CD4⁺ T cells support in vivo proliferation of CLL cells in a CD40L-independent manner. (A) Schematic representation of the experiment. Briefly, CD19⁺ cells were enriched from the spleens of young (<6 months) or old (>8 months) TCL-1^+/+ mice and injected (5 × 10⁶ cells per mouse) intraperitoneally into 8-week old C57, CD40L^−/−, or AB0 mice. After 5 months, mice were euthanized and analyzed by flow cytometry for accumulation of CD19⁺CD5⁺ B cells within the peritoneal cavity, blood, and spleen. (B) Gating strategy. (C) Mean ± standard error of the mean (SE) of the relative contribution of CD19⁺CD5⁺ cells to the entire B-cell pool in the indicated organs. Each point represents a single mouse. Data are expressed as a percentage and are representative of ≥3 independent experiments. CD19⁺ cells were enriched from the spleen of a TCL-1^+/+ mouse, labeled with CFSE, and injected (5 × 10⁶ cells per mouse) intraperitoneally into 8-week-old C57, AB0, and CD40L^−/− mice. Mice were euthanized 2 weeks later, and cells in the peritoneal cavity were analyzed by flow cytometry for dilution of CFSE within the gate of CD19⁺CD5⁺ B cells. Statistical analysis of all panels: Student t test, *P < .05, **P < .01, ***P < .001, ****P < .0001. (D) Representative dot plots of CD19⁺CFSE⁺ cells. The gate identifies proliferating cells that have diluted CFSE. (E) Mean ± SE of CFSE⁺ cells in the peritoneal cavity. Data are expressed as a percentage; each point represents a single mouse. Data are representative of ≥3 independent experiments. Mice were treated with anti-CD4 antibody or isotype control antibodies. (F) CD4⁺ cell depletion was verified in peripheral blood from treated mice using flow cytometry. Data are reported as a percentage of CD4⁺ cells; each point represents a single mouse. (G) After 3 doses of antibodies, the mice in (F) were challenged with CD19⁺ cells enriched from the spleen of a TCL-1^+/+ mouse, and leukemia in peripheral blood was checked by flow cytometry. Solid line, CTRL-treated mice; dashed red line, mice treated with anti-CD4 antibodies (depleted [DEP]). (H) C57BL/6 mice were challenged with CD19⁺ cells enriched from the spleen of a TCL-1^+/+ mouse and were left untreated (C57) or were treated with anti-CD40L or isotype control antibodies (CTRL). Leukemia development was analyzed and reported as described in panel B. Each point represents a single mouse. FSC, forward scatter; SSC, side scatter.