IMPORTANCE Glioblastoma is an incurable tumor, and the therapeutic options for patients are limited. OBJECTIVE To determine whether the systemic administration of HER2-specific chimeric antigen receptor (CAR)–modified virus-specific T cells (VSTs) is safe and whether these cells have antiglioblastoma activity. DESIGN, SETTING, AND PARTICIPANTS In this open-label phase 1 dose-escalation study conducted at Baylor College of Medicine, Houston Methodist Hospital, and Texas Children’s Hospital, patients with progressive HER2-positive glioblastoma were enrolled between July 25, 2011, and April 21, 2014. The duration of follow-up was 10 weeks to 29 months (median, 8 months). INTERVENTIONS Monotherapy with autologous VSTs specific for cytomegalovirus, Epstein-Barr virus, or adenovirus and genetically modified to express HER2-CARs with a CD28.ζ-signaling endodomain (HER2-CAR VSTs). MAIN OUTCOMES AND MEASURES Primary end points were feasibility and safety. The key secondary end points were T-cell persistence and their antiglioblastoma activity. RESULTS A total of 17 patients (8 females and 9 males; 10 patients ≥ 18 years [median age, 60 years; range, 30–69 years] and 7 patients <18 years [median age, 14 years; range, 10–17 years]) with progressive HER2-positive glioblastoma received 1 or more infusions of autologous HER2-CAR VSTs (1 × 106/m2 to 1 × 108/m2) without prior lymphodepletion. Infusions were well tolerated, with no dose-limiting toxic effects. HER2-CAR VSTs were detected in the peripheral blood for up to 12 months after the infusion by quantitative real-time polymerase chain reaction. Of 16 evaluable patients (9 adults and 7 children), 1 had a partial response for more than 9 months, 7 had stable disease for 8 weeks to 29 months, and 8 progressed after T-cell infusion. Three patients with stable disease are alive without any evidence of progression during 24 to 29 months of follow-up. For the entire study cohort, median overall survival was 11.1 months (95% CI, 4.1–27.2 months) from the first T-cell infusion and 24.5 months (95% CI, 17.2–34.6 months) from diagnosis. CONCLUSIONS AND RELEVANCE Infusion of autologous HER2-CAR VSTs is safe and can be associated with clinical benefit for patients with progressive glioblastoma. Further evaluation of HER2-CAR VSTs in a phase 2b study is warranted as a single agent or in combination with other immunomodulatory approaches for glioblastoma.

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Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.Enzymes of the thiol reductase family carry out reduction, oxidation, and isomerization of protein disulfide bonds in cytosol (for example, thioredoxin) (1, 2), mitochondria (3), endoplasmic reticulum (protein-disulfide isomerase) (2), and lysosomes (gamma interferon-inducible thiol reductase, GILT).2 The majority of these enzymes are functional at neutral or slightly alkaline conditions (4), they have similar three-dimensional structures, and all feature a conservative active site loop containing two cysteines in the sequence -CGPC-(5). GILT is a unique and unusual member of the thiol reductase family because its optimal enzymatic activity is at a low pH (4.5-5.5) (6 -8) and has an atypical active site (-CGAC-).GILT is synthesized as a 35-kDa soluble glycoprotein precursor and is transported to the endosomal compartment via the mannose-6-P receptor pathway (9). It is processed into the mature form (30 kDa) by proteolytic removal of N-and C-terminal peptides. The protein has an approximate molecular mass of 30 kDa and was therefore initially named IP-30 (6). In addition to endosomal/lysosomal localization, GILT is secreted in the tissue culture medium of the GILT-expressing cell lines (7,10), and is present in mouse sera.3 GILT is constitutively expressed in professional antigen-presenting cells (APCs), but it is also inducible by pro-inflammatory cytokines such as interferon ␥, tumor necrosis factor ␣, and interleukin 1␤ (9).Using GILT Ϫ/Ϫ mice as a model, we have shown that GILT catalyzes initial unfolding of antigenic protein (protein becomes more accessible for further processing by cathepsins) and therefore facilitates protein/peptide binding to MHC class II molecules (10). By changing the redox state of exogenous antigenic proteins with disulfide bonds, GILT initiates the adaptive immune response. However, we have shown that GILT is constitutively expressed in T cells and has a role in the regulation of T cell activation. This is so far the only known GILT function not related to MHC class II processing (11). GILT Ϫ/Ϫ T cells show increased proliferation and cytotoxic T cell activity in response to anti-CD3 stimulation. This observation suggests that GILT has a more fundamental role in cellular processes than just reduction of antigens...

