However, we found that populations of CD8+ T cells and T cells appeared (and disappeared) with the same kinetics, all expressing gut homing receptors. expression of a single rearranged immunoglobulin or TCR on each B or T cell. And that in general, single cells are the operational models or quanta of immunity. With respect to T lymphocytes, this means that understanding their role in immune responses requires comprehensive methods of interrogating the phenotypic and functional characteristics of individual T Tilorone dihydrochloride cells. In this regard, the use of circulation cytometry for high-throughput analysis of individual T cells has been the platinum standard for many years3. Gradual improvements in flow cytometry allowing simultaneous assessment of expression of surface and intracellular markers4 and the precise temporal patterns of cytokine expression by T cells5-7 have enabled studies on the relationships between T-cell phenotype/function and clinical status in a range of diseases8-14. The study of antigen-specificity, however, is complicated by enormous variability and unpredictability in terms of the epitopes targeted by T cells in any given T-cell Tilorone dihydrochloride response, especially considering the highly polymorphic nature of the MHC, and the fact that intact pathogens typically encode a wide variety of potential T cell epitopes15. Furthermore, as the breadth or number of epitopes targeted by the T cell response can be important, especially in rapidly evolving viral infections16-18, and the phenotypes of T cells targeting different epitopes from the same pathogen can vary significantly19,20, it is important to be able to monitor recognition of numerous epitopes in the response to each pathogen. As a result, the number of parameters analyzed in any given experiment continues to grow beyond the number of colors (12C15) available for fluorescence-based flow cytometry, making the latter type of analysis increasingly arduous or even impossible. Recent developments in methods for analyzing antigen-specific T cells that extend these limits exploit multiplexing and single-cell mass spectrometry-based mass cytometry20-24. Other emerging technologies that promise to dramatically increase Tilorone dihydrochloride both the speed and depth of information that one can obtain about T-cell responses include techniques allowing the analysis of single-cell mRNA transcripts25,26. In addition, unlike most mouse models of immunological diseases, wherein the identity of the antigenic epitopes that drive disease initiation and/or progression are known, the instances of human immunological diseases wherein the precise specificities of T cells involved are known remain relatively rare. Therefore, until precise antigenic epitope specificities can be determined, study of these human T cell responses requires alternative approaches; none appear to be more powerful than high-throughput sequencing of TCR repertoires. Data generated by this approach are providing insights into T-cell selection and the nature of repertoire diversity in various T-cell subsets in normal and pathological circumstances27,28. TCR sequencing approaches also allow the identification and tracking of TCR clonotypes or motifs involved in immune responses and various pathologies29-31. Moreover, high-throughput yeast-display approaches represent a way to identify pMHC ligands that bind to these TCR clonotypes or motifs32,33. IFNGR1 These approaches hold promise for identifying relevant antigens for immune responses for which relevant antigens are currently completely unknown. For instance, identification of antigens targeted by T cells in patients with auto-inflammatory diseases could facilitate the development of novel treatment options. In this Review we discuss the advantages, disadvantages and complementarity of these high-dimensional approaches for the study of antigen-specific T cells. Common to each approach is the goal of understanding and/or exploiting the specificity of the T-cell mediated immune response to manipulate or predict outcomes of immunological diseases or vaccine Tilorone dihydrochloride responses. These recent technological advances seem poised to finally make possible the comprehensive analyses of T-cell responses. Analyzing T-cell phenotypic and functional diversity Each individual T cell expresses one of as many >1014 different TCR heterodimers34 and each of these TCRs is.