Tumor-associated pDC Research Articles

Role of CCL21 on the Transcriptional Regulation of MHC II in Malignant Glioma Infiltrating Plasmacytoid Dendritic Cells

Background and Hypothesis: Glioblastoma (GBM) is a malignant brain tumor characterized by high tumor heterogeneity and an immunosuppressive tumor microenvironment (TME). Immunomodulation approaches have been investigated, but outcomes remain poor. Several studies describe the functional deregulation of immune cells including, T cells, dendritic cells (DC), and macrophages. Plasmacytoid dendritic cells (pDC), which accumulate in the GBM TME, are shown to have an immunosuppressive phenotype characterized by a lack of IFN-[Symbol] secretion and upregulation of MHC II. MHC II presentation is transcriptionally regulated by several factors produced by tumor cells including, TGFβ, TNFα, and IL10 through the modulation of CIITA, the catalytic component of the enhanceosome. GBM tumor cells secrete several chemokines/cytokines, which may regulate MHC II expression in pDCs. We hypothesize that chemokine CCL21 transcriptionally upregulates MHC II through the activation of CIITA in pDC. 
 Experimental Design/Project methods: We performed experiments using two GBM tumor cells models GL261 and CT2A and used western blot, PCR, immunohistochemistry, immunofluorescence, and flow cytometry to determine the levels of CCL21 and its ligands ACKR3/4 in tumor cells and pDC. 
 Results: We observed overexpression of CCL21 in GBM and upregulation of MHCII in tumor associated pDC. We predict that inhibition of CCL21 will lead to downregulation of MHC II in tumor associated pDC which could potentially lead to reversal of the immunosuppressive TME by presenting the antigens to T cells. 
 Conclusions/Potential Impact: The results of this study can elucidate novel mechanisms of MHCII regulation and identify CCL21 as a potential therapeutic target for immunotherapy development in GBM.

Abstract 3782: Transcriptional regulation of MHC Class II in tumor associated plasmacytoid dendritic cells in glioblastoma

Abstract Background: Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary brain tumor in adults and carry very poor prognosis. One of the hallmark features of GBM is tumor induced dense immunosuppression that renders immunotherapeutic strategies against GBM ineffective. In order to develop successful immunotherapeutic strategies for GBM, it is critical to understand the mechanism of immunosuppression in the tumor microenvironment (TME). In our previous publication, we have shown that plasmacytoid dendritic cells (pDCs) accumulate in TME and preferentially up-regulate MHC-II expression in comparison to myeloid DCs (mDCs). GBM infiltrating pDCs are immunosuppressive in nature and lack the ability to secrete IFN-alpha, and MHC-II upregulation by immunosuppressive pDCs lead to development of tumor tolerance. MHC-II molecules are transcriptionally regulated by the multiprotein complex enhanceosome consisting of a catalytic Class II transactivator (C2ta) in a cell type-dependent mechanism. There are no studies clearly showing differential enhanceosome and C2ta regulation in GBM associated pDC and mDC. In the present study, we investigated the mechanism of differential expression of enhanceosome multiprotein complex in GBM associated pDC and mDC and identify the upstream regulators and downstream effectors. Methods: GL261 cells were intracranially injected (i.c.) in C57BL6 mice and allowed to establish tumors. Tumor and normal brain (n=5), along with cervical lymph nodes (cLN), were collected and processed for isolation of pDC and mDC using flow sorting. Quantitative RT-PCR analysis was used to determine the enhanceosome components. IHC/IF and Western blot analysis were performed to confirm differential expression of enhanceosome components in mouse and human GBM specimens. Cytokine/chemokine protein array and ELISA methods were used to determine the differential expression of cytokines/chemokines in pDC and mDC. Results: We found differential expression of enhanceosome components in tumor associated pDC compared to mDC. Additionally, we found that GBM derived cytokines regulate differential expression of enhanceosome in tumor associated pDC and mDCs. Conclusions: In conclusion results from our studies show that GBM microenvironment regulates differential expression of enhanceosome transcription factors in pDC compared to mDC in a cytokine dependent process. This in turn helps to maintain an immunosuppressive microenvironment for continued tumor growth. Citation Format: Sreenivasulu Chintala, Brij N. Tewari, Mario Henriquez, Kaleigh Fetcko, Anna Filley, Mahua Dey. Transcriptional regulation of MHC Class II in tumor associated plasmacytoid dendritic cells in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3782.

