper T (Tfh) cell presence along with Th1, Th2 and Th17 effector memory and Treg subpopulations. TIL expression patterns revealed alterations in T cell signaling pathways that reflect a mixture of activation and suppression characterized by restricted cytokine/chemokine production, which inversely parallel lymphoid infiltration levels and can be reproduced in activated donor CD4+ T cells treated with primary tumor supernatant. Comparison of extensive versus minimally-infiltrated tumors led to a key finding that extensive immune infiltrates are distinguished by CD4+ Tfh cells principally located in the germinal centers of organized tertiary lymphoid structures (TLS). Similar to secondary lymphoid organs, the TLS are composed of a T cell zone containing a majority of CD4+ T cells and a B cell follicle that includes Tfh and follicular dendritic cells along with the B cells. The CD4+ Tfh cells (and not tumor cells) were identified as the major producer of the potent B cell chemoattractant CXCL13, known for its role in promoting lymphoid neogenesis. An increased peri-tumoral TLS presence parallels an elevated level of intra-tumoral leukocytes (principally CD8+ T cells). Our data suggest that the level of tumor-mediated T cell suppression reflects not just the intrinsic suppressive quality of the tumor itself but also the presence (or not) of TLS, which successfully sequester lymphocytes for activation in a protected microenvironment adjacent to the tumor. An eight gene Tfh signature derived from the expression data powerfully predicted long-term survival in a patient cohort with &amp;gt;10-year survival (n=794) or pathological complete response in patients treated with preoperative chemotherapy (n=996). Our identification of CD4+ Tfh cells as a new key immune element in breast cancer, linked with organized immunity and associated with a higher response rate to chemotherapy and/or excellent long-term clinical outcome, suggests that their detectable presence is an important prognostic factor. Citation Format: Chunyan Gu-Trantien, Karen Willard-Gallo. CXCL13-producing follicular helper CD4+ T cells infiltrating human breast cancer signal an organized immune response and predict a favorable clinical outcome. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B87.

Abstract A84: Altered monocyte-derived dendritic cell differentiation in the presence of tumor supernatant: Possible involvement the p38MAPK pathway.

Abstract Introduction: Dendritic cells (DC) are the most efficient antigen-presenting cells in the immune system. In cancer patients, however, these cells present functional and phenotypic disturbances that could be related to a stressful environment induced by the tumor. Heat Shock Proteins (HSP) and the p38MAPK pathway are involved in cell responses against stress and, interestingly, we noted that mature monocyte-derived dendritic cells (Mo-DC) from breast cancer patients present, indeed, higher levels of HSP27 mRNA. Furthermore, HSP27 is a phosphorylation target of the p38 pathway and high HSP27 levels have been associated with impaired Mo-DC differentiation. To investigate these phenomena we exposed monocytes from healthy donors to a tumor cell line supernatant and evaluated their differentiation into DCs and the activation of HSP27 and p38 in the differentiating cells. Methods and Results: Monocytes from healthy donors were negatively selected by magnetic beads and induced to differentiate into DCs (by treatment with GM-CSF and IL-4) either in the presence of 20% conditioned medium (CM) from the breast adenocarcinoma cell line SK-BR-3 (tDC) or not (cDC). The surface phenotype, the presence of total p38, total HSP27, phospho-p38, phospho-HSP27 and the levels of IL-1beta, IL-6, IL-8, IL-10, TNF-alpha and IL-12p70 were evaluated at different time points of the culture, by flow cytometry. At 24h tDC cells expressed less HLA-DR, CD11c and CD86 and showed higher levels of phospho-p38, but less phospho-HSP27. At 72h of culture tDC contained a higher frequency of PD-L1+ cells. Furthermore, during differentiation, tDCs expressed higher levels of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha, but were produced less IL-12p70 in response to lipopolysaccharide (LPS) in comparison with cDCs. Conclusions: CM from SK-BR-3 cells affected the differentiation of Mo-DCs, inducing a phenotype with lesser expression of molecules associated with the induction of immune responses (HLA-DR, CD11c and CD86) but higher expression of PD-L1, a molecule associated with the induction of immune suppression. Dendritic cells exposed to CM also produced high levels of cytokines since the first day of differentiation. This cytokine production exposes the cells simultaneously to signals of differentiation (GM-CSF and IL-4, added to the culture) and maturation (TNF-alpha and IL-1beta produced by the cells), what might impair their differentiation. This was concomitant to an early increase in p38 phosphorylation, but, surprisingly, lower levels of phospho-HSP27 in the same cells. Though preliminary, these data suggest that the p38MAPK pathway may be involved, indeed, in the negative effects of tumors upon the differentiation of Mo-DCs into effective inducers of anti-tumor immune responses. Financial Support: FAPESP. Citation Format: Bruna Zelante, José Alexandre Marzagão Barbuto. Altered monocyte-derived dendritic cell differentiation in the presence of tumor supernatant: Possible involvement the p38MAPK pathway. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A84.

