Research ArticleTHYMIC SELECTION

The early proximal αβ TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network

See allHide authors and affiliations

Science Immunology  15 Feb 2019:
Vol. 4, Issue 32, eaal2201
DOI: 10.1126/sciimmunol.aal2201
  • Fig. 1 PiSCES signatures reveal differential qualitative and quantitative TCR-proximal signaling activities.

    (A) PiSCES signature of LC13ab.huCD8ab.JRT3 cells stimulated with agonist peptide (FLRGRAYGL) for 5 min (mean log2 fold change, stimulated/basal; dotted lines indicate trend of nonsignificant protein pairs that appear as hits for data comparisons shown ahead). (B) Comparison of mean log2 fold changes in abundance of protein pair hits induced by agonist pMHC in LC13ab.huCD8ab.JRT3 cells versus SEE superantigen in Jurkat cells. Statistically significant hits that occurred in both stimuli are black, hits in LC13 system only are gray, and hits in SEE-Jurkat system are teal. (C) Subnetwork [from (B), teal points within the teal dashed box] visualized as the difference in mean log2 fold change in SEE versus LC13. (D) Surface staining with anti-CD28 or immunoglobulin G (IgG) negative control for Jurkat versus LC13ab.huCD8ab.JRT3 cells. (E) PiSCES signature of LC13ab.huCD8ab.JRT3 cells stimulated with antagonist peptide (FLRGRFYGL) for 5 min (mean log2 fold change, stimulated/basal; dotted lines indicate trend of nonsignificant protein pairs). (F) Comparison of mean log2 fold changes in abundance of protein pair hits induced by LC13 agonist versus antagonist stimulation. Compared with (B), note lack of teal “antagonist-only” hits. (G) PCA of four independent experiments shows separation of 5-min agonist versus antagonist PiSCES signatures.

  • Fig. 2 Predominant quantitative difference in PiSCES signatures comparing OT1 thymocyte response to positive and negative selection pMHC ligands.

    PiSCES signature of preselection OT1.β2m0.RAG20 thymocytes stimulated for 1 min with (A) a negative selection peptide, OVA (mean log2 fold change, OVA/FARL conditions), or (B) a positive selection peptide, Q7 (mean log2 fold change, Q7/FARL conditions). (C) PiSCES signature when response to OVA is directly normalized to the response to Q7 (mean log2 fold change, OVA/Q7 conditions). Dotted lines in (A) to (C) indicate trend of nonsignificant protein pairs that appear as hits in any of the three experimental comparisons performed. (D) Comparison of mean log2 fold changes in abundance of protein pair hits induced by OVA versus Q7 in preselection OT1.β2m0.RAG20 thymocytes. Data points are displayed for hits that were statistically significant in any of the OVA versus FARL, Q7 versus FARL, or OVA versus Q7 comparisons. A separate trajectory of orange points |x-axis value| > |y-axis value| that would clearly indicate positive selection–specific protein complexes is not observed. (E) PCA of three independent experiments shows separation of 1-min OVA/FARL versus Q7/FARL PiSCES signatures.

  • Fig. 3 Kinetics of PiSCES data for positive and negative selection stimuli.

    PiSCES data from preselection OT1.β2m0.RAG20 thymocytes stimulated for 1, 5, or 15 min with null peptide FARL, positive selection peptide Q7, or negative selection peptide OVA. (A and B) K-means clustering was performed using percent maximum log2 fold changes to define three kinetic patterns observed among the top 15 hits in response to OVA stimulation, categorized in groups 1 to 3. (A) A kinetic heat map of the log2 fold changes is shown for these hits defined by the OVA stimulation condition. The matching data points in response to Q7 stimulation were observed to display similar kinetic behavior, but lower intensity fold changes than those induced by OVA stimulation. (B) K-means clustering data displayed as percent maximum log2 fold change show that the three kinetic behavior groups defined by response to OVA stimulation (top) also described the overall kinetic behavior of the same protein pairs in response to Q7 stimulation (bottom). (C) With experimental n = 3 per time point, data across 1-, 5-, and 15-min time points were used to generate a kinetic PCA matrix. Subjectively, it appears that data for response to OVA versus Q7 are distinguishable but relatively close to each other at each time point, with the time point of stimulation playing a major role in data placement in three-dimensional analysis space, and OVA data appearing farther than Q7 data from a zero-stimulation point (*).

