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ABSTRACT: Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.
Vaccine 03/2013; · 3.77 Impact Factor
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ABSTRACT: Background. Generation of tumor-antigen specific CD4+ T helper (TH) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4+ T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematological cancers, to implement an in vitro priming platform allowing the generation of ESO-specific TH lines. Design and Methods. We isolated phenotypically defined CD4+ T cell subpopulations from circulating lymphocytes of DR52b+ healthy donors by flow-cytometry cell sorting and stimulated them in vitro with peptide ESO119-143, autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO119-143 tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer+ cells by co-staining with TCR variable beta chain (BV) specific antibodies. We isolated ESO-tetramer+ cells by flow-cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific TH lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Results. Using this approach, we could consistently generate ESO-tetramer+-TH lines from conventional CD4+CD25- naïve and central memory populations, but not from effector memory populations or CD4+CD25+ Treg. In vitro-primed TH lines recognized ESO with affinities comparable to ESO-tetramer+ cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. Conclusions. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal TH lines from non-immune individuals, an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.
Haematologica 08/2012; · 6.42 Impact Factor
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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
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Melita Irving,
Vincent Zoete,
Michael Hebeisen,
Daphné Schmid,
Petra Baumgartner,
Philippe Guillaume,
Pedro Romero,
Daniel Speiser, Immanuel Luescher,
Nathalie Rufer,
Olivier Michielin
[show abstract]
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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
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ABSTRACT: In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.
Cancer Immunology and Immunotherapy 11/2011; 61(6):817-26. · 3.70 Impact Factor
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Nassima Redjimi,
Karine Duperrier-Amouriaux,
Isabelle Raimbaud, Immanuel Luescher,
Danijel Dojcinovic,
Jean-Marc Classe,
Dominique Berton-Rigaud,
Jean-Sébastien Frenel,
Emmanuelle Bourbouloux,
Danila Valmori,
Maha Ayyoub
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ABSTRACT: Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.
PLoS ONE 01/2011; 6(7):e22845. · 4.09 Impact Factor
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ABSTRACT: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO(119-143) region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO(119-143) tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1).
We generated tetramers of DRB1*0101 incorporating peptide ESO(119-143) using a previously described strategy. We assessed ESO(119-143)-specific CD4 T cells in peptide-stimulated postvaccine cultures using the tetramers. We isolated DR1/ESO(119-143) tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO(119-143) tetramer(+) T cells ex vivo and characterized them phenotypically.
Staining of cultures from vaccinated patients with DR1/ESO(119-143) tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO(123-137) as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO(119-143) tetramer(+) cells using T cell receptor (TCR) β chain variable region (Vβ)-specific antibodies, we identified several frequently used Vβ. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells.
The development of DR1/ESO(119-143) tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients.
Clinical Cancer Research 09/2010; 16(18):4607-15. · 7.74 Impact Factor
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Daisuke Muraoka,
Takuma Kato,
Linan Wang,
Yuka Maeda,
Takuro Noguchi,
Naozumi Harada,
Kazuyoshi Takeda,
Hideo Yagita,
Philippe Guillaume, Immanuel Luescher,
Lloyd J Old,
Hiroshi Shiku,
Hiroyoshi Nishikawa
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ABSTRACT: CD8(+) CTLs play a critical role in antitumor immunity. However, vaccination with synthetic peptide containing CTL epitopes has not been generally effective in inducing protective antitumor immunity. In this study, we addressed the detailed mechanism(s) involved in this failure using a new tumor model of BALB/c transplanted tumors expressing NY-ESO-1, an extensively studied human cancer/testis Ag. Whereas peptide immunization with an H2-D(d)-restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81-88) induced NY-ESO-1(81-88)-specific CD8(+) T cells in draining lymph nodes and spleens, tumor growth was significantly enhanced. Single-cell analysis of specific CD8(+) T cells revealed that peptide immunization caused apoptosis of >80% of NY-ESO-1(81-88)-specific CD8(+) T cells at tumor sites and repetitive immunization further diminished the number of specific CD8(+) T cells. This phenomenon was associated with elevated surface expression of Fas and programmed death-1. When peptide vaccination was combined with an adjuvant, a TLR9 ligand CpG, the elevated Fas and programmed death-1 expression and apoptosis induction were not observed, and vaccine with peptide and CpG was associated with strong tumor growth inhibition. Selection of appropriate adjuvants is essential for development of effective cancer vaccines, with protection of effector T cells from peptide vaccine-induced apoptosis being a prime objective.
The Journal of Immunology 09/2010; 185(6):3768-76. · 5.79 Impact Factor
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ABSTRACT: MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4(+) T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b(+) cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4(+) T cells, include central and transitional memory polyfunctional populations, and do not include CD4(+)CD25(+)CD127(-) regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.