Langerhans Cells from Human Cutaneous Squamous Cell Carcinoma Induce Strong Type 1 Immunity

Langerhans cells (LCs) are dendritic cells (DCs) localized to the epidermis. They should be the first antigen-presenting cells to encounter squamous cell carcinoma (SCC). The aim of this study was to investigate the ability of LCs isolated from human SCC to induce T-cell proliferation and polarization. We investigated the ability of LCs from SCC and peritumoral skin to induce T-cell proliferation and polarization. We also studied the effect of SCC supernatant on the ability of LCs from normal skin, in vitro-generated LCs, and DCs to activate and polarize T cells. LCs from SCC were stronger inducers of allogeneic CD4(+) and CD8(+) T-cell proliferation and IFN-γ production than LCs from peritumoral skin. We found that tumor supernatants (TSNs) were rich in immunosuppressive cytokines; despite this, allogeneic CD4(+) and CD8(+) T-cell proliferation and IFN-γ induction by LCs were augmented by TSN. Moreover, TSN facilitated IFN-γ induction by in vitro-generated LCs, but suppressed the ability of in vitro-generated DCs to expand allogeneic CD4(+) and CD8(+) T cells. We have demonstrated that LCs from SCC can induce type 1 immunity. TSN induces IFN-γ induction by in vitro-generated LCs. This contrasts greatly with prior studies showing that DCs from SCC cannot stimulate T cells. These data indicate that LCs may be superior to DCs for SCC immunotherapy and may provide a new rationale for harnessing LCs for the treatment of cancer patients.

TSLP driven inflammation fosters breast tumor development (74.2)

Abstract Solid tumors are often associated with aseptic inflammation. There are two types of inflammation that have opposing effects on tumors, chronic inflammation that promotes cancer survival and metastasis, and acute inflammation which triggers cancer destruction. Chronic inflammation is often linked with the presence of type 2-polarized macrophages induced by Th2 cytokines, IL-4 and IL-13. Our studies have demonstrated the presence in breast cancer tumors of inflammatory Th2 cells, which produce IL-13, IL-4, and TNF. These CD4+ T cells appear to play a key role in the disease as they accelerate breast tumor development in a xenograft model through the production of IL-13. Breast tumors appear to play a critical role in conditioning the infiltrating myeloid DCs (mDCs) to induce such inflammatory Th2 cells. Our recent results suggest that thymic stromal lymphopoetin (TSLP) plays a role in mDCs conditioning. Breast cancer cell lines and primary tumors from patients show TSLP protein expression. TSLP-neutralizing antibodies block the upregulation of OX40L by mDCs exposed to tumor supernatant and consequently block mDCs capacity to generate inflammatory Th2 cells in vitro. Targeting pattern recognition receptors on mDCs renders them resistant to TSLP signal. These mDCs don't phosphorylate STAT6, don't express OX40L and turn on the secretion of IL12p70. The final outcome is the block of inflammatory Th2 and tumor rejection. Thus, TSLP could serve as therapeutic target in breast cancer.

Abstract 3994: Loss of STAT2 enhances the establishment of B16 melanoma tumors

Abstract STAT2 is a key component of type I interferon (IFN-I) induced activation of the JAK/STAT signaling pathway. Several studies including ours have shown that STAT2 is a critical signaling mediator in the apoptotic and cell growth inhibitory effects of IFN-I. These findings suggest that STAT2 has tumor suppressive function. To investigate this further, the B16-F1 mouse melanoma cell line was used in a tumor transplantation model to interrogate the role of STAT2 in tumorigenesis. We found that STAT2 null mice injected subcutaneously with B16-F1 melanoma tumor cells showed accelerated tumor growth rate and grew larger tumors compared to those formed in wild type mice. We next compared the antitumor effects of IFN-I in wild type and STAT2 null mice administered intraperitoneally 3 days after tumor injection and given three times weekly until day 21 of the study. IFN-[[Unsupported Character - Symbol Font &amp;#61538;]] therapy of STAT2 null mice was ineffective as tumors continued to grow at the same rate as in saline treated STAT2 null mice. In contrast, wild type mice that received IFN-[[Unsupported Character - Symbol Font &amp;#61538;]] developed smaller tumors compared to saline treated control mice. Evaluation of B16 tumors established in wild type mice by Western blot analysis showed a marked reduction in the levels of STAT2 and STAT1 proteins. Similarly, B16 tumors established in STAT2 null mice also showed low levels of STAT1 protein. To determine whether tumor derived soluble factors were involved in decreasing STAT2 protein; supernatants collected from B16-F1 tumors incubated overnight with serum free medium were used to treat B16-F1 tumor cells grown in vitro. A decrease in STAT2 protein was evident when tumor supernatants derived from tumors established in wild type and STAT2 null mice were used. Taken together our findings reveal that host STAT2 restricts the establishment of melanoma tumors and soluble factors secreted by melanoma tumors reduce STAT2 protein levels thereby diminishing the antitumor effects of IFN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3994. doi:1538-7445.AM2012-3994

