GVHD induces a loss of CB2R eGFP expression in T cells isolated from target organs. (A) Spleen sections from CB2R^eGFP reporter mice were stained with fluorescence-labeled antibodies directed against eGFP, CD19, or CD3. Topro served as a nuclear stain. Merged results are shown in final panels in each row. (B-C) eGFP expression on CD4⁺ T cells, CD8⁺ T cells, and B cells derived from the spleens of normal CB2R^eGFP mice (n = 16-17) are shown in panel B. Representative dot plots showing eGFP expression on these immune cell populations are depicted in panel C. (D) Lethally irradiated Balb/c mice received transplants of CB2R^eGFP BM alone (n = 4) or together with CB2R^eGFP spleen cells (adjusted to yield an αβ⁺ T-cell dose of 0.6 × 10⁶; n = 5). Animals were euthanized 14 days posttransplantation. The eGFP expression percentage on CD4⁺ and CD8⁺ T cells obtained from spleen, liver, lung, and colon of animals receiving transplants of BM alone (BM) or together with adjunctive spleen cells (GVHD) is depicted. The eGFP expression percentage on comparable cells obtained from the spleen of normal nontransplanted CB2R^eGFP mice (labeled naïve; n = 5) is shown for comparison. Statistical comparison is from all tissue sites relative to splenic CD4⁺ and CD8⁺ T cells from naïve mice. (E) Lethally irradiated Balb/c or B6.PL mice received transplants of CB2R^eGFP BM and spleen cells (adjusted to yield an αβ⁺ T-cell dose of 0.6 × 10⁶). The eGFP expression percentage on CD4⁺ and CD8⁺ T cells obtained from spleen, liver, and lung of animals receiving syngeneic vs allogeneic marrow grafts 14 days posttransplantation is depicted. Data are from 3 experiments, with a total of 15 mice per group. The eGFP expression percentage on T cells obtained from the spleen of normal CB2R^eGFP mice is shown for comparison. (F) Magnetically purified CD4⁺ or CD8⁺ T cells (1 × 10⁵) from CB2R^eGFP mice were cultured with anti-CD3 (2.5 μg/mL) and anti-CD28 (5 μg/mL) antibody for 3 days. Magnetically purified pan T cells (1 × 10⁵) from CB2R^eGFP mice were cultured with allogeneic Balb/c CD11c–enriched dendritic cells (5 × 10⁴) for 4 days. Data depict the percentage of CD4⁺ T cells and CD8⁺ T cells expressing eGFP after stimulation. Results are from 2 experiments, with 7 to 14 mice per group. (G) CB2R messenger RNA (mRNA) expression in B6 naïve and anti-CD3/anti-CD28 antibody activated CD4⁺ or CD8⁺ T cells. Data are presented as fold expression above 18S ribosomal RNA control and are derived from 10 to 13 mice per group. Results in panels B and D to G are presented as the mean ± standard deviation. **P < .01, ****P < .0001.