MIF deficiency leads to reduced numbers of TAMs and increased apoptosis of CLL cells. (A) MIF protein content determined by Western blotting in purified splenic macrophages and B cells and total splenocytes as control from TCL1^+/wtMIF^wt/wt and TCL1^+/wtMIF^−/− mice. β-actin as loading control. (B) Absolute numbers of CD68⁺ TAMs per mm² in sections of enlarged spleens (top panel) and bone marrow (bottom panel) are higher in TCL1^+/wtMIF^wt/wt (n = 18) versus TCL1^+/wtMIF^−/− (n = 14) mice (spleen: P < .0001; bone marrow: P = .066). (C) Representative immunohistochemistry for CD68 in splenic sections of TCL1^+/wtMIF^wt/wt (top panel) and TCL1^+/wtMIF^−/− mice (bottom panel). Macrophage agglomerates (red, arrow) are present in spleens of TCL1^+/wtMIF^wt/wt mice, whereas macrophages are only sparsely distributed in TCL1^+/wtMIF^−/− mice. Insets represent negative control without primary antibody (10×magnification). Representative CD68/Ki67 double staining of TCL1^+/wtMIF^wt/wt and TCL1^+/wtMIF^−/− spleen showing accumulation of proliferating cells (brown) in areas of macrophage agglomeration in TCL1^+/wtMIF^wt/wt but not in TCL1^+/wtMIF^−/− spleen. (D) TUNEL-positive cells in splenic sections of TCL1^+/wtMIF^wt/wt and TCL1^+/wtMIF^−/− mice (n = 5 per genotype; 62 ± 7 in TCL1^+/wtMIF^wt/wt mice vs 192 ± 20 in TCL1^+/wt MIF^−/− mice; P < .0001). (E) Spontaneous and drug-induced rate of apoptosis as detected by flow cytometric positivity for annexin V/7-amino-actinomycin D in cultured nonleukemic splenocytes of TCL1^+/wtMIF^−/− versus TCL1^+/wtMIF^wt/wt mice. Cells were treated ex vivo with cytostatic drugs (50μM fludarabine, 61nM vincristine, 1μM prednisolone) for 24 hours. For each group and genotype, n = 5 independent splenocyte preparations were analyzed.