Abstract 5412: CCR4+CCR6+Th17 cells suppress autologous CD8+ T cell responses in patients with hepatocellular carcinoma

Abstract The role of Th17 cells in cancer patients remains unclear and controversial. We have noticed an increase in Th17 related cytokines (IL-17 and IL-23) in tumor supernatants derived from primary liver cancer, which prompted us to examine Th17 cells in patients with hepatocellular carcinoma (HCC). First we analyzed the frequency of CD4+IL-17+ cells in peripheral blood from patients with HCC, which was increased in comparison to healthy controls. In order to further analyze the function of these cells, we generated Th17 cells in vitro in the presence of IL-1beta, IL-6 and IL-23 cytokines, anti-IL-4 and anti-IFN-gamma antibodies. Th17 cells were than stained using an IL-17 capture antibody and purified by FACS sorting. In vitro primed Th17 cells were further purified into CCR4+CCR6+ and CCR4negCCR6+ Th17 cells. When cultured with autologous CD8+ T cells, CCR4+CCR6+Th17 cells suppressed the lytic function, proliferation and cytokine secretion of both antigen specific and CD3/CD28/CD2 stimulated autologous CD8+ T cells. In contrast, CCR4negCCR6+ CD4+ T cells, which also secrete IL-17, did not affect the CD8+ T cells. Suppression of CD8+ T cells by CCR4+CCR6+Th17 cells was partially dependent on TGF-beta, since neutralization of TGF-beta in cocultures reversed their suppressor function. Based on these findings we evaluated the frequency CCR4+CCR6+Th17 and CCR4negCCR6+Th17 cells in patients with HCC separately. Our analysis revealed also an increase in the frequency of CCR4+CCR6+Th17 cells but not CCR4negCCR6+Th17 cells in peripheral blood of HCC patients and ex vivo isolated CCR4+CCR6+CD4+ T cells from peripheral blood of patients. Finally we demonstrated the suppressor activity of freshly isolated CCR4+CCR6+T cells on autologous CD8+ T cell proliferation. In summary our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in HCC. This has important implications when studying the mechanisms of carcinogenesis as well as designing effective immunotherapy protocols for patients with cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5412. doi:1538-7445.AM2012-5412

Abstract IA17: TSLP-driven inflammation fosters development of epithelial tumors

Abstract Solid tumors are often associated with aseptic inflammation. There are two types of inflammation that have opposing effects on tumors, chronic inflammation that promotes cancer cell survival, and metastasis, and acute inflammation which triggers cancer cell destruction. Chronic inflammation is often linked with the presence of type 2-polarized macrophages (M2), which are induced by Th2 cytokines, IL-4 and IL-13. Our recent studies have demonstrated the presence in breast cancer tumors of inflammatory Th2 cells, which produce IL-13, IL-4, and TNF. These CD4+ T cells appear to play a key role in the disease as they accelerate breast tumor development in a xenograft model through the production of IL-13. Breast tumors appear to play a critical role in conditioning the infiltrating myeloid DCs (mDCs) to induce such inflammatory Th2 cells. Our most recent results suggest that thymic stromal lymphopoetin (TSLP) secreted by cancer cells plays a role in mDCs conditioning. Breast cancer cell lines and primary tumors from patients show TSLP protein expression. TSLP-neutralizing antibodies block the upregulation of OX40L by mDCs exposed to tumor supernatant and consequently block mDCs capacity to generate inflammatory Th2 cells in vitro. The TSLP production is mediated by PAR2-signaling in cancer cells. PAR2 is expressed by all 4 breast cancer cell lines, and TSLP production can be induced upon PAR2-agonist peptide treatment. Similar observations were made by us and others in pancreatic cancer as well as lung cancer. Thus, TSLP could serve as therapeutic target in tumors of epithelial origin.

