Journal: bioRxiv

Article Title: TcrDesign: De novo design of epitope specific full-length T cell receptors

doi: 10.64898/2026.01.15.699824

Figure Lengend Snippet: ( A ) UMAP dimensionality reduction was performed on generated TCRs for two distinct epitopes, utilizing TCR embedding extracted by TcrDesign-B. ( B ) Binding assay of HLA-A*02:01-ELAGIGILTV tetramer to the nine generated TCRs expressing Jurkat cells was analyzed by flow cytometry. Three candidate TCRs ELA-TCR3, ELA-TCR4 and ELA-TCR6 exhibited specific binding to the tetramer. ( C ) Activation markers NFAT and CD69 levels of reporter Jurkat NFAT-ZsGreen cells expressing candidate TCRs ELA-TCR3, ELA-TCR4, ELA-TCR6 and ELApositive TCR after incubation with ELAGIGILTV-pulsed T2 cell for 24 hours, each sample was tested in triplicate. ( D ) The NFAT-ZsGreen signal and CD69 level of ELApositive-TCR and ELA-TCR3 were determined by fluorescence imaging and flow cytometry analysis. ( E ) Engineered Jurkat T cells were co-cultured with T2 cells and serially diluted peptides for 24 h. Co-cultured supernatants were analyzed by ELISA for secreted IL-2. ( F ) TCR-T cells at various E:T ratios were co-cultured with luciferase-transduced T2 cells. The % specific lysis of T2-luciferase (black) and 1ug/mL ELAGIGILTV pulsed T2-luciferase cells (blue) obtained by bioluminescence assay is plotted against multiple E:T ratios. Dots in the figure represents three replicates.

Article Snippet: For 1 x 10 6 T-lymphocytes, 25 μL of Dynabeads coated with anti-CD3 and anti-CD28 antibodies was added to the RPMI 1640 media supplemented with 200U human recombinant IL-2 (#11848-HNAH1-E, Sino Biological).

Techniques: Generated, Binding Assay, Expressing, Flow Cytometry, Activation Assay, Incubation, Fluorescence, Imaging, Cell Culture, Enzyme-linked Immunosorbent Assay, Luciferase, Lysis, ATP Bioluminescent Assay

Journal: Nature biotechnology

Article Title: Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses

doi: 10.1038/s41587-024-02418-6

Figure Lengend Snippet: a , Mice were s.c. engrafted with 0.5 × 10 6 and 0.3 × 10 6 MC38 cells on the left and right flanks. Starting from day 7, tumors on the left side were treated with PBS ( n = 8), OmpA–mDR18 (0.5 × 10 9 CFU, n = 10) or OmpA (0.5 × 10 9 CFU, n = 10) (i.t.) five times on days 7, 11, 14, 17 and 21. b , c , Mean tumor growth on the treated side ( b ) and untreated side ( c ) of mice. d , Mice engrafted with 10 6 MC38 cells were i.v. treated with PBS ( n = 9), OmpA ( n = 10), OmpA–mDR18 ( n = 10) or mDR18 ( n = 9) on days 8, 11, 15 and 18. e , f , Mean tumor growth ( e ) and Kaplan–Meier survival ( f ) curves of mice. g , On day 8 after engraftment of 10 6 MC38 cells, mice ( n = 8 each group) were treated once (i.v.). Plasma was isolated from blood collected from the submandibular vein on days 11 and 15 for cytokine measurement. Tissues were collected on days 7, 10 and 14 after injection for biodistribution. h , Bacterial distribution in tumor, liver, kidney and spleen on days 7, 10 and 14 after injection. CFU g −1 is bacterial concentration. i , The concentration of cytokines associated with CRS: IL1β, IL6, monocyte chemoattractant protein-1 (CCL2), IFNγ, IL2 and granulocyte macrophage colony-stimulating factor (GM-CSF) in the plasma (on days 3 and 7 after treatment unless otherwise mentioned) from mice treated with LPS (5 h after treatment), PBS, mDR18, OmpA and OmpA–mDR18. Mice were treated with PBS, 4 mg kg −1 mDR18, 10 9 CFU OmpA or OmpA–mDR18 ( d – h ). Two-way ANOVA test for growth curve ( b , c , e ), Mantel–Cox test for survival curve ( f ), one-way ANOVA test and two-sided unpaired t -test for comparison ( i ). Data represent mean ± s.d. ( b , c , e , i ).

