Research ArticleIMMUNOLOGY

Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion

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Science Advances  30 Apr 2021:
Vol. 7, no. 18, eabd2710
DOI: 10.1126/sciadv.abd2710
  • Fig. 1 Tim-3 signaling partially contributes to Bat3-mediated T cell exhaustion.

    (A) CD4 T cells from Bat3cko × 2D2 and WT 2D2 mice were in vitro differentiated into TH1 cells, and 4 × 106 cells were transferred into C57BL/6 recipient mice to induce EAE. Disease progression was monitored on daily basis until the end of the experiment. Mean disease scores are shown as indicated. (B to E) Ex vivo analyses on transferred 2D2 cells (Va3.2+Vb11+) in the central nervous system (CNS), spleen (SPN), and lymph node (LN) on day 12 (D12) and day 21 (D21) were performed to determine the phenotype of Bat3-deficient CD4 T cells during EAE induction. Error bars indicate mean SEM [*P = 0.01, **P = 0.0085, and ***P = 0.006, unpaired two-tailed t test; ****P = 0.0001, two-way analysis of variance (ANOVA)]. NS, not significant. (F) Naïve CD4 T cells were isolated from Bat3fl/fl, Tim-3cko, Bat3cko × 2D2, and Tim-3×Bat3dko × 2D2 mice and were in vitro differentiated into TH1 cells; 4 × 106 cells were transferred into C57BL/6 recipient mice to induce EAE. Disease progression was monitored on daily basis till the end of experiment. Mean disease scores were shown as indicated. Statistical significance between Tim-3cko × 2D2 and Tim-3×Bat3dko × 2D2 was reached on D12 (**), increasing in significance until cessation of experiment. Statistical significance between Bat3cko × 2D2 and Tim-3 × Bat3dko × 2D2 mice was reached on D8 (*), increasing in significance until cessation of experiment. (G) Ex vivo analyses on transferred 2D2 cells (Va3.2+Vb11+) in the CNS on D21 was performed to determine the phenotype of Bat3fl/fl, Tim-3cko, Bat3cko × 2D2, and Tim-3 × Bat3dko–deficient CD4 T cells during EAE induction. Data represent three independent experiments. Error bars indicate mean SEM (*P = 0.01 and ***P = 0.0012, unpaired two-tailed t test; ****P = 0.0001, two-way ANOVA).

  • Fig. 2 Tim-3+ TH1 cells represent terminal differentiated and dysfunctional T cells.

    (A) WT (FoxP3-GFP KI) mice were immunized with MOG35–55/CFA to induce EAE. At the peak of the disease (day 10), the immunized mice were intraperitoneally injected with BrdU and were euthanized the following day to isolate CNS-infiltrating CD4+FoxP3 T cells for flow cytometry analysis. Flow cytometry data (to the left) represent results from four mice from the same experiment. Further, the ratio (fold) differences between Brdu+PD-1+/Brdu+PD-1 and Brdu+Tim-3+/Brdu+Tim-3 cells were analyzed by ratio pair t test and were shown to the right (P = 0.0048). (B) FoxP3-GFP KI mice were immunized with MOG35–55/CFA to induce EAE. At the peak of the disease (day 10), Tim-3+FoxP3 and Tim-3FoxP3 CD4 T cells were isolated from the CNS of EAE mice by cell sorting. Total RNA samples were prepared for Nanostring analysis using in-house–designed Nanostring code set (14). Cells from three EAE mice were analyzed as indicated in each individual column. (C) WT mice were immunized with MOG35–55/CFA to induce EAE. On day 14, CNS-infiltrating T cells were isolated. CD4+ Foxp3; Tim-3hi/lo cells were analyzed by flowcytometry for expression of PD-1, CD160, and KLRG1. Data shown (C) as mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001 (Student two-tailed t test).

  • Fig. 3 Bat3 deficiency results in increased Akt phosphorylation at S473.

    Total CD4 T cells from Bat3fl/fl or Bat3cko mice were activated with plate bound anti-CD3 and anti-CD28 antibodies for 2 days. Cells were rested for additional 2 days afterward. After overnight serum starvation, the cells were stimulated with anti-CD3 and anti-CD28 antibodies for indicated time points. Whole-cell lysates were prepared, and Western blot was performed to analyze the TCR and mTOR signaling pathways. Results represent at least three independent experiments.

  • Fig. 4 Bat3 interacts with Rictor to regulate mTORC2 function.

