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Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3+ T cells are typical Treg based on their CD4+CD25highCD127lowFOXP3+ phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP+ dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3+, and CD8+ T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem. [Cancer Res 2009;69(5):2000–9]

Supplementary Text - PDF file 132K, Supplementary Material and methods, and Supplementary figure legends

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PDF file, 67K, Identification of pDC and mDC in blood and tissues.

PDF file, 64K, Breast TUMSN, but not BCCSN, inhibit IFN-α production by CpGA- or Flu-activated pDC in a dose-dependent manner.

Supplementary Data from Regulatory T Cells Recruited through CCL22/CCR4 Are Selectively Activated in Lymphoid Infiltrates Surrounding Primary Breast Tumors and Lead to an Adverse Clinical Outcome

PDF file, 46K, Purified healthy pDC were cultured with IL-3 either alone or in combination with 25% of TUMSN for 40 h and then incubated for 5 days with allogeneic naive CD4+ T cells.

Supplementary Figures 1-4 from Quantitative and Functional Alterations of Plasmacytoid Dendritic Cells Contribute to Immune Tolerance in Ovarian Cancer

PDF file, 114K, Overall survival and relapse-free survival of patients according to the presence of CD123+ pDC in primary breast carcinoma.

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Supplementary Tables 1-2 from Quantitative and Functional Alterations of Plasmacytoid Dendritic Cells Contribute to Immune Tolerance in Ovarian Cancer

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PDF file, 768K, IHC analysis on frozen BT sections was performed using anti-BDCA2 (brown) together with anti-CD3 (green, a) or anti- cytokeratine (green, b) antibodies (x10)

Supplementary Figures - PDF file 322K, S1. Spontaneous MMTVneu display high Her2/neu expression. S2. FoxP3 expression among CD4+ CD25+ T cells correlates with CD45RB negativity. S3. NEU15WT TApDC display unaltered uptake ability. S4. NEU15WT TApDC display unaltered TLR7 and TLR9 expression. S5. Fold changes in gene expression upon intratumoral TLR7L injection. S6. Intratumoral TLR7L or CpG injection induced distinct immune cell recruitment. S7. List of flurochrome-conjugated antibodies used for multi-parametric flow cytometry analysis

PDF file - 296K, Figure S1: ICOS is mainly expressed by TA-Treg. Figure S2: pDC expanded Treg sustain suppressive activity. Figure S3: ICOS-L is highly expressed on R848-activated HD-pDC. Figure S4: ICOS blockade reduces Treg but not Tconv proliferation induced by pDC without impacting mDC/T cell activation. Figure S5: Strong IL-10 secretion in culture of BT single cell suspension. Figure S6: Correlation between TA-pDC and TA-Treg percentages in fresh primary BT and in retrospective IHC cohort

PDF file - 103K, Table S1: Clinical characteristic of the cohort of BT patients enrolled for TMA analysis (n=120) Table S2: clones of mAbs used for the multi-parametric flow cytometry analyses

PDF file, 77K, Correlations between tumor characteristics and TA(p/m)DC.

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The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4+CD8+ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL. We thank Drs. Susan Schwab, Dan Littman, Sherif Ibrahim, Angel Pellicer, Susanne Tranguch and Adolfo Ferrando for helpful discussions and/or critically comments on the manuscript. Elisabetta Andermarcher professionally edited the manuscript. We are indebted to Dr. M. Yan (Genentech) for the anti-DLL4 antibody for cytometry. We are also in debt with Christopher Murriel from Oncomed who provided the therapeutic murine anti-DLL4 antibody and demcizumab (anti-human DLL4 antibody). We thank the NYU School of Medicine Flow Cytometry Core facility, particularly Dr. Peter Lopez, Keith Kobylarz and Michael Gregory, and also the NYU School of Medicine Confocal imaging facility, particularly Yan Deng. We also thank Henry Alexandre Michaud for his great help with the FACS analysis of PDTALL cells. We thank Nelly Pirot and the rest of members of the IRCM IHC platform for their fantastic work. M.M. is supported by a contract from Fondation ARC. The NYU Cancer Institute Center Support Grant partially funded this core through grant NIH/NCI 5 P30CA16087-31. Work in JJL's laboratory is supported by the NIH/NIAID, National Multiple Sclerosis Society, and the Helmsley Charitable Trust. Work in AM's laboratory is supported by the Fondation ARC (PJA 20131200405), the European Commission (CIG631431), the Institute de Cancer de Montpellier Fondation, and the Institut National du Cancer (INCa_9257 and INCa-DGOS-Inserm 12553). Sí

T-helper-17 (Th17) cells have critical roles in mucosal defense and in autoimmune disease pathogenesis 1-3. They are most abundant in the small intestine lamina propria (SILP), where their presence requires colonization of mice with microbiota 4-7. Segmented Filamentous Bacteria (SFB) are sufficient to induce Th17 cells and to promote Th17-dependent autoimmune disease in animal models 8-14. However, the specificity of Th17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T cell receptor (TCR) repertoire of intestinal Th17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4+ T cells and that most Th17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing Th17 cells, even if SFB-colonized mice also harbored a strong Th1 cell inducer, Listeria monocytogenes, in their intestine. The match of T cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

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.

