Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Andersen, Rikke; Kvistborg, Pia; Frøsig, Thomas Mørch; Pedersen, Natasja Wulff; Lyngaa, Rikke; Bakker, Anke; Shu, Chengyi J.; Straten, Per thor; Schumacher, Ton N.; Hadrup, Sine Reker

doi:10.1038/nprot.2012.037

Cited by 119 publications

(118 citation statements)

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“…All samples were analyzed by combinatorial coding (22,23), using a panel of peptide-major histocompatibility complex (pMHC) multimers loaded with 72 melanoma-associated epitopes (table S1). In addition, three viral epitopes, derived from Epstein-Barr virus (EBV), cytomegalovirus (CMV), and influenza A, were included to allow comparison with T cell reactivity against persistent and nonpersistent viruses.…”

Section: Resultsmentioning

confidence: 99%

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 ⁺ T cell response

et al. 2014

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Anti-CTLA-4 treatment improves the survival of patients with advanced-stage melanoma. However, although the anti-CTLA-4 antibody ipilimumab is now an approved treatment for patients with metastatic disease, it remains unknown by which mechanism it boosts tumor-specific T cell activity. In particular, it is unclear whether treatment amplifies previously induced T cell responses or whether it induces new tumor-specific T cell reactivities. Using a combination ultraviolet (UV)-induced peptide exchange and peptide-major histocompatibility complex (pMHC) combinatorial coding, we monitored immune reactivity against a panel of 145 melanoma-associated epitopes in a cohort of patients receiving anti-CTLA-4 treatment. Comparison of pre- and posttreatment T cell reactivities in peripheral blood mononuclear cell samples of 40 melanoma patients demonstrated that anti-CTLA-4 treatment induces a significant increase in the number of detectable melanoma-specific CD8 T cell responses (P = 0.0009). In striking contrast, the magnitude of both virus-specific and melanoma-specific T cell responses that were already detected before start of therapy remained unaltered by treatment (P = 0.74). The observation that anti-CTLA-4 treatment induces a significant number of newly detected T cell responses-but only infrequently boosts preexisting immune responses-provides strong evidence for anti-CTLA-4 therapy-enhanced T cell priming as a component of the clinical mode of action.

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Section: Resultsmentioning

confidence: 99%

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 ⁺ T cell response

et al. 2014

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“…In all cases, incubation with MHC multimers was done before mAb staining (either at 4°C, 25°C, or 37°C). Each multimer was used at 1–5 µg/ml when labeled with one single fluorochrome and at 2–10 µg/ml final when labeled with two different fluorochromes in the combinatorial approach (16,18). Staining with commercial multimers was performed as per manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells

et al. 2014

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Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5–16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry

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“…MHC complexes loaded with the indicated peptides of interest were generated by UV-induced ligand exchange. In brief, pMHC complexes with UV-sensitive peptide (100 mg/ml) were subjected to 366 nM UV light (Camag) for 1 h at 4˚C in the presence of rescue peptide (200 mM) (13,15).…”

Section: Generation Of Pmhc Complexesmentioning

confidence: 99%

HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses

Buuren

Dijkgraaf

Linnemann

et al. 2014

The Journal of Immunology

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Peptide–MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01–restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06–restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.

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Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Cited by 119 publications

References 20 publications

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 ⁺ T cell response

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 ⁺ T cell response

Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells

HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses

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Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Cited by 119 publications

References 20 publications

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 + T cell response

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 + T cell response

Cryopreservation of MHC multimers: Recommendations for quality assurance in detection of antigen specific T cells

HLA Micropolymorphisms Strongly Affect Peptide–MHC Multimer–Based Monitoring of Antigen-Specific CD8+ T Cell Responses

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Product

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Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 ⁺ T cell response

Anti–CTLA-4 therapy broadens the melanoma-reactive CD8 ⁺ T cell response