Allogeneic CD4⁺ T_EM cells of aged mice are highly alloreactive in vitro without causing GVHD in vivo. (A) MACS-enriched CD4⁺ T cells (FVB/N-L2G85, H-2^q) were sorted upon the expression of CD44^loCD62L^hi (naive CD4⁺ T cells) and CD44^hiCD62L^lo (effector memory CD4⁺ T cells [T_EM]) and transplanted with 5 × 10⁶ FVB/N wild-type bone marrow cells into lethally irradiated Balb/c recipients (H-2^d). Cell purity of these populations exceeded more than 99% (numbers on plots represent percentages of cells). (B) Naive CD4⁺ T cells emit less light than CD4⁺ T_EM cells. 10⁵ CD4⁺ T-cell subsets were FACS sorted and bioluminescence quantified (error bars indicate SD of triplicates). (C) FACS-sorted CD4⁺ T_EM cells of old donor animals showed a strong alloreactive response to irradiated splenocytes in MLR experiments (10⁵ sorted T_EM cells from 8-month-old FVB/N donors against Balb/c). Interestingly, CD4⁺ T_EM cells of young donor mice (8 weeks old) did not show an alloresponse in vitro. In contrast, naive CD4⁺ T cells were alloreactive in this MLR independent of age of the donor (8-10 weeks vs 8-10 months). The bars represent the means of triplicate values and the brackets indicate SDs. One of 3 experiments with similar results is shown. (D) All allogeneic recipients of transplants of either bone marrow cells alone or bone marrow cells plus CD4⁺ T_EM cells of 8-month-old donors survived and performed well without any signs of GVHD until the end of the observation period. Animals that received allogeneic naive CD4⁺ T cells and bone marrow all died of acute GVHD, either within 14 days or between days 40 to 60 after HCT. (E) Naive CD4⁺ T cells of 8-month-old donor mice displayed similar proliferation and migration patterns as animals given transplants of whole splenocytes. Early gut infiltration is correlated with severe acute GVHD and early mortality. Although mice that survived more than 2 weeks after HCT show a decrease of the abdominal BLI signal, they displayed clinical signs of GVHD and persistent luc⁺ T cells in liver and skin measured by BLI. One representative animal is shown from the latter category surviving until day 55. Allogeneic CD4⁺ T_EM cells undergo only a very limited cell proliferation phase until day 6 and home preferentially to the liver. (F) Liver sample of an animal that had received naive CD4⁺ T lymphocytes (day 6 after transfer) showed severe damage of the bile duct epithelia (arrow), indicative of grade IV GVHD of the liver. Dispersed in periductular and perivascular regions of portal triads, lymphocyte infiltration was observed (compare Figure 3N). In addition, the liver samples of this group showed mixed fatty changes of hepatocytes and hydropic degeneration. (G) Liver samples of mice that had received allogeneic CD4⁺ T_EM cells of 8-month-old donors displayed intact bile ducts with a regular epithelial lining (arrow). No signs of inflammation were detected and hepatocytes looked normal, while only single lymphocytes were visible in perivascular regions. Images in panels F and G show hematoxylin-eosin stains at 200× magnification.