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.

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

“…Our findings in this study further support a model in which calcium signals can play a possible quantitative role in this functional separation (for example, by setting different thresholds for cytotoxic and cytokine programs). We further hypothesize that the relationships between calcium signals and effector functions observed in NK cells could be extrapolatable to T cells and may help explain the dichotomy observed between cytotoxic activity and cytokine production reported for CD8 T cells in several studies (9,(44)(45)(46) (47), kinase activities (48), or transcriptional factors (49) using fusion proteins or reporter mouse (50)] should also allow better resolving the contributions of the dynamics and magnitudes of a wide variety of molecules to functional diversification at the single-cell level. Establishment of this new tool now opens the door to pursue these and many other previously intractable hypotheses on immune cell communication, activation, and function at the single-cell level.…”

“…made it feasible to resolve the relationships between different immune responses, particularly between cellular motility, cytotoxicity, and secretory activity (9,10,12), two primary limitations inherent in their operation principles have precluded the ability to study early signaling dynamics and their correlation to subsequent functional behaviors. First, generation of cell-cell interactions is governed by stochastic cell loading that limits conjugate formation efficiency and yields low-throughput-per-footprint area, which in turn severely limits the spatiotemporal resolution for dynamic imaging (i.e., number of cells that can be imaged with high temporal resolution).…”

“…A common approach has been to isolate discrete numbers of cells in spatial confinements [such as microwells (9)(10)(11)(12), microchambers (13), or droplets (14)] and monitor their interactions with multiple measurements. Although these approaches have…”

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

“…While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets 8 .Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes 9 . In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array.…”

“…Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes 9 . In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array.…”