Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Quantitative immunopeptidomics reveals a tumor stroma-specific target for T cell therapy.

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

A versatile system for rapid multiplex genome-edited CAR T cell generation.

The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.

Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition.

Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases.Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, ß-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Results: The CRISPR gene-edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene-edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells.Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255-66. ©2016 AACR.

A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors.

Chimeric antigen receptor (CAR)-modified adoptive T-cell therapy has been successfully applied to the treatment of hematologic malignancies, but faces many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced in both naturally occurring and genetically modified tumor-infiltrating lymphocytes (TIL) by inhibitory receptors (IR), namely PD1. We hypothesized that interfering with PD1 signaling would augment CAR T-cell activity against solid tumors. To address this possibility, we introduced a genetically engineered switch receptor construct, comprising the truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28, into CAR T cells. We tested the effect of this supplement, "PD1CD28," on human CAR T cells targeting aggressive models of human solid tumors expressing relevant tumor antigens. Treatment of mice bearing large, established solid tumors with PD1CD28 CAR T cells led to significant regression in tumor volume due to enhanced CAR TIL infiltrate, decreased susceptibility to tumor-induced hypofunction, and attenuation of IR expression compared with treatments with CAR T cells alone or PD1 antibodies. Taken together, our findings suggest that the application of PD1CD28 to boost CAR T-cell activity is efficacious against solid tumors via a variety of mechanisms, prompting clinical investigation of this potentially promising treatment modality.

Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice.

Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach.

Nature of tumor control by permanently and transiently modified GD2 chimeric antigen receptor T cells in xenograft models of neuroblastoma.

Chimeric antigen receptor (CAR) therapy has begun to demonstrate success as a novel treatment modality for hematologic malignancies. The success observed thus far has been with T cells permanently engineered to express chimeric receptors. T cells engineered using RNA electroporation represent an alternative with the potential for similar efficacy and greater safety when initially targeting novel antigens. Neuroblastoma is a common pediatric solid tumor with the potential to be targeted using immunotherapy. We performed xenograft studies in NSG mice in which we assessed the efficacy of both permanently modified and transiently modified CAR T cells directed against the neuroblastoma antigen GD2 in both local and disseminated disease models. Disease response was monitored by tumor volume measurement and histologic examination, as well as in vivo bioluminescence. RNA-modified GD2 CAR T cells mediated rapid tumor destruction when delivered locally. A single infusion of lentivirally modified GD2 CAR T cells resulted in long-term control of disseminated disease. Multiple infusions of RNA GD2 CAR T cells slowed the progression of disseminated disease and improved survival, but did not result in long-term disease control. Histologic examination revealed that the transiently modified cells were unable to significantly penetrate the tumor environment when delivered systemically, despite multiple infusions of CAR T cells. Thus, we demonstrate that RNA-modified GD2 CAR T cells can mediate effective antitumor responses in vivo, and permanently modified cells are able to control disseminated neuroblastoma in xenograft mice. Lack of long-term disease control by RNA-engineered cells resulted from an inability to penetrate the tumor microenvironment.

Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy.

BACKGROUND AIMS: Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in pre-clinical models, and this efficacy has translated to success in several clinical trials. Many early trials were disappointing in large part because of the lack of proliferation and subsequent persistence of transferred cells. Recent investigations have pointed to the importance of delivering highly proliferative cells, whether of naive or early memory phenotypes. METHODS: We investigated the influence of two common cell culturing methods used in early trials and their relationship to T-cell phenotype and pre-clinical efficacy. RESULTS: We observed that stimulation with soluble anti-CD3 antibody OKT-3 and high-dose interleukin-2 produces more effector memory-type T cells with shorter average telomeres when compared with cells generated with the use of CD3/CD28 beads. When used in xenograft models of leukemia, bead-stimulated cells proliferated earlier and to a higher degree than those generated with the use of OKT-3/IL2 and resulted in better disease control despite no difference in distribution or migration throughout the mouse. Inclusion of the known successful clinical 4-1BB endodomain in the CAR could not rescue the function of OKT-3/IL-2-cultured cells. T cells isolated from animals that survived long-term (>120 days) retained a central memory-like phenotype and demonstrated a memory response to a large re-challenge of CD19-positive leukemia. CONCLUSIONS: In summary, we confirm that cells with a younger phenotype or higher proliferative capacity perform better in pre-clinical models and that cell culturing influences cell phenotype seemingly independent of the 4-1BB endodomain in the CAR structure.

