fitc conjugated mouse anti canine 221 cd3  (Bio-Rad)

Journal: Cell Reports Medicine

Article Title: Unedited allogeneic iNKT cells show extended persistence in MHC-mismatched canine recipients

doi: 10.1016/j.xcrm.2023.101241

Figure Lengend Snippet: Comparative analysis of the canine, human, and murine (mu) iNKT TCRs and CD4/CD8 immunophenotypes (A) ClustalOmega identity and alignment of the amino acid sequence of the human TRAV10∗01 gene product (GenBank: AAB69005.1) and the predicted amino acid sequence of the canine TRAV10∗01 (IMGT: IMGT000004) accessed in the ImMunoGeneTics (IMGT) database. (B) Alignment of the human and predicted canine TRAV10-TRAJ18 germline junction defining iNKTs. (C) Canine and human circulating T cells labeled with PE-conjugated human CD1d tetramers loaded with PBS57 (huCD1d-tet/PBS57). (D) Frequency of circulating canine versus human iNKTs shown as percentage of T cells labeled by the huCD1d-tet/PBS57 reagent (canine, n = 18, mean = 0.042% ± 0.0.0063%; human, n = 12, mean = 0.074% ± 0.02120%; dog versus human, p = 0.102, unpaired t test). Error bars indicate SEM. (E) Canine, human, and murine CD3 + cells labeled with the indicated CD1d-tet/PBS57 reagent (murine iNKT frequency, n = 3; mean = 2.790% ± 0.459%). (F) Mean fluorescence intensity (MFI) of PE-conjugated tetramers as a surrogate of iTCR affinity to CD1d in tet + cells. (G) CD4 + , CD8α + , and double-negative (DN) subsets in PB canine and human iNKTs and splenic murine iNKTs, classically used for iNKT ACT preclinical studies. (H) Summary of CD4/CD8 expression in canine and human iNKTs. Canine, n = 18, CD8α = 14.09% ± 9.344%, double-positive (DP) = 2.207% ± 0.5402%, CD4 = 26.46% ± 3.034%, DN = 57.75% ± 3.567%; human, n = 12, CD8a = 20.05% ± 2.456%, DP = 2.698% ± 1.147%, CD4 = 28.75% ± 3.629%, DN = 48.48% ± 3.767%; dog versus human, p > 0.05, unpaired t test. (I and J) Flow cytometric analysis of splenic (I) and lymph node (J) canine iNKTs (n = 4 dogs; iNKT frequency: spleen mean ± SEM 0.1% ± 0.029% [range 0.02%–0.21%]; lymph nodes mean ± SEM 0.038% ± 0.020% [range 0.006–0.120]).

Article Snippet: Anti-Canine CD3 FITC (clone CA17.2A12) , Bio-Rad , Cat# MCA1774F; RRID: AB_2291174.

Techniques: Sequencing, Labeling, Fluorescence, Expressing

Journal: Cell Reports Medicine

Article Title: Unedited allogeneic iNKT cells show extended persistence in MHC-mismatched canine recipients

doi: 10.1016/j.xcrm.2023.101241

Figure Lengend Snippet: Interdonor variability in canine CAR13-iNKTs (A) CAR13 construct cloned into MSGV1 retroviral vector with a truncated CD19 marker gene (ΔCD19 ) and an intervening FMD2A gene to ensure co-expression. (B) Representative transduction efficiency. (C) Summary of transduction efficiency with optimized canine CAR engineering protocol. (D) CAR13-iNKT dual specific activation. (E) CAR13-iNKT versus untransduced iNKT cytotoxicity against the IL-13Rα2-expressing MC-KOSA cells (n = 3 donors). (F) CAR13-iNKT proliferative response after 4-day co-culture with K562 targets expressing either CD1d or IL-13Rα2 or both, with and without αGC, relative to parental, control K562 cells. (G) Summary of CAR13-iNKT proliferative response to CD1d + and/or IL-13Rα2 + K562 targets with and without αGC (n = 3 donors). (H) Individual donor proliferative response against CD1d + IL-13Rα2 + K562 targets without and with αGC. (I) Differential gene expression profile of pure CAR13-iNKTs expanded from two distinct donors. Heatmaps show normalized expression levels relative to housekeeping genes in resting CAR13-iNKTs and after a 4 and 8 h stimulation with anti-canine CD3/CD28-coated beads. (J) Volcano plot showing 4 h gene expression profile of CAR13-iNKTs from donor 5 relative to donor 6. Red, significantly upregulated genes (38 genes); blue, significantly downregulated genes (110 genes). (K) Top 12 pathways enriched in CAR13-iNKTs. Red, upregulated; blue: downregulated in donor 5 compared with donor 6; gray, no difference between donors 5 and 6 (p > 0.05). In bar graphs, bars indicate means. Error bars indicate SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Anti-Canine CD3 FITC (clone CA17.2A12) , Bio-Rad , Cat# MCA1774F; RRID: AB_2291174.

Techniques: Construct, Clone Assay, Retroviral, Plasmid Preparation, Marker, Expressing, Transduction, Activation Assay, Co-Culture Assay, Control, Gene Expression

Journal: Cell Reports Medicine

Article Title: Unedited allogeneic iNKT cells show extended persistence in MHC-mismatched canine recipients

doi: 10.1016/j.xcrm.2023.101241

Figure Lengend Snippet:

Article Snippet: Anti-Canine CD3 FITC (clone CA17.2A12) , Bio-Rad , Cat# MCA1774F; RRID: AB_2291174.

Techniques: Control, Virus, Recombinant, Sterility, Activation Assay, Gene Expression, Amplification, Cloning, Software, Modification, Saline, Cell Culture