contain These results indicate that neutrophils may represent an additional target for CD47-Var1 in inflamed colons and further demonstrate that cytokine expression is restricted to CD172a+ cells in hematopoietic cells, whether or not they express HLA-DR. Figure 5. CD47-Var1 identifies cytokine-producing HLA-DR+ and HLA-DR? CD172a+ cells in inflamed colons from CD patients. Ex vivo�Cisolated LP cells from inflamed colons of CD patients were stained for intracellular cytokines (IL-1��, TNF, … Finally, we addressed whether CX3CR1+ or CX3CR1?HLA-DR+CD172a+ cell subsets purified from the mLNs of CD patients could activate effector memory CD4 T cells isolated from the same mLN donor, and examined how CD47-Var1 interfered with Th cell fate (Fig. 6).

Effector memory CD45RA?CD25?CD62Llow CD4+ T cells were purified as high (CCR7?) and low (CCR7+) IL-17 plus IFN-�èCproducing cells (Kryczek et al., 2011) and co-cultured with CD14+CX3CR1+ (P1), CD14?CX3CR1+ (P2; CD1c+DCs), or CD14?CX3CR1? (P3, which may include E-Cadherin+ cells) HLA-DR+CD172a+ cells (Fig. 6, A to C). CD47-Var1 significantly impaired Th17 but not Th1 responses selectively in P3/CD62LlowCCR7? T cell co-cultures (Fig. 6 D). More specifically, CD47-Var1 suppressed the emergence of IL-17+IFN-��? and IL-17+IFN-��+, but not IL-17-IFN-��+ cells in CD62LlowCCR7? and CD62LlowCCR7+ effector memory CD4 T cells co-cultured with P3 cells (Fig. 6, E and F). Figure 6. CD47-Var1 impairs memory Th17 and Th17/Th1, but not Th1, responses by CD62LloCD4 effector T cells co-cultured with HLA-DR+CD172a+ cells isolated from mLNs of CD patients.

(A) Two subsets of effector memory CD62LloCD4 T cells (CCR7? and CCR7+) … In summary, we conclude that the human counterparts of the murine colitogenic CD172a+ DCs, identified as proinflammatory cytokine producing HLA-DR+CD172a+ cells, accumulate in the mLNs and intestinal mucosa of CD patients and can be targeted by CD47 fusion proteins. DISCUSSION Several studies in animal models support a role for DCs in the pathogenesis of IBD (Coombes and Powrie, 2008), yet the nature and functional properties of the human counterparts of these colitogenic CD172a+ DCs has not been reported. Here, we show that HLA-DR+CD172a+ cells accumulate in the mLNs and the inflamed intestinal mucosa of CD patients, and demonstrate that IL-1��, TNF, IL-6, IL-8, or IL-10 production is selectively produced by CD172a+ but not CD172a? cells.

In inflamed intestinal tissue explants, CD47-Var1, which specifically binds CD172a, profoundly inhibits the production of a wide array of proinflammatory cytokines, and in mLNs it impairs the ability of HLA-DR+CD172a+ cells to stimulate memory Th17 Drug_discovery but not Th1 responses. We conclude that HLA-DR+CD172a+ cells may contribute to CD pathogenesis at effector and inductive sites and can be targeted therapeutically by a CD47 fusion protein.