Hugues Duvillier

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.

Increased production of interleukin-10 and interleukin-1 receptor antagonist after extracorporeal photochemotherapy in chronic graft-versus-host disease

Background. Mechanisms of action of extracorporeal photochemotherapy (ECP) in graft-versus-host disease are incompletely understood. It has been proposed that phagocytosis of apoptotic bodies by monocytes and macrophages induces their activation, a process that increases production of immunosuppressive cytokines. We analyzed apoptosis and cytokine secretion in an autologous coculture system combining peripheral blood lymphocytes (PBL) obtained after ex vivo ECP treatment and nonirradiated peripheral blood mononuclear cells (PBMC). Methods. We studied 11 leukapheresis products treated by ECP from six patients with resistant limited or extensive chronic graft-versus-host disease. Purified PBL obtained by monocyte depletion of the buffy coat from leukapheresis products were mixed with nonirradiated PBMC. Nonirradiated PBL were used as control. Cytokine production was tested at the mRNA level by real-time reverse transcriptase-polymerase chain reaction for interleukin (IL)-10, IL-1 receptor antagonist (IL-1Ra), IL-1&bgr;, tumor necrosis factor-&agr;, and IL-12p40. Results. Morphologic analysis and flow cytometry displayed important lymphocyte apoptotic features culminating at 24 to 48 hr. Coculture of patient’s PBMC with ultraviolet-irradiated PBL as compared with nonirradiated PBL resulted in significant increase of IL-10 mRNA (3418±1015 versus 10596±3402 mRNA copy numbers;P =0.001) and IL-1Ra mRNA (23890±6166 versus 41767±10181 mRNA copy numbers;P =0.001). Incubation with a neutralizing anti-IL-10 monoclonal antibody resulted in a marked decrease of IL-1Ra mRNA levels. Conclusion. Our findings are consistent with the fact that ECP modifies patient’s autologous lymphocytes by inducing a process of apoptosis that activates monocytes and macrophages, leading to increased synthesis of IL-10 and IL-1Ra mRNAs. The induction of this latter mediator is dependent on IL-10.

Insights into Gene Expression Changes Impacting B-Cell Transformation: Cross-Species Microarray Analysis of Bovine Leukemia Virus Tax-Responsive Genes in Ovine B Cells

ABSTRACT Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. TaxBLV, the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify TaxBLV-responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of TaxBLV. Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported TaxHTLV-1-responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that TaxBLV deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.

Pharmacological inhibition of macrophage migration inhibitory factor interferes with the proliferation and invasiveness of squamous carcinoma cells

Recent clinical observations and experimental studies of our group indicate that macrophage migration inhibitory factor (MIF) may contribute to tumor progression in head and neck squamous cell carcinomas (HNSCC). The present study was undertaken to examine the effects of the irreversible MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) on proliferation and invasiveness of the squamous carcinoma cell line SCCVII. Cell counting, crystal violet assay and flow cytometry were used to analyze the effects of 4-IPP on SCCVII cell growth. The impact of 4-IPP on cell invasiveness was assessed by Boyden chamber assay. Knockdown of the MIF receptor CD74 was achieved by transduction with lentiviral vectors encoding anti-CD74 shRNAs. As shown by immunofluorescence staining, SCCVII cells express both MIF and CD74. Decreased MIF immunoreactivity as a result of exposure to 4-IPP suggested a covalent modification of the cytokine. 4-IPP inhibited SCCVII cell proliferation and invasiveness. Moreover, the cytostatic effect of 4-IPP was enhanced by CD74 knockdown. The inhibitory effects of 4-IPP on cell proliferation and invasiveness strongly suggest that MIF is involved in proliferative activity and invasive properties of squamous carcinoma cells. In conclusion, MIF inhibition may open possibilities for target-directed treatment of head and neck squamous cell carcinoma.

