Journal: bioRxiv

Article Title: An ER Retrieval-and-Release System Reveals Ligand-Independent Basal Signaling by Cell Surface TCRs

doi: 10.64898/2026.10.13.758962

Figure Lengend Snippet: (A) JKεKO Ii-εRUSH (left) and JKεKO KDEL-εRUSH cells (right) were cultured in medium without biotin and left untreated (0 min), treated for different times with 40 µM biotin or were cultured in biotin-containing RPMI medium (RPMI). Cells were stained with an AF647-labeled anti-GFP antibody and TCR surface levels were measured by flow cytometry. The MFI of surface GFP is shown. Significance to the 0 min samples was analyzed by ordinary one-way ANOVA (n=3 for Ii-εRUSH, n>3 for KDEL-εRUSH; one representative experiment in triplicates is shown). (B and C) Cells as in (A) were fixed, permeabilized and stained with an BV421-labeled anti-CD3ε antibody and measured by flow cytometry. The MFI of total GFP is shown (B) , and the MFI of the anti-CD3ε stain is depicted (C) . Significance to the 0 min samples was analyzed by ordinary one-way ANOVA (n>3 for B and n=3 for C; one experiment in triplicates is shown).

Article Snippet: For functional assessment, CD3ε-ERR and control mock T cells were stimulated with plate-bound anti-CD3 (clone OKT3; Miltenyi Biotec), as previously described .

Techniques: Cell Culture, Staining, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: An ER Retrieval-and-Release System Reveals Ligand-Independent Basal Signaling by Cell Surface TCRs

doi: 10.64898/2026.10.13.758962

Figure Lengend Snippet: (A) Scheme of the DNA encoding for the CD3ε-ERR and control constructs. LTR: long terminal repeat promoter; ERR: ER retrieval; ffluc: firefly luciferase. (B) Principle of the CD3ε-ERR system; please note that in the primary T cells wild type CD3ε and CD3ε-ERR are co-expressed. (C) Expanded human T cells from blood were retrovirally transduced to express mock or CD3ε-ERR. After five days of culture cells were stained with APC-labeled anti-CD3 antibodies and TCR surface levels were measured by flow cytometry. The MFI of CD3 in the GFP+ cells is shown. (D) Cells as in (C) were fixed, permeabilized and stained with an APC-labeled anti-CD3ε antibody and measured by flow cytometry. The MFI of CD3ε in the GFP+ cells is shown. (E) Cells as in (C) were stained with an PE-labeled anti-CD69 antibody and measured by flow cytometry. Cells stained with an isotype antibody serve as control. The MFI of CD69 in the GFP+ cells is shown. (C, D and E) Significance was analyzed by paired t-test (n=3 different donors).

Article Snippet: For functional assessment, CD3ε-ERR and control mock T cells were stimulated with plate-bound anti-CD3 (clone OKT3; Miltenyi Biotec), as previously described .

Techniques: Control, Construct, Luciferase, Staining, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: An ER Retrieval-and-Release System Reveals Ligand-Independent Basal Signaling by Cell Surface TCRs

doi: 10.64898/2026.10.13.758962

Figure Lengend Snippet: (A) JKεKO KDEL-εRUSH cells were cultured for one hour with (+ biotin) or without biotin. After cell lysis, peptides were generated using trypsin. Phospho-peptides were enriched and identified by tandem mass spectrometry. Their intensities in the +biotin condition were compared with those without biotin treatment and visualized as volcano plot based on moderated t-tests. Phosphorylations significantly enriched, p-value <0.05 and log 2 fold change >1.0, are shown in green and the ones enriched without biotin in brown. The black dots represent Y phosphorylations significantly changed in one condition. (B) All phospho-Y peptides enriched in the + biotin sample from (A) are shown. Sites known to be induced by ligand binding to the TCR are indicated in the “by TCR ligand” column and the ones known to have an activatory role in ligand-induced TCR signaling in the “activation” column. (C) JKεKO KDEL-εRUSH cells were cultured for one hour with (+ biotin) and left unstimulated or stimulated for 5 min with anti-CD3ε antibodies. Phosphoproteomics and display as in (A) comparing the +/- TCR stimulation conditions. (D) Comparison of the basal TCR signal (phosphopeptides from JKεKO KDEL-εRUSH cells with biotin for 1 h versus without biotin, X axis) with the activation signal (phosphopeptides from JKεKO KDEL-εRUSH cells with biotin and stimulated for 5 min with anti-CD3 antibodies versus unstimulated, Y axis). Phosphopeptides significantly enriched both in the basal TCR signature (Table S4) and with the activation signal are shown in purple, those depleted in the basal signal and enriched in the activation signal in blue, those enriched in the basal signal and depleted in the activation signal in cyan, and all others in grey.

Article Snippet: For functional assessment, CD3ε-ERR and control mock T cells were stimulated with plate-bound anti-CD3 (clone OKT3; Miltenyi Biotec), as previously described .

Techniques: Cell Culture, Lysis, Generated, Mass Spectrometry, Ligand Binding Assay, Activation Assay, Phospho-proteomics, Comparison, Significance Assay