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Phospho-flow cytometry based analysis of differences in T cell receptor signaling between regulatory T cells and CD4+ T cells.

ABSTRACT: CD4+ T regulatory cells (Tregs) are activated during auto-immune, injury, and inflammatory responses, however, the molecular events that trigger Treg activation are poorly understood. The purpose of this study was to investigate whether Tregs (FoxP3+ CD4+ T cells) and non-Treg CD4+ T cells might display differences in T cell receptor (TCR) dependent signaling responses following in vitro or in vivo stimulation. This study used phospho-flow cytometry as a tool to profile the kinetics and extent of TCR signaling (ZAP-70 and PKC-? phosphorylation and expression) in Tregs and non-Tregs. We found that in vitro stimulation with anti-CD3? induces early and transient activation of ZAP-70 and PKC-? in both Tregs and non-Tregs. However, the response in Tregs was more rapid and higher in magnitude than responses seen in non-Tregs. In contrast, bacterial superantigen or antigen-specific TCR stimulation did not significantly activate these signaling pathways in Tregs or non-Tregs. Additional experiments tested the kinetics of in vivo TCR signaling in Tregs and non-Tregs in mice challenged with bacterial superantigen. The results of these experiments showed that superantigen rapidly activated ZAP-70 and PKC-? in lymph node Tregs, but not in non-Tregs. In summary, we demonstrate the versatility of using phospho-flow cytometry to measure cell signaling in CD4+ T cells. The results of these in vitro and in vivo studies demonstrate that Tregs and non-Treg CD4+ T cells show marked differences in their reactivity to TCR-dependent stimulation and contribute new insights into basic mechanisms that lead to Treg activation.

SUBMITTER: Hanschen M

PROVIDER: S-EPMC4287240 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Phospho-flow cytometry based analysis of differences in T cell receptor signaling between regulatory T cells and CD4+ T cells.

Hanschen Marc M Tajima Goro G O'Leary Fionnuala F Hoang Kimberly K Ikeda Kimiko K Lederer James A JA

Journal of immunological methods 20110906 1-2

CD4+ T regulatory cells (Tregs) are activated during auto-immune, injury, and inflammatory responses, however, the molecular events that trigger Treg activation are poorly understood. The purpose of this study was to investigate whether Tregs (FoxP3+ CD4+ T cells) and non-Treg CD4+ T cells might display differences in T cell receptor (TCR) dependent signaling responses following in vitro or in vivo stimulation. This study used phospho-flow cytometry as a tool to profile the kinetics and extent o ...[more]

PMID: 21945004

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Project description:BackgroundKnowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR) and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and marginal zone lymphoma (MZL) patients.MethodsSamples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR), CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling.ResultsMalignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p)-SFKs, p-PLC?, p-ERK, p-p38, p-p65 (NF-?B), p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLC? levels. Impaired anti-BCR-induced p-PLC? was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-?B) was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells.ConclusionsBCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation-induced phosphorylation of signaling proteins in distinct cell populations can be used to identify aberrant signaling pathways.

Project description:BackgroundIn our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.MethodTargeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.ResultsThe copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.ConclusionBecause of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Dataset Information

Phospho-flow cytometry based analysis of differences in T cell receptor signaling between regulatory T cells and CD4+ T cells.

Publications

Phospho-flow cytometry based analysis of differences in T cell receptor signaling between regulatory T cells and CD4+ T cells.

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