Journal: ImmunoHorizons
Article Title: Maturation of thymocytes with a monoclonal TCR under control of Trac promoter elements in the absence of β-selection
doi: 10.1093/immhor/vlaf035
Figure Lengend Snippet: 1045 TRex Tcrb −/− CD8 + T cells populate the periphery and are functional. (A) Spleen weights from mice with indicated 1045 and Tcrb genotypes. (B, C) CD8 + T cell frequency of CD45 + splenocytes (B) or number per mg of spleen (C). (D, E) Representative plots (D) of Vβ9 + frequency of CD8 + T cells (E). (F) Vβ9 gMFI on CD8 + T cells. G) CD44 + frequency of Vβ9 + CD8 + T cells. (H–J) Representative plots (H) of γδTCR + CD4 − CD8 − frequency of CD45 + splenocytes (I) or number per mg of spleen (J). (K, L) Representative plots (K) of Vβ9 + and γδTCR + frequency of CD8 + or CD4 + T cells (L). (M) Number of Vβ9 + CD8 T cells from 1045 +/+ Tcrb + or 1045 +/+ Tcrb −/− splenocytes following 72 h of stimulation with varying concentrations of Msln 406–414 peptide. (N) Representative histograms of proliferation of Vβ9 + CD8 + T cells stimulated with 0.1 µg/mL Msln 406–414 peptide as assessed by CellTrace Violet (CTV) dilution. (O) Frequency of Vβ9 + CD8 + T cells by number of divisions in splenocytes stimulated with 0.1 µg/mL Msln 406–414 peptide. (P) Frequency of Vβ9 + CD8 + T cells that reached ≥4 divisions across varying concentrations of Msln 406–414 peptide. (Q) Schematic depicting in vitro priming and restimulation of 1045 +/+ Tcrb + or 1045 +/+ Tcrb −/− splenocytes for evaluation of cytokine production. (R) Representative plots of IFNγ, TNFα, and granzyme B (GzmB) production in Vβ9 + CD8 + T cells following restimulation with 0.001 µg/mL Msln 406–414 peptide. (S–U) IFNγ + (S), IFNγ + TNFα + (T), and GzmB + (U) frequency of Vβ9 + CD8 + T cells following restimulation with varying concentrations of Msln 406–414 peptide. (A–L) Each dot represents an individual animal with n = 3 to 8 animals per group and mean ± SEM shown. (M–U) Mean ± SD of technical triplicates, representative of 3 independent experiments. Comparisons evaluated using 1-way analysis of variance with Tukey posttest in panels A to J and unpaired Student’s t test in panels L to U. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.
Article Snippet: Cells were then stained with 1 μM CellTrace Violet (Thermo Fisher Scientific) in PBS for 20 min at 37 °C and subsequently quenched with 4 volumes of 4 °C T cell media.
Techniques: Functional Assay, In Vitro
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