Tumor-infiltrating lymphocyte composition, organization and PD-1/ PD-L1 expression are linked in breast cancer

ABSTRACT The clinical relevance of tumor-infiltrating lymphocytes (TIL) in breast cancer (BC) has been clearly established by their demonstrated correlation with long-term positive outcomes. Nevertheless, the relationship between protective immunity, observed in some patients, and critical features of the infiltrate remains unresolved. This study examined TIL density, composition and organization together with PD-1 and PD-L1 expression in freshly collected and paraffin-embedded tissues from 125 patients with invasive primary BC. Tumor and normal breast tissues were analyzed using both flow cytometry and immunohistochemistry. TIL density distribution is a continuum with 25% of tumors identified as TIL-negative at a TIL density equivalent to normal breast tissues. TIL-positive tumors (75%) were equally divided into TIL-intermediate and TIL-high. Tumors had higher mean frequencies of CD4+ T cells and CD19+ B cells and a lower mean frequency of CD8+ T cells compare with normal tissues, increasing the CD4+/CD8+ T-cell ratio. Tertiary lymphoid structures (TLS), principally located in the peri-tumoral stroma, were detected in 60% of tumors and correlated with higher TIL infiltration. PD-1 and PD-L1 expression were also associated with higher TIL densities and TLS. TIL density, TLS and PD-L1 expression were correlated with more aggressive tumor characteristics, including higher proliferation and hormone receptor negativity. Our findings reveal an important relationship between PD-1/PD-L1 expression, increased CD4+ T and B-cell infiltration, TIL density and TLS, suggesting that evaluating not only the extent but also the nature and location of the immune infiltrate should be considered when evaluating antitumor immunity and the potential for benefit from immunotherapies.

Trophic effect in MCF-7 cells of ERalpha17p, a peptide corresponding to a platform regulatory motif of the estrogen receptor alpha--underlying mechanisms.

As yet, estrogen receptor alpha (ERalpha) inhibitors used in clinical practice target a unique site, i.e. the hormone-binding pocket. With the aim of discovering other potential therapeutic targets in the receptor, we studied its AF-2a domain, a site that proves to be critical for ligand-independent ERalpha activity. Previous studies from our laboratory highlighted an auto-inhibitory action associated with a site included in this domain, i.e. the P295-T311 sequence. Accordingly, a deletion of this sequence produces a constitutively activated receptor mutant. More interestingly, a synthetic peptide with the P295-T311 sequence (ERalpha17p) elicits in breast cancer cell lines estrogenic responses that may be ascribed to a competitive mechanism towards the P295-T311-associated auto-inhibition of ERalpha. In the present study, we show that ERalpha17p sustains MCF-7 cell growth in estrogen-depleted culture medium by inducing molecular events promoting G1/S phase transition. We demonstrate, moreover, that this proliferative activity is associated with receptor down regulation (acceleration of ERalpha degradation and repression of ESR1 gene transcription), similar to that induced by estrogen agonists. Complementary studies suggest that our observations may be, at least in part, relevant to a competitive inhibition affecting ERalpha-Hsp70 association. Hence, the design of drugs able to stabilize ERalpha-Hsp70 complexes - where the receptor is in an inactive conformation - may be of therapeutic value.

CXCL13-producing TFH cells link immune suppression and adaptive memory in human breast cancer

T follicular helper cells (TFH cells) are important regulators of antigen-specific B cell responses. The B cell chemoattractant CXCL13 has recently been linked with TFH cell infiltration and improved survival in human cancer. Although human TFH cells can produce CXCL13, their immune functions are currently unknown. This study presents data from human breast cancer, advocating a role for tumor-infiltrating CXCL13-producing (CXCR5-) TFH cells, here named TFHX13 cells, in promoting local memory B cell differentiation. TFHX13 cells potentially trigger tertiary lymphoid structure formation and thereby generate germinal center B cell responses at the tumor site. Follicular DCs are not potent CXCL13 producers in breast tumor tissues. We used the TFH cell markers PD-1 and ICOS to identify distinct effector and regulatory CD4+ T cell subpopulations in breast tumors. TFHX13 cells are an important component of the PD-1hiICOSint effector subpopulation and coexpanded with PD-1intICOShiFOXP3hi Tregs. IL2 deprivation induces CXCL13 expression in vitro with a synergistic effect from TGFβ1, providing insight into TFHX13 cell differentiation in response to Treg accumulation, similar to conventional TFH cell responses. Our data suggest that human TFHX13 cell differentiation may be a key factor in converting Treg-mediated immune suppression to de novo activation of adaptive antitumor humoral responses in the chronic inflammatory breast cancer microenvironment.

