Detection, phenotyping, and quantification of antigen- specific T cells using a peptide-MHC dodecamer Jun Huanga,1,2, Xun Zenga, Natalia Sigala,3, Peder J. Lunda, Laura F. Sub, Huang Huanga, Yueh-hsiu Chiena, and Mark M. Davisa,c,d,2 aDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Medicine, Division of Rheumatology, University of Pennsylvania, Philadelphia, PA 19104; cStanford Institute for Immunity, Transplantation, and Infection, Stanford University, Stanford, CA 94305; and dHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305 Contributed by Mark M. Davis, February 18, 2016 (sent for review June 5, 2015; reviewed by Marc K. Jenkins and Andrew J. McMichael) Here we report a peptide-MHC (pMHC) dodecamer as a “next gen- of-flight mass spectrometry make the low-avidity MHC class II eration” technology that is a significantly more sensitive and ver- tetramers unsuitable for CyTOF studies. satile alternative to pMHC tetramers for the detection, isolation, and To augment the avidity of the tetramer, we have engineered phenotypic analysis of antigen-specific T cells. In particular, dodeca- abiotinylatedscaffoldproteinlinkedtofourstreptavidintet- mers are able to detect two- to fivefold more antigen-specific T cells in ramers, each capable of binding three biotinylated pMHC + + both human and murine CD4 and CD8 αβ T-cell compartments monomers. We then used the resulting dodecamer (Greek for compared with the equivalent tetramers. The low-affinity, tetramer- “12”) to detect low-affinity αβ and γδ T cells in blood and tissue negative, dodecamer-positive T cells showed comparable effector cy- samples from humans and mice. Compared with tetramers and tokine responses as those of high-affinity, tetramer-positive T cells. at least some multimers, this construct has better sensitivity, + + Dodecamers are able to detect early stage CD4 CD8 double-positive stronger signal strength, higher binding avidity, and a much thymocytes on which T-cell receptors are 10- to 30-fold less dense slower dissociation rate. For the various specificities that we have than mature T cells. Dodecamers also show utility in the analysis of analyzed, it can identify two- to fivefold more specific T cells γδ T cells and in cytometry by time-of-flight applications. This con- than an equivalent tetrameric reagent. struct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or Results other molecules. The dodecamer is straightforward and inexpensive Dodecamer Construction. To overcome the limitations of current to produce and is compatible with current tetramer technology and tetramer technology, we aimed to engineer a dodecamer with commercially available streptavidin conjugates. higher avidity to detect and quantify antigen-specific T cells, especially rare and low-affinity ones. To accomplish this, we added a peptide-MHC | dodecamer | avidity | antigen-specific T cell cysteine residue at the C terminus of an inactive streptavidin subunit that has no biotin binding sites (18). After expression, refolding, and -cell receptors (TCRs) detect antigens in the form of pep- purification, a tetrameric scaffold protein was assembled bear- Ttides bound to peptide-major histocompatibility complex ing four terminal cysteines, which were subsequently biotinylated (pMHC) molecules on the surface of antigen-presenting cells. – TCR pMHC interactions determine the selection, development, Significance differentiation, fate, and function of a T cell. However, TCRs bind monomeric pMHCs with very low binding affinities (K , ∼1–200 μM, d The recognition of foreign peptide-MHCs by T cells is a central 1,000- to 200,000-fold weaker than a typical antibody–antigen − event in adaptive immunity that triggers antigen-specific im- interaction) and with fast dissociation rates (k , ∼0.05 s 1)in off mune responses against infections and cancer. To study anti- solution (1). To increase the binding avidity and circumvent gen-specific T cells, we devised a peptide-MHC dodecamer that the problem of fast dissociation, we previously engineered a pMHC can sensitively detect and specifically stain these T cells, espe- tetramer to detect antigen-specific T cells by conjugating four bio- cially low-affinity and rare ones. This dodecamer technology is tinylated pMHC monomers to a single fluorescent-labeled strepta- superior to most current peptide-MHC multimers, compatible vidin (2). This fulfilled a critical need in both basic and clinical with existing reagents, inexpensive to make, and easy to use. It immunology to be able to identify and characterize often very rare has been successfully applied to studies of human and murine specific T cells in a population. Subsequent improvements in