Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 10; pp. 3885 - 3890
• Main Authors: Varadarajan, Navin, Kwon, Douglas S, Law, Kenneth M, Ogunniyi, Adebola O, Anahtar, Melis N, Richter, James M, Walker, Bruce D, Love, J. Christopher
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 06.03.2012; National Acad Sciences
• Subjects: Age; Antibodies; Antigen presenting cells; Antigens; Biological Sciences; Biopsy; Blood; CD3 Complex - biosynthesis; CD8 antigen; CD8-positive T-lymphocytes; CD8-Positive T-Lymphocytes - cytology; CD8-Positive T-Lymphocytes - immunology; Cloning, Molecular; Comparative studies; Computed tomography; Cytokines; Cytometry; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Epitopes; Epitopes - chemistry; functional diversity; Genotype; HIV; HIV - metabolism; HIV 1; HIV Infections - diagnosis; HIV Infections - immunology; HIV Infections - virology; HLA Antigens - chemistry; Human immunodeficiency virus; Humans; image analysis; Immunology; Infection; Infections; Interferon-gamma - metabolism; Lab-On-A-Chip Devices; Lymphocytes T; Micromanipulation; Mucosa; Nanotechnology - methods; Neonates; Pediatrics; Physical Sciences; Proteins; Recovery of function; T cell receptors; T lymphocytes; T-Lymphocytes - metabolism; Therapeutic applications; Tissue samples
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1111205109

The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 106 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 104–105 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called "microengraving"—to enumerate antigen-specific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T-cell responses demonstrates that it is possible to establish clonal CD8+ T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments.