Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Macrophages are required for localization of antigen-activated B cells to the follicular perimeter and the subsequent germinal center response 1

doi: 10.4049/jimmunol.1300350

Figure Lengend Snippet: Macrophages of the splenic MZ are required for B cell migration to the follicllar perimeter and subsequent formation of germinal centers. (A, B) Recipient B6 mice were injected i.p. with either empty or clodronate containing liposomes and primed with 100 μg of Ars-KLH in alum i.p. the next day. Five days post-liposome treatment, each mouse received 3 × 106 HKI65/Vκ10 splenic B cells, followed by a single i.p. injection of Ars-KLH in PBS as described in Materials and Methods. Mice were sacrificed at different time points and adjacent spleen sections were analyzed. (A) Day 1, left and middle sections show donor B cells (CD45.1+, red) concentrated at the interface of B (unstained) and T (blue) cell zones. MOMA-1 staining identifies MZ metallophilic macrophages and marks the follicular perimeter in the left panel. The location of follicular perimeters can be estimated in the middle panel using adjacent sections shown in the right panel where B and T zones are identified with B220 (blue) and TCRβ (red) staining, respectively. Day 2, the left panel shows donor B cells (red) accumulating at the follicle perimeter. Day 2, the middle panel shows donor B cells (red) randomly distributed within the B cell zone. Day 3, the left panel shows donor B cells (red) of increased numbers at the follicle perimeter and populating the follicles. The middle section shows donor B cells (red) randomly distributed within the follicles. (B) Day 5, GCs are visualized as GL7+ (green, left) and IgD low (red, right) areas in mice treated with either empty (top) or clodronate (bottom) containing liposomes. (C) B6 mice were injected with either empty or clodronate containing liposomes and immunized i.p. with 0.2 ml of 10% SRBC in PBS two days later. Mice were sacrificed on day 7 post-immunization and spleen sections were analyzed. GCs are visualized as GL7+ and IgD low areas in mice treated with empty (top) or clodronate (bottom) containing liposomes. (A, B, C) For each sample, serial sections were analyzed focusing on at least three distinct regions of the spleen. The data are representative of at least three mice per group.

Article Snippet: For T cell transfer, recipient B6.SJL (CD45.1+) mice were given liposomes, and four days later given OT-II T cells before immunization the next day with 100 μg Ovalbumin (OVA) in alum i.p. Alternatively, B6 mice were immunized i.p. with 0.2 ml of 10% sheep red blood cells (SRBC) (Colorado Serum Company, Denver, CO) in PBS two days after receiving liposomes.

Techniques: Migration, Injection, Staining