Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro-phage cytokine production and defective phagocytosis of apoptotic cells despite normal phagocytosis of other particles. We show here that c-mer–deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity appears to be driven by endogenous antigens, with little polyclonal B cell activation. These mice should be an excellent model for studying the role of apoptotic debris as an immunogenic stimulus for systemic autoimmunity.

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No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-c, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a c-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Purpose Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. EGFRvIII is a constitutively activated mutant of the naturally occurring epidermal growth factor receptor and is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues. Experimental Design We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immune-competent mouse model of malignant glioma. Results At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. Additionally, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Lastly, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIIINEG tumors, suggesting generation of host immunity against additional tumor antigens. Conclusion All together, these data support that third-generation, EGFRvIII specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immune-competent models in the preclinical evaluation of tumor immunotherapies.

Purpose Patients with glioblastoma (GBM) have a <15 month median survival despite surgical resection, high-dose radiation and chemotherapy with temozolomide (TMZ). We previously demonstrated that targeting Cytomegalovirus (CMV) pp65 using dendritic cells (DCs) can extend survival and, in a separate study, that dose-intensified (DI) TMZ and adjuvant GM-CSF potentiates tumor-specific immune responses in patients with GBM. Here, we evaluated pp65-specific cellular responses following DI-TMZ with pp65-DCs and determined the effects on long-term progression-free survival (PFS) and overall survival (OS). Experimental Design Following standard of care, 11 patients with newly diagnosed GBM received DI-TMZ (100 mg/m2/day × 21 days per cycle) with at least three vaccines of pp65-lysosome-associated membrane glycoprotein (LAMP) mRNA-pulsed DCs admixed with GM-CSF on Day 23 ± 1 of each cycle. Thereafter, monthly DI-TMZ cycles and pp65-DCs were continued if patients had not progressed. Results Following DI-TMZ cycle 1 and three doses of pp65-DCs, pp65 cellular responses significantly increased. After DI-TMZ, both the proportion and proliferation of regulatory T-cells (TRegs) increased and remained elevated with serial DI-TMZ cycles. Median PFS and OS were 25.3 months (CI95: 11.0-∞) and 41.1 months (CI95: 21.6-∞), exceeding survival using recursive partitioning analysis and matched historical controls. Four patients remained progression-free at 59 to 64 months from diagnosis. No known prognostic factors (age, KPS, IDH-1/2 mutation, and MGMT promoter methylation) predicted more favorable outcomes for the patients in this cohort. Conclusions Despite increased TReg proportions following DI-TMZ, patients receiving pp65-DCs showed long-term PFS and OS, confirming prior studies targeting CMV in GBM.

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The lpr gene encodes a defective form of Fas, a cell surface protein that mediates apoptosis. This defect blocks apoptotic deletion of autoreactive T and B cells, leading to lymphoproliferation and lupus-like autoantibody production. The effects of the lpr Fas mutation on other kinds of physiologically relevant apoptosis are largely undocumented. To assess whether some of the apoptosis known to occur after ionizing radiation might be mediated by Fas͞Fas ligand (FasL) interactions, we quantitated in vitro apoptosis by f low cytometry measurement of DNA content in splenic T and B cells from irradiated 5-to 8-month-old B6͞lpr mice. Total apoptosis of both lpr and control cells was substantial after treatment; however there was a significant difference between B6 (73%) and lpr (25%) lymphocyte apoptosis. Thy1, CD4, CD8, and IgM cells from lpr showed much lower levels of apoptosis than control cells after irradiation. Apoptosis induced by heat shock was also impaired in lpr. The finding that ␥-irradiation increased Fas expression on B6 cells and that irradiation-induced apoptosis could be blocked with a Fas-Fc fusion protein further supported the possible involvement of Fas in this form of apoptosis. Fas͞FasL interactions may thus play an important role in identifying and eliminating damaged cells after ␥-irradiation and other forms of injury.