TCR Trafficking in Resting and Stimulated T Cells

Geisler, Carsten

doi:10.1615/critrevimmunol.v24.30

Cited by 28 publications

(43 citation statements)

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“…Thus, novel labeling strategies for different imaging modalities should meet several requirements, such as reduced side effects to target cells, high intracellular retention, the possibility of long tracking periods, and easy transfer to clinical practice. We developed a new specific and direct intracellular labeling strategy for mouse T cells by applying TCR-specific mAbs that are internalized in the cells via endocytosis within 24 h. Because of the high regular turnover rate of the TCR and the subsequent expression of free TCR molecules on the cell membrane, TCR internalization enables intracellular labeling of T cells (14,15). Intracellular labeling via the application of radiolabeled mAbs provides a low efflux of the label and, consequently, a high in vivo signal-to-background contrast among PET studies.…”

Section: Discussionmentioning

confidence: 99%

⁶⁴ Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

Griessinger

Maurer

Kesenheimer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64 Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosisnecrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64 Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayedtype hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.PET imaging | mouse T cells | in vivo cell tracking | antibody-based cell labeling | airway DTHR

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Section: Discussionmentioning

confidence: 99%

⁶⁴ Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

Griessinger

Maurer

Kesenheimer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, the quality of CD4 APC-Alexa 750 fluorescence was acceptable, but relatively dim, even on singly-stained samples. When used in combination with other stains, as in panels 6,8,10,17,[20][21][22] (Fig .2), APC-Alexa750 CD4 1 subsets often appeared as diffuse regions that were difficult to distinguish from CD8 1 cells, a problem that was overcome only in the context of reagent panel 26.…”

Section: Discussionmentioning

confidence: 99%

Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach

et al. 2008

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Polychromatic flow cytometry offers the unprecedented ability to investigate multiple antigens per cell. Unfortunately, unwanted spectral overlaps and compensation problems increase when more than four colors are used, but these problems can be minimized if staining combinations are chosen carefully. We used an empiric approach to design, test and identify six-color T cell immunophenotyping reagent panels that can be expanded to include three or more functional or other markers in the FITC, PE, and APC channels without significant spectral limitations. Thirty different six-color T cell surface antigen reagent panels were constructed to identify major T cell subsets and maturational subtypes as defined by CCR7 and CD45RA expression, while excluding monocytes, B and non-viable cells. Staining performance of each panel was compared on cryopreserved cells from a single healthy donor recorded on a multiparameter cell sorter. Ten of the thirty reagent panels offered reliable resolution of T cell major and maturational surface markers. Of these, two panels were selected that showed the least spectral overlap and resulting background increase in the FITC, PE, and APC channels. These channels were left unoccupied for inclusion of additional phenotypic or functional markers, such as cytokines. Careful reagent titration and testing of multiple candidate panels are necessary to ensure quality results in multiparametric measurements. ' 2008 International Society for Advancement of Cytometry Key terms polychromatic flow cytometry; T cell immunophenotyping; fluorochrome conjugated antibody; compensation POLYCHROMATIC flow cytometry allows for detailed measurements even with small sample sizes and has recently been advanced by the development of new instrumentation, reagents and data analysis tools. Despite these significant improvements, it can be difficult to derive meaningful results when reagent combinations are expanded to include eight or more fluorescent markers. This is because unwanted spectral overlaps and measurement errors worsen as the number of fluorochromes used to label coordinately expressed cell markers increase (1-3). Using appropriate controls, software compensation algorithms can correct spillover problems post acquisition. However, due to the increased number of spectral overlaps in polychromatic reagent combinations, even properly compensated data can exhibit unwanted spreading into other measurement channels, complicating data analysis and interpretation (3,4). For example, dimly expressed markers, such as cytokines, are difficult to measure in channels where spreading in properly compensated data increases the background in the cytokine measurement channel. Such data spread in effect masks low intensity events; a problem not usually apparent when only a few stains are used simultaneously.A variety of sources contribute to this error in compensated data and are partially corrected by newer digital electronic configurations that collect and store data

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“…To transmit the signal through the plasma membrane, the TcR uses a multimeric CD3 complex whose subunits (γ-, δ-, ε-, and ζ chains) contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). Upon TcR ligation, the two tyrosine residues in the ITAMs (Yxx[L/I] x [6][7][8][9] Yxx[L/I]) are phosphorylated by the Src family kinases Lck and Fyn. The dually phosphorylated ITAMs serve as a binding platform for molecules involved in TcR/CD3-proximal signaling, most importantly the tyrosine kinase ZAP-70, which phosphorylates downstream targets (1).…”

mentioning

confidence: 99%

“…TcR/CD3 is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells (reviewed in ref. 6), but the function of this turnover as well as the phosphorylation state of internalized receptor remain unknown.…”

mentioning

confidence: 99%

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Yudushkin

Vale²

2010

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/ CD3 signaling after the receptor internalization. Recent studies using high-resolution imaging have demonstrated that signaling via TcR/CD3 displays complex spatial organization. TcR/CD3 molecules are preclustered at the plasma membrane and, upon ligation, nucleate downstream signaling components seconds after T-cell activation (4, 5). TcR/CD3 is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells (reviewed in ref. 6), but the function of this turnover as well as the phosphorylation state of internalized receptor remain unknown.Currently, phosphorylation of TcR/CD3 in cells and in vitro is monitored using phospho-specific antibodies, and there are no methods that enable dynamic monitoring of the spatial organization of CD3 phosphorylation in live cells. Here, we have constructed genetically encoded fluorescent reporters compatible with imaging of live and fixed cells and demonstrate that they accurately monitor the dynamics and intracellular organization of CD3ζ phosphorylation in Jurkat T cells. Using the reporters, we observed that in addition to the expected activationdependent phosphorylation at the plasma membrane, tyrosinephosphorylated CD3ζ accumulated in the perinuclear endosomal vesicles. Our results demonstrate that endosomal CD3ζ remains signaling-competent and suggest the possibility that internalized CD3ζ pool may help to sustain long-term signaling in T cells. Results Design and Characterization of the CD3ζ Phosphorylation Reporters.To generate a Förster's resonant energy transfer (FRET)-based monomolecular reporter for phosphorylation of the key CD3 signal-transducing subunit ζ, we fused the C terminus of CD3ζ to a pair of green and red fluorescent proteins, eGFP and mCherry, linked by a flexible spacer and followed by the tandem SH2 domains of human ZAP-70 (residues 1-259; tSH2 ). We reasoned that intramolecular binding of the SH2 domains to tyrosine-phosphorylated ITAMs of CD3ζ (7) would result in conformational rearrangement of the adjacent fluorescent proteins and change in the FRET efficiency between the dono...

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TCR Trafficking in Resting and Stimulated T Cells

Cited by 28 publications

References 0 publications

⁶⁴ Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

⁶⁴ Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

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TCR Trafficking in Resting and Stimulated T Cells

Cited by 28 publications

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64 Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

64 Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

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⁶⁴ Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

⁶⁴ Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET