Publications by authors named "Pierre van der Bruggen"

73 Publications

Identification of an Immature Subset of PMN-MDSC Correlated to Response to Checkpoint Inhibitor Therapy in Patients with Metastatic Melanoma.

Cancers (Basel) 2021 Mar 17;13(6). Epub 2021 Mar 17.

Immunity and Cancer Team, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068, CNRS, UMR7258, Institut Paoli-Calmettes, Aix Marseille University, UM105, 13009 Marseille, France.

PMN-MDSCs support tumor progression and resistance to ICI therapy through their suppressive functions but their heterogeneity limits their use as biomarkers in cancer. Our aim was to investigate the phenotypic and functional subsets of PMN-MDSCs to identify biomarkers of response to ICI therapy. We isolated low-density CD15 PMNs from patients with metastatic melanoma and assessed their immune-suppressive capacities. Expression of CD10 and CD16 was used to identify mature and immature subsets and correlate them to inhibition of T cell proliferation or direct cytotoxicity. Frequencies of the PMN-MDSCs subsets were next correlated to the radiological response of 36 patients receiving ICI therapy. Mature activated cells constituted the major population of PMN-MDSCs. They were found in a higher proportion in the pre-treatment blood of patients non responders to ICI. A subset of immature cells characterized by intermediate levels of CD10 and CD16, the absence of expression of SIRPα and a strong direct cytotoxicity to T cells was increased in patients responding to ICI. The paradoxical expansion of such cells during ICI therapy suggests a role of PMNs in the inflammatory events associated to efficient ICI therapy and the usefulness of their monitoring in patients care.
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http://dx.doi.org/10.3390/cancers13061362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002694PMC
March 2021

A Genetic Vaccine Encoding Shared Cancer Neoantigens to Treat Tumors with Microsatellite Instability.

Cancer Res 2020 09 20;80(18):3972-3982. Epub 2020 Jul 20.

Nouscom S.R.L., Rome, Italy.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an "off-the-shelf" cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. SIGNIFICANCE: These findings demonstrate the feasibility of an "off-the-shelf" vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1072DOI Listing
September 2020

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

Nat Commun 2020 03 19;11(1):1457. Epub 2020 Mar 19.

UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Croix du Sud 4-5, B-1348, Louvain-la-Neuve, Belgium.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.
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http://dx.doi.org/10.1038/s41467-020-15303-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081352PMC
March 2020

Protocol to assess the suppression of T-cell proliferation by human MDSC.

Methods Enzymol 2020 8;632:155-192. Epub 2019 Jul 8.

de Duve Institute, Université catholique de Louvain, Brussels, Belgium; WELBIO, Brussels, Belgium. Electronic address:

Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye.
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http://dx.doi.org/10.1016/bs.mie.2019.05.046DOI Listing
December 2020

Reverse immunology: From peptide sequence to tumor-killing human T-cell clones.

Methods Enzymol 2020 17;631:159-194. Epub 2019 Jun 17.

De Duve Institute, Université catholique de Louvain, Brussels, Belgium.

Recent advances in next generation sequencing expanded the availability of tumor mutanome data that list the mutations present in cancer cells. Mutated proteins are an interesting source of neoantigens that can be used to specifically target tumor cells in the context of immunotherapy. However, identifying new antigenic peptides from mutated proteins remains challenging. In this chapter, we present Reverse Immunology as an approach to identify potential antigens from any given polypeptide sequence. First, we explain the rationale behind the identification of candidate HLA-binding peptides through mass spectrometry or in silico approaches. Then, we describe the isolation of low-frequency T-cell precursors specific for the candidate peptides using peptide-HLA multimers. Finally, we discuss validation steps leading to the identification of a T-cell clone recognizing tumor cells that endogenously process the candidate peptide. We also present approaches to study the impact of the proteasome complex on candidate peptide processing.
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http://dx.doi.org/10.1016/bs.mie.2019.05.033DOI Listing
January 2021

The Vacuolar Pathway of Long Peptide Cross-Presentation Can Be TAP Dependent.

J Immunol 2019 01 17;202(2):451-459. Epub 2018 Dec 17.

