High-Throughput Characterization of Single Antigen-Specific T Lymphocytes by Microengraving

CD4⁺ T lymphocytes (T helper cells) are critical for coordinating immune responses against infections and in autoimmune conditions.  CD4⁺ T cells sustain the activation and proliferation of CD8⁺ cytotoxic T cells, as well as induce class switching of immunoglobulins and induction of memory B cells.  Given the significant role assumed by CD4⁺ T cells, evaluating the number and functional activities of antigen-specific CD4⁺ T cells is, therefore, critical for monitoring the progression of pathological conditions and responses to interventions like vaccines.  Here, we describe a new approach for identifying and assessing the functional responses of single, antigen-specific CD4⁺ T cells directly ex vivo using antigen-presenting cells (APCs) to induce activation.  Central to this approach, a process called microengraving enables the generation of microarrays for detection of secreted proteins captured from a large number of single cells (~10⁵ cells per assay).  Using this approach, it is possible to measure the rate of secretion for up to eight cytokines released, and by automated microscopy, the characteristic phenotypes of single CD4⁺ T cells stimulated with activated DCs.  Our data demonstrate the ability to link CD4⁺ T cell phenotypes to functional responses against pools of peptides derived from pathogenic agents restricted through MHC-II presentation. This integrated approach for single-cell analysis suggests a new strategy for ex vivo, high-throughput screening of antigen-specific human CD4⁺ T cells and may provide new insights in understanding the contributions of rare immune cells in defense of various immune diseases.