Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8+ T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8+ immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.
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We thank T. Seremet (Center for Cancer Immune Therapy, University Hospital Herlev) and M. Toebes (Netherlands Cancer Institute) for excellent technical assistance; and S. Walter (Immatics) and C. Gouttefangeas (Tübingen University) for supporting data on MHC multimer storage. This work was supported by the Danish Cancer Society (grant DP06031), The Danish Council for Strategic Research (grant 09-065152), the Center for Translational Molecular Medicine (grant 04I-301) and Integration of Biosynthesis and Organic Synthesis (grant 053.63.015).
Experimental setup in 384-well microplates. When multiple MHC multimer panels are generated simultaneously, we advise the use of the outlined setup. Each row on a 384 well plate (plus 3 additional wells on a second plate) holds one full panel of 27 MHC multimers with different color codes. The exchange reaction (step 4) is conducted in 384-well microplates, numbers indicate the different peptides. After the exchange reaction is completed pMHC monomers are transferred to two new 384-well microplates (step 7) for coupling to the different fluorochrome-streptavidin conjugates (step 8). Importantly, a given pMHC complex should always maintain the same position in the plate. Finally, the MHC multimers are mixed in a new plate to obtain the final color codes (step 12), such that each well contains one pMHC complex, conjugated to two different fluorochromes. Before T-cell staining each row is mixed to generate one complete panel of 27 MHC multimers for combinatorial encoding.
Flow cytometry analysis of a T-cell population containing MART-1-reactive T cells that was either stained with MHC (HLA-A0201) multimers made with MART-1 modified peptide (left), or with MART-1 wild type peptide (right). The MART-1 wildtype peptide has a very low affinity for HLA-A0201, whereas the MART-1 modified peptide binds with high affinity. Dot plots are gated on CD8+ T cells and the MART-1-MHC multimer stained T cells are indicated in red. The panel used for this analysis had been stored at 4°C in the dark for six days prior to T-cell staining. Note that staining intensity is comparable for the two peptides.
Typical PMT voltages for an experiment
Spectral overlap values from a typical compensation made with the PMT voltages shown in Supplementary Table 2