Reactivity to self-peptide/MHC complexes is required for selection of the TCR repertoire in the thymus but can also promote autoimmunity. Reduced TCR sensitivity of mature T cells is thought to help control the autoreactivity in peripheral T cells. The molecular basis for reduced sensitivity of peripheral T cells is not known. We found that peripheral T cells, but not immature thymocytes, lacking IFN-γ-inducible lysosomal thiol reductase (GILT) display increased sensitivity to TCR ligation. GILT−/− peripheral T cells express reduced levels of mitochondrial superoxide dismutase 2 and consequently display higher levels of reactive oxygen radicals and ERK1/2 phosphorylation following activation. The increased sensitivity of GILT-deficient T cells results in a more severe hyperglycemia associated with streptozotocin-induced diabetes. GILT expression levels progressively increase in T cells with maturation. These data suggest that regulation of GILT expression may be a mechanism of T cell differentiation-associated changes in sensitivity to TCR engagement.

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4+CD25+) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.

BackgroundThymic involution is a prominent characteristic of an aging immune system. When thymic function is reduced/absent, the peripheral T cell pool is subject to the laws of peripheral T cell homeostasis that favor survival/expansion of T cell receptors with relatively higher functional avidity for self-peptide/MHC complexes. Due to difficulties in assessing the TCR avidity in polyclonal population of T cells, it is currently not known whether high avidity T cells preferentially survive in aging individuals, and what impact this might have on the function of the immune system and development of autoimmune diseases.ResultsThe phenotype of T cells from aged mice (18-24 months) indicating functional TCR avidity (CD3 and CD5 expression) correlates with the level of preserved thymic function. In mice with moderate thymic output (> 30% of peripheral CD62Lhi T cells), T cells displayed CD3lowCD5hi phenotype characteristic for high functional avidity. In old mice with drastically low numbers of CD62Lhi T cells reduced CD5 levels were found. After adult thymectomy, T cells of young mice developed CD3lowCD5hi phenotype, followed by a CD3lowCD5low phenotype. Spleens of old mice with the CD3low/CD5hi T cell phenotype displayed increased levels of IL-10 mRNA, and their T cells could be induced to secrete IL-10 in vitro. In contrast, downmodulation of CD5 was accompanied with reduced IL-10 expression and impaired anti-CD3 induced proliferation. Irrespective of the CD3/CD5 phenotype, reduced severity of experimental allergic myelitis occurred in old mice. In MTB TCRβ transgenic mice that display globally elevated TCR avidity for self peptide/MHC, identical change patterns occurred, only at an accelerated pace.ConclusionsThese findings suggest that age-associated dysfunctions of the immune system could in part be due to functional erosion of T cells devised to protect the hosts from the prolonged exposure to T cells with high-avidity for self.

During the early phase of atherosclerosis, T cells and monocytes attach to and migrate through the endothelium into the vessel wall. To provide an insight into the potential cross talk between T cells and smooth muscle cells (SMC) in atherogenesis, we investigated changes in gene expression caused by CD40 ligation in cultured vascular SMC and their consequences for monocyte activation. CD40 expression in human-cultured SMC was induced by 24-h treatment with tumor necrosis factor-alpha plus interferon-gamma followed by 12-h exposure to mouse myeloma cells stably expressing human CD154 or the corresponding control cells. DNA microarray analysis (Affymetrix HG-U952A chip) indicated 33 up-regulated genes in three individual experiments of which 19 encoded pro-inflammatory adhesion molecules, cytokines, chemokines, and receptors. One functional consequence of this change in gene expression was an activation of transformed human promonocytic-1 monocytes exposed to the conditioned medium of the stimulated SMC. Subsequent antibody neutralization experiments identified granulocyte-macrophage colony-stimulating factor (GM-CSF) as the SMC-derived cytokine responsible for this effect. Thus, vascular SMC-like endothelial cells appear to contribute to the maintenance of an inflammatory response in the atherosclerotic vessel wall upon CD40-CD154 co-stimulation. Among 19 up-regulated pro-inflammatory gene products, GM-CSF plays an important role in SMC-dependent monocyte activation.