We examined the sera of six patients before and after i.v. infusions of autologous chronic lymphocytic leukemia (CLL) cells transduced ex vivo with an adenovirus encoding CD154 (Ad-CD154). Five patients made high-titer antibodies against adenovirus and three made IgG reactive with a leukemia-associated surface antigen, which we identified as ROR1. Anti-ROR1 antibodies were not detected in the sera of untreated patients. We generated anti-ROR1 mAbs and found they reacted specifically with the CLL cells of all patients, but not with nonleukemic leukocytes, a wide variety of normal adult tissues, or blood mononuclear cells, including CD5 ؉ B cells of healthy adults. ROR1 could bind Wnt5a, which induced activation of NF-B when coexpressed with ROR1 in HEK293 cells and enhanced the survival of CLL cells in vitro, an effect that could be neutralized by posttreatment anti-ROR1 antisera. We conclude that patients with CLL can break immune tolerance to ROR1, which is an oncofetal surface antigen and survival-signaling receptor in this neoplastic disease.chronic lymphocytic leukemia ͉ neoplasia P atients with chronic lymphocytic leukemia (CLL) typically develop hypogammmaglobulinemia and progressive immune deficiency, which impairs their immune response to vaccines (1-3). Implicated in the abnormal immune function are immunesuppressive factors (4, 5) and an acquired functional deficiency of the CD40 ligand (CD154) (6). Furthermore, CLL cells are particularly inept at antigen presentation, which appears in part secondary to inadequate leukemia cell expression of immune costimulatory and adhesion molecules (7-9).Activation of CLL cells via CD40 ligation can reverse this immune-suppressive phenotype (7,8,10). Although CLL cells express class I and II major histocompatibility complex antigens, CD54 (ICAM-1), CD27, and CD40, these cells have low-to-absent expression of important immune costimulatory molecules, such as CD80, and cannot stimulate even allogeneic T cells in mixed lymphocyte reactions. However, upon interaction with cells that express CD154, CLL cells are induced to express immune costimulatory molecules, allowing them to become effective antigenpresenting cells (7). Similarly, CLL cells transduced with an adenovirus encoding CD154 (Ad-CD154) can induce CD40 activation of both transduced and nontransduced CLL cells, which then can stimulate autologous, leukemia-reactive T cells both in vitro (8) and in vivo (10-13).Conceivably, Ad-CD154-transduced CLL cells also could induce an antibody response against CLL-associated antigens. To test for this, we examined the sera of six CLL patients before and after five biweekly infusions of autologous, Ad-CD154-transduced CLL cells. ResultsBefore the infusions of autologous, Ad-CD154-transduced CLLcells, the six patients had CLL-associated hypogammaglobulinemia with median serum levels of IgM, IgA, or IgG of 28 Ϯ 21 mg/dl (SD), 53 Ϯ 63 mg/dl, or 600 Ϯ 297 mg/dl, respectively. However, 2 weeks after the final infusion, the median serum levels of IgM and IgG had increased to 77...

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.

Therapeutic antibodies that target T-cell co-inhibitory molecules display potent antitumor effects in multiple types of cancer. LSECtin is a cell surface lectin of the DC-SIGN family expressed in dendritic cells that inhibits T-cell responses. LSECtin limits T-cell activity in infectious disease, but it has not been studied in cancer. Here we report the finding that LSECtin is expressed commonly in melanomas where it blunts tumor-specific T-cell responses. When expressed in B16 melanoma cells, LSECtin promoted tumor growth, whereas its blockade slowed tumor growth in either wild-type or LSECtin-deficient mice. The tumor-promoting effects of LSECtin were abrogated in Rag1 À/À mice or in response to CD4 þ or CD8 þ T-cell depletion. Mechanistic investigations determined that LSECtin inhibited the proliferation of tumor-specific effector T cells by downregulating the cell cycle kinases CDK2, CDK4, and CDK6. Accordingly, as expressed in B16, tumor cells LSECtin inhibited tumorspecific T-cell responses relying upon proliferation in vitro and in vivo. Notably, LSECtin interacted with the coregulatory molecule LAG-3, the blockade of which restored IFNg secretion that was reduced by melanomaderived expression of LSECtin. Together, our findings reveal that common expression of LSECtin in melanoma cells engenders a mechanism of immune escape, with implications for novel immunotherapeutic combination strategies. Cancer Res; 74(13); 3418-28. Ó2014 AACR.

Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6C hi CX 3 CR1 lo monocytes/macrophages to pro-resolving Ly6C lo CX 3 CR1 hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2 −/− neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.