Changes in the graft-infiltrating cell population (GIC) induced by Class I MHC pretreatment were characterized using flow cytometric analysis. C3H mice received 107 EL4 cells intravenously 14 days prior to transplant with C57BL/10 (H-2b) hearts. Transplanted hearts were removed from six recipients in both groups at 4, 6, 9, 12, 15, and 21 days after transplantation. GICs were harvested and incubated with FITC-conjugated Lyt-1 and Lyt-2 and phycoerythrin-conjugated L3T4 monoclonal antibodies. The proportion of GICs which were Lyt-1, L3T4, or Lyt-2 positive was similar in both control and pretreated groups at all times post-transplant, and all three cell populations exhibited similar changes over the course of the study in both groups. At Post-transplant Day (PTD) 9, there were significantly more double-staining cells (L3T4+, Lyt2+) in the pretreated group than in the control group (8.58 ± 2.49% vs 4.36 ± 1.32%, P < 0.05). By PTD 15, the double-staining cells had increased in pretreated mice to 17.46 ± 4.36% of the total GICs (P < 0.05). The percentage of GICs in EL4-pretreated mice which were Lyt-1- was significantly higher at all time points than the percentage of L3T4- or Lyt-2- cells, whereas in the controls, these cell populations were equivalent, implying that in pretreated mice, cells are present which are L3T4+ or Lyt2+ but Lyt-1-. These data are consistent with the hypothesis that prolonged allograft survival following EL4-associated Class I pretreatment is secondary to the induction of phenotypically unusual cells early after transplantation which may be clonally anergic or ultimately exert a suppressive effect on other mediators of graft rejection.
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