Eric Tycksen

Mining Exomic Sequencing Data to Identify Mutated Antigens Recognized by Adoptively Transferred Tumor-reactive T cells

Substantial regressions of metastatic lesions have been observed in up to 70% of patients with melanoma who received adoptively transferred autologous tumor-infiltrating lymphocytes (TILs) in phase 2 clinical trials. In addition, 40% of patients treated in a recent trial experienced complete regressions of all measurable lesions for at least 5 years following TIL treatment. To evaluate the potential association between the ability of TILs to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, we developed a new screening approach involving mining whole-exome sequence data to identify mutated proteins expressed in patient tumors. We then synthesized and evaluated candidate mutated T cell epitopes that were identified using a major histocompatibility complex–binding algorithm for recognition by TILs. Using this approach, we identified mutated antigens expressed on autologous tumor cells that were recognized by three bulk TIL lines from three individuals with melanoma that were associated with objective tumor regressions following adoptive transfer. This simplified approach for identifying mutated antigens recognized by T cells avoids the need to generate and laboriously screen cDNA libraries from tumors and may represent a generally applicable method for identifying mutated antigens expressed in a variety of tumor types.

Zika virus has oncolytic activity against glioblastoma stem cells

Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients.

Changes in Transcriptome-Wide Gene Expression of Anterior Cruciate Ligament Tears Based on Time From Injury

Background: Anterior cruciate ligament (ACL) tears are a common injury. The healing potential of the injured ACL is poorly understood and is considered limited. Therefore, most ACL tears that are treated surgically undergo reconstruction rather than repair. However, there has been renewed interest recently in repairing ACL tears despite unanswered questions regarding the healing capacity of the ACL. Hypothesis: Gene expression in the injured ACL varies with time from injury. Study Design: Descriptive laboratory study. Methods: Transcriptome-wide expression profiles of 24 human ACL remnants recovered at the time of surgical reconstruction were analyzed using the Agilent human 8x60K microarray platform. Gene ontology was performed on differentially expressed transcripts based on time from injury (acute, <3 months; intermediate, 3-12 months; chronic, >12 months). A subset of transcripts with large fold changes in expression between any 2 categories was validated via microfluidic digital polymerase chain reaction. Results: Numerous transcripts representing important biological processes were differentially expressed by time from injury. The most significant changes were noted between the acute and chronic groups. Expression of several extracellular matrix genes— namely, POSTN, COL5A1, COL1A1, and COL12A1—was lower in the chronic tears compared with acute and intermediate tears. In acute tears, processes representing angiogenesis and stem cell differentiation were affected. In intermediate tears, processes representing stem cell proliferation concomitant with cellular component organization/cellular localization were altered. In ACL tears more than 12 months out from injury, processes denoting myosin filament organization, cellular component organization/cell localization, and extracellular matrix organization were affected. Conclusion: These findings are consistent with initial repair activity in the injured ACL, which declines with time from injury. Individual genes identified in this study, such as periostin, deserve further investigation into their role in tissue repair. Clinical Relevance: The decreased healing capacity of ACL tears over time is relevant to the development of effective techniques for repairing ACL tears and may have some significance for ACL reconstruction techniques as well. The potential for healing appears to be greatest in acute ACL tears, suggesting this window should be the focus of research for ACL repair.

Advantages of RNA-seq Compared to RNA Microarrays for Transcriptome Profiling of Anterior Cruciate Ligament Tears†

Microarrays and RNA‐seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA‐seq and microarrays for genome‐wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA‐seq and microarrays and to assess potential benefits of RNA‐seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time‐from‐injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA‐seq (N = 8). The correlation of biological replicates in RNA‐seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA‐seq data and the individual microarrays were low, but correlations between the RNA‐seq values and the geometric mean of the microarrays values were moderate. The cross‐platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA‐Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA‐seq and microarrays data. These findings demonstrate that RNA‐seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance.

Presence of meniscus tear alters gene expression profile of anterior cruciate ligament tears.