The presence of regulatory T cells (Treg) has been described in a large panel of solid tumors. However, their impact on tumor progression differs according to the tumor type analyzed. We recently obtained evidence in breast carcinoma that Treg localized within lymphoid aggregates, but not in the tumor bed, have a negative impact on patients' survival. Moreover, we showed selective Treg recruitment through CCR4/CCL22 in the lymphoid aggregates upon contact with dendritic cells (DC), where they became strongly and selectively activated (ICOShigh) and block conventional T-cell response. Here, we discuss the meaning and potential implication of these novel findings. [Cancer Res 2009;69(20):7895–8]

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3+ T cells are typical Treg based on their CD4+CD25highCD127lowFOXP3+ phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP+ dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3+, and CD8+ T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem. [Cancer Res 2009;69(5):2000–9]

Regulatory T (Treg) cells, whose differentiation and function are controlled by X-chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg) and their deficiency results in fatal autoimmunity. Here we demonstrate that a Foxp3 enhancer, conserved non-coding sequence 1 (CNS1), essential for pTreg, but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that during evolution a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

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. Cancer Res; 72(23); 6130–41. ©2012 AACR.

Neuropilin-1 surface expression discriminates between nT reg cells with stable expression and Nrp1 low iT reg cells showing inducible expression under inflammatory conditions., Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-β. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1low. We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.

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. Cancer Res; 72(20); 5188–97. ©2012 AACR.

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (Treg) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1β], contrasting with CCL17. Primary breast tumors and CD45+ infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1β and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ–producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1β and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of Treg. As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits Treg to evade this early antitumor immune response. Cancer Res; 71(19); 6143–52. ©2011 AACR.

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. Cancer Res; 71(16); 5423–34. ©2011 AACR.

Lipid phosphate phosphatase 3 in endothelial and epithelial cells promotes efficient T cell emigration from the thymus to the periphery., The signaling lipid sphingosine-1-phosphate (S1P) stabilizes the vasculature, directs lymphocyte egress from lymphoid organs, and shapes inflammatory responses. However, little is known about how S1P distribution is controlled in vivo, and it is not clear how a ubiquitously made lipid functions as a signal that requires precise spatial and temporal control. We have found that lipid phosphate phosphatase 3 (LPP3) enables efficient export of mature T cells from the thymus into circulation, and several lines of evidence suggest that LPP3 promotes exit by destroying thymic S1P. Although five additional S1P-degrading enzymes are expressed in the thymus, they cannot compensate for the loss of LPP3. Moreover, conditional deletion of LPP3 in either epithelial cells or endothelial cells is sufficient to inhibit egress. These results suggest that S1P generation and destruction are tightly regulated and that LPP3 is essential to establish the balance.

We recently obtained evidences, in breast carcinoma, that regulatory T cells (Treg) localized within lymphoid aggregates, but not in the tumor bed, have a negative impact on patients’ survival. Immunohistochemical analysis on paraffin embedded sections of tumors demonstrated correlation between CCL22 production tumor cells and Treg infiltration thus identifying CCL22 as a key mediator of intratumoral Treg recruitment (Gobert M., Cancer Res vol 69, p2000-9, 2009). Herein we decipher the mechanisms regulating CCL22 production by tumor cells. In vitro, human breast epithelial tumor cell lines secreted low CCL22 levels that were strongly increased in response to inflammatory signals (TNFα, IFNγ and IL-1β or their combination). Using an in vitro model we demonstrated that breast tumor cell lines (MCF-7, MDA-MB453, CLB-SAV, BT-474) but not immortalized normal breast epithelial cell line (Telma) secreted functional CCL22 in response to culture with PBMC or their supernatants. Addition of IL-1RA and anti-TNFα abrogated the CCL22 secretion demonstrating the importance of Il-1β and TNFα in CCL22 production. Depletion experiments demonstrated a major role of monocytes within PBMC in i) the induction of CCL22 and ii) the production of IL-1β and TNFα. However, co-culture of tumor cells with purified monocytes was not sufficient to reproduce the PBMC effects. The addition of rhIFNγ to tumor cell and monocyte co-cultures restored the CCL22 production by tumor cells and that of IL-1β by monocytes suggesting a role of IFNγ producing cells (T cells or NK cells) within PBMC. Taken together, these results demonstrate a dialogue between monocytes, tumor cells and IFNγ producers (NK or T cells) leading, through monocyte-derived IL-1β and TNFα, to CCL22 production by tumor cells and Treg recruitment. These observations would suggest that early tumor cell detection by innate effectors (monocytes, NK) would trigger CCL22 production by tumor cells and not normal mammary epithelial cells leading to Treg recruitment as a mechanism to stop the anti-tumor immune reactivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1341.

Plasma sphingosine-1-phosphate (S1P) regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As the first differential requirement for lymph and blood S1P to our knowledge, Spns2 may be an attractive target for immune suppressive drugs.