InVivoMAb anti-mouse CD326 (EpCAM)

Extracellular cold-inducible RNA-binding protein (eCIRP) induces acute lung injury (ALI) in sepsis. Triggering receptor expressed on myeloid cells-1 (TREM-1) serves as a receptor for eCIRP to induce inflammation in macrophages and neutrophils. The effect of eCIRP on alveolar epithelial cells (AECs) remains unknown. We hypothesize that eCIRP induces inflammation in AECs through TREM-1. AECs were isolated from C57BL/6 mice and freshly isolated AECs were characterized as alveolar type II (ATII) cells by staining AECs with EpCAM, surfactant protein-C (SP-C), and T1 alpha (T1alpha) antibodies. AECs were stimulated with recombinant murine (rm) CIRP and assessed for TREM-1 by flow cytometry. ATII cells from WT and TREM-1(-/-) mice were stimulated with rmCIRP and assessed for interleukin-6 (IL-6) and chemokine (C-X-C motif) ligand 2 (CXCL2) in the culture supernatants. ATII cells from WT mice were pretreated with vehicle (PBS), M3 (TREM-1 antagonist), and LP17 (TREM-1 antagonist) and then after stimulating the cells with rmCIRP, IL-6 and CXCL2 levels in the culture supernatants were assessed. All of the freshly isolated AECs were ATII cells as they expressed EpCAM and SP-C, but not T1alpha (ATI cells marker). Treatment of ATII cells with rmCIRP significantly increased TREM-1 expression by 56% compared to PBS-treated ATII cells. Stimulation of WT ATII cells with rmCIRP increased IL-6 and CXCL2 expression, while the expression of IL-6 and CXCL2 in TREM-1(-/-) ATII cells were reduced by 14 and 23%, respectively. Pretreatment of ATII cells with M3 and LP17 significantly decreased the expression of IL-6 by 30 and 47%, respectively, and CXCL2 by 27 and 34%, respectively, compared to vehicle treated ATII cells after stimulation with rmCIRP. Thus, eCIRP induces inflammation in ATII cells via TREM-1 which implicates a novel pathophysiology of eCIRP-induced ALI and directs a possible therapeutic approach targeting eCIRP-TREM-1 interaction to attenuate ALI.

In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment.

Epithelial cell adhesion molecule (EpCAM) was reported to be cleaved into extracellular domain of EpCAM (EpEX) and intracellular domain of EpCAM (EpICD). We previously reported that EpCAM serves as a potent stem cell marker which is highly and selectively expressed by undifferentiated rather than differentiated hESC. However, the functional role of EpCAM remains elusive. Here, we found that EpEX and EpCAM enhance the efficiency of OSKM reprogramming. Interestingly, Oct4 or Klf4 alone, but not Sox2, can successfully reprogram fibroblasts into iPSCs with EpEX and EpCAM. Moreover, EpEX and EpCAM trigger reprogramming via activation of STAT3, which leads to the nuclear-translocation of HIF2alpha. This study reveals the importance of a novel EpEX/EpCAM-STAT3-HIF2alpha signal in the reprogramming process, and uncovers a new means of triggering reprogramming by delivery of soluble and transmembrane proteins.

Thymic epithelial cells (TECs) provide essential signals for alphabetaT-cell development, and medullary TECs (mTECs) control T-cell tolerance through both negative selection and Foxp3(+) regulatory T (Treg) cell development. Although heterogeneity within the mTEC compartment is well studied, the molecular regulators of specific stages of mTEC development are still poorly understood. Given the importance of the RANK-RANKL axis in thymus medulla formation, we have used RANK Venus reporter mice to analyze the ontogeny of RANK(+) TECs during development and correlated RANK expression with mTEC stem cells defined by SSEA-1. In addition, we have investigated how requirements for the key regulators Foxn1 and Relb map to specific stages of mTEC development. Here, we show SSEA-1(+) mTEC stem cells emerge prior to RANK expression and are present in both nude and Relb(-/-) mice, providing direct evidence that mTEC lineage specification occurs independently of Foxn1 and Relb. In contrast, we show that Relb is necessary for the effective production of downstream RANK(+) mTEC progenitors. Collectively, our work defines stage-specific requirements for critical TEC regulators during medulla development, including the timing of Relb dependency, and provides new information on mechanisms controlling mTEC specification.