Journal: Advanced Science

Article Title: BiTE‐Secreting CAR‐ γδ T as a Dual Targeting Strategy for the Treatment of Solid Tumors

doi: 10.1002/advs.202206856

Figure Lengend Snippet: Characterization of mRNA‐driven Nb‐CAR.BiTE‐ γδ T cells. A) Nb‐CAR expression was assessed after lentiviral transduction or mRNA electroporation. γδ T cells (1 × 10 6 ) were electroporated with 2 µg Nb‐CAR/CAR.BiTE IVT mRNA or transduced with lentiviral Nb‐CAR/CAR.BiTE particles (MOI = 3). The Nb‐CAR expression on the indicated days was determined through flow cytometry using a specific antibody against VHH. B,C) Superior cell growth and Nb‐BiTE secretion using the mRNA delivery strategy. B) Cell number and viability were recorded during 7 d after transfection of the mRNA or lentiviral Nb‐CAR.BiTE transgene. C) Contents of PD‐L1‐targeted Nb‐BiTE in the supernatants from mRNA‐ or lentiviral‐driven Nb‐CAR.BiTE‐ γδ T cell cultures were detected by an ELISA‐based coating with recombinant full‐length PD‐L1. D) Phenotype and purity of mRNA‐driven Nb‐CAR.BiTE‐ γδ T cells. T effector and central memory phenotype, activating marker, and purity of Nb‐CAR.BiTE mRNA‐electroporated γδ T cells were analyzed through flow cytometry using specific antibodies against CD27, CD45RA, CD3, NKG2D, V δ 2, V γ 9, αβ TCR, γδ TCR, CD4, CD8, CD19, CD14, CD66b, or CD56. Isotype staining was used as a gating control for positive staining. E) Increased secretion of granzyme B and IFN‐ γ , but not IL‐17A, from Nb‐CAR.BiTE‐ γδ T cells after coculture with A549 or MDA‐MB‐231 cells. The contents of granzyme B (upper panel), IFN‐ γ (middle panel), and IL‐17A (bottom panel) in culture supernatants were measured using ELISA. These results showed that mRNA‐driven Nb‐CAR.BiTE‐ γδ T cells would be an effective strategy for the manufacture of Nb‐CAR.BiTE‐ γδ T cells. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 3–4, * p < 0.05; ** p < 0.01; and *** p < 0.001 based on paired Student's t‐tests.

Article Snippet: Antibodies specific for CD3 ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals.

Techniques: Expressing, Transduction, Electroporation, Flow Cytometry, Transfection, Enzyme-linked Immunosorbent Assay, Recombinant, Marker, Staining, Control

Journal: Advanced Science

Article Title: BiTE‐Secreting CAR‐ γδ T as a Dual Targeting Strategy for the Treatment of Solid Tumors

