Daniel Speiser

Publications (35)223.73 Total impact

• Article: Interplay between T Cell Receptor Binding Kinetics and the Level of Cognate Peptide Presented by Major Histocompatibility Complexes Governs CD8+ T Cell Responsiveness

  Melita Irving, Vincent Zoete, Michael Hebeisen, Daphné Schmid, Petra Baumgartner, Philippe Guillaume, Pedro Romero, Daniel Speiser, Immanuel Luescher, Nathalie Rufer, Olivier Michielin
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  ABSTRACT: Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1157–165-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8+ T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8+ T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca2+ flux for CD8+ T cells expressing TCR within a dissociation constant (KD) range of ∼1–5 μm. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with KD < ∼1 μm, irrespective of CD8 co-engagement and of half-life (t1/2 = ln 2/koff) values. With increased peptide concentration, however, the activity levels of CD8+ T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8+ T cells, as well as for peptide vaccination strategies.
  Journal of Biological Chemistry 06/2012; 287(27):23068-23078. · 4.77 Impact Factor
• Article: Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.

  Melita Irving, Vincent Zoete, Michael Hebeisen, Daphné Schmid, Petra Baumgartner, Philippe Guillaume, Pedro Romero, Daniel Speiser, Immanuel Luescher, Nathalie Rufer, Olivier Michielin
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  ABSTRACT: Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.
  Journal of Biological Chemistry 05/2012; 287(27):23068-78. · 4.77 Impact Factor
• Article: Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma.

  Sergey I Nikolaev, Donata Rimoldi, Christian Iseli, Armand Valsesia, Daniel Robyr, Corinne Gehrig, Keith Harshman, Michel Guipponi, Olesya Bukach, Vincent Zoete, Olivier Michielin, Katja Muehlethaler, Daniel Speiser, Jacques S Beckmann, Ioannis Xenarios, Thanos D Halazonetis, C Victor Jongeneel, Brian J Stevenson, Stylianos E Antonarakis
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  ABSTRACT: We performed exome sequencing to detect somatic mutations in protein-coding regions in seven melanoma cell lines and donor-matched germline cells. All melanoma samples had high numbers of somatic mutations, which showed the hallmark of UV-induced DNA repair. Such a hallmark was absent in tumor sample-specific mutations in two metastases derived from the same individual. Two melanomas with non-canonical BRAF mutations harbored gain-of-function MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) mutations, resulting in constitutive ERK phosphorylation and higher resistance to MEK inhibitors. Screening a larger cohort of individuals with melanoma revealed the presence of recurring somatic MAP2K1 and MAP2K2 mutations, which occurred at an overall frequency of 8%. Furthermore, missense and nonsense somatic mutations were frequently found in three candidate melanoma genes, FAT4, LRP1B and DSC1.
  Nature Genetics 12/2011; 44(2):133-9. · 35.53 Impact Factor
• Article: Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

  Julien Schmidt, Philippe Guillaume, Melita Irving, Petra Baumgaertner, Daniel Speiser, Immanuel F Luescher
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  ABSTRACT: MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.
  Journal of Biological Chemistry 12/2011; 286(48):41723-35. · 4.77 Impact Factor
• Article: Reversible MHC I-peptide multimers containing Ni2+NTA peptides and his tags improve analysis and sorting of CD8+ T cells

  Julien Schmidt, Philippe Guillaume, Melita Irving, Petra Baumgaertner, Daniel Speiser, Immanuel F. Luescher
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  ABSTRACT: MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin streptavidin (PE SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni2+ nitrilotriacetic acid (NTA) with oligo-histidines. We synthesized biotinylated linear mono, di and tetra NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His6, His12 or a 2xHis6 tag by surface plasmon resonance on SA coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His6 < His12 < 2xHis6 and NTA1 < NTA2 < NTA4, respectively, depended on the configuration of the NTA moieties and increased to pico-molar KD for the combination of a 2xHis6 His tag and a 2xNi2+NTA2. We demonstrate that HLA-A2-2xHis6-peptide multimers containing either Ni2+ NTA4-biotin and PE-SA or PE-NTA4 stained influenza and Melan-A-specific CD8+ T cells equal of better than conventional multimers. While these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.
  Journal of Biological Chemistry 10/2011; · 4.77 Impact Factor
• Article: MAGE-A3 and MAGE-A4 specific CD4(+) T cells in head and neck cancer patients: detection of naturally acquired responses and identification of new epitopes.

