Evolution of MHC-Based Technologies Used for Detection of Antigen-Responsive T Cells

Cancer Immunol Immunother DOI 10.1007/s00262-017-1971-5 FOCUSSED RESEARCH REVIEW Evolution of MHC-based technologies used for detection of antigen-responsive T cells Amalie Kai Bentzen1 · Sine Reker Hadrup1 Received: 28 September 2016 / Accepted: 3 February 2017 © The Author(s) 2017. This article is an open access publication Abstract T cell-mediated recognition of peptide-major disease development and response to immunotherapy, and histocompatibility complex (pMHC) class I and II mol- extend fundamental knowledge related to T cell recognition ecules is crucial for the control of intracellular pathogens patterns and cross-recognition by TCRs. MHC multimer- and cancer, as well as for stimulation and maintenance of based technologies have now evolved from detection of efficient cytotoxic responses. Such interactions may also 1–2 different T cell specificities per cell sample, to include play a role in the development of autoimmune diseases. more than 1000 evaluable pMHC molecules using novel Novel insights into this mechanism are crucial to under- technologies. Here, we provide an overview of MHC mul- standing disease development and establishing new treat- timer-based detection technologies developed over two dec- ment strategies. MHC multimers have been used for detec- ades, focusing primarily on MHC class I interactions. tion of antigen-responsive T cells since the first report by Altman et al. showed that tetramerization of pMHC class Keyword MHC multimer · T cell receptor · MHC class I molecules provided sufficient stability to T cell receptor I · Antigen specificity · DNA barcode-labeled MHC (TCR)-pMHC interactions, allowing detection of MHC multimers multimer-binding T cells using flow cytometry. Since this breakthrough the scientific community has aimed for Abbreviations expanding the capacity of MHC multimer-based detection AG+ Antigen positive technologies to facilitate large-scale epitope discovery and CLIP Class II-associated invariant chain peptide immune monitoring in limited biological material. Screen- CyTOF Cytometry by time-of-flight ing of T cell specificity using large libraries of pMHC mol- NACS Nucleic acid cell sorting ecules is suitable for analyses of T cell recognition poten- p* UV-cleavable peptides tially at genome-wide levels rather than analyses restricted pMHC Peptide-MHC to a selection of model antigens. Such strategies provide SA Streptavidin novel insights into the immune specificities involved in This paper is a Focussed Research Review based on a Introduction presentation given at the Fourteenth Annual Meeting of the Association for Cancer Immunotherapy (CIMT), held in Mainz, Techniques for the detection of antigen-responsive T cells Germany, 10th–12th May, 2016. It is part of a series of Focussed exploit the interaction between a given TCR and its pMHC Cancer Immunology, Research Reviews and meeting report in recognition motif. T cell detection using multimerized Immunotherapy. pMHC molecules has evolved into the preferred method for * Sine Reker Hadrup detecting antigen-specific T cells. Over the past 20 years, [email protected] since the first multimerizations of pMHC molecules [1], several approaches have aimed at matching the heteroge- 1 Section for Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark, neity of T cells and epitope presentation to enhance our Copenhagen, Denmark ability to map immune responses. The evolution of these Vol.:(0123456789)1 3 Cancer Immunol Immunother approaches, in terms of increasing complexity, is shown in have been developed in the last decade. First, Schumacher Fig. 1. et al. described the use of conditional MHC ligands that MHC multimer technologies have primarily been devel- are cleaved upon exposure to 366 nm UV-light and can be oped and applied for analyses of CD8 T cell responses, exchanged with any MHC ligand of interest [4]. Using this because MHC class I molecules have proven easier to han- strategy, individual MHC class I molecules are correctly dle in terms of protein folding and expression. Additionally, refolded with carefully designed UV-cleavable peptides the MHC class I binding groove is more restricted in terms (p*), allowing sufficient stability of the complex. Indi- of the length of peptide bound—hence, it is more straight- vidual p*MHC molecules are purified, and stored to serve forward to predict MHC class I binding peptides. Most of as a source of stock molecules that can be exchanged with the technologies described in this review relate to detection any ligand of interest upon exposure to UV-light. The UV- of specific CD8 T cell responses, but they are in principle cleavable conditional ligand-strategy has enabled the pro- also applicable to MHC class II multimers and detection duction of large numbers of different pMHC molecules in of CD4 T cell responses. Specific challenges associated a high throughput manner [5, 6]. Today, such UV-ligands with the production and use of MHC class II multimers are have been designed for a number of different MHC class addressed in the final section. I alleles, of both