Research ResourceTechniques

Multiplex matrix network analysis of protein complexes in the human TCR signalosome

See allHide authors and affiliations

Science Signaling  02 Aug 2016:
Vol. 9, Issue 439, pp. rs7
DOI: 10.1126/scisignal.aad7279
  • Fig. 1 A protein-protein interaction web consisting of the proximal TCR signalosome.

    Visualization of many indirect and direct interactions among 20 proteins that mediate proximal TCR signaling, according to published reports or the Protein Interaction Network Analysis (PINA) database (43). Note that not all of the possible interactions among the collection are depicted, and this visualization provides no information regarding the activity of protein complexes under specific physiologic conditions.

  • Fig. 2 PiSCES workflow for network profiling of TCR signalosome activation.

    (A) Protein complexes immunoprecipitated on microsphere color classes coupled to capture antibodies were detected with a fluorophore-coupled detection (probe) antibody. (B) A Bio-Plex 200 instrument with a customized setup was used to refrigerate samples at 4°C through the data acquisition process. An insulated plexiglass divider enabled the upper flow cytometer portion to remain at room temperature, whereas the lower plate carrier and samples were maintained at 4°C. See fig. S2 for further details. (C) Bead classes were identified by the ratio of two classifier dyes. (D) PE fluorescence readings of example protein-protein associations that increased in intensity upon 5-min stimulation of Jurkat cells with SEE-loaded Raji cells. Black trace, unstimulated; red trace, stimulated. (E) Hierarchical clustering of PiSCES network median fluorescence intensity (MFI) values distinguished SEE-stimulated [S, red treatment (Tx) group] from unstimulated (U, black Tx group) conditions. Data from four independent experiments (labeled 1, 2, 3, and 4) were included in the analysis. P = 0.028, Fisher’s exact test. (F) PCA of log2 MFI from the four experiments (labeled 1, 2, 3, and 4) also distinguished SEE-stimulated from unstimulated conditions, displaying a stimulation axis that was largely dominated by principal component 1 (PC1).

  • Fig. 3 Comparison of ANC and WCNA analyses.

    (A) ANC analysis of four independent SEE stimulation experiments identified PiSCES that exhibited statistically significant changes in three of four experiments for the SEE data set. Edge color and thickness correspond to mean log2 fold change (color legend on the right). (B) WCNA analysis of the MFI values from the same SEE data set. PiSCES with MM > 0.7 in the turquoise stimulation network (see fig. S4 and Materials and Methods) are visualized in the node-edge diagram. Edge thickness corresponds to mean log2 fold change, whereas edge color corresponds to MM (color legend on the right). (C) Venn diagram of the overlap between both methods of analysis. Because most hits coincided between the two analyses, we concluded that the two analyses produced compatible results. (D) Hits identified by ANC (red), WCNA (blue), or both (dark purple) are graphed by the absolute value (Abs) of mean log2 fold change and the absolute value of MM in the stimulation (stim) (turquoise) module.

  • Fig. 4 High-confidence PiSCES network signature of SEE-induced proximal TCR signaling.

    Shared hits identified by both ANC and WCNA analyses (ANC ∩ WCNA) are displayed for the PiSCES data set introduced in Figs. 2 and 3, in which Jurkat cells were either left unstimulated or stimulated for 5 min with SEE-loaded Raji cells. Edge color and thickness correspond to the mean log2 fold change (color legend on the right) from four independent experiments.

  • Fig. 5 High-confidence PiSCES network signature of CD28 costimulation.

    Network activity profile is displayed for Jurkat cells stimulated by SEE-loaded Raji cells in the presence of physiological CD28 engagement (control human IgG–treated) normalized to the CD28-blocked condition (CTLA4-Ig–treated). Shared hits identified by both ANC and WCNA analyses (ANC ∩ WCNA) are displayed. Edge color and thickness correspond to mean log2 fold change (color legend on the right) from three independent experiments.

  • Fig. 6 PiSCES network analysis of clinical biopsies distinguishes the alopecia areata patient group from the control patient group and suggests enhanced basal TCR signaling network activity in alopecia areata.

