Fig 2: In Vivo Responses of SIRPa+/+ or SIRPa-/- CD8+ T Cells in LCMV-Infected Mice Treated with Anti-CD47(A–G) Representative flow-cytometric plots of SIRPa expression on (A) CD8+ T cells from spleens of naive WT (SIRPa+/+) and SIRPa KO (SIRPa-/-) C57BL/6 mice (left), and from the same strains infected with LCMV-WE at 13 dpi (right). (B–G) 2.5 × 106 naive CD8+ T cells were sorted from splenocytes of WT or SIRPa KO mice (indicated as donor type) and were transferred intravenously into TCRb-/- mice at day -1. The recipient TCRb-/- mice were then infected i.v. with 1 × 104 PFU LCMV-WE i.v. and then treated daily with 100 µg/mouse of anti-CD47 or isotype control antibody from day 2 to day 6 post-infection. Mice were euthanized for analyses at 10 dpi (n = 5 mice/group) and 13 dpi (n = 4 mice/group). Cumulative results from flow-cytometric analysis of (B) total splenic CD8+ T cells, (C) percentages of GP33-Tet+ CD8+ T cells, (D) percentages of CD107a+ CD8+ T cells, (E) percentages of IFN-?-producing CD8+ T cells as measured by intracellular cytokine flow cytometry, (F) plasma viremia analyzed at 10 and 13 dpi, and (G) kidney virus levels at 13 dpi. The dashed lines indicate the limit of detection for virus assays. Data shown are means ± SEM. Statistics were done by ordinary one-way ANOVA with Tukey’s multiple-comparisons post-test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The gating strategy for the spleen cell subsets is shown in Figure S1.

Fig 3: Effects of CD47 Blockade on LCMV Infection in Mice(A) CD47 cell surface expression on splenic cell subsets was analyzed by flow cytometry at 3 days post-infection with LCMV-WE-infected C57BL/6 mice. The gating strategy for the spleen cell subsets is shown in Figure S1. n = 4 mice per group. (B–I) C57BL/6 mice infected with 2 × 106 PFU LCMV-WE were treated by intraperitoneal injection of 100 µg of anti-CD47 or isotype at days 2, 3, 4, 5, and 6 post-infection and analyzed on the indicated days post-infection for (B) plasma virus titers, (C) kidney virus titers, (D) CD86 expression on splenic macrophages, and (E) dendritic cells analyzed from anti-CD47-treated and control mice. Mice were also analyzed for (F) total number of CD4+ and (G) CD8+ T cells in the spleens of mice treated with anti-CD47 or isotype control antibody. Two-way ANOVA analysis showed a statistically significant difference in the kinetics for CD4+ T cells (p = 0.004) and CD8+ T cells (p = 0.0002). The CD8+ T cells were further analyzed for the following: (H) total numbers of GP33-Tet+ CD8+ T cells at the indicated time points and (I) the numbers of CD107a+ and IFN-?+ cells at 7 dpi, the peak of the CD8+ T cell response. Data shown are consistent with two independent experiments (n = 7) and are shown as mean ± SEM. Statistics comparing treated versus control mice were done by Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 4: TCR Activation Fails to Induce Degranulation and Target Cell Killing(A) pERK activation of CTLs expressing mPIP5Kß, mPIP5Kß-K138A, or farnesyl-EGFP stimulated with plate-bound anti CD3 (black line) or unstimulated (shaded peaks) (n = 3 independent experiments).(B) Degranulation assay measured by CD107a uptake of CTLs expressing either mPIP5Kß or mPIP5Kß-K138A in response to targets pulsed with either OVA (bold line) or the null-peptide (NP68) (gray shaded peaks) after 2.5 hr (n = 4 independent experiments).(C) Percentage target cell lysis of NucRed-EL4 over 4 hr by CTL expressing either mPIP5K (dotted-line), mPIP5Kß-K138A (gray line) or farnesyl-EGFP (black line) (showing propagated SEM of triplicates; n = 3 independent experiments).See also Figure S4.