Regimen-specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia.

Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies have demonstrated activity in early phase clinical trials. While T cells bearing stably expressed CARs are efficacious and have potential long-term persistence, temporary expression of a CAR via RNA electroporation is also potentially efficacious in preclinical models. Temporary CAR expression using RNA presents a method of testing CARs clinically with additional safety where there may be concerns about possible chronic "on-target, off-tumor" toxic effects, as the degradation of RNA ensures complete removal of the CAR over time without relying on suicide induction systems. CD19-directed RNA CAR T cells were tested in vivo for efficacy and comparison to lentiviral vector (LV)-generated stable CAR T cells. We tested the hypothesis that multiple infusions of RNA CAR T cells preceded by lymphodepleting chemotherapy could mediate improved survival and sustained antitumor responses in a robust leukemia xenograft model. The saturation strategy using rationally designed multiple infusions of RNA CARs based on multiple model iterations approached the efficacy of a stable LV expression method. Two-color imaging revealed that relapse was a locoregional phenomenon in both the temporary and the stable expression models. In marked contrast to stably expressed CARs with retroviral or LV technology, the efficacy of RNA CARs appears independent of the costimulatory signaling endodomains likely because they more influence proliferation and persistence rather than short-term efficacy. The efficacy of the RNA CAR infusions may approach that of stably expressed CARs, offer theoretically safer initial clinical testing in addition to suicide systems, and allow for rapid and effective iterative preclinical modeling for the testing of new targets.

Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5.

Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation.

It is likely that the enhancement of signaling after antigenic stimulation, particularly in the tumor microenvironment, would improve the function of adoptively transferred T cells. Linker for activation of T cells (LAT) plays a central role in T cell activation. We hypothesized that the ubiquitylation-resistant form of LAT in cells would enhance T cell signaling and thus augment antitumor activity. To test this, human CD4(+) or CD8(+) T cells were electroporated with small interfering RNA (siRNA) to repress endogenous LAT and ubiquitylation-resistant LAT 2KR or wild-type LAT mRNA was introduced for reexpression. Significantly enhanced phosphorylation of LAT and phospholipase C-γ (PLCγ) was observed, and augmented calcium signaling after T cell receptor (TCR) triggering was observed in LAT 2KR-expressing T cells. TCR-induced calcium signaling was abrogated in LAT knockdown cells, but the baseline was higher than that of control siRNA-electroporated cells, suggesting a fundamental requirement of LAT to maintain calcium homeostasis. Redirected LAT 2KR T cells expressing a chimeric antigen receptor or an MHC class I-restricted TCR showed augmented function as assessed by enhanced cytokine secretion and cytotoxicity. These results indicate that interruption of LAT ubiquitylation is a promising strategy to augment effector T cell function for adoptive cell therapy.

The potent oncogene NPM-ALK mediates malignant transformation of normal human CD4(+) T lymphocytes.

With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

Treatment of advanced leukemia in mice with mRNA engineered T cells.

Cytotoxic T lymphocytes (CTLs) modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in preclinical models and are being tested in several clinical trials. Although CTLs bearing stably expressed CARs generated by integrating viral vectors are efficacious and have potential long-term persistence, this mechanism of CAR expression can potentially result in significant toxicity. T cells were electroporated with an optimized in vitro transcribed RNA encoding a CAR against CD19. These RNA CAR CTLs were then tested in vitro and in vivo for efficacy. We found that T cells expressing an anti-CD19 CAR introduced by electroporation with optimized mRNA were potent and specific killers of CD19 target cells. CD19 RNA CAR T cells given to immunodeficient mice bearing xenografted leukemia rapidly migrated to sites of disease and retained significant target-specific lytic activity. Unexpectedly, a single injection of CD19 RNA CAR T cells reduced disease burden within 1 day after administration, resulting in a significant prolongation of survival in an aggressive leukemia xenograft model. The surface expression of the RNA CARs may be titrated, giving T cells with potentially tunable levels of effector functions such as cytokine release and cytotoxicity. RNA CARs are a genetic engineering approach that should not be subject to genotoxicity, and they provide a platform for rapidly optimizing CAR design before proceeding to more costly and laborious stable expression systems.

Endothelial cells mediate the regeneration of hematopoietic stem cells.