The estrogen receptor alpha-derived peptide ERα17p (P295-T311) exerts pro-apoptotic actions in breast cancer cells in vitro and in vivo, independently from their ERα status

In recent years, our knowledge on estrogen receptors (ER) has been modified profoundly with the identification and the deciphering of the role of its protein effectors, as well as with the deeper insight of its molecular structure/function dynamics, characteristics associated with its nucleo‐cytoplasmic‐membrane shuttling properties. Also, significant progress has been made concerning its turn‐over and associated final proteasomal degradation processes. These advances could lead in the near future to the design and the synthesis of novel receptor‐interacting drugs. Recently, a number of receptor‐related peptides acting as specific ER ligands have been identified and extensively studied with respect to their estrogenic/antiestrogenic activities. Among them, ERα17p, a synthetic analog of the P295‐T311 sequence of ERα, has been shown to exert pseudo‐estrogenic effects by interacting in the close vicinity of its hinge region (BF3 domain). Remarkably, this sequence appears as the epicenter of a number of post‐transcriptional modifications as well as of the recruitment of co‐regulators, suggesting that it would play a key role in ERα functions. Here, we provide evidence that ERα17p induces apoptosis in ERα‐positive (MCF‐7, T47D) and ‐negative (MDA‐MB‐231, SK‐BR‐3) breast cancer cells by an ERα‐independent membrane mechanism, triggering major pro‐apoptotic signaling cascades. Finally, ERα17p induces the regression of breast ERα‐negative cancer tumor xenografts, without apparent toxicity, suggesting that it could represent a new attractive tool for the development of future promising therapeutic approaches, and providing a novel insight to ER regulation of cell fate.

Extracellular calcium increases bisphosphonate-induced growth inhibition of breast cancer cells

IntroductionBisphosphonates have become standard therapy for the treatment of skeletal complications related to breast cancer. Although their therapeutic effects mainly result from an inhibition of osteoclastic bone resorption, in vitro data indicate that they also act directly on breast cancer cells, inhibiting proliferation and inducing apoptosis.MethodsThe present study examined the effects of calcium (from 0.6 to 2.0 mmol/l) on the antitumour activity of the bisphosphonate ibandronate (1 to 1,000 nmol/l) on MDA-MB-231 and MCF-7 breast cancer cells. Cell culture densities were determined using crystal violet staining assay. Apoptotic cell death was assessed by annexin V-phycoerythrin and 7-amino-actinomycin double staining.ResultsAt low calcium concentration, 30 μmol/l ibandronate had no effect on MDA-MB-231 cells growth and only slightly inhibited MCF-7 cells growth. Higher calcium levels significantly increased growth inhibition as well as cell apoptosis induced by ibandronate. We observed similar effects with zoledronic acid. Of note, enhancement of ibandronate-induced growth inhibition was also observed in other breast cancer cell lines (T-47D, ZR-75, Hs-578T and BT-549 cells). The growth inhibitory effect of ibandronate in the presence of high concentrations of calcium was partly suppressed by the calcium chelator EGTA (ethylene glycol tetra-acetic acid). In addition, in the presence of calcium at high concentrations, cells accumulated more [14C]ibandronate than at low calcium concentrations. We obtained further evidence of enhancement of cellular ibandronate accumulation by calcium by demonstrating that high calcium levels increased the inhibition of protein prenylation induced by the bisphosphonate.ConclusionAltogether, our data suggest that extracellular calcium, probably through its binding to ibandronate, markedly increased its cellular accumulation and its inhibitory activity on breast tumour cells. Thus, calcium released during the process of tumour-induced osteolysis might enhance the antitumour effects of bisphosphonates and contribute to their therapeutic efficacy.

Dendritic Cells Generated in Clinical Grade Bags Strongly Differ in Immune Functionality When Compared With Classical Dcs Generated in Plates

Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor κ B and β-catenin). Using microarrays, we found underexpression of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electrophoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce TH1-type responses.