Origin of CD4/CD8 DP macrophages and function of CD4/CD8 DP monocytes. (A) GFP-positive spleen cells were transferred into GFP-negative recipients that had been immunized with porcine myosin. EGFP transgenic rats and nontransgenic Wistar rats (all rats were 4 weeks old) were immunized with myosin as described in “Immunization of rats with porcine heart myocin and induction of myocarditis.” Mononuclear cells were isolated from the spleen of EGFP transgenic rats 1 week after immunization and transferred into Wistar rats intravenously 2 weeks after immunization (1 × 10⁷ cells per animal). Five days later, the hearts of recipients were extirpated, and then tissue-infiltrating macrophages were isolated and used. Experiments were repeated twice, and representative results are shown. Arrowheads indicate cells expressing both GFP and CD68 (ED-1, red). Total magnification: × 100. (B) The cells isolated from the cardiac tissues were cultured in chamber slides at 37°C for 1 hour. Resultant adherent cells were fixed using cold acetone for 5 minutes and then stained for CD4 (OX-35, red) and CD8 (OX-8, blue). The merged image shows that the cell expressing both CD4 and CD8 is also positive for GFP. Total magnification: × 600. (C) FW-wt rats were immunized with myosin and the adjuvant containing killed tuberculosis germs. Mononuclear cells separated from the spleen or cardiac tissues 1 week or 3 weeks after immunization, respectively, were cultured in plastic dishes at 37°C for 1 hour, and then the adherent cells were divided into CD8^- and CD8⁺ populations, using the MACS system. Expression profiles of CD3, NKR-P2, Fas L, perforin, and granzyme B were compared by RT-PCR. The cDNA from CD8⁺ T cells served as a positive control. The negative control was the cDNA derived from the 1:9 mixture of CD8⁺ T cells and fibroblasts. (D) Six-week-old Wistar rats were immunized with adjuvants containing killed tuberculosis germs. One week later, mononuclear cells were separated from the spleen and then cultured in chamber slides at 37°C for 1 hour. Resultant adherent cells were fixed using cold acetone for 5 minutes, followed by staining for CD68 (ED-1, green), granzyme B (red), and CD8 (OX-8, blue). The merged image shows the cells stained with 3 colors. Total magnification: × 200. (E) Cytotoxic assay in vitro. Six-week-old Wistar rats were immunized with adjuvants containing killed tuberculosis germs. One week later, mononuclear cells were separated from the spleen and incubated in plastic dishes for 20 minutes at 37°C. Resultant adherent cells were collected and divided into CD8^- and CD8⁺ cells using the MACS system. These cells were added to the culture of allogenic epithelial thymoma cells with E/T ratios of 30, 10, 1, and 0.1 (4 × 10⁴ target cells per well in 24-well plates). After incubation for 18 hours, cytotoxicity was measured using the CytoTox 96 test kit. Data are represented as mean ± SD of experiments done in triplicate. ^*P < .05. (F) Phagocytosis assay. Yellow-green carboxylate-modified 1.0 μm latex beads were mixed with peripheral blood from Wistar rats that had been immunized with adjuvants containing killed tuberculosis germs one week before (1.5 × 10⁷ beads/300 μL blood). After incubation for 2 hours at 37°C, PE-conjugated anti-CD4 (OX-35) and PerCP-conjugated anti-CD8 (OX-8) Abs were added to the mixture, followed by depletion of erythrocytes. After 3 times wash with cold PBS, CD4⁺/CD8⁺ cells were gated to determine uptake of the fluorescence-labeled beads using FACScan. Experiments were done in triplicate. Representative results are shown. The filled and gray histograms represent the profiles of CD4⁺/CD8⁺ and CD4⁺/CD8^- monocytes, respectively.