Anterior cruciate ligament (ACL) tears occur in isolation or in tandem with other intra‐articular injuries such as meniscus tears. The impact of injury pattern on the molecular biology of the injured ACL is unknown. Here, we tested the hypothesis that the biological response of the ACL to injury varies based on the presence or absence of concomitant meniscus tear. We performed RNA‐seq on 28 ACL tears remnants (12 isolated, 16 combined). In total, 16,654 transcripts were differentially expressed between isolated and combined injury groups at false discovery rate of 0.05. Due to the large number of differentially expressed transcripts, we undertook an Ensembl approach to discover features that acted as hub genes that did not necessarily have large fold changes or high statistical significance, but instead had high biological significance. Our data revealed a negatively correlated module containing 5,960 transcripts (down‐regulated in combined injury) and a positively correlated module containing 2,260 transcripts (up‐regulated in combined injury). TNS1, MEF2D, NOTCH3, SOGA1, and MLXIP were highly‐connected hub genes in the negatively correlated module and SCN2A, CSMD3, LRC44, USH2A, and LRP1B were critical hub genes in the positively correlated module. Transcripts in the negatively correlated module were associated with biological adhesion, actin‐filament organization, cell junction assembly, and cell matrix adhesion. The positively correlated module transcripts were enriched for neuron migration and exocytosis regulation. These findings indicate genes and pathways reflective of healing deficiency and gain of neurogenic signaling in combined ACL and meniscus tears, suggesting their diminished repair potential. The biological response of ACL to injury could have implications for healing potential of the ligament and the long term health of the knee.

PPARγ Deficiency Suppresses the Release of IL-1β and IL-1α in Macrophages via a Type 1 IFN–Dependent Mechanism

Obesity and diabetes modulate macrophage activation, often leading to prolonged inflammation and dysfunctional tissue repair. Increasing evidence suggests that the NLRP3 inflammasome plays an important role in obesity-associated inflammation. We have previously shown that activation of the lipotoxic inflammasome by excess fatty acids in macrophages occurs via a lysosome-dependent pathway. However, the mechanisms that link cellular lipid metabolism to altered inflammation remain poorly understood. PPARγ is a nuclear receptor transcription factor expressed by macrophages that is known to alter lipid handling, mitochondrial function, and inflammatory cytokine expression. To undercover novel links between metabolic signaling and lipotoxic inflammasome activation, we investigated mouse primary macrophages deficient in PPARγ. Contrary to our expectation, PPARγ knockout (KO) macrophages released significantly less IL-1β and IL-1α in response to lipotoxic stimulation. The suppression occurred at the transcriptional level and was apparent for multiple activators of the NLRP3 inflammasome. RNA sequencing revealed upregulation of IFN-β in activated PPARγKO macrophages, and this was confirmed at the protein level. A blocking Ab against the type 1 IFNR restored the release of IL-1β to wild type levels in PPARγKO cells, confirming the mechanistic link between these events. Conversely, PPARγ activation with rosiglitazone selectively suppressed IFN-β expression in activated macrophages. Loss of PPARγ also resulted in diminished expression of genes involved in sterol biosynthesis, a pathway known to influence IFN production. Together, these findings demonstrate a cross-talk pathway that influences the interplay between metabolism and inflammation in macrophages.

The role of Twist1 in mutant huntingtin-induced transcriptional alterations and neurotoxicity

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the huntingtin protein (Htt). Transcriptional dysregulation is an early event in the course of HD progression and is thought to contribute to disease pathogenesis, but how mutant Htt causes transcriptional alterations and subsequent cell death in neurons is not well understood. RNA-Seq analysis revealed that expression of a mutant Htt fragment in primary cortical neurons leads to robust gene expression changes before neuronal death. Basic helix-loop-helix transcription factor Twist1, which is essential for embryogenesis and is normally expressed at low levels in mature neurons, was substantially up-regulated in mutant Htt–expressing neurons in culture and in the brains of HD mouse models. Knockdown of Twist1 by RNAi in mutant Htt–expressing primary cortical neurons reversed the altered expression of a subset of genes involved in neuronal function and, importantly, abrogated neurotoxicity. Using brain-derived neurotrophic factor (Bdnf), which is known to be involved in HD pathogenesis, as a model gene, we found that Twist1 knockdown could reverse mutant Htt–induced DNA hypermethylation at the Bdnf regulatory region and reactivate Bdnf expression. Together, these results suggest that Twist1 is an important upstream mediator of mutant Htt–induced neuronal death and may in part operate through epigenetic mechanisms.