Ludwig Institute for Cancer Research, Brussels B-1200, Belgium;

The intracellular pathway of cross-presentation, which allows MHC class I-restricted presentation of peptides derived from exogenous Ags, remains poorly defined and may vary with the nature of the exogenous Ag and the type of APC. It can be cytosolic, characterized by proteasome and TAP dependency, or vacuolar, usually believed to be proteasome and TAP independent. Cross-presentation is particularly effective with long synthetic peptides, and we previously reported that the HLA-A2-restricted cross-presentation of a long peptide derived from melanoma Ag gp100 by human monocyte-derived immature dendritic cells occurred in a vacuolar pathway, making use of newly synthesized HLA-A2 molecules that follow a nonclassical secretion route. In this article, we show that the HLA-A1-restricted cross-presentation of a long peptide derived from tumor Ag MAGE-A3 by human monocyte-derived immature dendritic cells also follows a vacuolar pathway. However, as opposed to the HLA-A2-restricted peptide, cross-presentation of the HLA-A1-restricted peptide is TAP dependent. We show that this paradoxical TAP-dependency is indirect and reflects the need for TAP to load HLA-A1 molecules with peptides in the endoplasmic reticulum, to allow them to escape the endoplasmic reticulum and reach the vacuole, where peptide exchange with the cross-presented peptide likely occurs. Our results confirm and extend the involvement of the vacuolar pathway in the cross-presentation of long peptides, and indicate that TAP-dependency can no longer be used as a key criterion to distinguish the cytosolic from the vacuolar pathway of cross-presentation. They also stress the existence of an alternative secretory route for MHC class I, which will be worthy of further studies.
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http://dx.doi.org/10.4049/jimmunol.1800353DOI Listing
January 2019

Propeptide glycosylation and galectin-3 binding decrease proteolytic activation of human proMMP-9/progelatinase B.

FEBS J 2019 03 30;286(5):930-945. Epub 2018 Nov 30.

Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Belgium.

Matrix metalloproteinases (MMPs) are secreted as proenzymes, containing propeptides that interact with the catalytic zinc, thereby controlling MMP activation. The MMP-9 propeptide is unique in the MMP family because of its post-translational modification with an N-linked oligosaccharide. ProMMP-9 activation by MMP-3 occurs stepwise by cleavage of the propeptide in an aminoterminal (pro-AT) and carboxyterminal (pro-CT) peptide. We chemically synthesized aglycosyl pro-AT and pro-CT and purified recombinant glycosylated pro-AT . First, we report new cleavage sites in the MMP-9 propeptide by MMP-3 and neutrophil elastase. Additionally, we demonstrated with the use of western blot analysis a higher resistance of glycosylated versus aglycosyl pro-AT against proteolysis by MMP-3, MMP-9, meprin α, neutrophil elastase and by protease-rich synovial fluids from rheumatoid arthritis patients. Moreover, we investigated the effect of glycosylation on proteolytic activation of human proMMP-9 with the use of zymography and dye-quenched gelatin cleavage analysis. Compared to recombinant Sf-9 proMMP-9 glycoforms, larger oligosaccharides of human neutrophil proMMP-9 increased resistance against proteolytic activation. Additionally, proMMP-9 from Congenital Disorder of Glycosylation patients, compared to healthy controls, showed a higher activation rate by MMP-3. Finally, we demonstrated that glycan-galectin-3 interactions reduced proMMP-9 activation. In conclusion, modification of MMP-9 propeptide glycosylation is a fine-tuning mechanism and co-determines the specific activity of MMP-9 in physiology and pathology. ENZYMES: MMP-9 EC 3.4.24.35, MMP-3 EC 3.4.24.17, meprin α EC 3.4.24.18, neutrophil elastase EC 3.4.21.37, trypsin EC 3.4.21.4 and PNGase F EC 3.5.1.52.
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http://dx.doi.org/10.1111/febs.14698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379967PMC
March 2019

Extracellular galectins as controllers of cytokines in hematological cancer.

Blood 2018 08 6;132(5):484-491. Epub 2018 Jun 6.

de Duve Institute, Université Catholique de Louvain, Brussels, Belgium; and.

