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


The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells from peripheral blood by combinatorial encoding is as efficient as detection with conventionally labeled multimers but results in a substantially increased sensitivity and, most notably, allows comprehensive screens to be performed. We obtained proof of principle for the feasibility of large-scale screening of human material by analysis of human leukocyte antigen A3–restricted T-cell responses to known and potential melanoma-associated antigens in peripheral blood from individuals with melanoma.

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Figure 1: Overview of the combinatorial encoding approach.
Figure 2: Feasibility of T-cell staining with dual-color–encoded pMHC multimers.
Figure 3: Multiplex detection of virus-specific T-cell responses through combinatorial encoding.
Figure 4: Correlation between different T-cell detection strategies.
Figure 5: T-cell responses to melanoma-associated peptides.


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We thank B. Rodenko and H. Ovaa (Netherlands Cancer Institute) for the kind gift of J, W. van de Kasteele for help with cell culture, T. Wirenfeldt for statistical assistance, and A. Pfauth, F. van Diepen, M. van der Hoorn and G. de Roo for technical support with flow cytometry. This work was supported by the Danish Cancer Society grant DP06031 and the Carlsberg Foundation grant 2005-1-641 (to S.R.H.), Landsteiner Foundation of Blood Transfusion research grant 0522 and a Melanoma Research Alliance established investigator award (to T.N.S.) and Dutch Cancer Society grant UL 2007-3825 (to M.H.H. and T.N.M.S.).

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S.R.H., A.H.B. and C.J.S. designed research, performed research, analyzed data and wrote the paper; R.S.A. performed research and analyzed data; J.v.V. performed research; P.H. and E.C. provided practical assistance; P.t.S., C.B. and J.B.H. contributed material from individuals with melanoma; M.H.H., designed research and analyzed data; T.N.S., designed research, analyzed data and wrote the paper.

Corresponding author

Correspondence to Ton N Schumacher.

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Competing interests

The technology described in this manuscript is the subject of an EU patent application. Based on Netherlands Cancer Institute policy on management of intellectual property, S.R.H., A.H.B, C.J.S. and T.N.S would be entitled to a portion of received royalty income.

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Hadrup, S., Bakker, A., Shu, C. et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods 6, 520–526 (2009).

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