Fig 1.
Interaction of CD47 with its ligand SIRPα is necessary for Ag-induced CD4+ T cell expansion in vivo.
(A) T cell adoptive transfer scheme. (B) Proliferation of adoptively transferred violet dye labeled CD4+ OTII T cells recovered from WT, Cd47KO or SKI recipient mice. Each peak indicates one cell division. (C-F) OTI cells recovered after 72 h or 6 days, and division and proliferation indices of transferred OTII T cells post OVA immunization. Means ± SEM, 4–6 animals per group, and representative of 2 independent experiments. * p<0.05, ***p<0.001 compared to WT by one-way ANOVA with Tukey test. (G) Representative CD62L and CD44 surface expression on gated double positive CD4+ WT, Cd47KO and SKI OTII T cells recovered from spleens of SKI, Cd47KO or WT mice. The number above the open rectangle is the average percentage of CD44hi effector T cells. Data are means ± SEM from 3–4 mice per group. * p< 0.05 compared to WT-OVA by one-way ANOVA with Tukey test.
Fig 2.
CD47 deficiency or loss of SIRPα signaling in recipient mice results in severe reduction in CD4+ T cell expansion in vivo.
(A) Proliferation of adoptively transferred naïve dye labeled CD4+ OTII T cells recovered from WT, Cd47KO or SKI recipient mice. Means ± SEM from 3–4 animals per group from 2 separate experiments. (B-E) CD4+ OTII T cell expansion in 72 h, % CD44hi activated T cells, and proliferation and division indexes relative to WT mice. Means ± SEM from 4 mice per group, results are representative of 2 independent experiments. * p<0.05, ** p< 0.01, *** p<0.001 versus WT mice by one-way ANOVA with Tukey test. (F) Proliferation of adoptively transferred naïve dye-labeled SKI or Cd47KO CD4+ OTII T cells recovered from WT, Cd47KO or SKI recipient mice. Means ± SEM from 4 animals per group. (G-I) CD4+ OTII T cell expansion in 72 h, % CD44hi activated T cells, and proliferation and division indexes relative to WT mice. Means ± SEM from 4 mice per group.
Fig 3.
Interaction of CD47 and SIRPα does not play a role in OVA-induced CD4+ T cell production in vitro.
(A) Conjugate formation between murine WT, Cd47KO and SKI OTII CD4+ T cells with BMDC from WT, SKI and Cd47KO mice at baseline and with OVA or PBS control. Means ± SEM from 3 independent studies, each performed with duplicate samples. *** p< 0.001, by one-way ANOVA with Tukey test. (B) Conjugate formation of WT BMDC and OTII CD4+ T cells in presence of various function blocking mAb specific for molecules indicated on x-axis. Means ± SEM from 3 independent studies, each performed with duplicate samples. ** p<0.01 by one-way ANOVA with Tukey test. (C,D) Conjugate formation between human CD4+ T cells and MDDC in presence of IgG control or blocking mAb specific for molecules indicated. Means ± SEM from n = 3 independent studies each performed with duplicate samples. * p<0.05, ** p<0.01, *** p< 0.001 by one-way ANOVA with Tukey test. (E) Analysis of WT, Cd47KO and SKI CD4+ T cell activation (CD44hi CD62Llo) and proliferation assessed by dye dilution after 48 h of coculture in vitro with indicated MDDCs, with or without OVA. Proliferation of Cd47KO and SKI was not significantly different from WT. PBS treated, purple line; Ova stimulated, red line. The results are representative of 3 independent experiments.
Fig 4.
OTII T cells home to and achieve similar densities in T cell zones of WT, Cd47KO and SKI mice.
(A) Dye-labeled WT OTII T cells were injected i.p. into WT, SKI and Cd47KO mice and 24 h later spleens were harvested, and the number of transferred OTII cells was determined. Mean ± SEM from 4 mice per group. (B) Areas of T cell zone within the white pulp of the spleens from WT, Cd47KO and SKI mice. Means ± SEM, n = 10–16 white pulp areas: 3 mice per group. * p < 0.05 by one-way ANOVA with Tukey test. (C) Immunofluorescence images of splenic white pulp of WT, Cd47KO and SKI mice demonstrating transferred CFSE CD4+ OTII T cells (green), CD4+ T cells (red), follicle containing B220+ B cells (blue) and CD169+ marginal zone metallophilic macrophages (white). Top images scale bar, 100 μm, 10x objective; bottom zoomed in images, scale bar, 25 μm, using a 20x objective. (D) Manual count of CFSE+ OTII T cells per T cell zone. Means ± SEM from 10–16 splenic white pulps; 3 mice per group. ** p < 0.01 by one-way ANOVA with Tukey test. (E) Density of CFSE+ OTII T cells [CFSE+ OTII T cells ÷ total T cell area = CFSE+ per 1000 μm2]. Means ± SEM from 10–16 splenic white pulp sections; 3 mice per group. (F) Ratio of transferred CFSE+ OTII T cells to endogenous CD4+ T cells by flow cytometric analysis. Mean ± SEM, n = 4 mice per group.
Fig 5.
SIRPα - CD47 axis regulates phenotype and function of DCs.
(A-C) Hock injected BMDC from Cd47KO and SKI, compared to WT mice, exhibited reduced migration. Data are expressed as the % recovery of labeled live DC (% of total recovered). Data are means ± SEM from 3 independent studies with 3–4 mice per group per study. ** p <0.01 by one-way ANOVA. (D, E) % DC and total DC number in spleens were reduced in Cd47KO and SKI compared to WT mice. (F) CD4+ DCs as % of total spleen DCs. (G) Expression of APA1/1 TCR activation epitope in CD4+ WT OTII cells recovered from spleen of WT, Cd47KO and SKI mice. Means ± SEM from 2 independent studies from 3–4 mice per group. *** p< 0.001 by one-way ANOVA with Tukey test. (H) Representative flow cytometry histograms of APA1 staining in WT, SKI and KO mice after OVA immunization.
Fig 6.
Reduced CD4+ OTII T cell proximity to CD11c+ DCs in Cd47KO and SKI splenic T cell zones.
(A) CFSE-labeled WT OTII T cells (4x106) were i.v. injected into Cd47KO, SKI and WT recipient mice. Twenty-four hrs later, spleens were harvested and processed into frozen blocks. Four-color immunofluorescence microscopy of transferred CFSE+ OTII T cells, CD11c+, B cell follicles and CD169+ marginal zone macrophages within the splenic white pulp. Data are representative of 3–5 replicate sections from 3 mice per group. Calibration bar, 100 μm. (B) Immunofluorescence microscopy of CFSE+ T cells (green) and CD11c+ DCs (red) within T cell zone. Arrows identify CD11c+ DCs. Representative of 3 mice per group. Scale bars, (A) 25 μm and (B) 5 μm. (C) Proximity of CFSE CD4+ OTII T cells (green) to splenic CD11c+ (red) DCs in T cell zones of splenic white pulp in mice immunized with OVA protein. The number of CFSE+ T cells in T cell zone analyzed were WT, 154; Cd47KO, 135; SKI, 109. *** p < 0.0001 by one-way ANOVA with Welch’s correction.