Galectins and cytokines are both secreted proteins whose levels are prognosis factors for several cancers. Extracellular galectins bind to the glycans decorating glycoproteins and are overproduced in most cancers. Accumulative evidence shows that galectins regulate cytokines during cancer progression. Although galectins alter cytokine function by binding to the glycans decorating cytokines or their receptors, cytokines could also regulate galectin expression and function. This review revises these complex interactions and their clinical impact, particularly in hematological cancers.
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http://dx.doi.org/10.1182/blood-2018-04-846014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073326PMC
August 2018

How to measure the immunosuppressive activity of MDSC: assays, problems and potential solutions.

Cancer Immunol Immunother 2019 Apr 21;68(4):631-644. Epub 2018 May 21.

Research Division, Department of Otorhinolaryngology, West German Cancer Center, University Hospital Essen, Hufelandstrasse 55, 45122, Essen, Germany.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of mononuclear and polymorphonuclear myeloid cells, which are present at very low numbers in healthy subjects, but can expand substantially under disease conditions. Depending on disease type and stage, MDSC comprise varying amounts of immature and mature differentiation stages of myeloid cells. Validated unique phenotypic markers for MDSC are still lacking. Therefore, the functional analysis of these cells is of central importance for their identification and characterization. Various disease-promoting and immunosuppressive functions of MDSC are reported in the literature. Among those, the capacity to modulate the activity of T cells is by far the most often used and best-established read-out system. In this review, we critically evaluate the assays available for the functional analysis of human and murine MDSC under in vitro and in vivo conditions. We also discuss critical issues and controls associated with those assays. We aim at providing suggestions and recommendations useful for the contemporary biological characterization of MDSC.
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http://dx.doi.org/10.1007/s00262-018-2170-8DOI Listing
April 2019

Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration.

Nat Commun 2017 10 6;8(1):793. Epub 2017 Oct 6.

Ludwig Institute for Cancer Research, de Duve Institute, Université catholique de Louvain, Avenue Hippocrate 74, 1200, Brussels, Belgium.

The presence of T cells in tumors predicts overall survival for cancer patients. However, why most tumors are poorly infiltrated by T cells is barely understood. T-cell recruitment towards the tumor requires a chemokine gradient of the critical IFNγ-induced chemokines CXCL9/10/11. Here, we describe how tumors can abolish IFNγ-induced chemokines, thereby reducing T-cell attraction. This mechanism requires extracellular galectin-3, a lectin secreted by tumors. Galectins bind the glycans of glycoproteins and form lattices by oligomerization. We demonstrate that galectin-3 binds the glycans of the extracellular matrix and those decorating IFNγ. In mice bearing human tumors, galectin-3 reduces IFNγ diffusion through the tumor matrix. Galectin antagonists increase intratumoral IFNγ diffusion, CXCL9 gradient and tumor recruitment of adoptively transferred human CD8 T cells specific for a tumor antigen. Transfer of T cells reduces tumor growth only if galectin antagonists are injected. Considering that most human cytokines are glycosylated, galectin secretion could be a general strategy for tumor immune evasion.Most tumours are poorly infiltrated by T cells. Here the authors show that galectin-3 secreted by tumours binds both glycosylated IFNγ and glycoproteins of the tumour extracellular matrix, thus avoiding IFNγ diffusion and the formation of an IFNγ-induced chemokine gradient required for T cell infiltration.
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http://dx.doi.org/10.1038/s41467-017-00925-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630615PMC
October 2017

Treatment of Patients With Metastatic Cancer Using a Major Histocompatibility Complex Class II-Restricted T-Cell Receptor Targeting the Cancer Germline Antigen MAGE-A3.

J Clin Oncol 2017 Oct 15;35(29):3322-3329. Epub 2017 Aug 15.

Yong-Chen Lu, Linda L. Parker, Tangying Lu, Zhili Zheng, Mary Ann Toomey, Donald E. White, Xin Yao, Yong F. Li, Paul F. Robbins, Steven A. Feldman, Christopher A. Klebanoff, Stephanie L. Goff, Richard M. Sherry, Udai S. Kammula, James C. Yang, and Steven A. Rosenberg, National Cancer Institute, Bethesda, MD; Pierre van der Bruggen, Ludwig Institute for Cancer Research; De Duve Institute, Université Catholique de Louvain, Brussels; and Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Wallonia, Belgium; Christopher A. Klebanoff, Memorial Sloan Kettering Cancer Center, Parker Institute for Cancer Immunotherapy, New York, NY.