Role of IL-4 in Inducing Immunosuppressive Tumor Microenvironment in Follicular Lymphoma

Abstract 771 Background:Recently, using proteomic profiling of tumor lysates, Calvo et al (Blood, 2008) found that IL-4 levels were significantly higher in follicular lymphoma (FL) tissues than in tissues from follicular hyperplasia. IL-4 present in the tumor microenvironment may promote the survival and growth of tumor B cells and may play a role in the pathogenesis of FL. However, the source of IL-4 production was not evaluated in these studies. Several reports have shown that regulatory T cells (Tregs) that can inhibit the function of antitumor CD4+ and CD8+ T cells are increased in number in the tumor microenvironment in FL. Tregs can be induced from conventional CD4+ T cells by the tumor cells but may also be actively recruited to the tumor site by CCL17 and CCL22 produced by tumor cells. However, B cells are not known to constitutively express either of these chemokines. As IL-4 is increased in the FL tumor microenvironment and can activate STAT6, and CCL17 and CCL22 are STAT6-responsive chemokine genes, we tested the possibility that these are interrelated. Methods:Surgical tumor samples from FL patients were processed into a single-cell suspension and cryopreserved for later use. Tumor cells isolated by magnetic cell separation were cultured in the presence or absence of IL-4 and chemokine levels were assessed in the supernatants after 48 hours by ELISA. Intracellular cytokine assay was performed on intratumoral T cells and tumor cells to determine the source of IL-4. Western blotting and phosflow was performed to assess the phosphorylation status of STAT6. Migration of Tregs in response to tumor supernatants was assessed in a transwell chemotaxis assay. Results:Using primary human FL samples we demonstrate that IL-4 is secreted by intratumoral follicular helper T cells (Tfh) and more importantly, IL-4-producing CD4+ T cells were increased in number in the FL tumor microenvironment as compared with benign follicular hyperplasia. While FL tumor cells produced CCL22 constitutively, the production of CCL17 was induced by IL-4 via activation of STAT6. Consistent with this, we observed a direct correlation between IL-4 and CCL17 mRNA transcripts in whole genome microarray studies of FL tissues (r = 0.41, P = <0.0001). The expression of CCL17 by tumor B-cells was dependent on STAT6 since knockdown of STAT6 using siRNA inhibited its production. IL-4 also stimulated CCL17 secretion from tumor cells from other B-cell lymphomas such as diffuse large cell lymphoma, mantle cell lymphoma, and splenic marginal zone lymphoma as well as intratumoral T cells in FL. CCL17 produced by these cells induced preferential migration of Tregs and IL-4–producing T cells than IFN-g–producing T cells. Antibodies to CCL17 and CCR4 significantly abrogated the migration of Tregs in response to tumor supernatants. Conclusions:Our results suggest that IL-4 produced by Tfh cells in FL induces CCL17 production through autocrine and paracrine effects on intratumoral T cells and tumor cells, respectively, and facilitates active recruitment of regulatory T cells and IL-4–producing T cells that in turn may stimulate more CCL17 production. Thus, IL-4 creates a vicious cycle and leads to an immunosuppressive tumor microenvironment that promotes immune evasion and tumor survival and growth. Our results provide novel insights into the role of IL-4 in immunosuppression in FL and other B-cell lymphomas and offer potential targets for development of therapeutic strategies to overcome immune evasion in these malignancies. Characters:2,993 (3,800 max without spaces) Disclosures:No relevant conflicts of interest to declare.

STAT3 Knockdown in B16 Melanoma by siRNA Lipopolyplexes Induces Bystander Immune Response In Vitro and In Vivo

Persistent activation of STAT3 plays a major role in cancer progression and immune escape. Therefore, targeting STAT3 in tumors is essential to enhance/reactivate antitumor immune response. In our previous studies, we demonstrated the efficacy of stearic acid-modified polyethylenimine (PEI-StA) in promoting small interfering RNA (siRNA) silencing of STAT3 in B16.F10 melanoma in vitro and in vivo. In the current study, we examine the immunologic impact of this intervention. Toward this goal, the infiltration and activation of lymphocytes and dendritic cells (DCs) in the tumor mass were assessed using flow cytometry. Moreover, the levels of IFN-γ, IL-12, and TNF-α in homogenized tumor supernatants were determined. Moreover, mixed lymphocytes reaction using splenocytes of tumor-bearing mice was used to assess DC functionality on siRNA/lipopolyplexes intervention. Our results demonstrated up to an approximately fivefold induction in the infiltration of CD3+ cells in tumor mass on STAT3 knockdown with high levels of CD4+, CD8+, and NKT cells. Consistently, DC infiltration in tumor milieu increased up to approximately fourfold. Those DCs were activated, in an otherwise suppressive microenvironment, as evidenced by a high expression of costimulatory molecules CD86 and CD40. ELISA analysis revealed a significant increase in IFN-γ, IL-12, and TNF-α. Moreover, mixed lymphocytes reaction demonstrated alloreactivity of these DCs as assessed by high T-cell proliferation and IL-2 production. Our results suggest a bystander immune response after local STAT3 silencing by siRNA. This strategy could be beneficial as an adjuvant therapy along with current cancer vaccine formulations.