  Valérie Cesson, Jean-Paul Rivals, Anette Escher, Elsa Piotet, Kris Thielemans, Vilmos Posevitz, Danijel Dojcinovic, Philippe Monnier, Daniel Speiser, Luc Bron, Pedro Romero
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  ABSTRACT: Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlapping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from healthy donors and seven head and neck cancer patients. CD4(+) T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from 7/7 cancer patients analyzed. Moreover, we identified six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3(111-125) and MAGE-A3(161-175) epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were specific for new helper epitopes. These data suggest that naturally acquired CD4(+) T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumor.
  Cancer Immunology and Immunotherapy 01/2011; 60(1):23-35. · 3.70 Impact Factor
• Source

  Available from: Takuya Yamamoto

  Article: Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets.

  Bruno Salaun, Takuya Yamamoto, Bassam Badran, Yasuko Tsunetsugu-Yokota, Antoine Roux, Lukas Baitsch, Redouane Rouas, Hussein Fayyad-Kazan, Petra Baumgaertner, Estelle Devevre, Anirudh Ramesh, Marion Braun, Daniel Speiser, Brigitte Autran, Philippe Martiat, Victor Appay, Pedro Romero
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  ABSTRACT: The differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown. In order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway. We found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease. This study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.
  Journal of Translational Medicine 01/2011; 9:44. · 3.41 Impact Factor
• Article: Correction: Frequent MAGE Mutations in Human Melanoma.

  Otavia L Caballero, Qi Zhao, Donata Rimoldi, Brian J Stevenson, Suzanne Svobodová, Sylvie Devalle, Ute F Röhrig, Anna Pagotto, Olivier Michielin, Daniel Speiser, [......], Cailian Liu, Tanja Pejovic, Kunle Odunsi, Francis Brasseur, Benoit J Van den Eynde, Lloyd J Old, Xin Lu, Jonathan Cebon, Robert L Strausberg, Andrew J Simpson
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  ABSTRACT: [This corrects the article on p. e12773 in vol. 5.].
  PLoS ONE 01/2010; 5(11). · 4.09 Impact Factor
• Source

  Available from: Francis Brasseur

  Article: Frequent MAGE mutations in human melanoma.

  Otavia L Caballero, Qi Zhao, Donata Rimoldi, Brian J Stevenson, Suzanne Svobodová, Sylvie Devalle, Ute F Röhrig, Anna Pagotto, Olivier Michielin, Daniel Speiser, [......], Cailian Liu, Tanja Pejovic, Kunle Odunsi, Francis Brasseur, Benoit J Van den Eynde, Lloyd J Old, Xin Lu, Jonathan Cebon, Robert L Strausberg, Andrew J Simpson
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  ABSTRACT: Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype. We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1. These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.
  PLoS ONE 01/2010; 5(9). · 4.09 Impact Factor
• Article: Recent advances and hurdles in melanoma immunotherapy.

  Camilla Jandus, Daniel Speiser, Pedro Romero
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  ABSTRACT: Worldwide incidence of malignant melanoma has been constantly increasing during the last years. Surgical excision is effective when primary tumours are thin. At later disease stages patients often succumb, due to failure of metastasis control. Therefore, great efforts have been made to develop improved strategies to treat metastatic melanoma patients. In the search for novel treatments during the last two decades, immunotherapy has occupied a prominent place. Numerous early phase immunotherapy clinical trials, generally involving small numbers of patients each time, have been reported: significant tumour-specific immune responses could often be measured in patients upon treatments. However, clinical responses remain at a dismal low rate. In some anecdotal cases, objective clinical benefit was more frequently observed among immune responders than immune non-responders. This clearly calls for a better understanding of protective immunity against tumours as well as the cross talk taking place between tumours and the immune system. Here we discuss advances and limitations of specific immunotherapy against human melanoma in the light of the literature from the last 5 yr.
  Pigment Cell & Melanoma Research 10/2009; 22(6):711-23. · 5.06 Impact Factor
• Article: Vaccination of melanoma patients with Melan-A/Mart-1 peptide and Klebsiella outer membrane protein p40 as an adjuvant.

