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: Journal of immunological methods

Article Title: Cross-reactivity of T cell-specific antibodies in the bank vole (Myodes glareolus).

doi: 10.1016/j.jim.2023.113524

Figure Lengend Snippet: Fig. 2. A) Tree showing phylogenetic relationships among rodent species discussed in the article. Tree topology was based on a rodent phylogenetic tree published by Steppan and Schenk (2017), with root scaled to the median divergence time between Myodes and Mus reported on Timetree (see the main text). B) Pairwise amino acid sequence identity (upper-diagonal, in blue) and similarity (lower diagonal, in grey) matrices for CD4, CD8α and CD8β molecules. Mus musculus – mouse, Rattus norvegicus – rat, Peromyscus maniculatus - eastern deer mouse, Sigmodon hispidus - hispid cotton rat, Cricetulus griseus - Chinese hamster, Mesocricetus auratus - Syrian hamster, Microtus ochrogaster - prairie vole, Myodes glareolus – bank vole, and Homo sapiens, human. For brevity, only the generic name is provided on the figure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: In addition, because anti-CD4 and anti-CD8α cotton rat-specific mAbs are commercially available, CD4 and CD8A hispid cotton rat (Sigmodon hispidus) sequences were obtained from commercially available constructs (Cotton Rat CD4 VersaClone cDNA, cat# RDC1063, R&D Systems; Cotton Rat CD8 alpha (AAL55392) VersaClone cDNA, cat# RDC0871, R&D Systems), which had been used A M. Migalska et al. Journal of Immunological Methods 520 (2023) 113524 in the construction of said antibodies.

Techniques: Sequencing

Journal: Journal of immunological methods

Article Title: Cross-reactivity of T cell-specific antibodies in the bank vole (Myodes glareolus).

doi: 10.1016/j.jim.2023.113524

Figure Lengend Snippet: Fig. 3. Expression of the CD4, CD8α and LCK genes in three sorted populations of cells: “CD4+” - CD3 + CD4+; “CD8+” - CD3 + CD4-; “neg” - CD3-CD4-. Relative normalized expression levels were measured with ΔΔCt method, with calibrator sample (“Calib”) prepared by mixing RNA from the three cell populations. TBP (TATA box binding protein) was used as a reference gene.

Article Snippet: In addition, because anti-CD4 and anti-CD8α cotton rat-specific mAbs are commercially available, CD4 and CD8A hispid cotton rat (Sigmodon hispidus) sequences were obtained from commercially available constructs (Cotton Rat CD4 VersaClone cDNA, cat# RDC1063, R&D Systems; Cotton Rat CD8 alpha (AAL55392) VersaClone cDNA, cat# RDC0871, R&D Systems), which had been used A M. Migalska et al. Journal of Immunological Methods 520 (2023) 113524 in the construction of said antibodies.

Techniques: Expressing, Binding 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