Use of overlapping peptide mixtures as antigens for cytokine flow cytometry

Maecker, Holden T.; Dunn, Holli S; Suni, Maria; Khatamzas, Elham; Pitcher, Christine J.; Bunde, Torsten; Persaud, Natasha; Trigona, Wendy L.; Fu, Tong-Ming; Sinclair, Elizabeth; Bredt, Barry M.; McCune, Joseph M.; Maino, Vernon C.; Kern, Florian; Picker, Louis J.

doi:10.1016/s0022-1759(01)00416-1

Cited by 333 publications

(280 citation statements)

References 43 publications

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“…Until recently, monitoring of HCMV-specific CD8 + T cell response remained elusive and difficult. To overcome this limitation, some authors [12,13] proposed the use of peptide pools covering the entire sequence of the two most immunogenic HCMV proteins (pp65 and IE-1 proteins). Using 15-mer peptide pools, both CD4 + and CD8 + IFN-c-producing T cell responses can be elicited, as with 8-12-mer mixes [12].…”

Section: Discussionmentioning

confidence: 99%

“…Peptides used in this study are pools of 15-mers [12,13] with an overlap of 11 amino acids spanning the entire pp65 or IE-1 proteins of HCMV (Jerini AG, Berlin, Germany). Peptides were stored according to the manufacturer's instructions and used at a final concentration of 1 lg/ml.…”

Section: Peptide Poolsmentioning

confidence: 99%

“…Recently some authors [12,13] developed a method involving the activation of both CD4 + and CD8 + T cell subsets, irrespective of the HLA type and known epitopes, using whole-protein-spanning peptide pools. This method is useful to detect protein-specific CD4 + or CD8 + T cell reactivity, but does not enable simultaneous quantification of HCMV-specific CD4 + and CD8 + T cell responses to multiple viral antigens.…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

et al. 2005

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Immature dendritic cells (DC) infected with an endotheliotropic (Huv + ) and leukotropic (Leuk + ) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4 + and CD8 + T cells. Infected DC were cocultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-c production. Efficient stimulation of HCMV-specific CD4 + and CD8 + T cell responses was achieved using DC productively infected with Huv + Leuk + VR1814 strain. On the contrary, a negligible CD8 + T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4 + and CD8 + T cell responses and may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.

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Section: Discussionmentioning

confidence: 99%

Section: Peptide Poolsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

et al. 2005

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“…One potential explanation is a technical one, since we used long peptides of 19 aa whereas in contrast, smaller peptides of 8-12 aa are more efficient for stimulating CD8 + T cells (Maecker et al, 2001). Recent evidence suggests that CD8 + T cells participate in MS (Babbe et al, 2000;Biddison et al, 1997Biddison et al, , 1998Correale et al, 1997b), and hence, future studies should optimize peptide size and selection.…”

Section: Discussionmentioning

confidence: 99%

A new approach for evaluating antigen-specific T cell responses to myelin antigens during the course of multiple sclerosis

Arbour

Holz

Sipe

et al. 2003

Journal of Neuroimmunology

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We used a flow cytometry assay to measure proliferation and cytokine production of self-antigenspecific T cells in individual patients during the clinical course of multiple sclerosis (MS). Myelinassociated oligodendrocytic basic protein (MOBP) was selected for proof of principles in the assay, along with myelin basic protein (MBP) to assess specific activated T cells in 10 MS patients over an 18-month period, in parallel with brain magnetic resonance imaging (MRI) scans and clinical rating scale. A positive correlation occurred between antigen-specific T cell proliferation and interferon-γ production with clinical relapses and MRI lesion activity that was absent when the same patients were in remission.

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“…Cellular immunity was measured by intracellular IFN-c staining 2 wk after the last injection. hEp-CAM-specific IFN-c secretion from stimulated splenocytes was evaluated with a pool of 15-mer peptides overlapping by 11 amino acids and encompassing the entire protein [18].…”

Section: Adeno and Dna-ep Vaccines Induce Immune Responses Against Hementioning

confidence: 99%

Genetic vaccines against Ep‐CAM break tolerance to self in a limited subset of subjects: Initial identification of predictive biomarkers

et al. 2006

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The epithelial cell adhesion molecule, Ep-CAM, has been historically considered a target of passive immunotherapy using monoclonal antibodies, and more recently, of a first Pox-vector-based cancer vaccine Phase I trial in colorectal cancer patients. To shed further light on the use of this antigen, we isolated the mouse and rhesus homologues of human Ep-CAM and explored different genetic vaccination modalities based on the use of adenoviral vectors as well as DNA electroporation (DNA-EP). Immune responses to Ep-CAM were measured by IFN-c ELISPOT and intracellular staining assays using overlapping sets of peptides covering the entire coding regions. We found the most powerful vaccination regimen to be constituted by DNA-EP-prime/Adeno-boost mixedmodality protocols. Vaccination in rhesus macaques resulted in breakage of immunological tolerance in a minority of cases. Similarly, a low frequency of responders was observed with the mouse Ep-CAM vaccine in outbred CD1 mice. When immunized CD1 mice were analyzed for MHC haplotype and TCR expression levels, we observed that immune responders all had the same q/q MHC class I haplotype and showed higher expression levels of the TCRVb4 and TCRVb8 T cell receptors. Our results underscore the current limitations in our capacity to induce efficient cancer vaccines against self antigens like Ep-CAM, but also represent a first effort to identify predictive biomarkers of response.See accompanying commentary: http://dx

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Use of overlapping peptide mixtures as antigens for cytokine flow cytometry

Cited by 333 publications

References 43 publications

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

A new approach for evaluating antigen-specific T cell responses to myelin antigens during the course of multiple sclerosis

Genetic vaccines against Ep‐CAM break tolerance to self in a limited subset of subjects: Initial identification of predictive biomarkers

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