B cell antigen receptor (BCR) and CD40 signaling are rewired in germinal center (GC) B cells (GCBCs) to optimize selection for high-affinity B cells. In GCBC, BCR signals are constrained, but the mechanisms are not well understood. Here we describe a GC-specific, AKT-kinase-driven negative feedback loop that attenuates BCR signaling. Mass spectrometry revealed that AKT target activity was altered in GCBCs compared with naive B cells. Retargeting was linked to differential AKT T308 and S473 phosphorylation, in turn controlled by GC-specific upregulation of phosphoinositide-dependent protein kinase PDK1 and the phosphatase PTEN. In GCBCs, AKT preferentially targeted CSK, SHP-1 and HPK1, which are negative regulators of BCR signaling. We found that phosphorylation enhances enzymatic activity of these proteins, creating a negative feedback loop that dampens upstream BCR signaling. AKT inhibition relieved this negative feedback and enhanced activation of BCR-proximal kinase LYN, as well as downstream BCR signaling molecules in GCBCs.
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The data that support the findings of this study are available from the corresponding author upon reasonable request. Reagents and methods used in this paper are described in the Nature Research Reporting Summary, available online.
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We thank L. Garrett-Sinha, L. Kane, G. Delgoffe and B. Su for critical reading of the manuscript. We thank R. Elsner for useful discussions. We thank S. Joachim for supporting experimental procedures. This work was supported by National Institutes of Health grant no. R01 AI105018 to M.J.S.
Integrated supplementary information
a, Different B cell populations were gated as shown for comparing phosphorylation of signaling proteins, related to Fig 1, 2 and 7. b, Gating strategy for purification and purity test post purification of different B cell populations (pre-gated on live singlets), related to Fig 3.
NBCs and GCBCs were purified from d14 immunized MEG mice. Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors followed by BCA protein assay to measure protein concentration. The same amount of protein from each cell type was loaded on SDS-PAGE gels, followed by immunoblotting with antibodies as indicated. Data represent cells from two experiments and in each experiment, cells were combined from three mice. The number on the right represents the average ratio of GCBC/NBC quantitated by image J.
The proteomic workflow for identifying AKT substrates during activation in NBCs and GCBCs.
Supplementary Figure 4 PTEN inhibition alters AKT signaling networks in GCBC, related to Fig. 6 and Supplementary Table 2.
NBCs and GCBCs were processed as described in Fig. 6. Shown is global change of AKT substrates. The average peak area determined by relative quantitation of the mass spectrometric data presented in Supplementary Table 2 was normalized to the highest value across the experimental group. The data were subjected to hierarchical clustering using the Morpheus software package (https://software.broadinstitute.org/morpheus).
Splenocytes from NP-CGG immunized MEG mice were treated with DMSO or AKT inhibitor for 40 min followed by anti-IgM stimulation for indicated time points. Cells were then analyzed by flow cytometry. Shown here is the analysis for NBCs. Data represent two independent experiments with a total of four mice tested. Data are mean ± SEM; P values are comparing treatments (DMSO vs AKT inhibitor) by two-way ANOVA (two factors: treatment and time). p-BTK: F=9.034, d.f.=30; p-PLC-γ2: F=13.15, d.f.=30. Related to Fig. 7b.
In comparison to NBCs, GCBCs express higher amounts of PTEN and PDK1, which changes the ratio of PtdIns(3,4,5)P3/ PtdIns(4,5)P2 and favors T308 over S473 phosphorylation on AKT. AKT, as differentially phosphorylated in GCBCs, in turn phosphorylates different substrates resulting in a GCBC-specific AKT signaling network. GCBC-specific AKT substrates include CSK, SHP-1 and HPK1, which when phosphorylated by AKT in GCBCs demonstrate enhanced activity that serves to inhibit upstream BCR signaling molecules such as Lyn, Syk and BLNK. This activation of regulators of proximal signaling further attenuates PtdIns(3,4,5)P3 generation. Taken together, this GCBC-specific feedback loop rewires BCR signaling, which can affect both GC selection and affinity maturation.
Proteins identified by mass spectrometry in the AKT substrate motif IP from naive inactivated B cells, naive activated B cells, inactivated GCBCs and activated CGBCs are presented. Cells were activated by BCR stimulation with 20 µg ml−1 anti-IgM for 5 min. For each protein in the analysis, the average peak area determined by relative quantitation of the mass spectrometric data was normalized to the lowest value across the experimental group. The criteria used to assign a protein to a specific experimental group required that the normalized abundance was greater than three.
Supplementary Table 2: Proteins identified by mass spectrometry in the AKT substrate motif IP from Naïve and GC B cells treated with PTEN inhibitor. Related to Fig. 6.
Cell treatment is described in Fig. 6. For each protein in the analysis, the average peak area determined by relative quantitation of the mass spectrometric data is reported here.
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Nature Immunology (2019)