A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis
Navin Varadarajan, … , Bruce D. Walker, J. Christopher Love
Navin Varadarajan, … , Bruce D. Walker, J. Christopher Love
Published October 3, 2011
Citation Information: J Clin Invest. 2011;121(11):4322-4331. https://doi.org/10.1172/JCI58653.
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Technical Advance Immunology

A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis

Abstract

CD8+ T cells are a key component of the adaptive immune response to viral infection. An inadequate CD8+ T cell response is thought to be partly responsible for the persistent chronic infection that arises following infection with HIV. It is therefore critical to identify ways to define what constitutes an adequate or inadequate response. IFN-γ production has been used as a measure of T cell function, but the relationship between cytokine production and the ability of a cell to lyse virus-infected cells is not clear. Moreover, the ability to assess multiple CD8+ T cell functions with single-cell resolution using freshly isolated blood samples, and subsequently to recover these cells for further functional analyses, has not been achieved. As described here, to address this need, we have developed a high-throughput, automated assay in 125-pl microwells to simultaneously evaluate the ability of thousands of individual CD8+ T cells from HIV-infected patients to mediate lysis and to produce cytokines. This concurrent, direct analysis enabled us to investigate the correlation between immediate cytotoxic activity and short-term cytokine secretion. The majority of in vivo primed, circulating HIV-specific CD8+ T cells were discordant for cytolysis and cytokine secretion, notably IFN-γ, when encountering cognate antigen presented on defined numbers of cells. Our approach should facilitate determination of signatures of functional variance among individual effector CD8+ T cells, including those from mucosal samples and those induced by vaccines.

Authors

Navin Varadarajan, Boris Julg, Yvonne J. Yamanaka, Huabiao Chen, Adebola O. Ogunniyi, Elizabeth McAndrew, Lindsay C. Porter, Alicja Piechocka-Trocha, Brenna J. Hill, Daniel C. Douek, Florencia Pereyra, Bruce D. Walker, J. Christopher Love

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Figure 4

Direct evidence that immediate cytotoxicity and short-term IFN-γ secretion are not correlated functions for ex vivo HIV-specific CD8+ T cells.

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Direct evidence that immediate cytotoxicity and short-term IFN-γ secreti...
(A) Representative scatter plots of the frequencies of HLA-B*27-KK10–specific CD8+ T cells in 4 different clinical samples (2 elite controllers [Fw056, CTR40] and 2 progressors [FEN033, 8222]) determined by flow cytometry and staining with recombinant MHC class I tetramers. The percentages of gated populations are indicated. (B) Matched data for concurrent measurement of cytolysis and IFN-γ secretion for ex vivo CD8+ T cells from 4 HIV+ individuals coincubated in arrays of microwells for 6 hours with single HLA-matched, KK10-pulsed target B cells. The data are shown as a heatmap and organized by unsupervised hierarchical clustering (Euclidean distance, complete linkage). Cytolytic activity is shown as discrete values (blue, negative; red, death). The relative rate of secretion is shown for IFN-γ (green). The dendrograms indicate the relative calculated distances and separation among clusters of wells. All events with any functional activity are shown; the total number of CD8+ T cells in wells with E/T = 1:1–5:1 is indicated for each sample. There were no cells among any of the 4 samples that released either IL-2 or TNF-α at a detectable rate (limit of detection, ~2 molecules/s). The Pearson’s product-moment correlation coefficient (r) and 95% CI were estimated for each dataset by random data resampling. The data are representative of at least 3 independent measurements for each subject.