Purpose Adoptive transfer of genetically modified T cells is being explored as a treatment for patients with metastatic cancer. Most current strategies use genes that encode major histocompatibility complex (MHC) class I-restricted T-cell receptors (TCRs) or chimeric antigen receptors to genetically modify CD8 T cells or bulk T cells for treatment. Here, we evaluated the safety and efficacy of an adoptive CD4 T-cell therapy using an MHC class II-restricted, HLA-DPB1*0401-restricted TCR that recognized the cancer germline antigen, MAGE-A3 (melanoma-associated antigen-A3). Patients and Methods Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4 T cells, retrovirally transduced with MAGE-A3 TCR plus systemic high-dose IL-2. A cell dose escalation was conducted, starting at 10 total cells and escalating at half-log increments to approximately 10 cells. Nine patients were treated at the highest dose level (0.78 to 1.23 × 10 cells). Results Seventeen patients were treated. During the cell dose-escalation phase, an objective complete response was observed in a patient with metastatic cervical cancer who received 2.7 × 10 cells (ongoing at ≥ 29 months). Among nine patients who were treated at the highest dose level, objective partial responses were observed in a patient with esophageal cancer (duration, 4 months), a patient with urothelial cancer (ongoing at ≥ 19 months), and a patient with osteosarcoma (duration, 4 months). Most patients experienced transient fevers and the expected hematologic toxicities from lymphodepletion pretreatment. Two patients experienced transient grade 3 and 4 transaminase elevations. There were no treatment-related deaths. Conclusion These results demonstrate the safety and efficacy of administering autologous CD4 T cells that are genetically engineered to express an MHC class II-restricted antitumor TCR that targets MAGE-A3. This clinical trial extends the reach of TCR gene therapy for patients with metastatic cancer.
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http://dx.doi.org/10.1200/JCO.2017.74.5463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652397PMC
October 2017

Expression and prognostic relevance of MAGE-A3 and MAGE-C2 in non-small cell lung cancer.

Oncol Lett 2017 Mar 1;13(3):1609-1618. Epub 2017 Feb 1.

Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.

Melanoma-associated antigen (MAGE)-A3 and MAGE-C2 are antigens encoded by cancer-germline genes, and have been recognized as potential prognostic biomarkers and attractive targets for immunotherapy in multiple types of cancer. The present study aimed to analyze the clinicopathological significance of MAGE-A3/C2 expression in non-small cell lung cancer (NSCLC). The association between MAGE-A3/C2 mRNA and protein expression, and the pathological characteristics and overall survival of patients with NSCLC was analyzed. In addition, the functional role of MAGE-A3 in human NSCLC cell line A549 was examined . MAGE-A3/C2 mRNA expression was identified in 73% (151/206) and 53% (109/206) of patients with NSCLC, respectively. MAGE-A3/C2 protein expression was identified in 58% (44/76) and 53% (40/76) of NSCLC cases, respectively. MAGE-A3 mRNA expression was observed to be associated with smoking history, disease stage and lymph node metastasis. However, no association was identified between MAGE-C2 mRNA expression and the clinicopathological characteristics of patients with NSCLC. MAGE-A3/C2-positive patients had a poorer survival rate compared with MAGE-A3/C2-negative patients. Multivariate analysis identified that MAGE-A3 expression may serve as an independent marker of poor prognosis in patients with NSCLC. Downregulation of MAGE-A3 mRNA expression in A549 cells resulted in lower migration and colony formation rates, and a higher amount of epithelial marker and lower amount of mesenchymal marker expression compared with the control group. These results indicate that MAGE-A3 serves a role in NSCLC cell metastasis through the induction of epithelial-mesenchymal transition. In conclusion, MAGE-A3 may serve as a diagnostic and prognostic biomarker for patients with NSCLC, due to its association with tumor progression and poor clinical outcome.
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http://dx.doi.org/10.3892/ol.2017.5665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403542PMC
March 2017

Correlation between the high expression levels of cancer-germline genes with clinical characteristics in esophageal squamous cell carcinoma.

