A new method for rapid detection of T lymphocyte decision to proliferate after encountering activating surfaces

Crétel, E.; Touchard, Dominique; Bongrand, Pierre; Pierrès, Anne

doi:10.1016/j.jim.2010.10.007

Cited by 20 publications

(39 citation statements)

References 31 publications

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“…Previous experimental studies suggested that the initiation of an activation program might require the triggering of hundreds (42) to thousands (43) of TCRs, depending on the stage of T cells and conditions of TCR engagement. This would result in a combination of events including calcium rise, cytoskeletal reorganization, adhesion stabilization, and spreading with a time scale of minutes (15,20,21,42,44). We suggest that our experiments revealed the unitary recognitions events of a few second duration that preceded the coordinated global cell responses that are described in aforementioned reports.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, it is well known that PLCg1 activation is an early consequence of TCR activation. Also, when we performed a series of preliminary experiments to monitor the phosphorylation of known components of TCR-triggered cascades with immunofluorescence (20), only PLCg1 displayed robust (2-fold) phosphorylation increase in contrast with lck, ZAP-70, LAT, SLP-76, rac-1, cdc42, and Itk (not shown). Further, there are numerous potential links between PLCg1 activation and the events we observed: indeed, membrane retraction might be a consequence of inactivation of ERM protein interaction with membranes (45) as a consequence of PIP 2 elimination.…”

Section: Discussionmentioning

confidence: 99%

“…PBMCs were purified by density gradient centrifugation using Lymphocyte Separation Medium (Eurobio, Les Ulis, France) following standard techniques. CD4 + T lymphocytes were isolated by negative selection using magnetic cell sorting (Miltenyi Biotec) (20,21).…”

Section: Cd4mentioning

confidence: 99%

“…In contrast, encounter with surfaces made adherent to lymphocytes with anti-HLA class I Abs resulted in the slow growth of molecular contacts (∼0.2 mm 2 /s) after a lag of ∼1 min. When surfaces were coated with anti-CD3 Abs, thus inducing strong delayed proliferation (20,21), the 1-min lag was followed by a spreading response that was ∼8-fold more rapid than that found on anti-HLA-coated surfaces (∼1.5 mm 2 /s) and generated ∼4-fold higher contact area 5 min later. Thus.…”

mentioning

confidence: 96%

“…This may be viewed as a global event involving the whole cell (16) and a hallmark of analog-to-digital signal processing (17,18). Also, strong evidence supports the hypothesis that extensive morphological changes may act as early reporters of the triggering of a specific activation program (19,20). However, the precise time scale of and relationship among membrane motion, membrane receptor engagement, and triggering of a coordinated mechanical response remain incompletely understood.…”

mentioning

confidence: 97%

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T Lymphocytes Sense Antigens within Seconds and Make a Decision within One Minute

Brodovitch

Bongrand

Pierrès

2013

The Journal of Immunology

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Adaptive immune responses are triggered by the rapid and sensitive detection of MHC-bound peptides by TCRs. The kinetics of early TCR/APC contacts are incompletely known. In this study, we used total internal reflection fluorescence microscopy to image human T cell membranes near model surfaces: contact was mediated by mobile protrusions of <0.4 μm diameter. The mean lifetime of contacts with a neutral surface was 8.6 s. Adhesive interactions increased mean contact time to 27.6 s. Additional presence of TCR ligands dramatically decreased contact to 13.7 s, thus evidencing TCR-mediated triggering of a pulling motion within seconds after ligand encounter. After an interaction typically involving 30–40 contacts formed during a 1-min observation period, TCR stimulation triggered a rapid and active cell spreading. Pulling events and cell spreading were mimicked by pharmacological phospholipase Cγ1 activation, and they were prevented by phospholipase Cγ1 inhibition. These results provide a quantitative basis for elucidating the earliest cell response to the detection of foreign Ags.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Cd4mentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 97%

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T Lymphocytes Sense Antigens within Seconds and Make a Decision within One Minute

Brodovitch

Bongrand

Pierrès

2013

The Journal of Immunology

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Flow cytometric measurement of STAT5 phosphorylation in cytomegalovirus‐stimulated T cells

et al. 2020

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Cytomegalovirus (CMV)‐specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. Given the critical role STAT5A phosphorylation (pSTAT5A) in T cell proliferation, this study presents a simple and sensitive flow cytometric‐based pSTAT5A assay to quickly identify CMV‐specific T cell proliferation. We determined pSTAT5A in T cells treated with CMV‐specific peptide mix (pp65 + IE1 peptides) from 20 healthy adult subjects and three immunodeficient patients with CARMIL‐2 mutation. After stimulation, the percentage of pSTAT5A+ T cells in CMV‐seropositive (CMV+) subjects significantly increased from 3.0% ± 1.9% (unstimulated) to 11.4% ± 5.9% (stimulated) for 24 h. After 7 days of stimulation, the percentage of expanded T cells amounted to 26% ± 17.2%. Conversely, the percentage of pSTAT5A+ T cells and T cell proliferation from CMV‐seronegative (CMV−) subjects hardly changed (from 3.0% ± 1.3% to 3.7% ± 1.8% and from 4.3% ± 2.1% to 5.7% ± 1.7%, respectively). We analyzed the correlation between the percentage of pSTAT5A+ T cells versus (1) CMV‐IgG concentrations versus (2) the percentage of expanded T cells and versus (3) the percentage of initial CMV‐specific T cells. In immunodeficient patients with CARMIL‐2 mutation, CMV‐specific pSTAT5A and T cell proliferation were completely deficient. In conclusion, flow cytometric‐based pSTAT5A assay represents an appropriate tool to quickly identify CMV‐specific T cell proliferation and helps to understand dysfunctions in controlling other pathogens. Flow cytometric‐based pSTAT5A assay may be a useful test in clinical practice and merits further validation in large studies.

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T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR‐binding parameters

et al. 2015

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T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide–MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

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A new method for rapid detection of T lymphocyte decision to proliferate after encountering activating surfaces

Cited by 20 publications

References 31 publications

T Lymphocytes Sense Antigens within Seconds and Make a Decision within One Minute

T Lymphocytes Sense Antigens within Seconds and Make a Decision within One Minute

Flow cytometric measurement of STAT5 phosphorylation in cytomegalovirus‐stimulated T cells

T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR‐binding parameters

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