Publications by authors named "Adam J Litterman"

10 Publications

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RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

Cell Rep 2019 09;28(11):2795-2806.e3

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.
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http://dx.doi.org/10.1016/j.celrep.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752737PMC
September 2019

miR-15/16 Restrain Memory T Cell Differentiation, Cell Cycle, and Survival.

Cell Rep 2019 08;28(8):2169-2181.e4

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Sandler Asthma Basic Research Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Coordinate control of T cell proliferation, survival, and differentiation are essential for host protection from pathogens and cancer. Long-lived memory cells, whose precursors are formed during the initial immunological insult, provide protection from future encounters, and their generation is the goal of many vaccination strategies. microRNAs (miRNAs) are key nodes in regulatory networks that shape effective T cell responses through the fine-tuning of thousands of genes. Here, using compound conditional mutant mice to eliminate miR-15/16 family miRNAs in T cells, we show that miR-15/16 restrict T cell cycle, survival, and memory T cell differentiation. High throughput sequencing of RNA isolated by cross-linking immunoprecipitation of AGO2 combined with gene expression analysis in miR-15/16-deficient T cells indicates that these effects are mediated through the direct inhibition of an extensive network of target genes within pathways critical to cell cycle, survival, and memory.
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http://dx.doi.org/10.1016/j.celrep.2019.07.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715152PMC
August 2019

A massively parallel 3' UTR reporter assay reveals relationships between nucleotide content, sequence conservation, and mRNA destabilization.

Genome Res 2019 06 31;29(6):896-906. Epub 2019 May 31.

Department of Microbiology and Immunology and Sandler Asthma Basic Research Center, University of California San Francisco, San Francisco, California 94143, USA.

Compared to coding sequences, untranslated regions of the transcriptome are not well conserved, and functional annotation of these sequences is challenging. Global relationships between nucleotide composition of 3' UTR sequences and their sequence conservation have been appreciated since mammalian genomes were first sequenced, but the functional relevance of these patterns remain unknown. We systematically measured the effect on gene expression of the sequences of more than 25,000 RNA-binding protein (RBP) binding sites in primary mouse T cells using a massively parallel reporter assay. GC-rich sequences were destabilizing of reporter mRNAs and come from more rapidly evolving regions of the genome. These sequences were more likely to be folded in vivo and contain a number of structural motifs that reduced accumulation of a heterologous reporter protein. Comparison of full-length 3' UTR sequences across vertebrate phylogeny revealed that strictly conserved 3' UTRs were GC-poor and enriched in genes associated with organismal development. In contrast, rapidly evolving 3' UTRs tended to be GC-rich and derived from genes involved in metabolism and immune responses. Cell-essential genes had lower GC content in their 3' UTRs, suggesting a connection between unstructured mRNA noncoding sequences and optimal protein production. By reducing gene expression, GC-rich RBP-occupied sequences act as a rapidly evolving substrate for gene regulatory interactions.
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http://dx.doi.org/10.1101/gr.242552.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581050PMC
June 2019

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses.

PLoS One 2015 1;10(5):e0125565. Epub 2015 May 1.

Department of Immunology, Mayo Clinic, Rochester, MN, United States of America; Department of Neurology, Mayo Clinic, Rochester, MN, United States of America.

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125565PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416934PMC
February 2016

Antigen-specific culture of memory-like CD8 T cells for adoptive immunotherapy.

Cancer Immunol Res 2014 Sep 22;2(9):839-45. Epub 2014 May 22.

Masonic Cancer Center, Brain Tumor Program, Departments of Pediatrics, Genetics, Cell Biology and Development, and Center for Genome Engineering, and

Cytotoxic T cells typically are expanded ex vivo in culture with IL2 for adoptive immunotherapy. This culture period leads to a differentiated phenotype and acquisition of effector function, as well as a loss of in vivo proliferative capability and antitumor efficacy. Here, we report antigen-specific and polyclonal expansion of cytotoxic T cells in a cocktail of cytokines and small molecules that leads to a memory-like phenotype in mouse and human cells even during extended culture, leading to enhanced in vivo expansion and tumor control in mice.
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http://dx.doi.org/10.1158/2326-6066.CIR-14-0038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156517PMC
September 2014

Alkylating chemotherapy may exert a uniquely deleterious effect upon neo-antigen-targeting anticancer vaccination.

Oncoimmunology 2013 Oct 15;2(10):e26294. Epub 2013 Oct 15.

Masonic Cancer Center; University of Minnesota; Minneapolis; MN USA ; Brain Tumor Program; University of Minnesota; Minneapolis, MN USA ; Department of Pediatrics; University of Minnesota; Minneapolis, MN USA.

Alkylating chemotherapy exerts both antineoplastic and immunostimulatory effects. However, in addition to depleting regulatory T cells (Treg), alkylating agents also mediate a long lasting antiproliferative effect on responder lymphocytes. Our recent findings indicate that this antiproliferative effect profoundly impairs vaccination-induced immune responses, especially in the case of vaccines that target specific tumor-associated neo-antigens that do not require Treg depletion.
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http://dx.doi.org/10.4161/onci.26294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827073PMC
October 2013

Minicircle DNA is superior to plasmid DNA in eliciting antigen-specific CD8+ T-cell responses.

