Protocol for the assessment of human T cell activation by real-time metabolic flux analysis

Abstract Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8+ T cells and an interferon-response state for CD4+ T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8+ compared to CD4+ T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.

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Physiological strength electric fields modulate human T cell activation and polarisation

Scientific Reports ◽

10.1038/s41598-019-53898-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Christina E. Arnold ◽

Ann M. Rajnicek ◽

Joseph I. Hoare ◽

Swechha Mainali Pokharel ◽

Colin D. Mccaig ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Electric Fields ◽

T Cell Activation ◽

Cell Activation ◽

Cell Activity ◽

Research Area ◽

Functional Changes ◽

Cell Functions ◽

Human T Cell

AbstractThe factors and signals driving T cell activation and polarisation during immune responses have been studied mainly at the level of cells and chemical mediators. Here we describe a physical driver of these processes in the form of physiological-strength electric fields (EFs). EFs are generated at sites where epithelium is disrupted (e.g. wounded skin/bronchial epithelia) and where T cells frequently are present. Using live-cell imaging, we show human primary T cells migrate directionally to the cathode in low strength (50/150 mV/mm) EFs. Strikingly, we show for the first time that EFs significantly downregulate T cell activation following stimulation with antigen-activated APCs or anti-CD3/CD28 antibodies, as demonstrated by decreased IL-2 secretion and proliferation. These EF-induced functional changes were accompanied by a significant dampening of CD4+ T cell polarisation. Expression of critical markers of the Th17 lineage, RORγt and IL-17, and the Th17 polarisation mediator phospho-STAT3 were reduced significantly, while STAT1, ERK and c-Jun phosphorylation were comparatively unaffected suggesting STAT3 modulation by EFs as one mechanism driving effects. Overall, we identify electrical signals as important contributors to the co-ordination and regulation of human T cell functions, paving the way for a new research area into effects of naturally occurring and clinically-applied EFs in conditions where control of T cell activity is paramount.

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Definition of discrete signals involved in human T-cell activation

Molecular Immunology ◽

10.1016/0161-5890(86)90146-x ◽

1986 ◽

Vol 23 (11) ◽

pp. 1157-1163 ◽

Cited By ~ 8

Author(s):

S. Meuer ◽

M. Hauer ◽

U. Moebius ◽

E. Schiedhelm ◽

K. Deusch ◽

...

Keyword(s):

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Discrete Signals ◽

Human T Cell ◽

Definition Of

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Human T cell activation. IV. T cell activation and proliferation via the early activation antigen EA 1.

Journal of Experimental Medicine ◽

10.1084/jem.169.3.677 ◽

1989 ◽

Vol 169 (3) ◽

pp. 677-689 ◽

Cited By ~ 43

Author(s):

S Nakamura ◽

S S Sung ◽

J M Bjorndahl ◽

S M Fu

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Mrna Levels ◽

Dependent Manner ◽

Early Activation ◽

Human T Cell ◽

Accessory Cells ◽

Activation Antigen

A new mAb G38 was generated against purified EA 1, an early activation antigen. In immunoprecipitation, it was reactive with the same complex precipitated by the initial anti-EA 1 mAb P8. mAb G38 augmented PMA-induced proliferation of PBMC. It was shown to be mitogenic for purified T cells in collaboration with PMA in a dose-dependent manner. This effect was independent of monocytes and other accessory cells. mAb G38 augmented PMA-induced IL-2-R expression. In conjunction with PMA, it induced IL-2 synthesis and secretion. Its effects on IL-2-R and IL-2 expression were documented at both protein and mRNA levels. Both anti-EA 1 mAbs did not induce Ca2+ influx by themselves in PMA-treated T cells. However, the addition of second anti-mouse Ig antibodies induced readily detectable increases in [Ca2+]i. Ca2+-mediated pathways may be utilized as the transduction signal mechanisms. mAb Leu-23 was shown to be reactive with EA 1. mAb Leu-23 was also mitogenic for T cells in the presence of PMA. These findings provide evidence for a functional role for EA 1 in T cell activation and proliferation.

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