Intratumoral CpG-B Promotes Antitumoral Neutrophil, cDC, and T-cell Cooperation without Reprograming Tolerogenic pDC

Cancer immunotherapies utilize distinct mechanisms to harness the power of the immune system to eradicate cancer cells. Therapeutic vaccines, aimed at inducing active immune responses against an existing cancer, are highly dependent on the immunological microenvironment, where many immune cell types display high levels of plasticity and, depending on the context, promote very different immunologic outcomes. Among them, plasmacytoid dendritic cells (pDC), known to be highly immunogenic upon inflammation, are maintained in a tolerogenic state by the tumor microenvironment. Here, we report that intratumoral (i.t.) injection of established solid tumors with CpG oligonucleotides-B (CpG-B) inhibits tumor growth. Interestingly, control of tumor growth was independent of tumor-associated pDC, which remained refractory to CpG-B stimulation and whose depletion did not alter the efficacy of the treatment. Instead, tumor growth inhibition subsequent to i.t. CpG-B injection depended on the recruitment of neutrophils into the milieu, resulting in the activation of conventional dendritic cells, subsequent increased antitumor T-cell priming in draining lymph nodes, and enhanced effector T-cell infiltration in the tumor microenvironment. These results reinforce the concept that i.t. delivery of TLR9 agonists alters the tumor microenvironment by improving the antitumor activity of both innate and adaptive immune cells.Significance: Intratumoral delivery of CpG-B disrupts the tolerogenic tumor microenvironment and inhibits tumor growth. Cancer Res; 78(12); 3280-92. ©2018 AACR.

Tumor Promotion by Intratumoral Plasmacytoid Dendritic Cells Is Reversed by TLR7 Ligand Treatment

Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.

Breast cancer‐derived transforming growth factor‐β and tumor necrosis factor‐α compromise interferon‐α production by tumor‐associated plasmacytoid dendritic cells

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon-α (IFN-α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor-associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN-α, the production by Toll-like receptor (TLR)-activated healthy pDC of IFN-β and TNF-α but not IP-10/CXCL10 nor MIP1-α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF-β and TNF-α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF-β1 and TNF-α synergistically blocked IFN-α production of TLR-activated pDC, and neutralization of TGF-β and TNF-α in tumor-derived supernatants restored pDCs' IFN-α production. The involvment of tumor-derived TGF-β was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF-7 expression and nuclear translocation in pDC after their exposure to tumor-derived supernatants or recombinant TGF-β1 and TNF-α. Our findings indicate that targeting TApDC to restore their IFN-α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9-based immunotherapy with TGF-β and TNF-α antagonists.

ICOS-Ligand Expression on Plasmacytoid Dendritic Cells Supports Breast Cancer Progression by Promoting the Accumulation of Immunosuppressive CD4+ T Cells

Human breast tumors are infiltrated by memory CD4(+) T cells along with increased numbers of regulatory T cells (Treg) and plasmacytoid dendritic cells (pDC) that facilitate immune escape and correlate with poor prognosis. Here, we report that inducible costimulatory molecule (ICOS), a T cell costimulatory molecule of the CTLA4/PD1/CD28 family, is expressed mostly by tumor-associated Treg in primary breast tumors. A large proportion of these ICOS(+) Treg were Ki67(+) and this evident proliferative expansion was found to rely on interactions with tumor-associated pDC. Indeed, tumor-associated Treg highly expanded in presence of pDC but failed to proliferate under CD3/CD28 signal. In vitro experiments revealed that the addition of a neutralizing anti-ICOS antibody blocked pDC-induced Treg expansion and interleukin-10 secretion by memory CD4(+) T cells, establishing a pivotal role for ICOS in this process. Supporting these findings, the presence of ICOS(+) cells in clinical specimens of breast cancer correlated with a poor prognosis. Together, our results highlight an important relationship between Treg and pDC in breast tumors, and show that ICOS/ICOS-L interaction is a central event in immunosuppression of tumor-associated memory CD4(+) T cells. These findings strongly rationalize antibody-mediated ICOS blockade as a powerful clinical strategy to correct immune escape and promote therapeutic responses in breast cancer.

Impaired IFN-α Production by Plasmacytoid Dendritic Cells Favors Regulatory T-cell Expansion That May Contribute to Breast Cancer Progression

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.

