Figure 2.
Figure 2. TCR affinity inversely correlates with onset of effector gene expression and cytotoxic activity in vivo. (A) mRNA levels of Batf, Irf4, Gzmb, and Prf1 in divided (e670low) GFP+ OT-I T cells sorted 60 h after transfer into recipient mice that contained N4-, Q4-, or T4-pulsed DCs. Relative mRNA levels were normalized to naive OT-I T cell mRNA levels (set to 1). Fold-changes in mRNA levels are shown as mean ± SEM. Data are representative of two independent experiments with RNA from LNs pooled from two to three mice per condition. (B) Flow cytometry of gzmB-positive OT-I T cells and gzmB MFI 60 h after T cell transfer. Each dot represents data from one mouse. Data are pooled from three independent experiments with a total of 8–11 mice per group. (C) Relative mRNA levels of GzmB and Prf1 in N4-stimulated OT-I T cells at 60 h (as in A) and 108 h after transfer. Data are of one experiment with RNA pooled from LNs of two to three mice per condition. Error bars represent mean ± SEM. (D) Experimental layout of DC elimination assay. (E) Flow cytometry plots gated on CD45.1+ DCs injected 1 d before into mice that contained activated OT-I T cells (readout at 84 and 108 h after OT-I T cell transfer). e670low DCs were pulsed with peptides, and e670hIgh unpulsed DCs (U) were cotransferred as controls. Data are representative of six independent experiments. (F) Ratio of unpulsed DCs to N4-, Q4-, or T4-pulsed DCs recovered from LNs 84 or 108 h after OT-I T cell transfer. Bars show the mean, with each dot representing a single LN. (G) Killing efficacy as assessed by dividing data in F by the percentage of OT-I T cells in reactive LNs. Calculated values were normalized to the N4/DC ratio divided by the 84-h OT-I T cell percentage. The box extends from the 25th to 75th percentile with the median; whiskers indicate upper and lower range. (F and G) Data are pooled from six independent experiments with total of seven mice per condition. Data were analyzed using Kruskal-Wallis tests with Dunn’s posttest (A, B, F, and G) or Student’s t test (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TCR affinity inversely correlates with onset of effector gene expression and cytotoxic activity in vivo. (A) mRNA levels of Batf, Irf4, Gzmb, and Prf1 in divided (e670low) GFP+ OT-I T cells sorted 60 h after transfer into recipient mice that contained N4-, Q4-, or T4-pulsed DCs. Relative mRNA levels were normalized to naive OT-I T cell mRNA levels (set to 1). Fold-changes in mRNA levels are shown as mean ± SEM. Data are representative of two independent experiments with RNA from LNs pooled from two to three mice per condition. (B) Flow cytometry of gzmB-positive OT-I T cells and gzmB MFI 60 h after T cell transfer. Each dot represents data from one mouse. Data are pooled from three independent experiments with a total of 8–11 mice per group. (C) Relative mRNA levels of GzmB and Prf1 in N4-stimulated OT-I T cells at 60 h (as in A) and 108 h after transfer. Data are of one experiment with RNA pooled from LNs of two to three mice per condition. Error bars represent mean ± SEM. (D) Experimental layout of DC elimination assay. (E) Flow cytometry plots gated on CD45.1+ DCs injected 1 d before into mice that contained activated OT-I T cells (readout at 84 and 108 h after OT-I T cell transfer). e670low DCs were pulsed with peptides, and e670hIgh unpulsed DCs (U) were cotransferred as controls. Data are representative of six independent experiments. (F) Ratio of unpulsed DCs to N4-, Q4-, or T4-pulsed DCs recovered from LNs 84 or 108 h after OT-I T cell transfer. Bars show the mean, with each dot representing a single LN. (G) Killing efficacy as assessed by dividing data in F by the percentage of OT-I T cells in reactive LNs. Calculated values were normalized to the N4/DC ratio divided by the 84-h OT-I T cell percentage. The box extends from the 25th to 75th percentile with the median; whiskers indicate upper and lower range. (F and G) Data are pooled from six independent experiments with total of seven mice per condition. Data were analyzed using Kruskal-Wallis tests with Dunn’s posttest (A, B, F, and G) or Student’s t test (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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