Density gradient fractionation of mouse lymphoid tissues: I. Plaque-forming and rosette-forming cells in normal, sensitized and tolerant spleen

Abstract

Spleen from non-sensitized, primary sensitized, secondary sensitized and tolerant mice was separated by density gradient centrifugation on bovine serum albumin (BSA) into 5 subpopulations of cells. These subpopulations were analyzed for the presence of antigen-reactive cells (ARC), with the rosette-test, and for the presence of antibody-forming cells (AFC), with the plaque test. The thymic origin of the cells was tested with anti-theta serum. Sheep red blood cells were used as antigen. The results indicate that: 1. (1) Spleen cells are distributed in a characteristic pattern in the gradient, with most of the cells localizing in the middle “C” fraction. 2. (2) The top “A” fraction (light density) contains relatively more macrophages and large lymphocytes. The bottom “D” fraction (high density) contains relatively more small lymphocytes. 3. (3) Non-sensitized spleen has very few plaque-forming cells (PFC). Most of the rosette-forming cells (RFC) are localized in the “C” fraction but enrichment of RFC is highest in the “A” fraction. 4. (4) In primary sensitized spleen, the number of RFC and PFC has increased. The PFC secrete IgM antibody. Most of the RFC are localized in the top 3 fractions (“A”, “B”, “C”) of the gradient. Most of the PFC are localized in the top 2 fractions (“A”, “B”) of the gradient. The enrichment of both RFC and PFC is greatest in the top “A” fraction. 5. (5) In secondary sensitized spleen, some of the RFC and PFC (IgG-secreting) have increased in density. Most of the RFC and PFC are localized in the “B” and “C” fraction. The enrichment of RFC is still greatest in the “A” fraction. The enrichment of PFC is equally divided among the “A” and “B” fraction of the gradient. 6. (6) Tolerant spleen lacks both RFC and PFC. 7. (7) Anti-theta serum is cytotoxic only to cells in the “A” fraction of non-sensitized, primary sensitized and secondary sensitized spleen. Antitheta serum is not cytotoxic for any fraction of tolerant cells. 8. (8) Anti-theta serum only reduces the RFC in the top “A” fraction of the gradient. Anti-theta serum does not affect the PFC. The results indicate that RFC and PFC from primary and secondary sensitized spleen localize primarily in the light density fraction(s) of a BSA gradient and that the RFC in the top (light density) fraction are thymusderived. The results also indicate that tolerant spleen lacks thymus-derived cells.