Fig. 4
Restoration of lymphocyte proliferation in spleen cells from tumor-bearing mice depleted of cells with Mac-1 surface antigen in response to stimulation by allogeneic spleen cells
(A) Spleen cells were incubated with the primary rat anti-mouse antibodies IgG2a (control), CD11b/Mac-1, CD90/Thy-1.2, or CD45R/B220. The cells were washed and incubated with magnetic beads coated with anti-rat antibody. After a 30-min incubation, the magnetic beads with attached cells were removed. The nondepleted cells incubated with the control antibody were brought up to 4 × 106 cells/ml. and the other groups were brought up to equivalent volumes. One hundred microliters of these cells were then incubated with 4 × 105 irradiated allogeneic stimulator cells. [3H]thymidine was added on Day 4 of culture and incorporation was measured 16 hr later. Results are reported as CPM ± SEM. Values are the mean CPM from triplicate wells. Experiments are representative of results from at least three independent experiments. (B) Mixing experiments were performed to evaluate the effects of CD90/Thy-1.2 positive spleen cells derived from TBM on naive spleen cells. Responder spleen cells from naive mice were incubated alone, with TBM spleen cells, or with TBM spleen cells depleted of cells with IgG2b, CD90/Thy-1.2, or CD11b/Mac-1 and CD45R/B220 primary antibodies and magnetic beads coated with secondary antibodies. The wells contained 3.6 × 105 naive spleen cells. The TBM spleen cells and TBM spleen cells incubated with the control antibody were brought up to 3.6 × 106 cells/ml and the other groups were brought up to equivalent volumes. One hundredth of a milliliter of these cells were added to the normal spleen cells. The mixtures were then incubated with 4 × 105 irradiated allogeneic stimulator cells. [3H] thymidine was added on Day 4 of culture and incorporation was measured 16 hr later. Results are reported as CPM ± SEM. Values are the mean CPM from triplicate wells. Control responder cells from normal mouse spleen without stimulation demonstrated less than 2.5 times the amount of stimulation as the naive mouse spleen cells stimulated with irradiated allogeneic spleen cells.