*Purpose: Decay-accelerating factor (DAF, CD55) is a cell surface expressed, glycophosphatidylinositol-(GPI)-anchored, complement regulator. Previous studies showed that during cognate T cell/antigen presenting cell (APC) interactions DAF is downregulated on the surface of both partners, permitting local complement activation which is crucial for T cell immunity. Absence of DAF enhances local complement activation that provides survival signals and drives effector T cell responses. Whether DAF downregulation on T cells and/or APCs is required for effective T cell immunity is unclear.

*Methods: To test this, we used Crispr/Cas9 to target insertion of a construct comprising a flox stop flox cassette followed by a modified DAF gene into the Rosa26 locus, to permit conditional overexpression of a DAF transgene (Tg) with a stable, GPI cleavage-resistant transmembrane helix anchor (DAF-Tg fl/fl).

*Results: PCR confirmed site-specific insertion of the construct. Flow cytometry analysis of DAF-Tg fl/fl mice crossed to a ubiquitously expressed Cre transgenic mouse (DAF-Tg+ Cre+) revealed significant (up to ~7-fold) overexpression of DAF on all major immune cell types including T cells and APCs in naïve animals. While DAF surface levels decreased on stimulated WT T cells, DAF levels did not change on stimulated T cells of DAF-Tg+ Cre+ mice. Proportions of splenic CD4+ T cells, CD8+ T cells and DCs were not different between naïve WT and DAF-Tg+ Cre+ mice, indicating that T cell development is not affected by DAF overexpression. When we transferred equal numbers of either CFSE-labeled B6 WT or DAF-Tg+ Cre+ T cells into allogeneic (bxd)F1 mice, we observed a 30% lower proportion of DAF-Tg+ Cre+ T cells (2.7% vs 1.9% of spleen cells, p<.01), and 50% fewer proliferating DAF-Tg+ Cre+ CD8+ T cells (0.3 vs 0.15 million/spleen, p<.01) vs. WT T cells (n=5-6/grp). In conversely designed experiments, we transferred CFSE-labeled BALB/c T cells into irradiated allogeneic B6 control or DAF-Tg+ Cre+ recipients and observed 30% fewer CFSE-negative CD4+ and CD8+ T cells in the DAF-Tg+ Cre+ hosts (n=3-5/grp). Together the findings support the conclusion that preventing DAF downregulation on T cells and APCs limits T cell expansion in vivo.

*Conclusions: In addition to providing novel mechanistic insight, the findings suggest that therapies aimed at increasing stable DAF expression on immune cells could be an effective strategy for limiting alloreactive, T cell dependent, transplant injury.

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