Proceedings of the National Academy of Sciences 04/2010; 107(16):7437-42. · 9.68 Impact Factor
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ABSTRACT: The CD8alphabeta heterodimer is integral to the selection of the class I-restricted lineage in the thymus; however, the contribution of the CD8beta chain to coreceptor function is poorly understood. To understand whether the CD8beta membrane proximal stalk region played a role in coreceptor function, we substituted it with the corresponding sequence from the CD8alpha polypeptide and expressed the hybrid molecule in transgenic mice in place of endogenous CD8beta. Although the stalk-swapped CD8beta was expressed on the cell surface as a disulfide-bonded heterodimer at equivalent levels of expression to an endogenous CD8beta molecule, it failed to restore selection of CD8(+) class I MHC-restricted T cells and it altered the response of peripheral T cells. Thus, the stalk region of the CD8beta polypeptide has an essential role in ensuring functionality of the CD8alphabeta heterodimer and its replacement compromises the interaction of CD8 with peptide-MHC complexes.
The Journal of Immunology 02/2009; 182(1):121-9. · 5.79 Impact Factor
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ABSTRACT: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders.
We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL.
CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them.
Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.
Clinical Cancer Research 02/2009; 15(1):299-306. · 7.74 Impact Factor
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ABSTRACT: T cell tolerance depends on the T cell receptor's affinity for peptide/major histocompatibility complex (MHC) ligand; this critical parameter determines whether a thymocyte will be included (positive selection) or excluded (negative selection) from the T cell repertoire. A quantitative analysis of ligand binding was performed using an experimental system permitting receptor-coreceptor interactions on live cells under physiological conditions. Using three transgenic mouse strains expressing distinct class I MHC-restricted T cell receptors, we determined the affinity that defines the threshold for negative selection. The affinity threshold for self-tolerance appears to be a constant for cytotoxic T lymphocytes.
Journal of Experimental Medicine 11/2007; 204(11):2553-9. · 13.85 Impact Factor
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ABSTRACT: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides.
First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice.
In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016).
The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.
Clinical Cancer Research 01/2007; 12(24):7422-30. · 7.74 Impact Factor
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Heather Jackson,
Nektaria Dimopoulos,
Nicole A Mifsud,
Tsin Yee Tai,
Qiyuan Chen,
Suzanne Svobodova,
Judy Browning, Immanuel Luescher,
Lisa Stockert,
Lloyd J Old,
Ian D Davis,
Jonathan Cebon,
Weisan Chen
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ABSTRACT: Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.
The Journal of Immunology 06/2006; 176(10):5908-17. · 5.79 Impact Factor
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ABSTRACT: Longitudinal studies on the kinetics of viral antigen specific CD8 T cell responses have led to a model whereby a relatively small subset of the primary effector CD8 T cells expanding after the first week of acute viral infection initiate a program of cell survival and differentiation into long lived memory T cells. These T cells are then critical for maintaining protective immunity to subsequent viral infection. Recent observations, using fluorescent tetramers of the MHC class Ib molecule TL, link transient expression of CD8alphaalpha homodimers on expanding primary effector CD8 T cells to the generation of memory cells. At present it is controversial what the role of CD8alphaalpha is in the generation of memory CD8 T cells. The involvement of the high affinity CD8alphaalpha ligand, the TL molecule, is not understood either. However, evidence from two viral infection models in mice, including one paper in this issue of the European Journal of Immunology, suggest a role for CD8alphaalpha in this process and call for additional research focus into these issues.
European Journal of Immunology 12/2005; 35(11):3092-4. · 5.10 Impact Factor
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ABSTRACT: Longitudinal studies on the kinetics of viral antigen specific CD8 T cell responses have led to a model whereby a relatively small subset of the primary effector CD8 T cells expanding after the first week of acute viral infection initiate a program of cell survival and differentiation into long lived memory T cells. These T cells are then critical for maintaining protective immunity to subsequent viral infection. Recent observations, using fluorescent tetramers of the MHC class Ib molecule TL, link transient expression of CD8αα homodimers on expanding primary effector CD8 T cells to the generation of memory cells. At present it is controversial what the role of CD8αα is in the generation of memory CD8 T cells. The involvement of the high affinity CD8αα ligand, the TL molecule, is not understood either. However, evidence from two viral infection models in mice, including one paper in this issue of the European Journal of Immunology, suggest a role for CD8αα in this process and call for additional research focus into these issues.See accompanying article: http://dx.doi.org/10.1002/eji.200535162
European Journal of Immunology 11/2005; 35(11):3092 - 3094. · 5.10 Impact Factor
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Olivier Michielin,
Jean-Sebastien Blanchet,
Theres Fagerberg,
Danila Valmori,
Verena Rubio-Godoy,
Daniel Speiser,
Maha Ayyoub,
Pedro Alves, Immanuel Luescher,
Jean-Edouard Gairin,
Jean-Charles Cerottini,
Pedro Romero
Cancer treatment and research 02/2005; 123:267-91.
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Olivier Michielin,
Jean-Sebastien Blanchets,
Theres Fagerberg,
Danila Valmori,
Verena Rubio-Godoy,
Daniel Speiser,
Maha Ayyoub,
Pedro Alves, Immanuel Luescher,
Jean-Edouard Gairin,
Jean-Charles Cerottini,
Pedro Romero
12/2004: pages 267-291;
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ABSTRACT: We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.
Immunity 11/2004; 21(4):515-26. · 21.64 Impact Factor
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ABSTRACT: The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.
The Journal of Immunology 09/2003; 171(4):1844-9. · 5.79 Impact Factor