Histol Histopathol 2017 Aug 21;32(8):793-803. Epub 2016 Nov 21.

Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China.

Antigens encoded by cancer-germline genes are attractive targets for cancer immunotherapy. In this study, we aimed to evaluate the mRNA expression of cancer-germline genes, expression of the encoded proteins in patients with esophageal squamous cell carcinoma (ESCC) and their correlations with clinical characteristics. In addition, the effects of downregulation cancer-germline genes on ESCC cells were assessed in vitro. Our results showed that cancer-germline genes were frequently expressed in ESCC samples. The positive rates of in ESCC samples were: 87% of MAGE-A3, 60% of MAGE-A4, 65% of MAGE-C2, and 20% of NY-ESO-1 at mRNA level. MAGE-A3 expression was associated with age, lymph node metastasis and tumor stage (all P<0.05), while MAGE-C2 expression was only associated with tumor stage (P<0.05). Furthermore, the MAGE-A3 expressing patients had a poorer overall survival (P<0.05). Multivariate analysis identified MAGE-A3 as an independent poor prognostic marker in ESCC. In vitro assay, ESCC cell lines treated with specific siRNAs to down-regulate MAGE-A3 and MAGE-C2 resulted in decreased colony-formation and migration ability (P<0.05). Epithelial marker E-cadherin was up-regulated in siRNA-MAGE-A3/C2 cells compared to controls, whereas mesenchymal markers Vimentin, N-cadherin and Slug were downregulated (all P<0.05), suggesting a role for MAGE-A3/C2 in ESCC metastasis through inducing epithelial-mesenchymal transition. The present study revealed that cancer-germline genes and their encoded proteins were frequently expressed in ESCC tumor samples and were related to poor prognosis. Thus, cancer-germline genes may serve as useful biomarkers and potential targets for ESCC patients.
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http://dx.doi.org/10.14670/HH-11-847DOI Listing
August 2017

Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells.

Part Fibre Toxicol 2016 08 23;13(1):46. Epub 2016 Aug 23.

Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Experimentale et Clinique (IREC), Université catholique de Louvain, Avenue Mounier 53 bte B1.52.12, 1200, Brussels, Belgium.

Background: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells.

Methods: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells.

Results: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development.

Conclusions: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer.
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http://dx.doi.org/10.1186/s12989-016-0158-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4994252PMC
August 2016

A major secretory defect of tumour-infiltrating T lymphocytes due to galectin impairing LFA-1-mediated synapse completion.

Nat Commun 2016 07 22;7:12242. Epub 2016 Jul 22.

Ludwig Institute for Cancer Research, WELBIO and de Duve Institute, Université catholique de Louvain, B-1200 Brussels, Belgium.

Surface galectin has been shown to contribute to dysfunctions of human tumour-infiltrating lymphocytes (TILs). We show here that galectin-covered CD8 TILs produce normal amounts of intracellular cytokines, but fail to secrete them because of defective actin rearrangements at the synapse. The non-secreting TILs also display reduced adhesion to their targets, together with defective LFA-1 recruitment and activation at the synapse. These defects are relieved by releasing surface galectin. As mild LFA-1 blockade on normal blood T cells emulate the defects of galectin-covered TILs, we conclude that galectin prevents the formation of a functional secretory synapse by preventing optimal LFA-1 triggering. Our results highlight a major secretory defect of TILs that is not revealed by widely used intracellular cytokine immunomonitoring assays. They also provide additional insights into the T-cell response, by showing that different thresholds of LFA-1 triggering are required to promote the intracellular production of cytokines and their secretion.
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http://dx.doi.org/10.1038/ncomms12242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961845PMC
July 2016

CD8 T-cell responses against the immunodominant Theileria parva peptide Tp249-59 are composed of two distinct populations specific for overlapping 11-mer and 10-mer epitopes.

Immunology 2016 10 25;149(2):172-85. Epub 2016 Jul 25.

Division of Immunity and Infection, The Roslin Institute, The University of Edinburgh, Midlothian, UK.

Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide-MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106 ), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249-59 -reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross-reactivity. The Tp249-59 and Tp250-59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide-MHC I complexes adopt structurally different conformations and Tcell receptor β sequence analysis showed that Tp249-59 and Tp250-59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant.
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http://dx.doi.org/10.1111/imm.12637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011678PMC
October 2016

Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.

