Impairment of T cell zone development in the spleen of SIRPα MT mice. A, Paraffin sections of the spleen from WT or SIRPα MT mice were stained with H&E. The boxed regions of the left panels are shown at higher magnification in the right panels. Scale bars, 500 μm (left panels) and 200 μm (right panels). B, Frozen sections of the spleen from WT or MT mice were double-stained with an mAb to B220 (green) and an mAb to Thy1.2 (red). Scale bar, 500 μm. C, By the use of Image J software (National Institutes of Health), the areas for Thy1.2-positive T cell zones or B220-positive B cell zones in the spleen sections prepared as in B was measured per each image. Data are means ± SE for a total of five mice per group in three independent experiments. D, The absolute numbers of B cells (B220⁺), CD4⁺ T cells (CD4⁺), and CD8⁺ T cells (CD8⁺) in the spleen of WT or MT mice were determined by flow cytometry. Data are means ± SE for four mice per group and representative of three independent experiments. **p < 0.01 (Student t test).

Reduced expression of CCL19, CCL21, and IL-7 in the spleen of SIRPα MT mice. Expression of CCL19 (Ccl19), CCL21 (Ccl21), or CXCL13 (Cxcl13) mRNA (A) and IL-7 (Il7) mRNA (B) in the spleen of WT or SIRPα MT mice was evaluated by quantitative PCR. The level of expression of each mRNA was normalized to that of GAPDH mRNA and presented as fold increase relative to the value for WT mice. Data are means ± SE for a total of 10 mice per group in three independent experiments. C, Frozen sections of the spleen from WT or SIRPα MT mice were double-stained with pAbs to CCL19 (left panels, red), CCL21 (middle panels, red), or CXCL13 (right panels, red) and an mAb to B220 (left and middle panels, green) or Thy1.2 (right panels, green). Scale bar, 200 μm. D, Frozen sections of the spleen from WT or SIRPα MT mice were stained with mAbs to gp38 (red) and B220 (green). Scale bar, 200 μm (left panels). The area for gp38-positive region was measured per each image. Data are means ± SE for a total of four (WT) or three (MT) mice per group (right panel). *p < 0.05, **p < 0.01 (Student t test).

Importance of CD47 for development of the splenic T cell zone. A, Paraffin sections of the spleen from WT or CD47-deficient (KO) mice were stained with H&E. Scale bar, 200 μm. B, Frozen sections of the spleen from WT or CD47 KO mice were stained with mAbs to B220 (green) and Thy1.2 (red). Scale bar, 500 μm (left panels). The area for Thy1.2-positive T cell zone or B220-positive B cell zone was measured per each image by the use of Image J software (National Institutes of Health). Data are means ± SE for a total of six mice per group (right panel). C, The absolute numbers of B cells (B220⁺), CD4⁺ T cells (CD4⁺), and CD8⁺ T cells (CD8⁺) in the spleen of WT or CD47 KO mice were determined by flow cytometry. Data are means ± SE from three mice per group and representative of three independent experiments. Expression of CCL19 (Ccl19), CCL21 (Ccl21), or CXCL13 (Cxcl13) mRNA (D) or IL-7 (Il7) mRNA (E) in the spleen of WT or CD47 KO mice was evaluated by quantitative PCR. The level of expression of each mRNA was normalized to that of GAPDH mRNA and presented as fold increase relative to the value for WT mice. Data are means ± SE for a total of five mice per group in three independent experiments. *p < 0.05, **p < 0.01 (Student t test).

Importance of hematopoietic SIRPα for development of the splenic T cell zone. A, WT or SIRPα MT mice were lethally irradiated and then reconstituted with 5 × 10⁵ BM cells from WT or MT mice for generating WT→WT, WT→MT, or MT→WT chimeras. Eight weeks after transplantation, spleens were harvested, and frozen sections of the spleen from each chimera were stained with mAbs to B220 (green) and to Thy1.2 (red). Scale bar, 500 μm (upper panels). The area for Thy1.2-positive T cell zone or B220-positive B cell zone was measured per each image by the use of Image J software (National Institutes of Health). Data are means ± SE of eight to nine mice per group in two independent experiments (lower panels). Expression of CCL19 (Ccl19), CCL21 (Ccl21) or CXCL13 (Cxcl13) mRNA (B) or IL-7 (Il7) mRNA (C) in the spleen of WT→WT, WT→MT, or MT→WT chimeras was evaluated by quantitative PCR. The level of expression of each mRNA was normalized to that of GAPDH mRNA and presented as fold increase relative to the value for WT→WT chimeras. Data are means ± SE of four mice per group and are representative in two independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, followed by the Tukey-Kramer test).

Reduced expression of LTα, LTβ, or LTβR and impaired LT signaling in the spleen of SIRPα MT mice. A, Expression of mRNA for LTα (Lta), LTβ (Ltb), LIGHT (Tnfsf14), or LTβR (Ltbr) in the spleen of WT of SIRPα MT mice was evaluated by quantitative PCR. The level of expression of each mRNA was normalized to that of GAPDH mRNA and presented as fold increase relative to the value for WT mice. Data are means ± SE for a total of six mice per group in three independent experiments. B, Total RNA was extracted from T cells or B cells isolated from the spleen of WT or SIRPα MT mice, and the expression of LTα or LTβ mRNA was analyzed by quantitative PCR. The level of expression of each mRNA was normalized to that of GAPDH mRNA and presented as fold increase relative to the value for WT T cells or B cells. Data are means ± SE for a total three mice per group in three independent experiments. C, WT or SIRPα MT mice were injected i.p. with the agonistic mAb to LTβR or control IgG. Twenty-four hours after injection, spleens were harvested, and the expression of CCL21 (Ccl21) or NFκb2 p100 (Nfkb2) mRNA was analyzed by quantitative PCR. The level of expression of each mRNA was normalized to that of GAPDH mRNA and presented as fold increase relative to the value for WT mice treated with control IgG. Data are means ± SE for three mice per group and are representative from two independent experiments. *p < 0.05, **p < 0.01 (Student t test).