Research ArticleTCR Signaling

Alternative ZAP70-p38 signals prime a classical p38 pathway through LAT and SOS to support regulatory T cell differentiation

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Science Signaling  23 Jul 2019:
Vol. 12, Issue 591, eaao0736
DOI: 10.1126/scisignal.aao0736
  • Fig. 1 p38 activity is essential for TH cell differentiation, and two distinct pathways connect TCR to p38 activation.

    (A) Flow cytometry analysis of the indicated cytokine and transcription factors abundance in naïve CD4+ T cells cultured in vitro TH1 (IFN-γ+TBet+), TH2 (Gata3+IL-4+), TH17 (IL-17A+FoxP3), and iTreg (IL-17AFoxP3+) polarizing conditions after control DMSO or p38 inhibitor SB203583 (p38i) treatment. Data are representative of two independent experiments. (B) Model of the distinct p38 pathways that exist in T cells and B cells, where a ZAP70-dependent alternative pathway that is unique to T cells. (C and D) Flow cytometry analysis of pZAP70 (Tyr319) and pp38 (Thr180Tyr182) in wild-type Jurkat cells and LAT-deficient J.Cam2 cells at rest (filled) or at 3 min after TCR activation (open). Activation of p38 after TCR stimulation was compared among cells that activated ZAP70 comparably (pZAP70+ gate), and the fold increase relative to unstimulated cells is indicated (right). Histograms (C) and quantified data (D) are representative of two independent experiments (see also fig. S1, A and B). (E and F) Flow cytometry analysis of LAT, pERK1/2, and pp38 in wild-type and CRISPR-Cas9–mediated LAT knockout human CD4+ T cells. Gates separating LAT-hi and LAT-low cells (E) were determined by LAT staining of unedited control cells (red histogram) and staining control (blue histogram). The activation of ERK1/2 and p38 (F) were determined in LAT-hi and LAT-low cells after TCR stimulation, and insets indicate the fold increase after activation (open histogram) relative to unstimulated cells (shaded histogram). Histograms are representative of two independent experiments (see also fig. S1E).

  • Fig. 2 Strong activation of p38 is delayed and requires p38 kinase activity.

    (A and B) Flow cytometry analysis of p38 activation in Jurkat T cells stimulated with two doses of antibody against mouse TCR (A) or DT40 B cells stimulated with two doses of antibody against mouse BCR (B). Histograms are representative of two independent experiments (see also fig. S4). (C) Flow cytometry analysis of p38 activation DT40 B cells treated with DMSO, P38i (SB203580), or MEKi (U0126) and stimulated with antibody against mouse BCR for the indicated times. Dashed lines indicate p38 activation in the resting cells, and insets indicate the mean fluorescence intensity (MFI) of pp38. Histograms are representative of two independent experiments (also, fig. S5). (D and E) Western blot analysis of pZAP70 and pp38 in lysates from primary naïve CD4+ T cells (D) and CD4+ T blast cells (E) stimulated with antibodies against CD3ε. Blots are representative of two independent experiments (see also fig. S6A). (F) Flow cytometry analysis of p38 activation of CD4+ T blast cells stimulated with antibodies against CD3ε. Histograms are representative of two independent experiments (see also fig. S6B). (G) Western blot analysis of phosphorylation of the indicated proteins in lysates from CD4+ T cell blasts treated with DMSO or P38i and stimulated with antibodies against CD3ε. Blots are representative of two independent experiments (see also fig. S6C). (H) In the alternative pathway, TCR-induced ZAP70 activity phosphorylates Tyr323 in the p38 C terminus. Phosphorylation at that site induces autophosphorylation at Thr180 within the Thr180-X-Tyr182 activation loop. The p38 inhibitor SB203580 but not the MEK inhibitor U0126 blocks phosphorylation of p38 at Thr180 and Tyr182.

  • Fig. 3 In silico modeling of p38 activation predicts distinct roles for alternative and classical pathways.