J Immunother 2016 06;39(5):191-201

*Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD †Cellular Biomedicine Group Inc., Cupertino, CA ‡Ludwig Institute for Cancer Research and WELBIO §De Duve Institute, Université Catholique de Louvain, Brussels, Belgium.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.
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http://dx.doi.org/10.1097/CJI.0000000000000123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4947411PMC
June 2016

Long-Peptide Cross-Presentation by Human Dendritic Cells Occurs in Vacuoles by Peptide Exchange on Nascent MHC Class I Molecules.

J Immunol 2016 Feb 20;196(4):1711-20. Epub 2016 Jan 20.

Ludwig Institute for Cancer Research, Brussels B-1200, Belgium; Walloon Excellence in Life Sciences and Biotechnology, Brussels B-1200, Belgium; de Duve Institute, Université Catholique de Louvain, Brussels B-1200, Belgium;

Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides.
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http://dx.doi.org/10.4049/jimmunol.1501574DOI Listing
February 2016

Consensus nomenclature for CD8 T cell phenotypes in cancer.

Oncoimmunology 2015 Apr 25;4(4):e998538. Epub 2015 Feb 25.

National Center for Cancer Care & Research , Doha, Qatar.

Whereas preclinical investigations and clinical studies have established that CD8 T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8 T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8 T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8 T cell immunity, leading to the emergence of dysfunctional CD8 T cells. The existence of different types of CD8 T cells in cancer calls for a more precise definition of the CD8 T cell immune phenotypes in cancer and the abandonment of the generic terms "pro-tumor" and "antitumor." Based on recent studies investigating the functions of CD8 T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8 T cells in cancer.
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http://dx.doi.org/10.1080/2162402X.2014.998538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485711PMC
April 2015

Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice.

Nat Biotechnol 2015 Apr 16;33(4):402-7. Epub 2015 Mar 16.

1] Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. [2] Institute of Immunology, Charité Campus Buch, Berlin, Germany.

Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⁺ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.
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http://dx.doi.org/10.1038/nbt.3147DOI Listing
April 2015

Sugars boost exhausted tumor-infiltrating lymphocytes by counteracting immunosuppressive activities of galectins.

Oncoimmunology 2014;3:e28783. Epub 2014 Apr 29.

Ludwig Institute for Cancer Research Brussels; WELBIO; and de Duve Institute; Université Catholique de Louvain; Brussels, Belgium.

Galectins released by tumor cells and macrophages can bind surface glycoproteins of tumor-infiltrating lymphocytes (TILs), forming glycoprotein-galectin lattices with immunosuppressive activities. Specifically, TILs covered by galectin-3 are unable to secrete cytokines after stimulation. Treating TILs ex vivo with galectin antagonists for a few hours boosts their functions. Several galectin antagonists are currently available for clinical trials.
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http://dx.doi.org/10.4161/onci.28783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091051PMC
February 2021

Absence of recognition of common melanocytic antigens by T cells isolated from the cerebrospinal fluid of a Vogt-Koyanagi-Harada patient.

Mol Vis 2014 2;20:956-69. Epub 2014 Jul 2.

Ludwig Institute for Cancer Research and de Duve Institute, Université catholique de Louvain, 74 av. Hippocrate, Brussels, Belgium.

Purpose: Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disease characterized by inaugural uveomeningitidis and hearing loss and at late stages a depigmentation in eyes and skin. Melanocytes are the cells common to the four affected tissues, namely eye, brain, inner ear, and skin. Melanocytes are therefore considered as the source of self-antigens. The melanocytic proteins tyrosinase-related protein-1 (TRP1), TRP2, tyrosinase, and gp100 have been proposed as the proteins targeted by autoreactive T cells from VKH patients bearing human leukocyte antigen (HLA)-DRB1*04:05, the HLA allele classically associated with VKH disease. The objective of this work was to determine the antigens recognized by a large number of potentially autoreactive CD4 T lymphocytes obtained from the cerebrospinal fluid of one VKH patient who did not express HLA-DRB1*04:05.