human and murine origin [7, 8]. An MHC molecules are largely unstable when they are not alternative strategy is the preferential folding of correctly part of a complex with peptide. For this reason, pMHC- oxidized MHC class I heavy chains. This allows efficient based technologies were initially restricted by the tedi- folding-reactions in small volumes, reduces the need for ous production of pMHC molecules, where each peptide further optimization and can be used to create large librar- required an individual folding and purification procedure ies of diverse pMHC complexes [9]. More recently, it was [2, 3]. Thus, the development of high-throughput strate- discovered that certain di-peptides can assist folding and gies for T cell identification was constrained by the lim- peptide exchange of MHC class I molecules [10, 11]. Di- iting step involving the generation of large libraries of peptides bind specifically to the F pocket of MHC class I pMHCs. A number of potential solutions to this challenge molecules to facilitate peptide exchange and have so far Fig. 1 Evolution of MHC-based detection of antigen-responsive for antigen-specific recognition among 2 × 106 viable cells, though the T cells. The methodologies are shown on a Log scale with increas- mass cytometry and matrix-based approaches require a higher num- ing complexity in terms of the reported number of distinct antigen- ber of input cells. The DNA barcode-based methodology has been responsive T cell populations that may by identified per sample. Most reported to have an increased sensitivity with an increased number of + of the strategies offer the potential to screen for T cell reactivity at input cells. AG antigen positive, CyTOF cytometry by time-of-flight higher complexities. The listed sensitivities are based on screening 1 3 Cancer Immunol Immunother been described and validated for peptide exchange in HLA- activation markers once the T cells are captured at pMHC A*02:01, HLA-B*27:05, and H-2Kb molecules. The di- specific spots may improve the sensitivity of the assay [15, peptide exchange technology has not yet been applied in 16]. The study by Chen relied solely on T cell activation larger T cell epitopes mapping strategies. Together, these from multimeric pMHC:TCR interaction; however, multi- technologies have enabled efficient production of large meric pMHC:TCR interaction cannot be expected to acti- libraries of pMHC molecules, and consequently high- vate all relevant T cells. Thus, counting on a lower detec- throughput detection of CD8 T cell recognition using tion limit due to the capture and visualization of activation pMHC-based reagents. markers would also increase the risk of missing those T cell populations that were not activated in such a system or mis- interpreting their frequency. Strategies for high-throughput detection A general limitation with such array-based strategies of antigen-responsive T cells is the propensity of a given T cell to pursue all potential pMHC interactions displayed on a given array. As a conse- All the MHC-based strategies described throughout this quence, the frequency of antigen-responsive T cells in the report are summarized in Table 1. cell preparations should be >0.1% to allow a robust read- out. Deviren et al. [17] particularly addressed the issue of Detecting antigen-responsive T cells using MHC assay reproducibility and homogeneity, and by introducing microarrays a mild flow shear they obtained a more uniform distribution of captured T cells and a higher reproducibility. Consist- Early attempts to improve the complexity of MHC mul- ent with the issue of ensuring cell interaction at all possi- timer-based detection of antigen-responsive T cells were ble pMHC positions, they noted a greater variability asso- based on the success of gene-expression arrays, including ciated with decreased surface area of each spotted pMHC. the availability of efficient systems capable of quantify- The nucleic acid cell sorting (NACS) approach developed ing fluorescent probes when spatially separated as spots by Kwong et al. [18] employed unique sequences of sin- on a microarray [12]. Protein-based microarrays have been gle-stranded DNA conjugated to distinct pMHC tetramers, developed [13] and the idea of a pMHC microarray allow- which in turn were site-specifically assembled via hybridi- ing cell-capture based on specificity is appealing. Although zation onto glass slides printed with complementary DNA several such systems have been developed [14–19], their probes. This strategy offers an advantage over other MHC application seems to be limited on account of a poor detec- arrays in that T cells can be recovered from the glass slide tion limit and low reproducibility compared to existing by selective detachment using restriction enzymes. cytometry-based analyses. There is considerable overlap between MHC microarray approaches, where most work Detecting antigen-responsive T cells using has focused on optimizing the supporting surface and combinatorially labeled MHC multimers modifying the conditions applied during binding and/or washing.

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