Mol Ther 2013 Aug 21;21(8):1526-35. Epub 2013 May 21.

Department of Laboratory Medicine and Pathology, Center for Immunology and Masonic Cancer Center, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA.

Clinical trials reveal that plasmid DNA (pDNA)-based gene delivery must be improved to realize its potential to treat human disease. Current pDNA platforms suffer from brief transgene expression, primarily due to the spread of transcriptionally repressive chromatin initially deposited on plasmid bacterial backbone sequences. Minicircle (MC) DNA lacks plasmid backbone sequences and correspondingly confers higher levels of sustained transgene expression upon delivery, accounting for its success in preclinical gene therapy models. In this study, we show for the first time that MC DNA also functions as a vaccine platform. We used a luciferase reporter transgene to demonstrate that intradermal delivery of MC DNA, relative to pDNA, resulted in significantly higher and persistent levels of luciferase expression in mouse skin. Next, we immunized mice intradermally with DNA encoding a peptide that, when presented by the appropriate major histocompatibility complex class I molecule, was recognized by endogenous CD8(+) T cells. Finally, immunization with peptide-encoding MC DNA, but not the corresponding full-length (FL) pDNA, conferred significant protection in mice challenged with Listeria monocytogenes expressing the model peptide. Together, our results suggest intradermal delivery of MC DNA may prove more efficacious for prophylaxis than traditional pDNA vaccines.
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http://dx.doi.org/10.1038/mt.2013.85DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734653PMC
August 2013

Profound impairment of adaptive immune responses by alkylating chemotherapy.

J Immunol 2013 Jun 17;190(12):6259-68. Epub 2013 May 17.

Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.

Overall, cancer vaccines have had a record of failure as an adjuvant therapy for malignancies that are treated with alkylating chemotherapy, and the contribution of standard treatment to that failure remains unclear. Vaccines aim to harness the proliferative potential of the immune system by expanding a small number of tumor-specific lymphocytes into a large number of antitumor effectors. Clinical trials are often conducted after treatment with alkylating chemotherapy, given either as standard therapy or for immunomodulatory effect. There is mounting evidence for synergy between chemotherapy and adoptive immunotherapy or vaccination against self-Ags; however, the impact of chemotherapy on lymphocytes primed against tumor neoantigens remains poorly defined. We report that clinically relevant dosages of standard alkylating chemotherapies, such as temozolomide and cyclophosphamide, significantly inhibit the proliferative abilities of lymphocytes in mice. This proliferative impairment was long-lasting and led to quantitative and qualitative defects in B and T cell responses to neoantigen vaccines. High-affinity responder lymphocytes receiving the strongest proliferative signals from vaccines experienced the greatest DNA damage responses, skewing the response toward lower-affinity responders with inferior functional characteristics. Together, these defects lead to inferior efficacy and overall survival in murine tumor models treated by neoantigen vaccines. These results suggest that clinical protocols for cancer vaccines should be designed to avoid exposing responder lymphocytes to alkylating chemotherapy.
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http://dx.doi.org/10.4049/jimmunol.1203539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680135PMC
June 2013

Vaccine injection site matters: qualitative and quantitative defects in CD8 T cells primed as a function of proximity to the tumor in a murine glioma model.

J Immunol 2013 Jan 17;190(2):613-20. Epub 2012 Dec 17.

Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.

Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with tumor-associated Ags efficiently primes T cells to clear gliomas. In clinical trials, cancer vaccines have been less effective at priming T cells and extending survival. Generalized immune suppression in the tumor draining lymph nodes has been documented in multiple cancers. However, a systematic analysis of how vaccination at various distances from the tumor (closest to farthest) has not been reported. We investigated how the injection site chosen for vaccination dictates CD8 T cell priming and survival in an OVA-transfected murine glioma model. Glioma-bearing mice were vaccinated with Poly:ICLC plus OVA protein in the neck, hind leg, or foreleg for drainage into the cervical, inguinal, or axillary lymph nodes, respectively. OVA-specific CD8 T cell number, TCR affinity, effector function, and infiltration into the brain decreased as the vaccination site approached the tumor. These effects were dependent on the presence of the tumor, because injection site did not appreciably affect CD8 T cell priming in tumor-free mice. Our data suggest the site of vaccination can greatly impact the effectiveness of cancer vaccines. Considering that previous and ongoing clinical trials have used a variety of injection sites, vaccination site is potentially a critical aspect of study design that is being overlooked.
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http://dx.doi.org/10.4049/jimmunol.1201557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538956PMC
January 2013

Oxygen is a master regulator of the immunogenicity of primary human glioma cells.

Cancer Res 2011 Nov 9;71(21):6583-9. Epub 2011 Sep 9.

Department of Pediatrics, Graduate Program in Neuroscience, Biostatistics and Bioinformatics, University of Minnesota, Masonic Cancer Center, Minneapolis, Minnesota, USA.

With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (∼20% O₂) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (∼5% O₂) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O₂ more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O₂ took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O₂ or physiologic 5% O₂. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-1166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360979PMC
November 2011