Methods: T cells were isolated from the cerebrospinal fluid of a newly diagnosed HLA-DRB1*14:01,*15:03;-DPB1*01:01,*04:02 patient in the acute phase of the VKH disease and cloned by limiting dilution. Each of the 107 T cell clones, of which 90% were CD4(+), was tested for its ability to secrete cytokines upon contact with autologous antigen-presenting cells loaded with either of the melanocytic proteins TRP1, TRP2, tyrosinase, gp100, Melan-A and KU-MEL-1. The sensitivity of our recombinant bacteria-based approach was validated with a CD4 T cell clone with known antigen specificity. The ability of each of the 107 clones to secrete cytokines upon nonspecific stimulation was verified.

Results: None of the 107 T cell clones was able to secrete tumor necrosis factor-α, interferon-γ, interleukin (IL)-5, or IL-17 upon contact with autologous B cells loaded with any of the six common melanocytic proteins. Nine clones secreted high-level IL-17 upon stimulation with beads coated with antibodies.

Conclusions: The self-antigens that triggered the VKH disease in this patient probably derive from proteins other than the six melanocytic proteins mentioned above. Further study of antigens that are recognized by potential autoreactive T cells from VKH patients is likely to benefit from testing a broader set of melanocytic proteins.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077848PMC
September 2014

A short treatment with galactomannan GM-CT-01 corrects the functions of freshly isolated human tumor-infiltrating lymphocytes.

Clin Cancer Res 2014 Apr 13;20(7):1823-33. Epub 2014 Feb 13.

Authors' Affiliations: Ludwig Institute for Cancer Research Brussels and WELBIO; de Duve Institute; Departments of Gynecology and Oncology, Cliniques Universitaires Saint-Luc, Université catholique de Louvain; Laboratory of Molecular and Cellular Therapy, Department of Immunology-Physiology, Medical School of the Vrije Universiteit Brussel, Brussels; and Department of Oncology, Grand Hôpital de Charleroi, Charleroi, Belgium.

Purpose: Several galectins are released by tumor cells and macrophages and accumulate in the tumor microenvironment. Galectin-1 and -3 were found to bind to glycosylated receptors at the surface of tumor-infiltrating lymphocytes (TIL), forming glycoprotein-galectin lattices that could reduce the motility and therefore the functionality of surface molecules. In contrast to blood T cells, human TIL show defective IFN-γ secretion upon ex vivo stimulation. We have previously shown that extracellular galectin-3 participates in the impairment of TIL functions. Indeed, disruption of glycoprotein-galectin-3 lattices using anti-galectin-3 antibodies, or N-acetyllactosamine as a competing sugar, boosted cytokine secretion by TIL. Here we have tested a clinical grade galectin antagonist: GM-CT-01, a galactomannan obtained from guar gum reported to be safe in more than 50 patients with cancer.

Experimental Design: TIL were isolated from human tumor ascites, treated for 2 to 20 hours with galectin antagonists and tested for function.

Results: We found that GM-CT-01 boosts cytotoxicity of CD8(+) TIL and their IFN-γ secretion in a dose-dependent manner. Treating TIL obtained from patients with various cancers, during a few hours, resulted in an increased IFN-γ secretion in up to 80% of the samples.

Conclusions: These observations pave the way for investigating the potential benefit of this galectin antagonist in patients with cancer, alone or combined with cancer vaccination, in order to correct in vivo impaired functions of TIL.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-2459DOI Listing
April 2014

Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy.

Nat Rev Cancer 2014 Feb;14(2):135-46

1] de Duve Institute and the Université catholique de Louvain, B-1200 Brussels, Belgium. [2] Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium.

In this Timeline, we describe the characteristics of tumour antigens that are recognized by spontaneous T cell responses in cancer patients and the paths that led to their identification. We explain on what genetic basis most, but not all, of these antigens are tumour specific: that is, present on tumour cells but not on normal cells. We also discuss how strategies that target these tumour-specific antigens can lead either to tumour-specific or to crossreactive T cell responses, which is an issue that has important safety implications in immunotherapy. These safety issues are even more of a concern for strategies targeting antigens that are not known to induce spontaneous T cell responses in patients.
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http://dx.doi.org/10.1038/nrc3670DOI Listing
February 2014

Database of T cell-defined human tumor antigens: the 2013 update.