Quantitative and Functional Alterations of Plasmacytoid Dendritic Cells Contribute to Immune Tolerance in Ovarian Cancer

In ovarian cancer, the immune system fails to eradicate established tumors partly due to the induction of immune tolerance within tumor microenvironment. In this study, we investigated the contribution of plasmacytoid dendritic cells (pDC) in the establishment of immune tolerance in a cohort of 44 ovarian cancer patients. In the tumor and malignant ascites, CD4(+)CD123(+)BDCA2(+) pDC were the most abundant dendritic cell subset; however, they were profoundly depleted in peripheral blood. The presence of pDC in primary ovarian cancer, but not ascites, was an independent prognostic factor associated with early relapse. Following chemotherapy, we observed a partial restoration of blood pDC levels in patients in complete remission. These findings show preferential recruitment of pDC into tumors where they express a partially mature phenotype that may reflect an in situ activation. Importantly, compared with pDC found in ascites or blood, tumor-associated pDC (TApDC) produced less IFN-α, TNF-α, IL-6, macrophage inflammatory protein-1β, and RANTES in response to toll-like receptor stimulation, and alterations in pDC functions were mainly mediated through tumor-derived TNF-α and TGF-β. Unlike ascites-derived pDC, TApDC induced IL-10 production from allogeneic naive CD4(+) T lymphocytes, suggesting the existence of a paracrine immunosuppressive loop. Taken together, our findings indicate that both local and systemic dysfunction of pDC play a critical role in the progression of ovarian cancer via induction of immune tolerance.

Abstract 472: In vivo activation of plasmacytoid dendritic cells using TLR7-L induced an efficient antitumoral effect in a murine mammary preclinical tumor model

Abstract Dendritic cells (DC) are professional antigen presenting cells involved in both the induction and the polarization of adaptive immune response. Among them figure conventional myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC), the last ones being involved in the induction of antiviral immune response through type I interferons production. In human breast cancer, we have previously shown that pDC infiltration is an independent factor correlated with a poor prognosis. Although pDC can induce efficient T cell responses against viral or endogenous antigens, it remains unclear whether pDC are efficient for cross presentation of tumor antigens acquired from dying tumor cells. Moreover, pDC have been described in some contexts to be involved in the induction of immune tolerance by promoting regulatory T cell (Treg) differentiation. Thus herein we evaluate their ability to induce efficient antitumoral response after activation by viral signals such as TLR7L or TLR9L. First, we developed several preclinical murine mammary tumor models derived from spontaneous mammary tumors arose in transgenic mice MMTV-Her2/neu expressing the rat proto-oncogene neu under the control of MMTV promoter. Several tumor cell lines (NEU) expressing HER2/Neu were derived from these spontaneous mammary tumors and were implanted in the 4th fad pad of immunotolerant transgenic mice MMTV-Her2/neu or in immunocompetent FVB wild type mice witch developed an antibody immune response against the rat Her2/neu proto-oncogene. All NEU cell lines were highly tumorigeneic in immunotolerant mice but interestingly when implanted in FVBwt mice some were rejected, some escape by immunoediting with a selection of a NEU negative variant and one (NEU15) escape by another mechanism showing as in human breast cancer the maintenance of Her2/Neu expression despite an anti-tumoral immune response. We showed that NEU15 tumors in FVBwt mice (NEU15wt) were significantly more infiltrated by pDC and Treg than NEU15 tumors in immunotolerant transgenic mice (NEU15Tg). Secondly, we focused on this NEU15wt preclinical model and analyzed the functional status of tumor associated pDC (TApDC) and TAmDC. We showed that TApDC are altered for their capacity to secrete type I IFN in response to TLR9 engagement while preserving their ability to respond to viral signals such as Influenza and TLR7 ligands. TAmDC undergo a spontaneous maturation in culture independently of TLR engagement and secrete inflammatory cytokines (TNFα, RANTES) but also IL-10 and no IL12. In vivo immuno intervention using intra-tumoral injection of TLR-7L (50μg D13 and D20) in pre-established NEU15wt tumors allowed 50% of complete tumor rejection. IFNα production was detected in mice sera 3 hours after TLR-7L intratumoral injection. The impact of in vivo depletion of pDC on the therapeutic efficiency of TLR7L is under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2011-472