    (A) Equal concentration of EL4 cell lysate was used in co-IP using antibodies to Rictor, Raptor, mTOR, and isotype rabbit immunoglobulin G (IgG), respectively. Protein samples were analyzed by Western blot to examine the signal for Bat3. IB, immunoblot. (B) Whole-cell lysates were prepared from EL4 cells transduced with Bat3-expressing retrovirus (Bat3-RV), empty vector (GFP-RV), Bat3-sh RNA-expressing RV (Bat3-sh-RV), and empty vector (ctrl-sh-RV), respectively. Cell lysates were incubated with anti-mTOR antibody (Ab), respectively, to immunoprecipitate mTOR complex to examine the signal of coprecipitated Rictor.

  • Fig. 5 Bat3 suppresses Hsc70-mediated activation of mTORC2.

    (A) CD4 T cells from Bat3fl/fl and Bat3cko mice were activated with plate-bound anti-CD3 and anti-CD28 antibodies for 2 days. Cells were rested for an additional 2 to 3 days, and whole-cell lysates were prepared for anti-Rictor and anti-mTOR co-IP. When anti-Rictor co-IP was performed, 1 mM adenosine 5′-triphosphate (ATP) was added in a control sample to trigger adenosine triphosphatase (ATPase) activity of Hsc70. Eluted protein samples were analyzed by Western blot to determine the binding of Hsc70 to the mTORC2 complex. (B) Bat3fl/fl and Bat3cko CD4 T cell whole-cell lysates were prepared for anti-Rictor or isotype IgG co-IP with or without the presence of indicated doses of Hsc70. Immunoblot was performed to analyze the phosphorylation at mTOR S2481 (top), and total mTOR coprecipitated by anti-Rictor (bottom). (C) Co-IP experiment was performed as described in (B), and Akt phosphorylation at S473 by the mTORC2 complex was analyzed by Western blot.

  • Fig. 6 Bat3 regulates FoxO1–Blimp-1 activity.

    Naïve Bat3fl/fl and Bat3cko CD4+ T cells were in vitro differentiated into TH1 cells. (A) qPCR to determine the transcripts of Prdm1, Bcl6, and Tbx21 after 3 days. (B) Whole-cell lysates (WCLs) were prepared for Western blot to detect Blimp-1 expression. (C) Cells were activated with anti-CD3/CD28 for 2 days and rested for 2 days afterward. After overnight serum starvation, cells were stimulated with anti-CD3/CD28 for indicated time points. WCL were prepared and immunoblotted for pFoxO1 S256. (D) Four ChIP-PCR primer sets were used to confirm binding at selected sites of FoxO1 to Blimp-1 locus. (E) Naïve CD4+ T cells were activated with anti-CD3/CD28 for 1 day and retrovirally transduced with control (GFP-RV) or Blimp-1–expressing vector (Prdm1-RV). Tim-3 expression was analyzed 3 days later. (F) Naïve Prdm1fl/fl or Prdm1fl/fl cko CD4+ T cells were stimulated under TH0 and TH1 conditions with anti-CD3/CD28. Tim-3 expression was assessed after 72 hours. (G) CD4+ T cells from Bat3cko × 2D2, Bat3fl/fl × Prdm1fl/flcko × 2D2, and Bat3fl/fl × Prdm1fl/fl dko × 2D2 mice were differentiated into TH1 cells and transferred into C57BL/6 recipient mice. Disease progression was monitored daily; mean disease scores are shown as indicated. Results represent at least two independent experiments. Error bars indicate means ± SEM (*P = 0.01, unpaired two-tailed t test).

  • Fig. 7 Bat3 deficiency promotes an exhaustion/dysfunctional profile in T cells.

    Naïve CD4 T cells were isolated from Bat3cko × 2D2 and Tim-3cko × 2D2 and in vitro differentiated into TH1 cells; 4 × 106 cells were transferred into C57BL/6 recipient mice to induce EAE. (A) Gene expression in Tim-3cko and Bat3cko 2D2 cells (Va3.2+Vb11+) isolated from CNS at peak of disease (day 14) for population RNA-seq. DE genes (289) are shown as a heatmap. (B) Enrichments of different signatures from literature in Bat3cko versus Tim-3cko cells, determined using GSEA preranked analysis. Selected signatures with an adjusted P < 0.05 are shown, and references for signatures are numbered in italics. (C) Descriptive leading-edge plots for some of the signatures. NES, normalized enrichment score. (D) Graphical representation of the selected overlap genes between the Prdm1cko and Bat3cko CD4 T cells. (E) A t-distributed stochastic neighbor embedding (t-SNE) plot of the 316 CD4+ TILs obtained from WT mice bearing B16F10 melanoma tumors (14), colored by the relative signature score for Bat3cko versus Tim-3cko module (36 genes; table S2) and the IL-27 TIL signature (14).

  • Fig. 8 Graphical abstract.

    Model for Bat3-mediated mTORC2–Akt–Blimp-1–Tim-3 pathway regulation in T cell terminal differentiation and exhaustion.

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