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

Navin Varadarajan, Douglas S. Kwon, Kenneth M. Law, Adebola O. Ogunniyi, Melis N. Anahtar, James M. Richter, Bruce D. Walker, J. Christopher Love

Research output: Contribution to journalArticlepeer-review

92 Scopus citations

Abstract

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 10 6cells. Herewe present a process to identify antigen-specific responses efficiently ex vivo from 10 4-10 5 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 antigenspecific responses by single T cells in amanner 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 humanT-cell immunology across ages and anatomic compartments.

Original languageEnglish
Pages (from-to)3885-3890
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number10
DOIs
StatePublished - 6 Mar 2012
Externally publishedYes

Keywords

  • Microarrays
  • Nanowells
  • Single-cell analysis
  • Soft lithography
  • T-cell cloning

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