Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use. METHODS: In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3. RESULTS: Where the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non-tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo. CONCLUSIONS: By combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.

Chronic ethanol consumption: role of TLR3/TRIF-dependent signaling.

Chronic ethanol consumption stimulates neuroimmune signaling in the brain, and Toll-like receptor (TLR) activation plays a key role in ethanol-induced inflammation. However, it is unknown which of the TLR signaling pathways, the myeloid differentiation primary response gene 88 (MyD88) dependent or the TIR-domain-containing adapter-inducing interferon-ß (TRIF) dependent, is activated in response to chronic ethanol. We used voluntary (every-other-day) chronic ethanol consumption in adult C57BL/6J mice and measured expression of TLRs and their signaling molecules immediately following consumption and 24 hours after removing alcohol. We focused on the prefrontal cortex where neuroimmune changes are the most robust and also investigated the nucleus accumbens and amygdala. Tlr mRNA and components of the TRIF-dependent pathway (mRNA and protein) were increased in the prefrontal cortex 24 hours after ethanol and Cxcl10 expression increased 0 hour after ethanol. Expression of Tlr3 and TRIF-related components increased in the nucleus accumbens, but slightly decreased in the amygdala. In addition, we demonstrate that the IKKε/TBK1 inhibitor Amlexanox decreases immune activation of TRIF-dependent pathway in the brain and reduces ethanol consumption, suggesting the TRIF-dependent pathway regulates drinking. Our results support the importance of TLR3 and the TRIF-dependent pathway in ethanol-induced neuroimmune signaling and suggest that this pathway could be a target in the treatment of alcohol use disorders.

Peroxisome Proliferator Activated Receptor Agonists Modulate Transposable Element Expression in Brain and Liver.

Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors that act as transcription factors in response to endogenous lipid messengers. The fibrates and thiazolidinediones are synthetic PPAR agonists used clinically to treat dyslipidemia and Type 2 Diabetes Mellitus, respectively, but also improve symptoms of several other diseases. Transposable elements (TEs), repetitive sequences in mammalian genomes, are implicated in many of the same conditions for which PPAR agonists are therapeutic, including neurodegeneration, schizophrenia, and drug addiction. We tested the hypothesis that there is a link between actions of PPAR agonists and TE expression. We developed an innovative application of microarray data by mapping Illumina mouse WG-6 microarray probes to areas of the mouse genome that contain TEs. Using this information, we assessed the effects of systemic administration of three PPAR agonists with different PPAR subtype selectivity: fenofibrate, tesaglitazar, and bezafibrate, on TE probe expression in mouse brain [prefrontal cortex (PFC) and amygdala] and liver. We found that fenofibrate, and bezafibrate to a lesser extent, up-regulated probes mapped to retrotransposons: Short-Interspersed Elements (SINEs) and Long-Interspersed Elements (LINEs), in the PFC. Conversely, all PPAR agonists down-regulated LINEs and tesaglitazar and bezafibrate also down-regulated SINEs in liver. We built gene coexpression networks that partitioned the diverse transcriptional response to PPAR agonists into groups of probes with highly correlated expression patterns (modules). Most of the differentially expressed retrotransposons were within the same module, suggesting coordinated regulation of their expression, possibly by PPAR signaling. One TE module was conserved across tissues and was enriched with genes whose products participate in epigenetic regulation, suggesting that PPAR agonists affect TE expression via epigenetic mechanisms. Other enriched functional categories included phenotypes related to embryonic development and learning and memory, suggesting functional links between these biological processes and TE expression. In summary, these findings suggest mechanistic relationships between retrotransposons and PPAR agonists and provide a basis for future exploration of their functional roles in brain and liver.

CNS cell-type localization and LPS response of TLR signaling pathways.

Background: Innate immune signaling in the brain has emerged as a contributor to many central nervous system (CNS) pathologies, including mood disorders, neurodegenerative disorders, neurodevelopmental disorders, and addiction. Toll-like receptors (TLRs), a key component of the innate immune response, are particularly implicated in neuroimmune dysfunction. However, most of our understanding about TLR signaling comes from the peripheral immune response, and it is becoming clear that the CNS immune response is unique. One controversial aspect of neuroimmune signaling is which CNS cell types are involved. While microglia are the CNS cell-type derived from a myeloid lineage, studies suggest that other glial cell types and even neurons express TLRs, although this idea is controversial. Furthermore, recent work suggests a discrepancy between RNA and protein expression within the CNS. Methods: To elucidate the CNS cell-type localization of TLRs and their downstream signaling molecules, we isolated microglia and astrocytes from the brain of adult mice treated with saline or the TLR4 ligand lipopolysaccharide (LPS). Glial mRNA and protein expression was compared to a cellular-admixture to determine cell-type enrichment. Results: Enrichment analysis revealed that most of the TLR pathway genes are localized in microglia and changed in microglia following immune challenge. However, expression of Tlr3 was enriched in astrocytes, where it increased in response to LPS. Furthermore, attempts to determine protein cell-type localization revealed that many antibodies are non-specific and that antibody differences are contributing to conflicting localization results. Conclusions: Together these results highlight the cell types that should be looked at when studying TLR signaling gene expression and suggest that non-antibody approaches need to be used to accurately evaluate protein expression.