sen- antigen-specific αβ and γδ T cells by flow cytometry and mass sitivity, manufacture, and combinatorial labeling have made this cytometry. Thus, this dodecamer constitutes an important tool methodology even more useful (3–7). However, there is a sharp ∼ μ for the investigation of antigen-specific T cells in basic and drop-off in tetramer binding in the lower affinity range ( 150 M) clinical research. (8) so there have been a number of higher valency alternatives in- cluding pentamers (ProImmune), lipid vesicles (9), octamers (10), Author contributions: J.H. conceived, designed, and engineered the pMHC dodecamer dextramers (11), and quantum dot (QD)-based multimers (12). reagent; J.H. and M.M.D. designed staining experiments; J.H., X.Z., N.S., and L.F.S. per- Some of these clearly have improved sensitivity, and this is im- formed staining experiments; P.J.L., H.H., and Y.-h.C. contributed important ideas and portant in detecting T cells with especially low affinities (13, 14). reagents; J.H. and X.Z. analyzed data; and J.H., P.J.L., and M.M.D. wrote the paper. For example, naive T cells and thymocytes, which express low- Reviewers: M.K.J., University of Minnesota; and A.J.M., University of Oxford. level and/or low-affinity TCRs, show little-to-no tetramer stain- Conflict of interest statement: J.H. and M.M.D. are inventors on a patent application (US αβ γδ Provisional Application No. 62/194,726) that applies to detection, phenotyping, and quan- ing. Furthermore, T cells and T cells that do not bind tification of cells with multimeric binding reagent. antigen-specific tetramers can still produce significant antigen- 1Present address: Institute for Molecular Engineering, The University of Chicago, Chicago, specific cytokine responses (15, 16). MHC class II tetramers are IL 60637. also problematic in cytometry by time-of-flight mass spectrome- 2To whom correspondence may be addressed. Email: [email protected] or huangjun@ try (CyTOF), which is an advanced version of flow cytometry that uchicago.edu. can simultaneously measure more than 40 parameters on single 3Present address: Fluidigm, South San Francisco, CA 94080. cells (17). The extensive washing steps, harsh fixation conditions, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complicated sample injection process, and sensitivity of the time- 1073/pnas.1602488113/-/DCSupplemental. E1890–E1897 | PNAS | Published online March 15, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1602488113 Downloaded by guest on October 2, 2021 by 1-Biotinamido-4-[4′-(maleimidomethyl)cyclohexanecarboxami- to form pMHC dodecamers. Dodecamers labeled with either PNAS PLUS do]butane (BMCC-biotin) (molecular weight: 534). The scaffold pro- the large fluorescent protein PE (∼250 kDa) or the small fluo- tein with four biotin sites is the centerpiece of the dodecamer. As rescent dye A555 (∼1 kDa) specifically stained antigen-specific illustrated in Fig. 1A, a biotinylated scaffold protein associates naive T cells in preparations of transgenic 5C.C7 splenocytes. with four commercial or homemade fluorescent/metal-labeled The PE-labeled dodecamer gave ∼10 times better signal than streptavidin molecules, each of which further associates with three the A555-labeled dodecamer as illustrated in Fig. 2A and Fig. biotinylated pMHC monomers. Thus, a biotinylated scaffold pro- S1A. This is expected because PE is a large fluorescent protein tein allows the formation a pMHC dodecamer. The expression consisting of multiple fluorescent units whereas A555 is a small, and biotinylation of the scaffold protein were confirmed by singly fluorescent molecule. Both control dodecamers produced SDS/PAGE. This tetrameric scaffold protein has a molecular negligible nonspecific staining (Fig. 2 A and B). These data show weight of ∼53.4 and ∼55.5 kDa before and after biotinylation that the dodecamer is compatible with different types of fluo- (Fig. 1B). Consistently, a monomeric subunit has a molecular rophores for detecting antigen-specific T cells. We next compared weight of ∼13.3 and ∼13.9 kDa before and after biotinylation (Fig. dodecamers with tetramers. As shown in Fig. 2C, dodecamers 1C). The increase in the molecular weight of the scaffold protein gave a fivefold better signal than tetramers. Dodecamers yielded in both the tetrameric and the monomeric forms is consistent with minimal background staining at 4 °C, comparable to tetramers the expected results (Fig. 1 B and C). over a broad range of concentrations (Fig. 2 C and D and Fig. S1B). We next compared dodecamers with dextramers (Fig. 2 E and F). Comparison of Dodecamers to Different Multimer Formats. We first Dextramers are hetereogeneous mixtures of polymers