T-cell epitope mapping by flow cytometry

Kern, Florian; Surel, Ingolf; Brock, Carsten; Freistedt, Bernd; Radtke, H.; Scheffold, Alexander; Blasczyk, Rainer; Reinke, Petra; Schneider‐Mergener, Jens; Radbruch, Andreas; Walden, Peter; Volk, Hans‐Dieter

doi:10.1038/nm0898-975

Cited by 266 publications

(245 citation statements)

References 14 publications

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“…APC such as monocytes and macrophages preferentially process whole proteins via the exogenous pathway of antigen presentation (Braciale et al, 1987;Monaco, 1995). In contrast, short peptides spanning immunodominant epitopes can bind directly into the MHC groove, bypassing the requirement for protein internalization and processing (Kern et al, 1998;He et al, 2001). Previous research has shown that CD4 + T cell responses induced by peptide mixes are equal to those induced by whole proteins whilst CD8 + T cell responses can be significantly higher after peptide compared to whole protein stimulation, presumably a result of bypassing the antigen internalization and processing requirement.…”

Section: Discussionmentioning

confidence: 99%

Loss of T cell responses following long-term cryopreservation

Owen

Sinclair

Emu

et al. 2007

Journal of Immunological Methods

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Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined.Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4 + T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8 + T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8 + T cell responses to peptides and peptide pools were well preserved. Loss of both CD4 + and CD8 + T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4 + T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein.Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4 + T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4 + and CD8 + T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4 + T cell responses and mild skewing of T cell phenotypic marker expression.

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Section: Discussionmentioning

confidence: 99%

Loss of T cell responses following long-term cryopreservation

Owen

Sinclair

Emu

et al. 2007

Journal of Immunological Methods

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“…Flexibility due to independence from certain MHC alleles and defined antigenic peptides is one of the major advantages of such functional assays. The analysis of specific T cells according to the expression of cytokines upon in vitro re-stimulation is currently the state-of-theart for the analysis of physiologic CD4 T cell responses [5][6][7]30]. The introduction of CD154 as a universal marker for all antigen-reactive CD4 T cells for the murine system will significantly improve these analyses.…”

Section: Discussionmentioning

confidence: 99%

“…However, to access this extremely small population of antigenspecific T cells within the large population of nonspecific T cells is still a major challenge. Current technologies employ MHC/peptide multimers [1][2][3][4] or methods to identify cytokine-secreting T cells upon in vitro re-stimulation with the antigen [5][6][7][8][9]. Both methods allow the analysis of live cells on the singlecell level but are limited by several restrictions: MHC multimers require defined antigenic peptide epitopes, and for many pathogens and autoimmune diseases no epitopes are known.…”

Section: Introductionmentioning

confidence: 99%

Identification and isolation of murine antigen‐reactive T cells according to CD154 expression

et al. 2007

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T helper (Th) cells are central regulators of adaptive immune responses. However, the detection of the small number of Th cells specific for a particular antigen or pathogen is still a major challenge. CD154 was recently introduced as a marker for antigen-specific Th cells. To date, this technology was not applicable for mice -arguably the most important immunological model system. CD154 is difficult to detect due to its rapid removal from the cell surface upon binding to CD40 during antigen-specific activation by APC. We present an efficient strategy to block the degradation of murine CD154 by combined use of antibodies against CD40 and CD154. This strategy makes CD154 easily accessible for surface staining, which allows isolation and expansion of rare antigen specific T cells. Importantly, CD154 identified all specific T cells in strongly Th1-or Th2-polarized immune responses against pathogens like Salmonella typhimurium and Heligmosomoides polygyrus, independent of their potential to produce cytokines. We demonstrate that CD154 can in fact be used as a reliable marker for antigen-specific CD4 T cells in mice, offering a unique option to analyze, isolate and rapidly expand the entire pool of Th-cells generated during a physiological T cell response in vivo.

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“…For epitope mapping experiments, T cell lines from three donors with different HLA types were stimulated with autologous cells loaded with different sub-pools and single peptides of the pp65 peptide library, as described [27]. The IFN-c production of the specifically activated T cells was then analyzed by intracellular cytokine staining.…”

Section: Surface and Intracellular Cytofluorometry (Facs)mentioning

confidence: 99%

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

Hammer

Meyer²,

Brestrich³

et al. 2005

Eur. J. Immunol.

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Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4 + and CD8 + T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-c-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4 + and CD8 + lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

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T-cell epitope mapping by flow cytometry

Cited by 266 publications

References 14 publications

Loss of T cell responses following long-term cryopreservation

Loss of T cell responses following long-term cryopreservation

Identification and isolation of murine antigen‐reactive T cells according to CD154 expression

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy

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