  Danielle Lienard, Marie-Françoise Avril, Frédérique-Anne Le Gal, Petra Baumgaertner, Wim Vermeulen, Astrid Blom, Christine Geldhof, Donata Rimoldi, Sonia Pagliusi, Pedro Romero, Pierre-Yves Dietrich, Nathalie Corvaia, Daniel E Speiser
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  ABSTRACT: Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.
  Journal of immunotherapy (Hagerstown, Md.: 1997) 09/2009; 32(8):875-83. · 3.20 Impact Factor
• Article: Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides.

  Camilla Jandus, Gilles Bioley, Jean-Paul Rivals, Jean Dudler, Daniel Speiser, Pedro Romero
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  ABSTRACT: A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases. Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis. We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production. These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.
  Arthritis & Rheumatism 09/2008; 58(8):2307-17. · 7.87 Impact Factor
• Article: Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood.

  Camilla Jandus, Gilles Bioley, Daniel E Speiser, Pedro Romero
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  ABSTRACT: Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.
  Cancer Immunology and Immunotherapy 05/2008; 57(12):1795-805. · 3.70 Impact Factor
• Article: Sensitive gene expression profiling of human T cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages.

  Victor Appay, Andreas Bosio, Stefanie Lokan, Yvonne Wiencek, Christian Biervert, Daniel Küsters, Estelle Devevre, Daniel Speiser, Pedro Romero, Nathalie Rufer, Serge Leyvraz
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  ABSTRACT: The differentiation of CD4(+) or CD8(+) T cells following priming of naive cells is central in the establishment of the immune response against pathogens or tumors. However, our understanding of this complex process and the significance of the multiple subsets of differentiation remains controversial. Gene expression profiling has opened new directions of investigation in immunobiology. Nonetheless, the need for substantial amount of biological material often limits its application range. In this study, we have developed procedures to perform microarray analysis on amplified cDNA from low numbers of cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4(+) or CD8(+) T cell differentiation. Surprisingly, our data revealed that while CD4(+) and CD8(+) T cell gene expression programs diverge at early stages of differentiation, they become increasingly similar as cells reach a late differentiation stage. This suggests that functional heterogeneity between Ag experienced CD4(+) and CD8(+) T cells is more likely to be located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.
  The Journal of Immunology 01/2008; 179(11):7406-14. · 5.79 Impact Factor
• Article: New approaches in metastatic melanoma: biological and molecular targeted therapies.

  Ferdy J Lejeune, Donata Rimoldi, Daniel Speiser
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  ABSTRACT: Classical metastatic melanoma therapy is disappointing but important progress has been made in the understanding of melanoma biology. Genetic lesions and several intracellular signaling pathways that could serve as targets for novel therapy have been identified and a number of new agents are under evaluation. Promising tumor cell targets were identified in the cell membrane, cytoplasm and nucleus. New therapeutic approaches, besides monoclonal antibodies and vaccination, include an increasing number of small molecules that have been shown to interfere restrictively with intracellular signaling pathways in melanoma and decrease proliferation, survival, migration or invasion. Other agents can interfere with stromal components of melanoma, such as angiogenesis and components of the immune system.
  Expert Review of Anti-infective Therapy 06/2007; 7(5):701-13. · 2.65 Impact Factor
• Article: Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor.

  Julie Tabiasco, Estelle Devêvre, Nathalie Rufer, Bruno Salaun, Jean-Charles Cerottini, Daniel Speiser, Pedro Romero
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  ABSTRACT: TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.
  The Journal of Immunology 01/2007; 177(12):8708-13. · 5.79 Impact Factor
• Article: Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.

