To generate T cells throughout adult life, the thymus must import hemopoietic progenitors from the bone marrow via the blood. In this study, we establish that thymus settling is selective. Using nonirradiated recipient mice, we found that hemopoietic stem cells were excluded from the thymus, whereas downstream multipotent progenitors (MPP) and common lymphoid progenitors rapidly generated T cells following i.v. transfer. This cellular specificity correlated with the expression of the chemokine receptor CCR9 by a subset of MPP and common lymphoid progenitors but not hemopoietic stem cells. Furthermore, CCR9 expression was required for efficient thymus settling. Finally, we demonstrate that a prethymic signal through the cytokine receptor fms-like tyrosine kinase receptor-3 was required for the generation of CCR9-expressing early lymphoid progenitors, which were the most efficient progenitors of T cells within the MPP population. We conclude that fms-like tyrosine kinase receptor-3 signaling is required for the generation of T lineage-competent progenitors, which selectively express molecules, including CCR9, that allow them to settle within the thymus.

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b+CD14+ TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

Interactions with thymic stromal cells are known to be critical for the development of T cells from progenitors entering the thymus, yet the molecular mechanisms of stromal cell function remain poorly understood. Accumulating evidence has highlighted the importance of gcatenin-mediated activation of T cell factor (TCF)/lymphoid enhancer factor (LEF) transcription during thymocyte development. As regulation of this signaling pathway is controlled by binding of soluble Wnt proteins to cell surface Frizzled (Fz) receptors, we studied components of Wnt/Fz-mediated signaling in the context of stromal cell regulation of thymocyte development. We show that mRNA for a variety of Wnt family members, notably Wnt-4, Wnt7a and 7b, and Wnt-10a and 10b, are expressed by thymic epithelium rather then by thymocytes, while thymocytes demonstrate a developmentally regulated pattern of Fz receptor expression. Collectively these findings suggest (1) a functional role for Wnt-producing thymic epithelium in determining TCF/LEF-mediated transcriptional regulation in Fz-bearing thymocytes, and (2) a role for defined Wnt-Fz interactions at successive stages of thymocyte maturation. In support of this we show that separation of thymocytes from Wnt-producing epithelial cells and the thymic microenvironment, triggers g -catenin phosphorylation and degradation in thymocytes. Thus, sustained exposure to Wnt in the context of an intact stromal microenvironment is necessary for stabilization of g -catenin-mediated signaling in thymocytes.

PurposeTo evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs).MethodsRelative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys.ResultsIn human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1–5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing).ConclusionsIn adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.