    (A) A coarse grain model of p38 activation pathways in T cells where the alternative (alternate) pathway is directly connected to ZAP70 activation, and the classical pathway requires LAT signalosome formation and MAPK cascade activation. Temporal delay is introduced to reflect p38 activity–dependent full p38 activation. (B) In silico prediction of p38 activation with intermediate contribution from both the alternative and classical pathways. (C to F) Predicted p38 activation after varying the strength (none, weak, and strong) of alternative pathway (C) or classical pathway (D) activation. Projection in each model is graphically represented for alternative pathway (E) or classical pathway (F). The rate constant characterizing the phosphorylation of p38 by active ZAP70 was varied, from a value of zero (no contribution of alternative pathway) to stronger values (0.005 and 0.02).

  • Fig. 4 Basal ZAP70–dependent alternative pathway lowers the activation threshold to facilitate strong activation of p38.

    (A) Flow cytometry analysis of p38 activation Jurkat and DT40 B cells (top) or Jurkat cells treated with DMSO or the Src kinase inhibitor PP2. Histograms are representative of two independent experiments (see also fig. S7A). (B and C) Iteration of the p38 activation model that includes basal ZAP70 kinase activity (B) and in silico predictions of p38 activation with or without basal ZAP70 activity (C). (D) Flow cytometry analysis of pZAP70 and pp38 in DT40 B cells transfected with expression constructs for human ZAP70 or SYK kinases. Three subpopulations were arbitrarily defined as low (black), medium (blue), and high (red) for pZAP70 or pSYK (left), and histograms indicate pp38 of the indicated subpopulation with the inset of MFI values. The black vertical divider marks the basal p38 activation in parental DT40 cells. Data are representative examples of two independent experiments (see also fig. S7B). (E) Western blot analysis of the indicated proteins in lysates from ZAP70–deficient Jurkat T cells (P116) transfected with ZAP70 or SYK expression constructs and stimulated with antibody against CD3ε for the indicated times. Band intensity values are given relative to the indicated control. Blots are representative examples of two independent experiments (see also fig. S7C). (F to H) Flow cytometry analysis of pZAP70 and pp38 in ZAP70–deficient Jurkat T cells (P116) transfected with ZAP70 or SYK expression constructs (F) and stimulated with antibody against CD3ε for the indicated times (G). Subpopulations with low (black), medium (blue), or high (red) pZAP70 or pSYK abundance were identified (left), and histograms (right) indicate p38 activation in cells with equivalent ZAP70 (filled histogram) or SYK (open histogram) activation. Flow cytometry plots and quantified p38 values after CD3ε stimulation (H) are representative of two independent experiments (see also fig. S7, C and D).

  • Fig. 5 SOS1/2 deficiency reduces p38 activation and regulatory T cell differentiation.

    (A) Iteration of the p38 activation model that includes SOS and Grb2 molecules added as a component of the LAT signalosome. (B) Flow cytometry analysis of pp38 in wild-type (filled) and SOS1/2−/− (open) DT40 B cells stimulated with antibody against the BCR for the indicated times. Histograms are representative of two independent experiments (see also fig. S8, A and B). (C) Flow cytometry analysis of pp38 in wild-type and SOS1/2 dKO naïve CD4+ T cells stimulated with antibody against CD3ε for the indicated times. Histograms are representative of two independent experiments (see also fig. S8C). (D) Western blot analysis of the indicated proteins in lysates from wild-type or Sos1/2 dKO naïve CD4+ T cells with antibody against CD3ε for the indicated times. Blots are representative of three independent experiments (see also fig. S8D). (E to H) Flow cytometry analysis of the indicated cytokine and transcription factor expression in wild-type (top) and Sos1/2 dKO (bottom) naïve T cells cultured under TH1 (E), TH2 (F), TH17 (G, left), or iTreg (G, right) polarizing conditions. Dot plots (E to G) are representative of two independent experiments (see also figs. S8, E and F, and S9). The frequency of iTreg induced by stimulation of wild-type (filled) and Sos1/2 dKO (open) CD4+ T cells with increasing anit-CD3ε doses (H) of at least five mice are from two independent experiments. *P < 0.05, **P < 0.01, and n.s., not significant by two-tailed Mann-Whitney test. WT, wild type.