Recent studies suggest that endothelial cells are a critical component of the normal hematopoietic microenvironment. Therefore, we sought to determine whether primary endothelial cells have the capacity to repair damaged hematopoietic stem cells. Highly purified populations of primary CD31(+) microvascular endothelial cells isolated from the brain or lung did not express the pan hematopoietic marker CD45, most hematopoietic lineage markers, or the progenitor marker c-kit and did not give rise to hematopoietic cells in vitro or in vivo. Remarkably, the transplantation of small numbers of these microvascular endothelial cells consistently restored hematopoiesis following bone marrow lethal doses of irradiation. Analysis of the peripheral blood of rescued recipients demonstrated that both short-term and long-term multilineage hematopoietic reconstitution was exclusively of host origin. Secondary transplantation studies revealed that microvascular endothelial cell-mediated hematopoietic regeneration also occurs at the level of the hematopoietic stem cell. These findings suggest a potential therapeutic role for microvascular endothelial cells in the self-renewal and repair of adult hematopoietic stem cells.

Hematopoietic stem cells contribute to lymphatic endothelium.

BACKGROUND: Although the lymphatic system arises as an extension of venous vessels in the embryo, little is known about the role of circulating progenitors in the maintenance or development of lymphatic endothelium. Here, we investigated whether hematopoietic stem cells (HSCs) have the potential to give rise to lymphatic endothelial cells (LEC). METHODOLOGY/PRINCIPAL FINDINGS: Following the transfer of marked HSCs into irradiated recipients, donor-derived LEC that co-express the lymphatic endothelial markers Lyve-1 and VEGFR-3 were identified in several tissues. HSC-derived LEC persisted for more than 12 months and contributed to approximately 3-4% of lymphatic vessels. Donor-derived LECs were not detected in mice transplanted with common myeloid progenitors and granulocyte/macrophage progenitors, suggesting that myeloid lineage commitment is not a requisite step in HSC contribution to lymphatic endothelium. Analysis of parabiotic mice revealed direct evidence for the existence of functional, circulating lymphatic progenitors in the absence of acute injury. Furthermore, the transplantation of HSCs into Apc(Min/+) mice resulted in the incorporation of donor-derived LEC into the lymphatic vessels of spontaneously arising intestinal tumors. CONCLUSIONS/SIGNIFICANCE: Our results indicate that HSCs can contribute to normal and tumor associated lymphatic endothelium. These findings suggest that the modification of HSCs may be a novel approach for targeting tumor metastasis and attenuating diseases of the lymphatic system.

Adoptive immunotherapy: good habits instilled at youth have long-term benefits.

Many recent advances in basic cell biology and immunology are a harbinger of progress in adoptive cell therapy (ACT) including (1) the finding that host lymphodepletion enhances engraftment and efficacy, (2) the recognition that in vitro T cell functions may not correlate with in vivo efficacy, and (3) the development of advanced ex vivo culture methods to expand lymphocytes to therapeutically effective numbers. In this article, we focus on the development of artificial antigen presenting cells (aAPCs) in our laboratory and their applicability to augment ACT protocols. We also describe how aAPCs can be used to broaden ACT to treat patients with a wide variety of cancers, chronic infectious diseases, and autoimmune manifestations.

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-18Ralpha 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.

Transvection mediated by the translocated cyclin D1 locus in mantle cell lymphoma.

In mantle cell lymphoma (MCL) and some cases of multiple myeloma (MM), cyclin D1 expression is deregulated by chromosome translocations involving the immunoglobulin heavy chain (IgH) locus. To evaluate the mechanisms responsible, gene targeting was used to study long-distance gene regulation. Remarkably, these targeted cell lines lost the translocated chromosome (t(11;14)). In these MCL and MM cells, the nonrearranged cyclin D1 (CCND1) locus reverts from CpG hypomethylated to hypermethylated. Reintroduction of the translocated chromosome induced a loss of methylation at the unrearranged CCND1 locus, providing evidence of a transallelic regulatory effect. In these cell lines and primary MCL patient samples, the CCND1 loci are packaged in chromatin-containing CCCTC binding factor (CTCF) and nucleophosmin (NPM) at the nucleolus. We show that CTCF and NPM are bound at the IgH 3' regulatory elements only in the t(11;14) MCL cell lines. Furthermore, NPM short hairpin RNA produces a specific growth arrest in these cells. Our data demonstrate transvection in human cancer and suggest a functional role for CTCF and NPM.