  • Fig. 4 PiSCES signature makes quantitative signaling prediction, which passes functional test in FTOC.

    (A to C) Fetal thymi of genotype OT1.RAG20.β2m0 were cultured for 7 days in the presence of exogenous β2m and specific peptides at the stated concentrations. (A) FARL peptide (10 μM) loads with high affinity into H-2Kb but has no functional affinity for the OT1 TCR and represents the background no-selection condition. (B) Q7 (10 μM) induced positive selection of a substantial portion of CD8 SP cells. (C) OVA (10 μM) induced deletion and loss of CD8+ cells. (D and E) Lowering the dose of OVA peptide to 0.75 nM made its PiSCES signature almost indistinguishable from that of 10 μM Q7. (D) When PiSCES data resulting from 5-min stimulation of preselection OT1.β2m0.RAG20 thymocytes with 0.75 nM OVA were normalized to data from stimulation with 10 μM Q7, the signatures virtually cancelled out, eliminating almost all hits (mean log2 fold change, OVA/Q7 comparison; dotted lines indicate trend of nonsignificant protein pairs). (E) PCA of PiSCES data in four experiments described in (D), where separation of the two stimulatory conditions was no longer observable. (F) OVA (0.75 nM) in FTOC induced positive selection of CD8 SP cells (lower right quadrant). (G) Gating the data in (F) on CD4(−) cells, the positively selected CD8 SP cells were largely conventional αβ T cells marked by CD8αβ expression. (H) Raising OVA concentration to 3 nM in FTOC still induced positive selection of CD8 SP cells. (I) Gating the data in (H) on CD4(−) cells, many of the positively selected cells were seen to represent unconventional CD8α+ CD8β cells. (J) Live CD8β+ SP T cell counts from two independent experiments [including the experiment depicted in (A) to (C) and (F) to (I)], with each FTOC normalized to the mean of its corresponding FARL 0.75 nM condition and reported as fold change. Number of thymus lobes per condition: 9 (FARL 0.75 nM), 9 (OVA 0.75 nM), 4 (Q7 10 μM), and 6 (OVA 3 nM). Unpaired two-tailed Student’s t test, P < 0.01.

  • Fig. 5 MHC-dependent signaling generates residual αβ T cells in CD3δ0mice.

    Live Thy1.2+ thymocytes were analyzed for CD4 and CD8 surface expression by flow cytometry from (A) wild-type B6, (B) CD3δ0, and (C) MHC II0.β2m0.CD3δ0 mice, and counts were obtained for (D) CD4 SP cells and (E) CD8 SP cells. The percentage of CD4+CD8+ DP thymocytes that were Thy1.2+ and CD69+ was obtained from the same three genotypes (F to I). Live Thy1.2+ splenocytes were analyzed for CD4 and CD8 surface expression by flow cytometry from the same three genotypes (J to L), and live counts were calculated for (M) CD4 T cells and (N) CD8 T cells. Mouse n was ≥ 4 per genotype. Statistical significance was determined by unpaired two-tailed t test, P < 0.05.

  • Fig. 6 Positive selection signaling in the context of CD3δ0.

