human t lymphocyte molt 4 cells  (ATCC)

Journal: Frontiers in Immunology

Article Title: Split intein-mediated reconstitution of regulatable CAR T-cells

doi: 10.3389/fimmu.2026.1844972

Figure Lengend Snippet: IMPDH.1 N -CD1a scFv is functional and competent for PTS. (A) CD1a + MOLT-4 cells were incubated with IMPDH.1 N -CD1a scFv, and polypeptide attachment was detected by staining with an anti-His-tag antibody. (B) PTS activity of IMPDH.1 N -CD1a scFv was initially assessed using a model Int C partner designed to transfer a variable heavy domain of a heavy chain (VHH) onto the CD1a scFv upon successful trans-splicing. Upper: Schematic representation of the reaction: His-tagged IMPDH.1 N -CD1a scFv (MW = 39,6 kDa) + small ubiquitin-like modifier (SUMO)-His-IMPDH.1 C -VHH (MW = 33 kDa) → CD1a scFv-VHH (MW = 40,5 kDa; red asterisk) + His-tagged IMPDH.1 N (MW = 13,2 kDa; black asterisk) + SUMO-His-IMPDH.1 C (MW = 19 kDa; black asterisk). Reactants (green box); products (blue boxes). Scheme designed with FigureLabs™. To specifically identify the trans-splicing product (TSP), the reaction mixture was applied to a Ni resin (IMAC). Because the TSP lacks a His-tag, it does not bind the resin, like the other reaction components do. Bottom: Coomassie-stained SDS-PAGE gel showing reaction products obtained from the PTS assay: I N : His-tagged IMPDH.1 N -CD1a scFv; I C : SUMO-His-IMPDH.1 C -VHH; t 0 : initial time; 1h and 3h refer to the reaction time; FT: flow-through; R: Ni resin-bound proteins. The anticipated molecular weights (MW) are specified in parentheses. (C) JURKAT-cells transduced with IMPDH.1 C - t -CAR (J-IMPDH.1 C , upper ) or NRDJ.1 C - t -CAR (J-NRDJ.1 C , bottom ). Transduction efficacy was determined by GFP reporter and anti-His-tag antibody. (percentage shown in the inset). (D, E) JurkaT-cells were used for preliminary technical optimization of the conditions required for split-intein-mediated protein trans-splicing. These experiments were exploratory and intended to identify conditions for subsequent validation in primary human T-cells; therefore, the Jurkat optimization data were not subjected to inferential statistical analysis. Cells were subjected to PTS with IMPDH.1 N -CD1a scFv under reducing conditions (+DTT). PTS occurred efficiently in J-IMPDH.1 C cells (D) but not in J-NRDJ.1 C cells (E) , in a concentration-dependent manner after 3h of incubation. Green denotes the CD1a scFv-negative cell population, whereas cyan blue represents the CD1a scFv-positive population detected using anti-scFv Ab (percentage shown in the inset).

Article Snippet: The human T-ALL cell lines JURKAT and MOLT-4 cells were purchased from the DSMZ cell line bank (Braunschweig, Germany) and cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS.

Techniques: Functional Assay, Incubation, Staining, Activity Assay, Ubiquitin Proteomics, SDS Page, Transduction, Biomarker Discovery, Analysis, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Split intein-mediated reconstitution of regulatable CAR T-cells

doi: 10.3389/fimmu.2026.1844972

Figure Lengend Snippet: Functional reconstitution of CD1a CAR through IMPDH.1-mediated PTS. (A) Primary lymphocytes from three independent donors were transduced with IMPDH.1 C - t -CAR, as identified by GFP + His-tag + co-expression ( left ). Cells were then subjected to PTS by incubation with either untreated IMPDH.1 N -CD1a scFv ( middle ) or DTT-pretreated IMPDH.1 N -CD1a scFv ( right ). Successful CD1a scFv reconstitution was detected using an anti-scFv antibody. (B) Quantification of IMPDH.1 C - t -CAR + T-cells prior to PTS (green circles) and the proportion of reconstituted full-length CD1a CAR + T-cells following PTS with DTT-pretreated IMPDH.1 N -CD1a scFv (magenta circles) (n=3 donors). (C, D) Cytotoxic activity of reconstituted CD1a CAR T-cells generated through IMPDH.1-mediated PTS against CD1a + MOLT-4 cells, measured after 24 h (C) and 48 h (D) of co-culture, respectively (n=3 donors). DTT-untreated conditions mark as empty columns and DTT-treated counterparts as filled color-columns. (E, F) IFN-γ secretion into culture supernatants at 24 h (E) and 48 h (F) post co-culture with CD1a + MOLT-4 cells, respectively (n=3 donors). (G) Analysis of reconstituted CD1a CAR T-cells following DTT washout ( magenta ). As a control, His-tag expression in the corresponding IMPDH.1 C - t -CAR T-cells not subjected to PTS is shown ( green ). Statistical analyses were performed using two-way ANOVA with Tukey’s multiple-comparisons post hoc test. Data are shown as mean ± SEM. Significance levels were defined as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****); NS denotes not significant.

Article Snippet: The human T-ALL cell lines JURKAT and MOLT-4 cells were purchased from the DSMZ cell line bank (Braunschweig, Germany) and cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS.