MicroRNA-138 Inhibits Osteogenic Differentiation and Mineralization of Human Dedifferentiated Chondrocytes by Regulating RhoC and the Actin Cytoskeleton: MICRORNA-138 INHIBITS OSTEOGENESIS BY REGULATING RHOC AND ACTIN CYTOSKELETON

MicroRNAs (miRNAs) are known to play critical roles in many cellular processes including those regulating skeletal development and homeostasis. A previous study from our group identified differentially expressed miRNAs in the developing human growth plate. Among those more highly expressed in hypertrophic chondrocytes compared to progenitor chondrocytes was miR‐138, therefore suggesting a possible role for this miRNA in regulating chondrogenesis and/or endochondral ossification. The goal of this study was to determine the function of miR‐ 138 in regulating osteogenesis by using human osteoarthritic dedifferentiated chondrocytes (DDCs) as source of inducible cells. We show that over‐expression of miR‐138 inhibited osteogenic differentiation of DDCs in vitro. Moreover, cell shape was altered and cell proliferation and possibly migration was also suppressed by miR‐138. Given alterations in cell shape, closer analysis revealed that F‐actin polymerization was also inhibited by miR‐138. Computational approaches showed that the small GTPase, RhoC, is a potential miR‐138 target gene. We pursued RhoC further given its function in regulating cell proliferation and migration in cancer cells. Indeed, miR‐138 over‐expression in DDCs resulted in decreased RhoC protein levels. A series of rescue experiments showed that RhoC over‐expression could attenuate the inhibitory actions of miR‐138 on DDC proliferation, F‐actin polymerization and osteogenic differentiation. Bone formation was also found to be enhanced within human demineralized bone scaffolds seeded with DDCs expressing both miR‐138 and RhoC. In conclusion, we have discovered a new mechanism in DDCs whereby miR‐138 functions to suppress RhoC which subsequently inhibits proliferation, F‐actin polymerization and osteogenic differentiation. To date, there are no published reports on the importance of RhoC in regulating osteogenesis. This opens up new avenues of research involving miR‐138 and RhoC pathways to better understand mechanisms regulating bone formation in addition to the potential use of DDCs as a cell source for bone tissue engineering.

Erratum: Zika virus has oncolytic activity against glioblastoma stem cells [The Journal of Experimental Medicine, 214, 10, (2017)] DOI: 10.1084/jem.20171093

Correction: Zika virus has oncolytic activity against glioblastoma stem cells Zhe Zhu, Matthew J. Gorman, Lisa D. McKenzie, Jiani N. Chai, Christopher G. Hubert, Briana C. Prager, Estefania Fernandez, Justin M. Richner, Rong Zhang, Chao Shan, Eric Tycksen, Xiuxing Wang, Pei-Yong Shi, Michael S. Diamond, Jeremy N. Rich, and Milan G. Chheda Vol. 214, No. 10, October 2017. https://doi.org/10.1084/jem.20171093

Human primary macrophages derived in vitro from circulating monocytes comprise adherent and non-adherent subsets with differential expression of Siglec-1 and CD4 and permissiveness to HIV-1 infection

Macrophages are a major target for human immunodeficiency virus type 1 (HIV-1) infection. However, macrophages are largely heterogeneous and may exhibit differences in permissiveness to HIV-1 infection. This study highlights the interplay of macrophage heterogeneity in HIV-1 pathogenesis. We show that monocyte-derived macrophages (MDMs) could be divided into two distinct subsets: CD14+Siglec-1hiCD4+ (non-adherent MDM) and CD14+Siglec-1LoCD4− (adherent MDM). The CD14+Siglec-1hiCD4+MDM subset represented the smaller proportion in the macrophage pool, and varied among different donors. Fractionation and subsequent exposure of the two MDM subsets to HIV-1 revealed opposite outcomes in terms of HIV-1 capture and infection. Although the CD14+Siglec-1hiCD4+MDM captured significantly more HIV-1, infection was significantly higher in the CD14+Siglec-1LoCD4−MDM subset. Thus, CD14+Siglec-1hiCD4+MDM were less permissive to infection. Depletion of CD14+Siglec-1hiCD4+MDM or a decrease in their percentage, resulted in increased infection of MDM, suggestive of a capacity of these cells to capture and sequester HIV-1 in an environment that hinders its infectivity. Increased expression of innate restriction factors and cytokine genes were observed in the non-adherent CD14+Siglec-1hiCD4+MDM, both before and after HIV-1 infection, compared to the adherent CD14+Siglec-1LoCD4−MDM. We speculate that the differential expression of gene expression profiles in the two macrophage subsets may provide an explanation for the differences observed in HIV-1 infectivity.