Stromal and hematopoietic spleen remodeling in Fas-mutant mice.

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Stromal and hematopoietic spleen remodeling in Fas-mutant mice. Stromal and hematopoietic spleen remodeling in Fas-mutant mice. A, confocal microscopy analysis of spleens from control Sparc+/+ and Sparc−/− mice and Fas-mutant lpr/lpr/Sparc+/+ and lpr/lpr/Sparc−/− mice were stained with the following mAbs: anti-B220 (green), anti-CD3 (red), and anti-collagen type IV (blue). CD3+ and B220+ lymphocytes were confined to the follicular structures of the spleen in Sparc+/+ and Sparc−/− mice. In autoimmune lpr/lpr/Sparc+/+ mice, the expanding double-positive B220+CD3+ cells (arrows) enlarged the splenic follicles, whose structures remain defined, and the different cell types were separated and compartmentalized. In contrast, a loss of compartmentalization was observed in lpr/lpr/Sparc−/− spleens, where B and T lymphocytes as well as double-positive B220+CD3+ cells were interspersed. Scale bars, 10 μm. B, confocal microscopy analysis of spleens from control Sparc+/+ and Sparc−/− mice and Fas-mutant lpr/lpr/Sparc+/+ and lpr/lpr/Sparc−/− mice were stained with the following mAbs: anti-B220 (green), anti-CD3 (blue), and -CD169 (red). CD169+ cells were enriched in lpr/lpr/Sparc−/− mice. In these mice, CD169+ cells were also mixed with B and T lymphocytes. In control Sparc+/+ and Sparc−/− mice and lpr/lpr/Sparc+/+ mice, CD169+ cells remained confined to the marginal zone area of the splenic follicles (arrows). Scale bars, 20 μm. C, immunohistochemical analysis of SPARC expression in the spleens from Sparc+/+ and lpr/lpr/Sparc+/+ mice showed that SPARC was expressed by scattered stromal elements of the B-cell areas of Sparc+/+ mice, and SPARC staining also extended to the T cell–rich areas of the spleens in lpr/lpr/Sparc+/+ mice. Scale bars, 20 μm. D, flow cytometry analysis of spleen cell suspension was performed on control Sparc+/+ and Sparc−/− mice and Fas-mutant lpr/lpr/Sparc+/+ and lpr/lpr/Sparc−/− mice to evaluate splenic granulocytes and precursors (LK, GMP). Myeloid progenitors were identified on the basis of the expression of CD34 and CD16/CD32 within the gate of Lin-CD117+ cells. At different stages of maturation, granulocytes were identified according to their surface expression of the CD11b and GR-1 markers. The total number of GMPs was obtained by multiplying the fraction of LK cells by the total number of spleen cells. The total number of granulocytes was obtained from the fraction of each population multiplied by the fraction of CD45+ cells and then the total number of spleen cells (*, P < 0.05; Student t test). Overall, lpr/lpr/Sparc−/− spleens were characterized by the increased expansion of myeloid precursors and granulocytes compared with lpr/lpr/Sparc+/+ mice. Scale bars, 20 μm. E, confocal microscopy analysis of spleens from control Sparc+/+ and Sparc−/− mice and Fas-mutant, lpr/lpr/Sparc+/+ and lpr/lpr/Sparc−/− mice stained with mAbs to GR-1, CD19, and CD5 showed granulocyte enrichment in the spleens of lpr/lpr/Sparc−/− mice compared with lpr/lpr/Sparc+/+ mice. In lpr/lpr/Sparc−/− spleens, GR-1+ cells (green) made contact with CD5+ B cells (CD19+CD5+ double-positive, arrows). F, BAFF production evaluated by ELISA assay in splenic granulocytes from lpr/lpr/Sparc+/+and lpr/lpr/Sparc−/− mice. Granulocytes were obtained by magnetic bead separation and seeded onto a 96-well plate. Their supernatants were harvested 24 hours after cell culture (*, P < 0.05; Student t test). G, flow cytometry analysis of IL-21 production by the same splenic granulocytes used above. The representative image shows increased production of IL-21 by granulocytes from lpr/lpr/Sparc−/− mice compared with lpr/lpr/Sparc+/+ or normal mice (*, P < 0.05; Student t test). H, in situ immunohistochemical analysis of IL-21 expression in paraffin-embedded spleen sections showed increased IL-21 expression and production in granulocytes (arrows) from the spleens of lpr/lpr/Sparc−/− mice. Scale bars, 20 μm, right; 50 μm, left. Sabina Sangaletti et al. Cancer Discovery 2014;4:110-129 ©2014 by American Association for Cancer Research