Techniques: Functional Assay, Transduction, Expressing, Incubation, Activity Assay, Generated, Co-culture Assay, Analysis, Control

Journal: iScience

Article Title: Activation-gated, T cell-restricted silencing of Dapk1 enhances Bacille Calmette-Guérin-elicited protective CD4 + memory

doi: 10.1016/j.isci.2026.115593

Figure Lengend Snippet: Memory phenotype CD4 + T cells downregulate DAPK1 (A and B) Dapk1 is transcriptionally downregulated in CD44 hi CD4 + T cells and converges on apoptotic pathways. (A) Volcano plot displaying DEGs (CD44 hi vs. CD44 lo ). Key apoptosis-related genes are labeled with log 2 FC and p values. (B) Venn diagram illustrating that Dapk1 is uniquely shared across three apoptotic pathways. (C) Dapk1 mRNA expression was quantified by RT-qPCR in CD44 lo , CD44 int , and CD44 hi CD4 + T cell fractions and normalized to Gapdh ( n = 5). (D) Validation of DAPK1 knockdown by RT-qPCR (upper side) and immunoblotting (lower side) in Jurkat cells stably expressing sh DAPK1 or a scramble control ( n = 3). (E) FCM quantification of intracellular active caspase-3 (upper side) and cell viability (using FVS780 dye, lower side) in Jurkat cells 24 h after activation with anti-CD3/CD28 antibodies ( n = 6). (F) Doxycycline-inducible knockdown of DAPK1. DAPK1 mRNA and protein levels were assessed by RT-qPCR and immunoblotting, respectively, in MOLT-4 cells stably expressing a Dox-inducible sh DAPK1 construct 48 h after Dox treatment ( n = 3). (G) FCM quantification of cell viability in the same MOLT-4 cells as in (F) ( n = 3). Data are presented as the mean ± SD. Comparisons between two groups were analyzed using unpaired two-tailed Student’s t test. Comparisons among three or more groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Jurket cells MOLT-4 , ATCC ATCC , Cat# TIB-152 Cat# CRL-1582.

Techniques: Labeling, Expressing, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Western Blot, Stable Transfection, Control, Activation Assay, Construct, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Biosensor-guided discovery of peptide inhibitors targeting the ribosomal protein uS5-PDCD2 chaperone interaction

doi: 10.1016/j.jbc.2026.111415

Figure Lengend Snippet: Inhibition of the uS5-PDCD2 interaction using residues 21 to 50 of uS5. A , Western blot analysis of total extracts (Input, Top ) and anti-Flag purifications (IP:Flag, Bottom ) prepared from HeLa cells that stably expressed Flag-PDCD2 and that were previously transfected with constructs encoding either GFP alone (lane 1), wild-type (WT, lane 2) or F29Y (MT, lane 3) versions of uS5 21-50 -GFP. The blots were analyzed for GFP, Actin, uS5, and Flag-PDCD2. B , quantification of relative uS5 levels copurified from anti-Flag precipitates normalized to Flag-PDCD2 levels. Values were expressed relative to the GFP control vector, which was set to 1.0. The data and error bars represent the average and SD from at least three independent experiments. p -value is indicated, as determined by a one-way ANOVA with Dunnett’s multiple comparisons test. ( C ) U2OS cells that conditionally express Flag-PDCD2 were transfected with either GFP control vector (panels a-d), wild-type (panels e-h) or F29Y (panels i-l) versions of uS5 21-50 -GFP. At the time of transfection, doxycycline was added to the media to induce the expression of Flag-PDCD2. 48 h post-transfection, cells were fixed and simultaneously analysed by direct fluorescence (b, f and j) and immunostaining for Flag-PDCD2 (c, g and k). DNA staining with DAPI shows the nucleus of each cell (a, e and i). Scale bars correspond to 20 μm. D , quantification of nucleus-to-cytoplasmic ratios of Flag-PDCD2 shows a significant decrease in cells that expressed the wild-type version of uS5 21-50 -GFP. More than 60 cells were analyzed for each condition from three independent immunofluorescence experiments. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. p -value is indicated. E , Peptide sequences corresponding to residues 21 to 50 of uS5, showing conserved phenylalanines in blue (WT) and substitutions to alanine residues in the mutant in red (MT). F , schematic of the peptide competition experiments using purified uS5-GFP/PDCD2 complex. See text for details. G , Western blot analysis of an uS5-GFP immunoprecipitate that were washed, divided, and treated with increasing concentrations of either wild-type ( Top , WT) or mutant ( Bottom , MT) uS5 21-50 peptides (lanes 2–5) or with no peptide (lane 1). Blots were analyzed for uS5-GFP and endogenous PDCD2. ( H ) Quantification of PDCD2 levels copurified from anti-GFP precipitates normalized to uS5-GFP levels. The values were then set to 1.0 for the control purification in the absence of peptide. Solid lines mark the average binding from three independent replicates, with error bars corresponding to standard deviations. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. p -value is indicated.

Article Snippet: Human MOLT-4, HEK293T, HeLa, and U2OS cell lines were acquired from ATCC.

Techniques: Inhibition, Western Blot, Stable Transfection, Transfection, Construct, Control, Plasmid Preparation, Expressing, Fluorescence, Immunostaining, Staining, Immunofluorescence, Mutagenesis, Purification, Binding Assay