  Philippe Guillaume, Petra Baumgaertner, Georgi S Angelov, Daniel Speiser, Immanuel F Luescher
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  ABSTRACT: The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.
  The Journal of Immunology 10/2006; 177(6):3903-12. · 5.79 Impact Factor
• Article: Tumoral and immunologic response after vaccination of melanoma patients with an ALVAC virus encoding MAGE antigens recognized by T cells.

  Nicolas van Baren, Marie-Claude Bonnet, Brigitte Dréno, Amir Khammari, Thierry Dorval, Sophie Piperno-Neumann, Danielle Liénard, Daniel Speiser, Marie Marchand, Vincent G Brichard, [......], Pierre-Yves Dietrich, Dominique Maraninchi, Susanne Osanto, Ralf G Meyer, Gerd Ritter, Philippe Moingeon, Jim Tartaglia, Pierre van der Bruggen, Pierre G Coulie, Thierry Boon
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  ABSTRACT: To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3(168-176) and MAGE-1(161-169), which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3(168-176) and MAGE-1(161-169) peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient.
  Journal of Clinical Oncology 01/2006; 23(35):9008-21. · 18.37 Impact Factor
• Article: CD4+ T cell responses to SSX-4 in melanoma patients.

  Maha Ayyoub, Andrea Merlo, Charles S Hesdorffer, Donata Rimoldi, Daniel Speiser, Jean-Charles Cerottini, Yao-Tseng Chen, Lloyd J Old, Stefan Stevanovic, Danila Valmori
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  ABSTRACT: Genes of the synovial sarcoma X breakpoint (SSX) family are expressed in different human tumors, including melanomas, but not in adult somatic tissues. Because of their specific expression at the tumor site, SSX-encoded Ags are potential targets for anticancer immunotherapy. In this study, we have analyzed CD4+ T cell responses directed against the Ag encoded by SSX-4. Upon in vitro stimulation of PBMC from four melanoma patients bearing Ag-expressing tumors with a pool of long peptides spanning the protein sequence, we detected and isolated SSX-4-specific CD4+ T cells recognizing several distinct antigenic sequences, mostly restricted by frequently expressed HLA class II alleles. The majority of the identified sequences were located within the Krüppel-associated box domain in the N-terminal region of the protein, indicating a high potential immunogenicity of this region. Together our data document the existence of CD4+ T cells specific for multiple SSX-4 derived sequences in circulating lymphocytes from melanoma patients and encourage further studies to assess the impact of SSX-4-specific T cell responses on disease evolution in cancer patients.
  The Journal of Immunology 05/2005; 174(8):5092-9. · 5.79 Impact Factor
• Article: Distinct but overlapping T helper epitopes in the 37-58 region of SSX-2.

  Maha Ayyoub, Andrea Merlo, Charles S Hesdorffer, Daniel Speiser, Donata Rimoldi, Jean-Charles Cerottini, Gerd Ritter, Yao-Tseng Chen, Lloyd J Old, Stefan Stevanovic, Danila Valmori
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  ABSTRACT: Because of their specific expression in tumors of different histological types, the products of the SSX genes are important candidate targets for development of cancer vaccines. We have previously identified two immunodominant SSX-2-derived T cell epitopes recognized by HLA-A2-restricted CD8+ T cells (SSX-2 41-49) and HLA-DR11-restricted CD4+ T cells (SSX-2 45-59), respectively. In this study, we report the identification of an HLA-DR3-restricted epitope mapping to the 37-51 region of SSX-2, overlapping both previously identified epitopes. As about one fifth of individuals from several major ethnic groups express HLA-DR3, the identification of this epitope significantly increases the percent of patients that are expected to mount specific CD4+ T cell responses following vaccination with peptides in this region of SSX-2. Retrieval of multiple overlapping epitopes in a defined region of SSX-2 protein suggests the presence of a "hot spot" for T cell recognition that may prove sufficient for the induction of immune responses.
  Clinical Immunology 02/2005; 114(1):70-8. · 4.05 Impact Factor