doi: 10.1002/advs.202206856

Figure Lengend Snippet: Evaluation of Nb‐BiTE secreted from Nb‐CAR‐ γδ T cells. A–D) Functional binding of Nb‐BiTE released from Nb‐CAR‐ γδ T cells. A) Diagram shows the cell non‐contact coculture system for Nb‐CAR.BiTE‐ γδ T cells and target cells (left panel). Secreted Nb‐BiTE from Nb‐CAR‐ γδ T cells was detectable on target cells. We seeded 1 × 10 5 isolated primary CD3‐positive cells from PBMCs, A549, H1975, or MDA‐MB‐231 cells on the bottom, with or without exposure to 5 × 10 5 Nb‐CAR.BiTE‐ γδ T cells on the top, in impenetrable wells for 24 h. Subsequently, the bottom cells were harvested and Nb‐BiTE signals were detected by flow cytometry using specific antibody against VHH (right panel). B) Secreted Nb‐BiTE from Nb‐CAR‐ γδ T cells promoted CD3‐positive cell proliferation. We cocultured 1 × 10 6 isolated primary CD3 + cells, parental, or Nb‐CAR‐ γδ T cells with or without 1 × 10 6 parental, Nb‐CAR, or Nb‐CAR.BiTE‐ γδ T cells on the top well for 6 days. Subsequently, the CD3 + cell numbers were normalized to the non‐coculture group. C) Nb‐BiTE released from Nb‐CAR‐ γδ T cells strengthened effector cells against PD‐L1‐expressing tumor cells. Schematic illustration of the non‐contact coculture system for Nb‐CAR.BiTE‐ γδ T cells, which release Nb‐BiTE to engage CD3 + effectors and PD‐L1‐overexpressing tumor cells (left panel). The PD‐L1 level on PD‐L1‐overexpressing A549 and PD‐L1 knockdown MDA‐MB‐231 stable clones were examined by flow cytometry analysis (right panel). D) Purity of the isolated CD4‐ and CD8‐positive and CD3‐/CD56 + cells from PBMCs were checked by flow cytometry. E) We cocultured 1 × 10 5 PD‐L1‐overexpressing A549 cells with an individual healthy donor‐derived CD4 + , CD8 + , NK, parental γδ T, or Nb‐CAR‐ γδ T cells on the bottom well and with or without 5 × 10 5 Nb‐CAR.BiTE‐ γδ T cells on the top well for 72 h. F) PD‐L1 determined the cell‐killing ability of effector cells when exposed to released Nb‐BiTE. PD‐L1‐stable knockdown or scramble‐control MDA‐MB‐231 cells (1 × 10 5 ) were cocultured with an individual healthy donor‐derived CD4 + , CD8 + , NK, parental γδ T, or Nb‐CAR‐ γδ T cells on the bottom well and with 5 × 10 5 Nb‐CAR.BiTE‐ γδ T cells on the top well for 72 h. G,H) PD‐L1‐targeted Nb‐BiTE enhanced anti‐tumor responses of T cells. MDA‐MB‐231 or PD‐L1‐overexpressing A549 cells were cocultured with or without the isolated CD4 + or CD8 + cells at an E:T ratio of 5:1; or γδ T cells at an E:T ratio of 3:1 in the presence/absence of recombinant Nb‐BiTE (1 ng mL ‐1 ). G) After 24, 48, or 72 h of coculture, we determined the cytotoxicity induced by these effector cells. H) After 48 h of coculture, the supernatants were harvested for detecting the contents of granzyme B and IFN γ by ELISA. Specific lysis was determined using LIVE/DEAD cell‐mediated cytotoxicity assay. These results suggested that the secreted PD‐L1 targeting Nb‐BiTE from Nb‐CAR.BiTE‐ γδ T could trigger bystander effector cells active against PD‐L1‐expressing tumor cells. Results are representative of at least three independent experiments. Data represent the mean ± SD, n = 4, *, # , X p < 0.05; **, ## , XX p < 0.01; and ***, ### , XX X p < 0.001 based on paired Student's t‐test. For sub‐Figure G, * represents significant differences between Nb‐BiTE and effector cells (Nb‐BiTE vs effector cells); # represents significant differences between Nb‐BiTE and effector cells+Nb‐BiTE; X represents significant differences between effector cells and effector cells+ Nb‐BiTE.

Article Snippet: Antibodies specific for CD3 ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals.

Techniques: Functional Assay, Binding Assay, Isolation, Flow Cytometry, Expressing, Knockdown, Clone Assay, Derivative Assay, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Lysis, Cytotoxicity Assay

Journal: Frontiers in Microbiology

Article Title: A Functional Slow Recycling Pathway of Transferrin is Required for Growth of Chlamydia

doi: 10.3389/fmicb.2010.00112

Figure Lengend Snippet: Analysis of host cell trafficking pathways during treatment with FR179254 . HEp-2 cells were treated as indicated, and trafficking pathways were analyzed as described in Section (A) Lipid droplet (LD) visualization with the neutral lipid stain Bodipy 493. (B) Fluid-phase uptake as measured by fluorescent dextran. (C) Live cell visualization of multivesicular bodies/late endosomes using CD63-GFP expressing HEp-2 cells. (D) Analysis of late endosomes/lysosomes in LAMP1-YFP transfected cells. (E) Exocytic trafficking as measured by incorporation of fluorescent sphingomyelin (SM) in Golgi and chlamydiae. (F) Exocytic trafficking of glycoproteins as measured by localization of CD4 on the surface of transfected cells. (G,H) Analysis of retrograde trafficking using fluorescently tagged cholera toxin subunit B (CTxB) after pulse-chase labeling. Arrows within (C) and (E) indicate inclusions. Note the lack of significant staining in (C) (inset) and the presence of staining in (E) , which is contrast to a published study (Beatty, ).