Cancer Immun 2013 15;13:15. Epub 2013 Jul 15.

Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium.

The plethora of tumor antigens that have been--and are still being--defined required systematization to provide a comprehensive overview of those tumor antigens that are the most relevant targets for cancer immunotherapy approaches. Here, we provide a new update of a peptide database resource that we initiated many years ago. This database compiles all human antigenic peptides described in the literature that fulfill a set of strict criteria needed to ascertain their actual "tumor antigen" nature, as we aim at guiding scientists and clinicians searching for appropriate cancer vaccine candidates (www.cancerimmunity.org/peptide). In this review, we revisit those criteria in light of recent findings related to antigen processing. We also introduce the 29 new tumor antigens that were selected for this 2013 update. Two of the new peptides show unusual features, which will be briefly discussed. The database now comprises a total of 403 tumor antigenic peptides.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718731PMC
May 2014

IRF1 and NF-kB restore MHC class I-restricted tumor antigen processing and presentation to cytotoxic T cells in aggressive neuroblastoma.

PLoS One 2012 5;7(10):e46928. Epub 2012 Oct 5.

Paediatric Haematology/Oncology Department, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046928PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465322PMC
April 2013

Inefficient exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be improved by acid treatment or fixation of target cells.

Eur J Immunol 2012 Jun;42(6):1417-28

Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.
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http://dx.doi.org/10.1002/eji.201141954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766947PMC
June 2012

TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets.

Clin Dev Immunol 2012 12;2012:586314. Epub 2012 Feb 12.

Laboratory of Experimental Tumor Immunology, Department of Medical Oncology, Erasmus MC, 3015 GE, Rotterdam, The Netherlands.

Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαβ genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vβ28 and TCR-Vα38/Vβ2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.
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http://dx.doi.org/10.1155/2012/586314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287115PMC
March 2013

Dendritic cells loaded with mRNA encoding full-length tumor antigens prime CD4+ and CD8+ T cells in melanoma patients.

Mol Ther 2012 May 28;20(5):1063-74. Epub 2012 Feb 28.

Laboratory of Molecular and Cellular Therapy, Department of Immunology-Physiology, Medical School of the Vrije Universiteit Brussel (VUB), Brussels, Belgium.

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.
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http://dx.doi.org/10.1038/mt.2012.11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345975PMC
May 2012

Loss of effector function of human cytolytic T lymphocytes is accompanied by major alterations in N- and O-glycosylation.

J Biol Chem 2012 Mar 13;287(14):11240-51. Epub 2012 Feb 13.

Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom.

Most human tumors are not eliminated by the immune system, and therapeutic vaccination shows poor results, a fact that can be explained at least partially by an immunosuppressive tumor microenvironment that is abundant in galectin-3. On cytolytic T lymphocyte (CTL) clones, maintained in culture by regular stimulation, recently activated CTLs present low effector functions. However, these functions are restored after a short treatment with LacNAc. The latter, which is in agreement with the glycoprotein-galectin lattice concept involving reduced motility, poses the question why galectin-3 ligands improve effector functions. We employed ultrasensitive MALDI-TOF-MS on resting and recently activated CTL clones combined with various glycosidase digestions and GC-MS linkage analyses. Our results showed that compared with the resting CTLs, the N-glycans of the recently activated CTLs consisted of (i) larger LacNAc oligomers of which a significant portion was longer than four-units and (ii) more multi-antennary structures. Interestingly, our results showed that the poly-LacNAc appeared to be equally distributed on all available N-glycan branches and not selectively enriched on a specific branch. The above structural alterations in the recently activated CTLs are expected to increase the galectin-3-LacNAc lattices and multivalent interactions and, therefore, reduce the motility of surface glycoproteins, such as the T-cell receptor. These findings suggest that the loss of effector functions on CTLs may be linked to reduced motility of surface glycoproteins. In addition, our results showed that recently activated CTLs had a reduced abundance of NeuAcα2,6-linked N-glycans and an increased abundance of disialylated core 1 and monosialylated core 2 O-glycan structures.
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http://dx.doi.org/10.1074/jbc.M111.320820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322850PMC
March 2012