Xiaochuan Shan

CD28 Costimulation Is Essential for Human T Regulatory Expansion and Function

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8–12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.

Immunosurveillance and Survivin-Specific T-Cell Immunity in Children With High-Risk Neuroblastoma

PURPOSE Tumor immunosurveillance influences oncogenesis and tumor growth, but it remains controversial whether clinical failure of immunosurveillance is a result of lymphocyte dysfunction or tumor escape. In this study, our goal was to characterize the physiology of tumor immunosurveillance in children with high-risk neuroblastoma (HR-NBL). PATIENTS AND METHODS Immunohistopathologic studies were carried out on 26 tumor samples from a cohort of HR-NBL patients diagnosed at Childrens Hospital of Philadelphia for the 2-year period from May 2003 to May 2005. Blood from nine HLA-A2+ patients in this cohort was analyzed for T cells specific for the antiapoptotic protein survivin. RESULTS Survivin protein was expressed by 26 of 26 tumors. In HLA-A2+ patients, circulating cytotoxic T lymphocytes (CTLs) specific for survivin were detected by peptide/major histocompatibility complex tetramer analysis in the blood of eight of nine children with HR-NBL at the time of diagnosis. Rather than being selectively rendered anergic in vivo, circulating survivin-specific CTLs were highly functional as shown by cytotoxicity and interferon gamma enzyme-linked immunospot assays in six of nine patients. Survivin-specific CD107a mobilization by T cells was found in five of five patients. By immunohistochemistry, tumor-infiltrating T cells were few or absent in 26 of 26 tumors. CONCLUSION Children with HR-NBL harbor robust cellular immune responses to the universal tumor antigen survivin at the time of diagnosis, but intratumoral T cells are strikingly rare, suggesting a failure of cellular immunosurveillance. Efforts to develop novel therapies that increase T-cell trafficking into tumor nests are warranted.

Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases.

Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells.

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8+ effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Rα was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.

Interferon-gamma dependent infiltration of human T cells into neuroblastoma tumors in vivo

Purpose: To investigate the impact of interferon-γ–mediated upregulation of major histocompatibility complex class I expression on tumor-specific T-cell cytotoxicity and T-cell trafficking into neuroblastoma tumors in vivo. Experimental Design: Restoration of major histocompatibility complex class I expression by interferon-γ treatment enhances killing of neuroblastoma cells. To understand the potential of this approach in vivo, we developed a novel model of neuroblastoma in which NOD/scid/IL2Rγnull immunodeficient mice are engrafted with both human T cells and tumor cells. Results: Here, we show enhanced killing of neuroblastoma cells by patient-derived, tumor-specific T cells in vitro. In addition, interferon-γ treatment in vivo induces efficient upregulation of major histocompatibility complex class I expression on neuroblastoma tumor cells, and this is accompanied by significantly enhanced infiltration of T cells into the tumor. In a pilot clinical trial in patients with high-risk neuroblastoma, we similarly observed augmented T-cell trafficking into neuroblastoma nests in tumor biopsy specimens obtained from patients after 5 days of systemic interferon-γ therapy. Conclusions: Interferon-γ overcomes critical obstacles to the killing of human neuroblastoma cells by specific T cells. Together, these findings provide a rationale for the further testing of interferon-γ as an approach for improving the efficacy of T cell–based therapies for neuroblastoma and other major histocompatibility complex class I–deficient malignancies. In addition, we describe a model that may expedite the preclinical screening of approaches aimed at augmenting T-cell trafficking into human tumors. (Clin Cancer Res 2009;15(21):6602–8)

MULTIPLE INITIAL CULTURE CONDITIONS ENHANCE THE ESTABLISHMENT OF CELL LINES FROM PRIMARY OVARIAN CANCER SPECIMENS

SummaryTo increase the efficiency of stable cell line establishment from primary ovarian cancer specimens, we simultaneously initiated cultures under, multiple conditions, varying extracellular matrices and the inclusion of supplements (e.g., serum or serum albumin), while minimizing exposure to xenogeneic antigens (e.g., fetal calf serum). Primary cultures were initiated from 30 specimens; cell lines were established from 10 of these for a success rate of 33%. In some instances, multiple cell lines were established from the same specimen. Five lines were characterized extensively with respect to growth properties, antigen expression, and genomic alterations. Although these lines are all low-passage, marked heterogeneity was observed, even between lines derived from the same specimen. The culture approach outlined herein will facilitate generation of reagents useful for many aspects of ovarian cancer biology.

Registered report: androgen receptor splice variants determine taxane sensitivity in prostate cancer

The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from “Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer” by Thadani-Mulero and colleagues (2014) published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR), and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

Registered report: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukemia.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from ‘Inhibition of bromodomain and extra terminal (BET) recruitment to chromatin as an effective treatment for mixed-lineage leukemia (MLL)-fusion leukemia’ by Dawson and colleagues, published in Nature in 2011 (Dawson et al., 2011). The experiments to be replicated are those reported in Figures 2A, 3D, 4B, 4D and Supplementary Figures 11A-B and 16A. In this study, BET proteins were demonstrated as potential therapeutic targets for modulating aberrant gene expression programs associated with MLL-fusion leukemia. In Figure 2A, the BET bromodomain inhibitor I-BET151 was reported to suppress growth of cells harboring MLL-fusions compared to those with alternate oncogenic drivers. In Figure 3D, treatment of MLL-fusion leukemia cells with I-BET151 resulted in transcriptional suppression of the anti-apoptotic gene BCL2. Figures 4B and 4D tested the therapeutic efficacy of I-BET151 in vivo using mice injected with human MLL-fusion leukemia cells and evaluated disease progression following I-BET151 treatment. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.08997.001

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells

Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.

Replication study: androgen receptor splice variants determine taxane sensitivity in prostate cancer

In 2015, as part of the Prostate Cancer Foundation–Movember Foundation Reproducibility Initiative, we published a Registered Report (Shan et al., 2015) that described how we intended to replicate selected experiments from the paper “Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer” (Thadani-Mulero et al., 2014). Here we report the results of those experiments. Growth of tumor xenografts from two prostate cancer xenograft lines, LuCaP 86.2, which expresses wild-type androgen receptor (AR) and AR variant 567, and LuCaP 23.1, which expresses wild-type AR and AR variant 7, were not affected by docetaxel treatment. The LuCaP 23.1 tumor xenografts grew slower than in the original study. This result is different from the original study, which reported significant reduction of tumor growth in the LuCaP 86.2. Furthermore, we were unable to detect ARv7 in the LuCaP 23.1, although we used the antibody as stated in the original study and ensured that it was detecting ARv7 via a known positive control (22rv1, Hörnberg et al., 2011). Finally, we report a meta-analysis of the result.