    (A to G) Fetal thymi were cultured for 7 days in the presence of exogenous β2m (5 μg/ml) and specific peptides. For OT1.RAG20.β2m0 thymocytes (A to C), (A) 20 μM FARL peptide loads with high affinity into H-2Kb but has no functional affinity for the OT1 TCR and represents the background no-selection condition. (B) Q7 peptide (20 μM) induced positive selection of a substantial portion of CD8 SP cells. (C) OVA peptide (20 μM) induced deletion and loss of CD8+ cells. For OT1.β2m0.RAG20.CD3δ0 thymocytes (D to G), (D) 20 μM FARL peptide was inert and (E) Q7 peptide was inert, whereas (F) 20 μM OVA induced positive selection of a substantial portion of CD8 SP cells (lower right quadrant). (G) The positively selected CD8 SP cells from the OT1.β2m0.RAG20.CD3δ0 genotype were largely conventional αβ T cells as marked by CD8αβ expression. (H) Live CD8β+ SP T cell counts from two independent experiments [including the experiment depicted in (A) to (G)], with each FTOC normalized to the mean of its corresponding FARL 20 μM condition and reported as fold change. Number of thymus lobes per condition: 7 (FARL 20 μM) and 7 (OVA 20 μM). Unpaired two-tailed Student’s t test, P = 0.0069. (I and J) PiSCES analysis of signaling proteins that joined shared complexes in response to positive selection pMHC antigens. Dotted lines indicate trend of nonsignificant protein pairs. (I) Induction of protein pairs from OT1.RAG20.β2m0 thymocytes when assessing 5-min stimulation with 20 μM Q7 peptide over negative-control FARL peptide. (J) Induction of protein pairs from OT1.β2m0.RAG20.CD3δ0 thymocytes when assessing 5-min stimulation with 20 μM OVA peptide over negative-control FARL peptide.

  • Fig. 7 Diverse TCR repertoire in B6 and CD3δ0mice.

    A qPCR matrix-based method was used to assess relative representation of all possible TCRβ V-J combinations expressed as transcripts in splenocytes from two (A) B6 or (B) CD3δ0 mice. To approximate the maximum diversity generation potential per mouse for each genotype, the mean cycle threshold (Ct) value from the two mice with highest Shannon entropy for each genotype is displayed (top 2 of four B6 mice and top 2 of six CD3δ0 mice tested, when 15-ng total splenic RNA was used as input). As expected, because of higher T cell representation among splenocytes, B6 transcripts for TCR were more abundant than was found in CD3δ0. Greater quantities of specific transcripts appear darker due to fewer PCR amplification cycles required to reach Ct.

  • Fig. 8 T cells in CD3δ0mice provide immune activity against PCP and TMEV.

    (A) To assess the extent of T cell immune activity in a CD3δ0 setting, we infected mice from the listed genotypes with P. murina (mouse n ≥ 4 per genotype). Kaplan-Meier curves display survival defined by mice being euthanized upon loss of 20% weight, where CD3δ0 was statistically different from susceptible genotypes (P ≤ 0.004) by log-rank Mantel-Cox tests. (B) To test the role of MHC in mediating protection from PCP in CD3δ0 setting, mice from the listed genotypes were infected with PCP (mouse n ≥ 4 per genotype). Kaplan-Meier curves display survival defined by mice being euthanized upon loss of 20% weight, where CD3δ0 and MHC II0.β2m0.CD3δ0 were statistically different (P = 0.0018) by log-rank Mantel-Cox test. (C) Mice from the listed genotypes were infected with TMEV [mouse n ≥ 4 for all genotypes, except for CD3ε0ζ0 (n = 3)]. Kaplan-Meier curves display survival defined by mice being euthanized upon immobilization due to functional deficit or loss of 20% weight, where CD3δ0 was statistically different from susceptible genotypes (P ≤ 0.01) by log-rank Mantel-Cox tests. (D and E) To assess CD4 and CD8 T cell immune activity, B6 and CD3δ0 mice were either depleted of CD4 cells with GK1.5 anti-CD4 mAb injections, depleted of CD8 cells with 2.43 anti-CD8 mAb injections, depleted of both CD4 and CD8 cells, or PBS-control injected as indicated, and were infected with TMEV [mouse n = 4 for all groups, except for CD3δ0 + PBS (n = 3)]. Kaplan-Meier curves display survival defined by mice being euthanized upon immobilization due to functional deficit or loss of 20% weight, where CD3δ0 + PBS was statistically different from any of the three subset-depleted conditions (P < 0.02) by log-rank Mantel-Cox tests.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/32/eaal2201/DC1

    Results and Discussion

    Materials and Methods

    Fig. S1. Theoretical framework to categorize network patterns of TCR-proximal signaling protein complexes that could specify positive versus negative selection.

    Fig. S2. The response of preselection OT1.β2m0.RAG20 DP thymocytes to H2O2, and comparison with PV stimulation.

    Fig. S3. The response of preselection OT1.β2m0.RAG20 DP thymocytes to PV stimulation.

