Elsevier

Clinical Immunology

Volume 149, Issue 1, October 2013, Pages 55-64
Clinical Immunology

Medulloblastoma expresses CD1d and can be targeted for immunotherapy with NKT cells

https://doi.org/10.1016/j.clim.2013.06.005Get rights and content

Highlights

  • Tumor cells in a subset of patients with medulloblastoma express CD1d.

  • CD1d is expression is higher in Shh molecular subgroup compared with Group 4.

  • CD1d-positive medulloblastoma cells cross-present glycolipids to NKT cells.

  • Treatment with NKT cells results in regression of intracranial tumors in mice.

  • NKT cells are preserved in peripheral blood of children with medulloblastoma.

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Current therapies are toxic and not always curative that necessitates development of targeted immunotherapy. However, little is known about immunobiology of this tumor. In this study, we show that MB cells in 9 of 20 primary tumors express CD1d, an antigen-presenting molecule for Natural Killer T cells (NKTs). Quantitative RT-PCR analysis of 61 primary tumors revealed an elevated level of CD1d mRNA expression in a molecular subgroup characterized by an overactivation of Sonic Hedgehog (SHH) oncogene compared with Group 4. CD1d-positive MB cells cross-presented glycolipid antigens to activate NKT-cell cytotoxicity. Intracranial injection of NKTs resulted in regression of orthotopic MB xenografts in NOD/SCID mice. Importantly, the numbers and function of peripheral blood type-I NKTs were preserved in MB patients. Therefore, CD1d is expressed on tumor cells in a subset of MB patients and represents a novel target for immunotherapy.

Introduction

Medulloblastoma (MB) originates from neuronal precursors in the cerebellum and is the most common malignant brain tumor of childhood. Despite overall improvement in MB outcome in the last 30 years due to advancement in surgical techniques, radiotherapy and chemotherapy [1], many survivors suffer from debilitating long-term therapeutic toxicity, especially cognitive and endocrinal impairment caused by craniospinal irradiation at young age [2], [3]. New targeted therapies are necessary to improve outcome and reduce treatment-related morbidities in children with MB.

MB has been historically classified based on clinical markers (patient age, presence of metastases at diagnosis, extent of resection) and histopathological characteristics (classic, desmoplastic/nodular, and large cell/anaplastic) [4]. However, recent advances in gene expression profiling of large number of tumors from multiple studies have provided evidence for four MB molecular subgroups (Wnt, SHH, Group 3, and Group 4) that are associated with prognosis and provide targets for therapeutic intervention [5], [6], [7], [8]. The emerging evidence including two recent reports on MB exome sequencing [9], [10] reveals further heterogeneity within these four molecular subgroups among which are genetic alterations in known oncogenic pathways that can be targeted for therapy. However, the relationship between the new molecular classification and the immunobiology of MB has not been addressed.

The identification of antigens that are selectively expressed in MB cells could lead to the development of effective immunotherapy without major side effects. A few studies examined the expression of tumor-associated antigens in MB cells such as IL13Ralpha2 or HER2 that can be targeted for immunotherapy with therapeutic antibodies or T cells [11], [12]. However, MB has not been evaluated as a potential target for immunotherapy with Vα24-invariant (type-I) Natural Killer T (NKT) cells [13], which have potent anti-tumor properties [14] and have been associated with good outcome in several types of cancer both in children and adults [15].

Type-I NKT cells are an evolutionary conserved sub-lineage of T cells that are characterized by the expression of an invariant TCR α-chain, Vα24-Jα18 and reactivity to self- and microbial-derived glycolipids presented by monomorphic HLA class-I-like molecule CD1d [13]. NKT cell cytotoxicity is CD1d-restricted although NKT cells have been shown to suppress growth or metastases of CD1d-negative tumors indirectly via the activation of NK cells or killing of tumor-associated macrophages [15]. There are also type-II NKT cells that express a diverse TCR repertoire and react to other CD1d-bound glycolipids such as sulfatide [13], [16]. In this study we investigate only type-I NKT cells for potential immunotherapy applications.

CD1d is preferentially expressed in hematopoietic cells, especially those of myelomonocytic and B-cell lineages and malignancies originating from the corresponding tissues often express CD1d [17], [18], [19]. Although the majority of non-hematopoietic solid tumors are CD1d-negative, CD1d expression by tumor cells has been reported in malignant glioma and prostate cancer [20], [21]. However, neither CD1d expression nor the susceptibility to NKT-cell cytotoxicity has been examined in MB or any other pediatric brain tumors. In this study, we analyzed CD1d expression in MB cell lines and primary tumors. Our results demonstrate that CD1d is expressed on the tumor cell surface in a subset of primary MB tumors and transcriptional analysis revealed a preferential CD1d gene expression in SHH molecular subgroup compared with Group 4. Importantly, CD1d-positive MB cell lines were highly sensitive to direct NKT cell cytotoxicity, and intracranial injection of human NKT cells resulted in regression of established orthotropic human NB xenografts in NOD/SCID mice. These findings may lead to the development of an effective NKT-cell based immunotherapy of MB.

Section snippets

Human specimens

PBMC or frozen tumor specimens from MB patients (mean age of 7.8, range 2–16 years old) were obtained at diagnosis at Texas Children's Cancer Center, Baylor College of Medicine or Children's Hospital Los Angeles, respectively, according to the institution's approved IRB protocols. Informed consent was obtained in accordance with institutional review board policies and procedures for research dealing with human specimens. PBMCs of healthy donors (at least 17 years old) were isolated by gradient

CD1d is expressed in human MB cell lines and primary tumors

To examine CD1d expression in MB cells, we performed flow cytometry analysis of four established human MB cell lines and found that two of them (DAOY and MED8A) expressed CD1d on the cell surface (Fig. 1A). RT-PCR analysis confirmed CD1d expression in both DAOY and MED8A cell lines while two CD1d-negative cell lines (D341 and D283) did not express CD1d at the mRNA level (Fig. 1B). The immunohistochemical staining with an anti-CD1d mAb detected CD1d expression in nine of the twenty primary MB

Discussion

Limited knowledge of the immunobiology of MB prevents development of targeted immunotherapies against this most common malignant brain tumor of childhood. The results of this study demonstrate for the first time that primary tumors in a subset of MB patients express CD1d, an antigen-presenting molecule for NKT cells. Importantly, CD1d-positive MB cells effectively cross-present exogenous and endogenous ligands to NKT cells that trigger potent NKT-cell cytotoxicity and IFNγ production. Moreover,

Conclusions

We demonstrate for the first time that CD1d, an antigen-presenting molecule for NKT cells is expressed on the surface of human MB cells in cell lines and primary tumor specimens. CD1d-positive MB cells effectively cross-present glycolipid antigens and can be killed by NKT cells in vitro and in vivo. These findings may lead to an effective immunotherapy of MB and other CD1d-positive brain tumors.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by grants from the National Institutes of Health (RO1 CA116548), Cancer Prevention and Research Institute of Texas (RP1 100528 and RP1 110129), The Caroline Wiess Law Scholar Award (LSM); American Brain Tumor Association and Alliance for Cancer Gene Therapy (NA); American Cancer Society, Alex's Lemonade Stand Foundation, and St. Baldrick's Foundation (SA).

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    Present address: Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, 6431 Fannin Street, Houston, TX 77030, USA.

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