LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN2 as a regulator of effector functions

Abstract T‐cell receptor (TCR) ligation‐mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time‐resolved high‐resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T‐cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast‐track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T‐cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T‐cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.

A Left: number of unambiguously identified proteins across the following data sets: UniProt reviewed entries (www.uniprot.org); a proteome of CD4 + T cells (Voisinne et al, 2019) (PXD012826); the phosphoproteome and the set of TCR-regulated phosphoproteins. Right: repartition of unambiguously identified proteins across the proteome, the phosphoproteome, and the set of TCR-regulated phosphoproteins. The area of the circles in the Euler diagram illustrates the relative number of proteins per data set without being strictly proportional. B Output of the statistical analysis of protein abundances during TCR stimulation. Number of proteins significantly regulated upon TCR stimulation presented in bar plots for each time point next to the total number of regulated proteins across the entire time course (total). The corresponding percentage of the proteome that is regulated is indicated above the bars. Right panel: representative volcano plot presenting the statistical significance distribution against the log 2 -transformed fold change between 30 s (maximum number of regulated proteins) and the unstimulated control. For each condition, proteins were considered significantly up-regulated (red) or down-regulated (blue) when displaying a corrected P-value ≤ 0.05 (ANOVA test) and an absolute log 2 -transformed fold change ≥ 1 (see Materials and Methods for more detailed information). C Enriched UniProt-keywords (hypergeometric test P-value ≤ 0.05, fold change ≥ 1.5, number of annotated proteins ≥ 2) in the set of regulated phosphosites compared with the set of all identified phosphosites. Fold changes and P-values are indicated for each term.
▸ Figure EV3. Dynamics of phosphorylation and selected functional modules induced by TCR stimulation.
A Dynamics and cluster distribution of regulated phosphotyrosines, phospho-serines, and phosphothreonines. B Illustration of TCR-regulated phosphosites within the polycomb-repressive complex 1 (PRC1) (Ub: ubiquitination; Ac: acetylation). The corresponding log 2 -transformed MS intensities measured upon TCR activation for each of the biological replicates are shown below. Box plot elements: Center line corresponds to median, box limits correspond to the first and third quartiles, and whiskers indicate variability from Q1-1.5. IQR to Q3 + 1.5 IQR. C Short-time TCR stimulation induces FOXO3 phosphorylation and NFATC2 dephosphorylation and promotes nucleus exit or entry, respectively. CD4 + T cells left unstimulated (À) or stimulated for 2 and 5 min with anti-CD3 plus anti-CD4 antibodies were subjected to nuclear/cytoplasmic fractionation before immunoblot analysis with antibodies specific for NFATC2 and FOXO3. Arrows indicate phosphorylation (P) and dephosphorylation (deP) forms of the transcription factors. Lamin-B2 and GAPDH are used to control purity of nuclear and cytoplasmic extracts. D t-SNE plot highlighting phosphosites associated with the UniProt keywords "Protein biosynthesis" and "Translation regulation". Dot transparency is scaled according to the P-value corresponding to the local enrichment of the annotation term (hypergeometric test, see method for more detailed information). E Left: Dynamics of TCR-regulated phosphosites associated with the UniProt keywords "Protein biosynthesis" and "Translation regulation" (dots color-coded by cluster).
Right: Schematic representation of the translational initiation and elongation complexes. TCR-regulated phosphosites are represented as small dots color-coded by cluster.  A Euler diagram indicating the number of phosphosites identified (3612) and regulated (640) upon TCR stimulation in CD4 + and OT-I CD8 + T cells. B Comparison of phosphosite intensities between CD4 + and OT-I CD8 + T cells. Only sites regulated upon TCR stimulation in CD4 + T cells and identified in OT-I CD8 + T cells were considered. Log 2 -transformed phosphosite intensities with imputed missing values were normalized across biological replicates using the mean intensity and subsequently averaged for each condition of stimulation. Pearson correlation coefficient R = 0.54. C Distribution of Pearson's correlation coefficients across phosphosites regulated upon TCR stimulation in CD4 + T cells and identified in OT-I CD8 + T cells. Phosphosite intensities were normalized as in (B). The correlation coefficient was computed only for phosphosites with intensity values available in both data sets for a minimum of four stimulatory conditions (n = 584). D Overlay dynamics of selected phosphosites in CD4 + and OT-I CD8 + T cells. Cas9-EGFP OT-I CD8 + T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 and sgITSN2-2).
A EGFP expression was assessed by flow cytometry in cells transfected with sgEGFP or without guide (none). B Equal amounts of total lysates from cells transfected with sgEGFP or sgITSN2 were analyzed by immunoblot using anti-ITSN2 or anti-LCK antibodies. The arrows indicate short and long ITSN2 isoforms. Data are presented as mean AE SD from three independent experiments. C Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody or with IL-7 as control. Surface expression of CD69 and CD5 in cells was analyzed by flow cytometry. A representative FACS profile is shown.