Article Snippet: To support human CAR NK cells, the mice received 75 kU of human recombinant IL2 (Miltenyi Biotec, 130–097-748) i.p. every other day.

Techniques: Clinical Proteomics, Isolation, Injection, Concentration Assay, Comparison

Journal: Nature biotechnology

Article Title: Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses

doi: 10.1038/s41587-024-02418-6

Figure Lengend Snippet: a , NSG mice were s.c. engrafted with 5 × 10 6 H226 cells. Starting on day 30, mice ( n = 10 each group) were treated with PBS, YiaT232 (10 9 CFU), YiaT232–hDR18 (10 9 CFU) or purified hDR18 (4 mg kg −1 ) (i.t.) three times (on days 30, 37 and 44). In total, 3–5 million MSLN-CAR NK cells were i.v. administrated except for mice in the tumor-only groups. All mice were i.p. injected with 75 kU of human recombinant IL2 every other day to support the survival of human NK cells in vivo. b , c , Mean tumor growth ( b ) and Kaplan–Meier survival ( c ) curves for the tumor-bearing mice after treatment. Mean tumor growth and survival curves are the combination of two independent experiments, with n = 10 mice per group. d , NSG mice were s.c. engrafted with 5 × 10 6 H226 cells on day 40, and mice ( n = 10 each group) were treated with PBS, YiaT232 (10 9 CFU), YiaT232–hDR18 (10 9 CFU) or purified hDR18 (4 mg kg −1 ) (i.t.). In total, 5 million MSLN-CAR NK cells were i.v. administrated except in the mice from tumor-only groups. All mice were i.p. injected with 75 kU of human recombinant IL2 every other day. On day 47, mice were euthanized, and organs (tumor, liver, lung, spleen and bone marrow) of all mice were collected for analysis by flow cytometry. e , The percentage of human CD45 + cells in livers, tumors, spleens, lungs and bone marrow after treatment. Two-way ANOVA test for tumor growth curve ( b ), Mantel-Cox test for survival curve ( c ) and two-sided unpaired t -test for NK percentage ( e ). Data represent mean ± s.d. ( b , e ).

Article Snippet: To support human CAR NK cells, the mice received 75 kU of human recombinant IL2 (Miltenyi Biotec, 130–097-748) i.p. every other day.

Techniques: Bacteria, In Vivo, Control, Purification, Injection, Recombinant, Flow Cytometry

Journal: Nature biotechnology

Article Title: Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses

doi: 10.1038/s41587-024-02418-6

Figure Lengend Snippet: a , NSG mice were subcutaneously (s.c.) engrafted with 5 × 10 6 H226 cells, on day 40, mice (n = 5 each group) were treated with PBS, YiaT232 (10 9 CFU), YiaT232-hDR18 (10 9 CFU) and purified hDR18 (4 mg/kg) (i.t.). 5 million MSLN-CAR NK were administrated intravenously (i.v.). All mice were injected with 75kU human recombinant IL2 every other day intraperitoneally (i.p.). On Day 47, mice were sacrificed and organs (tumor, liver, lung, spleen, and bone marrow) of all mice were collected for analysis by flow cytometry and CFU. b , The concentration of bacteria (CFU/g) in tumors, livers, spleens, lungs, and bone marrow post-treatment. c , Percentage of Ki67 + cells gated on human CD45 + cells in livers, tumors, spleens, lungs, and bone marrow. Two-sided unpaired t-test for percentage of Ki67 + cells ( c ). Data represent means ± SD ( b , c ).