  • Fig. 6 SOS1/2 deficiency leads to reduced IL-2 production and increased sensitivity to EAE.

    (A and B) Flow cytometry analysis of CD25 and IL-2 abundance in wild-type (left) and Sos1/2 dKO (right) naïve T cells treated with DMSO or p38i after TCR stimulation. The relative percentage of cells in each quadrant is indicated. Histograms (A) are representative of two independent experiments, and quantified frequency of CD25+IL-2+ (B) are means ± SEM from two experiments performed in duplicate. (C to G) EAE disease severity in wild-type, SOS2 sKO, and SOS1/2 dKO mice was clinically scored at the indicated times after immunization with MOG35–55 peptide and CFA, which correlated with the induction (C), peak severity (D), or recovery (E) phases. Data with means ± SEM (D to F) of seven mice are from two independent experiments. Cumulative clinical score over time (F and G) are means ± SEM from all experiments. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test.

  • Fig. 7 The balance between distinct p38 pathways tunes pro- and anti-inflammatory T cell responses.

    (A) Selective disruption of the alternative p38 pathway by Y323F substitution in P38α and P38β (P38αβY323F) impairs proinflammatory TH1 and TH17 cell IFN-γ and IL-17 production (35, 36). (B) Uncoupling of the classical p38 pathway by SOS1/2 deletion reduces iTreg cell differentiation, which counteracts the proinflammatory T cell functions of p38 activation. (C) The functional dichotomy between pro- and anti-inflammatory T cell responses is determined by the cooperation of two distinct p38 activation pathways.

  • Table 1 Initial numbers of species.
    SpeciesInitial number of molecules
    ZAP70100
    GPS100
    LAT200
    GPS1000
  • Table 2 Rate constants.
    ReactionsRate constant k (1/s)
    Activated ZAP70 phosphorylates LAT, kzl0.05
    Activated ZAP70 phosphorylates p38, kzp0.005
    pLAT self-decays to the inactive form, kdl1
    GPS binds to pLAT, kbg0.0001
    GPS unbinds to pLAT, kug5
    GPS-LAT complex phosphorylates p38, ka0.1
    pp38 self-decays to the inactive form, kdp40
    The positive feedback of pp38 to GPS-LAT
    complex, kf
    0.01

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/591/eaao0736/DC1

    Fig. S1. The role of LAT in p38 activation in Jurkat T cells and human PBMC CD4+ T cells.

    Fig. S2. Experimental validation of anti-phospho p38 antisera for pTpY p38 and pY323 p38 detection.

    Fig. S3. Testing anti-pY323 p38 antibody for pFLOW assay.

    Fig. S4. Full activation of p38 through the classical pathway is thresholded and time delayed regardless of stimulus strength.

    Fig. S5. Impact of p38 or MEK1 inhibition on BCR-induced ERK activation.

    Fig. S6. p38 activation kinetics in primary T cells.

    Fig. S7. Zap70 expression facilitates p38 activation.

    Fig. S8. The role of SOS in p38 activation and iTreg formation.

    Fig. S9. Sos1/2 double deficiency has more substantial impact on iTreg than TH17 cell differentiation.

  • This PDF file includes:

    • Fig. S1. The role of LAT in p38 activation in Jurkat T cells and human PBMC CD4+ T cells.
    • Fig. S2. Experimental validation of anti-phospho p38 antisera for pTpY p38 and pY323 p38 detection.
    • Fig. S3. Testing anti-pY323 p38 antibody for pFLOW assay.
    • Fig. S4. Full activation of p38 through the classical pathway is thresholded and time delayed regardless of stimulus strength.
    • Fig. S5. Impact of p38 or MEK1 inhibition on BCR-induced ERK activation.
    • Fig. S6. p38 activation kinetics in primary T cells.
    • Fig. S7. Zap70 expression facilitates p38 activation.
    • Fig. S8. The role of SOS in p38 activation and iTreg formation.
    • Fig. S9. Sos1/2 double deficiency has more substantial impact on iTreg than TH17 cell differentiation.

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