    Fig. S4. Kinetics of PiSCES signatures for positive and negative selection stimuli.

    Fig. S5. Kinetics of PiSCES signatures for agonist and antagonist stimuli in LC13ab.huCD8ab.JRT3 cells.

    Fig. S6. Clustering analysis for top hits observed across the kinetic for agonist versus antagonist stimuli in LC13ab.huCD8ab.JRT3 cells.

    Fig. S7. Kinetics of PiSCES signatures for agonist and antagonist stimuli in OT1ab.muCD8ab.JRT3 cells.

    Fig. S8. Clustering analysis for top hits observed across the kinetic for OVA versus Q7 stimuli in OT1ab.muCD8ab.JRT3 cells.

    Fig. S9. Assessment of CD8β and CD8αα (TLA+) expression in OT1.RAG20.β2m0 FTOC cells.

    Fig. S10. FTOCs from positive selection conditions can be induced by antigenic stimulation to proliferate and kill target cells.

    Fig. S11. MHC-dependent signaling generates residual αβ T cells in CD3δ0 mice.

    Fig. S12. Developmental and maturation markers on peripheral T cells of wild-type and mutant mice.

    Fig. S13. The few peripheral T cells in OT1.RAG20.CD3δ0 mice require MHC class I for their generation/survival.

    Fig. S14. Diverse TCR repertoire in individual B6 and CD3δ0 mice.

    Fig. S15. Multiple peripheral TCRα transcripts in B6 and CD3δ0 mice.

    Fig. S16. T cells in CD3δ0 mice provide immune activity against PCP.

    Fig. S17. T cells in CD3δ0 mice provide immune activity against TMEV.

    Fig. S18. Example gating used for flow cytometry data.

    Table S1. Validated Ab pairs used to identify each mouse protein target.

    Table S2. Raw data for experiments with n ≤ 25.

    References (7582)

  • Supplementary Materials

    The PDF file includes:

    • Results and Discussion
    • Materials and Methods
    • Fig. S1. Theoretical framework to categorize network patterns of TCR-proximal signaling protein complexes that could specify positive versus negative selection.
    • Fig. S2. The response of preselection OT1.β2m0.RAG20 DP thymocytes to H2O2, and comparison with PV stimulation.
    • Fig. S3. The response of preselection OT1.β2m0.RAG20 DP thymocytes to PV stimulation.
    • Fig. S4. Kinetics of PiSCES signatures for positive and negative selection stimuli.
    • Fig. S5. Kinetics of PiSCES signatures for agonist and antagonist stimuli in LC13ab.huCD8ab.JRT3 cells.
    • Fig. S6. Clustering analysis for top hits observed across the kinetic for agonist versus antagonist stimuli in LC13ab.huCD8ab.JRT3 cells.
    • Fig. S7. Kinetics of PiSCES signatures for agonist and antagonist stimuli in OT1ab.muCD8ab.JRT3 cells.
    • Fig. S8. Clustering analysis for top hits observed across the kinetic for OVA versus Q7 stimuli in OT1ab.muCD8ab.JRT3 cells.
    • Fig. S9. Assessment of CD8β and CD8αα (TLA+) expression in OT1.RAG20.β2m0 FTOC cells.
    • Fig. S10. FTOCs from positive selection conditions can be induced by antigenic stimulation to proliferate and kill target cells.
    • Fig. S11. MHC-dependent signaling generates residual αβ T cells in CD3δ0 mice.
    • Fig. S12. Developmental and maturation markers on peripheral T cells of wild-type and mutant mice.
    • Fig. S13. The few peripheral T cells in OT1.RAG20.CD3δ0 mice require MHC class I for their generation/survival.
    • Fig. S14. Diverse TCR repertoire in individual B6 and CD3δ0 mice.
    • Fig. S15. Multiple peripheral TCRα transcripts in B6 and CD3δ0 mice.
    • Fig. S16. T cells in CD3δ0 mice provide immune activity against PCP.
    • Fig. S17. T cells in CD3δ0 mice provide immune activity against TMEV.
    • Fig. S18. Example gating used for flow cytometry data.
    • Table S1. Validated Ab pairs used to identify each mouse protein target.
    • References (7582)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data for experiments with n ≤ 25.

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article