Article Snippet: To support human CAR NK cells, the mice received 75 kU of human recombinant IL2 (Miltenyi Biotec, 130–097-748) i.p. every other day.

Techniques: Bacteria, Purification, Injection, Recombinant, Flow Cytometry, Concentration Assay

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia decreases the amount of mature perforin in NK cells. A, The level of total perforin in NK cells incubated with ammonia determined by Western blot using an anti-perforin antibody (B-D48 clone; n = 3). β-Actin is presented as a loading control. Representative blot from one donor is shown. B, The concentration of extracellular perforin secreted by NK cells in response to contact with target cells (K562) in different concentrations of NH 4 Cl ( n = 4). C, The level of perforin detected in NK cells incubated with NH 4 Cl determined by intracellular staining using an anti-perforin antibody (δG9 clone) and flow cytometry ( n = 4). In some groups, cells were washed and incubated in a medium without NH 4 Cl, as indicated in the figure. MFI, mean fluorescence intensity. D, A schematic representation of perforin maturation. Green and red circles show forms of perforin recognized by B-D48 and δG9 antibodies, respectively. E, The level of perforin in NK cells incubated for 4 hours with NH 4 Cl determined by Western blot methods using an anti-perforin antibody (Pf-344 clone; n = 3). Two forms of perforin, immature (70 kDa) and mature (60 kDa), were detected. F, The level of perforin detected in NK cells incubated with NH 4 Cl determined by intracellular staining using an anti-perforin antibody (δG9 clone) and flow cytometry ( n = 2). NK cells were primed with IL2 (200 U/mL) and IL15 (10 ng/mL) for 24 hours before the experiment. G, The level of perforin detected in NK cells incubated with NH 4 Cl and other lysosomotropic agents [chloroquine (CQ), concanamycin (CMA), and monensin] for 4 hours determined by intracellular staining using antibodies detecting total perforin (B-D48 clone) and lysosomal perforin (δG9 clone; n = 2). H, The cells were loaded with the lysosomal fluorescent probes LysoPrime Green (the pH-resistant probe) and pHLys Red (the pH-sensitive probe; Dojindo Laboratories) and treated with increasing concentrations of NH 4 Cl in the imaging medium or bafilomycin A as a control. Bars show calculated mean ratios of the LysoPrime Green signal divided by the pHLys Red signal ( n = 3). Images show cells that were untreated (top row) and treated with 5 mmol/L NH 4 Cl (bottom row). Left column, staining with the pH-resistant LysoPrime Green probe (green; scale bar, 10 μm); middle column, staining with the pH-sensitive pHLys Red probe (red); right column, merge of the LysoPrime Green signal, pHLys Red signal, and transmitted light image (gray). I, pH-dependent processing of perforin by human recombinant cathepsins B and L at concentrations ranging from 1 to 100 nmol/L, analyzed by Western blot methods using an anti-perforin antibody (B-D48 clone; n = 2). J, Processing of perforin by human recombinant granzyme B at concentrations of 0.5, 1, and 2.5 μmol/L, assessed at pH 7.4 in two different buffers ( n = 2). K, Raji tumor–bearing mice were intratumorally injected with 3–5 × 10 6 human NK cells. After 4 hours, tumors were dissected and enzymatically dissociated, followed by NK cell analysis for perforin levels using intracellular staining with an anti-perforin antibody (δG9 clone) and flow cytometry ( n = 9). Human NK cells incubated with Raji cells for 4 hours in control medium in vitro were used as controls. Data show means ± SEM. The n values represent the numbers of biological replicates in in vitro experiments or the number of mice used to obtain the data. D, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/g06z848 .

Article Snippet: T cells were cultured in full RPMI 1640 medium with 100 U/mL of recombinant human IL2 (PeproTech) unless otherwise described for specific experimental procedures.

Techniques: Incubation, Western Blot, Control, Concentration Assay, Staining, Flow Cytometry, Fluorescence, Imaging, Recombinant, Injection, Cell Analysis, In Vitro