Importantly, CD47low status was maintained on CD4 effectors cells in inflamed lymphoid and mucosal tissues of patients with Crohn��s disease (CD). We thus propose that CD47/SIRP-��/TSP-1 axis is involved in the resolution of the sellectchem inflammatory response. Results 1. CD47 Status is Differentially Regulated on TCR- Activated Human CD4 T Cell Subsets We first investigated the modulation of CD47 expression on in vitro activated human CD4 T cell subsets. To this end, we thought to use a SIRP-��-Fc fusion protein and two anti-CD47 monoclonal antibodies (mAbs) that identify different CD47 conformations [15], [17], [18], [19], [20] and/or distinct CD47 epitopes [21]. Hence, B6H12 mAb and SIRP-��-Fc compete for a similar CD47 binding site since B6H12 but not 2D3 inhibits SIRP-��-Fc binding to CD47 [22].

We showed that CD47 expression, as detected by SIRP-��-Fc binding, decreased on a majority of divided na?ve CD4 T cells (TN; CD45RA+CCR7+) following stimulation with anti-CD3 and anti-CD28 mAbs (Fig. 1A). The reduced CD47 expression was not observed when activated CD4 T cells were stained with B6H12 anti-CD47 mAb. Thus, decreased SIRP-��-Fc binding to CD47 on activated TN cells was hereafter referred to as CD47low status when compared to SIRP-��-Fc binding to CD47 on undivided TN cells as well as on 50% of activated central memory (TCM; CD45RA-CCR7+CD27+) T cells hereafter referred to as CD47high status (Fig. 1A). Divided CD47low CD4 T cells displayed an effector phenotype (CCR7low) when compared to undivided CD47high CD4 T cells (Fig. 1B).

Figure 1 CD47 status is differentially regulated on TCR- activated human CD4 T cell subsets. Further studies demonstrated that CD47 status was differentially modulated in ex vivo isolated circulating human CD4 T cell subsets (Fig. 1C). Effector memory (TEM; CD45RA?CCR7?CD27?) T cells, which represent chronically activated T cells by repeated exposure to Ag in the peripheral blood of healthy individuals, displayed a CD47low status when compared to CD47high TN and TCM T cells (Fig. 1D). Transitional memory (TTM, CD45RA?CCR7?CD27+) and terminally Batimastat differentiated (TTD, CD45RA+CCR7?CD27?) cells were detected as CD47low cells. Alike in vitro TCR-activated CD4 T cells, TN, TEM and TCM expressed similar levels of CD47 expression when they were stained with B6H12 and 2D3 anti-CD47 mAbs, suggesting a change in the conformation rather than in the amount of CD47 protein. Indeed, western blot analysis showed that the three circulating CD4 T cells subsets possessed similar CD47 protein content (Fig. 1E). We next investigated whether differences seen in SIRP-��- Fc binding to CD47 may reflect a differential distribution of CD47 on the cell surface of TN, TEM and TCM.