Article Snippet: For CD4, non-permeabilized cells were incubated with primary Ab mouse anti-human CD4 (R&D Systems, Minneapolis, MN, USA) followed by secondary goat anti-mouse Alexa488 (Invitrogen).

Techniques: Staining, Expressing, Transfection, Pulse Chase, Labeling

Journal: Nature Communications

Article Title: Loss of HIV candidate vaccine efficacy in male macaques by mucosal nanoparticle immunization rescued by V2-specific response

doi: 10.1038/s41467-024-53359-2

Figure Lengend Snippet: a , b Comparison of mucosal B cells in rectal mucosa producing a IgG (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9) and b IgA (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9) antibodies that bind to ∆V1 gp120. c Comparison of plasma and mucosal IgG binding titer (AUC) against different SIV immunogens in all groups of animals (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9). d , e Comparison of d ADCC titers (the endpoint reciprocal dilution at which the ADCC killing was greater than control killing + 3 SD) and e ADCC activity among different groups of animals (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9; systemic vaccine, n = 14). f–h Correlation of f ADCC titer in the V2-TTB NP group ( n = 12, p = 0.02), g ADCC activity in the V2-TTB NP group ( n = 12, p = 0.02), and h ADCC titer in all animal groups combined ( n = 44, p < 0.0001) with number of intrarectal challenges. In h , red dots represent V2-TTB NP, black dots represent systemic vaccine, and blue squares represent TTB NP. Enlarged shapes represent multiple animals, as indicated. i , j Comparison of i V2 (NCI05)-specific ADCC activity and j frequency of V2 (NCI05)-specific ADCC activity in total ADCC among different groups of animals (V2-TTB NP, n = 12; TTB NP, n = 9; empty NP, n = 9; systemic vaccine, n = 14). k , l Correlation of V2 (NCI05)-specific ADCC activity in k the V2-TTB NP group ( n = 12, p = 0.003) and l in all animal groups combined ( n = 44, p = 0.003) with number of intrarectal challenges. m Correlation of V2 (NCI05)-specific ADCC activity at week 17 with VL at 15 wpi in the systemic vaccine group ( n = 9, p = 0.03). n Comparison of plasma antibody avidity score against ∆V1gp120 protein in different groups of animals (V2-TTB NP, n = 10; TTB NP, n = 7; empty NP, n = 8; systemic vaccine, n = 14). o Correlation of antibody response units against ∆V1gp120 (higher RU values indicated stronger binding of antibody to antigen) with number of intrarectal challenges in the V2-TTB NP+vaccine group ( n = 12, p = 0.02). Data shown in a – c , e , i , j , n were analyzed with the two-sided Mann-Whitney test. Data shown in d were analyzed with two-sided Cochran-Armitage tests for the comparisons between V2-TTB NP and TTB NP and Empty NP, separately. The two-sided Mann-Whitney test was used for the comparison between V2-TTB NP and systemic vaccine. Data shown in f – h , k – m , o were analyzed with the two-sided Spearman correlation test. Horizontal and vertical bars denote mean and SD. Source data are provided as a Source Data file. Here, the black, red, blue, and purple symbols represent male macaques immunized with a systemic vaccine, vaccine combined with V2-TTB NP, vaccine combined with TTB NP, and vaccine combined with empty NP, respectively.

Article Snippet: The primary antibodies were mouse (IgG2a) monoclonal anti-CD4 (clone OTI5D9, Novus Biologicals) and rabbit anti-Ki67 (Abcam).

Techniques: Comparison, Clinical Proteomics, Binding Assay, Control, Activity Assay, MANN-WHITNEY