TIM-4 expressed on peritoneal macrophages and B-1 cells mediates the uptake of apoptotic cells in vitro. (A) Peritoneal cells were harvested from WT C57BL/6 mice by lavage, stained with CD11b-Pacific blue, CD19-allophycocyanin, and TIM-4-PE, and analyzed by flow cytometry. B cells were gated on CD19⁺CD11b⁻ cells, B-1 cells on CD19⁺CD11b⁺ cells, and macrophages on CD19⁻CD11b⁺ cells. Dashed gray lines represents isotype control staining, and blacks line are TIM-4 staining. (B) Peritoneal exudate cells were cultured with anti-TIM-4 mAb 5G3, 3A1, or 3H11 for 30 min before CMFDA-labeled AB were added to the cultures. Cells were harvested 30 min later, labeled with CD11b-Pacific blue and CD19-allophycocyanin, and analyzed by flow cytometry. Numbers in the upper right quadrants are the frequencies of AB⁺ peritoneal cells. (C) Results from three experiments performed as in B are summarized as percentage CMFDA-AB⁺ peritoneal subsets.

Targeting Tim-4 by homologous recombination to generate TIM-4^−/− mice. (A) A 1.7-kb short arm upstream of Tim-4 exon 2 and a 6.5-kb long arm downstream of exon 4 were subcloned into the pKSTKneo targeting vector. Homologous recombination leading to replacement of exons 2–4 with PGK-neo would disrupt Tim-4 gene expression as depicted. (B) Confirmation of the TIM-4^−/− genotype by PCR. Arrows in A represent primers for the PCR amplification strategy used to distinguish between WT, heterozygous, and homozygous TIM-4-mutant mice. WT band = 416 bp; KO band = 201 bp. Lane 1, 100-bp DNA ladder; lane 2, WT; lane 3, TIM-4^+/−; lane 4, TIM-4^−/−. (C) Splenic cells from WT and TIM-4^−/− mice were stained with CD11c-allophycocyanin, CD11b-FITC, and TIM-4-PE. Dashed gray line represents isotype control staining, and black line is TIM-4 staining.

TIM-4^−/− T and B cells have a hyperactive phenotype. (A) WT and TIM-4^−/− mice were immunized with MOG_35–55/IFA plus M. tuberculosis s.c., and spleens and lymph nodes were harvested 8 days later. Cells were reactivated with MOG_35–55 peptide, and proliferation was measured after 48 h by ³[H]-thymidine incorporation in triplicate wells. Results shown are the mean of three independent experiments (total n = 5 to 6 mice per group). (B) Supernatants were collected from the experiments described in A and analyzed by ELISA for IL-17 and IFNγ production. (C) CD19⁺ cells purified from peritoneal exudates and cultured in the presence of titrated anti-IgM or with PMA plus ionomycin. Proliferation was measured after 48h by ³[H]-thymidine incorporation in triplicate wells. Results shown are representative of 4 independent experiments. d) Ig isotype titers were measured by ELISA using serum from peripheral blood of naïve WT and TIM-4^−/− mice. Data are representative of two independent experiments (total n = 10–11 mice per group).

TIM-4^−/− peritoneal macrophages cannot efficiently engulf apoptotic cells in vitro or in vivo. (A) Peritoneal cells were cultured with CMFDA-labeled AB for various time points and analyzed by flow cytometry. Gates included CD11b⁺F4/80⁺ cells (Top) and CD11b⁺CD19⁺ cells (Middle). The average frequency of F4/80⁺CD11b⁺ that were CMFDA-AB⁺ after 30, 60, or 90 min is shown (Bottom). Data represent six independent experiments. (B) Peritoneal cells were cultured with CMFDA-labeled AB (green) for 3 h. Cells were stained with CD11b-PE (red) and analyzed by immunofluorescence microscopy. Arrows indicate nonengulfed AB associated with macrophage cell surface. Frequencies of CD11b⁺ cells that engulfed AB in two independent experiments are summarized (Right). (C) Images representing a 90-min time-lapse video of peritoneal cells cultured with CMFDA-labeled AB. Arrows indicate peritoneal cells associating with AB. (D) Representative images from a Z-stack analysis performed on peritoneal cells that were treated as in B. Images in C and D are representative of two independent experiments. (E) Mice were injected i.p. with CMFDA-labeled AB and killed 30 min later. Peritoneal cells were harvested and analyzed by flow cytometry (Top). Gate includes CD11b⁺F4/80⁺ cells. Data from three independent experiments are summarized (Bottom; *P < 0.05).

Apoptotic body clearance by WT and TIM-4^−/− splenic APC does not differ. Splenocytes from WT or TIM-4^−/− mice were depleted of Thy1.2⁺ cells and then used for isolating CD19⁺, CD11c⁺, or CD11b⁺ cells. Isolated cell subsets were incubated with CMFDA-labeled AB for 30 min and then analyzed by flow cytometry. Data shown are representative of three independent experiments.

TIM-4^−/− mice produce antibodies to dsDNA. Serum from naïve WT and TIM-4^−/− female and male mice was tested for the presence of anti-dsDNA antibodies by ELISA. Data shown are from four to five independent experiments (n = 16 to 17 mice per group).