    (A) A 4-mm scalp punch biopsy from which T cells were isolated. (B) Hierarchical clustering of the normalized fold changes in MFI predominately separates the seven alopecia areata patient samples from the five control patient samples. S, exogenously stimulated; U, unstimulated. P = 0.015, extension of Fisher’s exact test. (C) PCA of basal (not exogenously stimulated) PiSCES activity among all stimulation-responsive protein complexes (identified in fig. S5) separates alopecia areata patient samples from control patient samples. (D) Difference in the basal activity of stimulation-responsive network PiSCES in the alopecia areata patient group as compared to the control patient group (red indicates greater activity in the alopecia areata cells; blue indicates greater activity in the control patient cells) suggests that more stimulation-responsive PiSCES were basally increased in relative abundance in the alopecia areata patient group versus the control patient group. P = 0.039 with control-clustered patient AA4 removed; P = 0.115 with patient AA4 included, using a resampling-based test.

  • Fig. 7 PiSCES network activity suggests a disease-associated signaling profile in T cells from alopecia areata patients.

    (A) Using PiSCES data from patient and control T cells that were exogenously stimulated for 5 min with plate-bound anti-CD3 and anti-CD28 antibodies, WCNA analysis of normalized MFI fold changes identified a PiSCES subnetwork module that distinguished the alopecia areata patient group from the control patient group. Red, enhanced in the alopecia areata condition; blue, enhanced in the control condition. (B) An alternative view of the subnetwork module, with the nodes positioned on the basis of the activity in alopecia areata patient samples (n = 7) versus control patient samples (n = 5).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/439/rs7/DC1

    Equations

    Fig. S1. Screening strategy for multiplex panel antibodies, using SLP-76 as an example.

    Fig. S2. Instrument setup for optimal analysis of protein complexes.

    Fig. S3. Development and evaluation of ANC analysis.

    Fig. S4. Application and evaluation of WCNA analysis for the SEE-stimulated and unstimulated Jurkat cell data.

    Fig. S5. Stimulation-induced PiSCES network is similar between control and alopecia areata patient groups.

    Table S1. Validated antibody pairs used to identify each target in Jurkat cells.

    Table S2. Phenotypic characteristics of alopecia areata patients and controls and their T cell populations.

  • Supplementary Materials for:

    Multiplex matrix network analysis of protein complexes in the human TCR signalosome

    Stephen E. P. Smith, Steven C. Neier, Brendan K. Reed, Tessa R. Davis, Jason P. Sinnwell, Jeanette E. Eckel-Passow, Gabriel F. Sciallis, Carilyn N. Wieland, Rochelle R. Torgerson, Diana Gil, Claudia Neuhauser,* Adam G. Schrum*

    *Corresponding author. Email: schrum.adam{at}mayo.edu (A.G.S.); neuha001{at}umn.edu (C.N.)

    This PDF file includes:

    • Equations
    • Fig. S1. Screening strategy for multiplex panel antibodies, using SLP-76 as an example.
    • Fig. S2. Instrument setup for optimal analysis of protein complexes.
    • Fig. S3. Development and evaluation of ANC analysis.
    • Fig. S4. Application and evaluation of WCNA analysis for the SEE-stimulated and unstimulated Jurkat cell data.
    • Fig. S5. Stimulation-induced PiSCES network is similar between control and alopecia areata patient groups.
    • Table S1. Validated antibody pairs used to identify each target in Jurkat cells.
    • Table S2. Phenotypic characteristics of alopecia areata patients and controls and their T cell populations.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 3.12 MB


    Citation: S. E. P. Smith, S. C. Neier, B. K. Reed, T. R. Davis, J. P. Sinnwell, J. E. Eckel-Passow, G. F. Sciallis, C. N. Wieland, R. R. Torgerson, D. Gil, C. Neuhauser, A. G. Schrum, Multiplex matrix network analysis of protein complexes in the human TCR signalosome. Sci. Signal. 9, rs7 (2016).

    © 2016 American Association for the Advancement of Science

Navigate This Article