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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

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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 1

High-throughput, single-cell assay for cytolysis.

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High-throughput, single-cell assay for cytolysis.
(A) Schematic for the ...
(A) Schematic for the microwell-based assay. Unlabeled CD8+ T cells (blue) and labeled antigen-presenting target cells (red) are loaded sequentially onto an array, immersed in media, and then imaged at t = 0 hours and t = 4 hours. Dead target cells are identified by loss of integrity of the nuclear membrane (SYTOX, green; composite with CellTracker Red is superimposition of red and green (shown as yellow). (B) Representative composite micrographs of microwells containing individual KK10-specific CD8+ effector cells (clone E501) coincubated with naive or KK10-pulsed HLA-B*27+HLA-A*02+ B cells. Dashed lines indicate the perimeters of the wells. (C) Target cell scatter plots for wells containing 1 effector and 1 target at 4 hours without (left) and with (right) KK10; effectors not shown. For inclusion, targets in wells were gated as CellTracker Red positive (target fluorescence, >103) and SYTOX negative at 0 hours. Dashed line indicates the threshold for dead target cells, and the percentage is noted. AFU, arbitrary fluorescence units. (D) Plots of bulk cytolysis determined by 51Cr release for KK10-pulsed B cells mediated by E501 after either 2 or 4 hours as a function of E/T.
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