Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells

Schmidt, Julien; Guillaume, Philippe; Irving, Melita; Baumgaertner, Petra; Speiser, Daniel E.; Luescher, Immanuel F.

doi:10.1074/jbc.m111.283127

Cited by 33 publications

(53 citation statements)

References 36 publications

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“…Recently, we developed a novel approach combining reversible peptide-MHC multimers (NTAmers; ref. 15) and real-time flow cytometry (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

“…We previously developed a method for the isolation and analysis of antigen-specific cytotoxic T cells by reversible NTAmers (10,15). Upon addition of imidazole at low, nontoxic concentration, the SA-PE-NTA 4 moieties rapidly decay (average dissociation half-life of 2.5 seconds), thereby releasing the pMHC monomers bound at the cell surface.…”

Section: Direct Measurements Of Monomeric Tcr-pmhc Dissociation Kinetmentioning

confidence: 99%

“…Consequently, NTAmers allow FACS-sorting of CD8 þ T cells without inducing adverse effects on the cell integrity (e.g., activation-induced cell death; refs. 10,15). Taking advantage of the fact that NTAmers can be switched from stable binding to rapid dissociation, a two-color NTAmer was engineered to assess monomeric TCR-pMHC dissociation kinetics directly on living CD8 þ T cells (Fig.…”

Section: Direct Measurements Of Monomeric Tcr-pmhc Dissociation Kinetmentioning

confidence: 99%

“…15 (17). Dually labeled NTAmers containing SA-PE and Cy5-labeled monomers were used for dissociation kinetic measurements as described below.…”

Section: Ntamer Production and Stainingmentioning

confidence: 99%

“…To overcome these limitations, we here applied pMHC multimers built on reversible chelate complexes of Ni 2þ -nitriloacetic acid (NTA) with oligohistidines (15) allowing the efficient and direct cytometry-based assessment of monomeric TCR-pMHC dissociation kinetics on the surface of (self) tumor-specific CD8 T cells. We found that the NTAmer technology accurately predicted T-cell biologic responses within a large panel of tumorspecific T-cell clones, providing novel means for the direct isolation of rare functionally relevant CD8 þ T cells for adoptive cell transfer therapy.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR–Ligand Off-Rates Measurements on Living Cells

et al. 2015

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The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the peptide-MHC (pMHC) on cells is a key parameter for cell-mediated immunity. Yet a fundamental feature of most tumor antigen-specific CD8 þ T cells is that this avidity is low.In this study, we addressed the need to identify and select tumorspecific CD8 þ T cells of highest avidity, which are of the greatest interest for adoptive cell therapy in patients with cancer. To identify these rare cells, we developed a peptide-MHC multimer technology, which uses reversible Ni 2þ -nitrilotriacetic acid histidine tags (NTAmers). NTAmers are highly stable but upon imidazole addition, they decay rapidly to pMHC monomers, allowing flow-cytometric-based measurements of monomeric TCRpMHC dissociation rates of living CD8 þ T cells on a wide avidity spectrum. We documented strong correlations between NTAmer kinetic results and those obtained by surface plasmon resonance. Using NTAmers that were deficient for CD8 binding to pMHC, we found that CD8 itself stabilized the TCR-pMHC complex, prolonging the dissociation half-life several fold. Notably, our NTAmer technology accurately predicted the function of large panels of tumor-specific T cells that were isolated prospectively from patients with cancer. Overall, our results demonstrated that NTAmers are effective tools to isolate rare high-avidity cytotoxic T cells from patients for use in adoptive therapies for cancer treatment. Cancer Res; 75(10); 1983-91. Ó2015 AACR.

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“…Recently, we developed a novel approach combining reversible peptide-MHC multimers (NTAmers; ref. 15) and real-time flow cytometry (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

Section: Direct Measurements Of Monomeric Tcr-pmhc Dissociation Kinetmentioning

confidence: 99%

Section: Direct Measurements Of Monomeric Tcr-pmhc Dissociation Kinetmentioning

confidence: 99%

“…15 (17). Dually labeled NTAmers containing SA-PE and Cy5-labeled monomers were used for dissociation kinetic measurements as described below.…”

Section: Ntamer Production and Stainingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR–Ligand Off-Rates Measurements on Living Cells

et al. 2015

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Multivalent Chelators for In Vivo Protein Labeling

Wieneke

Tampé

2019

Angewandte Chemie

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With the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.

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