Hla-dr-initiated inhibition of hematopoiesis involves apoptosis via a fas/fas-ligand pathway

Treatment with anti-HLA-DR MAb (H81.9; IgG2a) prevents hematopoietic recovery in irradiated dogs given autotogous marrow grafts and inhibits hernatopoiesis in vitro. In vitro results suggest that inhibition involves apoptosis [API in a small portion of hematopoietic and non-hematopoitic calls. In assays for long-term culture-Mating cells (LTC-ICs) using CD34+ cells, the presence of MAb H81.9 (10μg/ml) prevented the generation of colonies as compared to untreated control (frequency of LTC-IC: control, 1:50 to 1:75, H81.9-treated., 0), Since MAb H81.9 upregulated TNFer which may upregulate Fas on hematopoietic cells, we investigated whether the mechanism of inhibition involved the Fas/Fas-ligand IFas-L) pathway. DNA histograms from 5-day cultured control 8M mononuclear cells IBMMNCsI showed 2 ±1.2% of AP, whereas 14,3 ±4.4% of cells were apoptotic after 8-hour incubation wrm MAb H81.9, The addition of Fas-lg, a soluble fusion protein that neutralizes Fas-L, prevented AP of BMMNCs which had been induced by MAb H81.9 alone, suggesting that Fas/Fa s-L interaction was involved in mediating HLA-DR-induced AP. Expression of Fas on marrow cells did not change with H81.9 treatment regardless of culture period, but Fas-L expression from 2-day cultured BMMNCs treated with H81.9 was 2.5fold higher than control (7.5% vs 3%), particularly on small cells (lymphocyte gate). AP in BMMNCs treated with H81.9 plus anti-Fas MAb (IgM) increased compared to H81.9 alone (27% vs 13%) as determined by FACS-TUNEL test, indicating that ligation of HLA-DR with MAb enhanced Fas/Fas-L mediated AP. These findings suggest that HLA-DR-mediated inhibition of hematopoiesis involves Fas/Fas-L-dependent signals.