Citations

Abstract Cytotoxic T lymphocytes (CTL's) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. However, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, we sought to identify key factors associated with failure of PD-1 blockade in order to develop strategies that could significantly improve response rates. We performed single-cell RNA-seq analysis of peripheral CD8+ T cells from responding and non-responding patients and found that the non-responders had less NKG7 expression. Functional assays revealed that knock-down of NKG7 compromised cytolytic granule localization and trafficking within CTL's. Since decreasing NKG7 reduced the cytolytic efficiency of CD8+ T cells, we next hypothesized that by increasing expression of NKG7 we could promote better T cell killing of tumor and enhance the effects of immune checkpoint inhibitors. To test this, we utilized peripheral blood samples from melanoma patients who failed to respond to anti-PD-1 treatment and transfected the PBMC's with NKG7 mRNA. The addition of NKG7 mRNA significantly increased T cell killing of melanoma tumor cells in an in vitro cytotoxicity assay, and this effect was even more robust in combination with anti-PD-1 or anti-PD-L1 pre-treatment. To confirm that over-expression of NKG7 could improve CD8+ T cell killing in an antigen-specific manner, we transfected OT-1 transgenic T cells with murine sequence of NKG7 mRNA or control mRNA. OT-1 CD8+ T cells with increased NKG7 expression exhibited enhanced cytolytic ability when challenged with either EL4 cells pulsed with OVA peptide or B16-OVA tumor cells in vitro. In addition, NKG7 mRNA-transfected OT-1 CD8+ T cells suppressed the aggressive growth of B16-OVA cells in vivo significantly better than control OT-1 CD8+ T cells. These results indicate that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/L1 blockade and establish NKG7 as a new therapeutic target for enhancing response to cancer immunotherapy. Citation Format: Whitney J. Barham, Ti Wen, Ying Li, Henan Zhang, Cindy Liu, Jacob Hirdler, Joanina Gicobi, Fabrice Lucien-Matteoni, Yiyi Yan, Haidong Dong. NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 504.

Excitatory descending glutamatergic (Glu) synaptic input to phrenic motor neurons (PhMNs) emanates from the ventrolateral medulla and is responsible for inspiratory drive and a range of higher force behaviors. Disruption of this excitatory input following unilateral C2 hemisection (C2SH) results in cessation of inspiratory‐related diaphragm muscle (DIAm) activity ipsilateral to injury, but DIAm activity during higher force behaviors persists, reflecting the contribution of spared contralateral inputs. In a model of PhMN (DIAm motor unit) recruitment, we proposed that smaller PhMNs innervating more fatigue resistant DIAm motor units are recruited to accomplish ventilatory behaviors, whereas larger PhMNs innervating more fatigable motor units are recruited to accomplish more forceful expulsive and straining behaviors. We hypothesized that differences in the distribution of ipsilateral and contralateral Glu synaptic inputs to PhMNs contribute to the effects of C2SH on the range of motor behaviors accomplished by the DIAm. Accordingly, we hypothesized that C2SH primarily disrupts ipsilateral Glu synaptic inputs to smaller PhMNs, whereas Glu synaptic inputs to larger PhMNs are minimally impacted after C2SH. To address this hypothesis, we examined changes in Glu presynaptic input onto retrogradely labeled PhMNs using immunohistochemistry for VGLUT1 and VGLUT2. We found that following C2SH (by 7 days), there was a ~45% reduction in ipsilateral Glu synaptic input to PhMNs compared to a ~20% reduction contralaterally. In addition, ipsilateral to C2SH, Glu synaptic input to smaller PhMNs was reduced to a greater extent compared to that to larger PhMNs (55 vs. 35% reduction, respectively). Contralateral to C2SH, inputs to PhMNs were similarly reduced independent of motor neuron size. These results are consistent with more uniformly distributed contralateral inputs to all PhMNs and predominant input to smaller PhMNs ipsilaterally. These results also indicate that the source of Glu synaptic input to PhMNs varies depending on motor neuron size and likely reflects different motor control, including possibly distinct central pattern generator and premotor circuits. Support or Funding Information Grant Support: NIH Grant R01 HL96750 and Mayo Clinic Graduate School of Biomedical Sciences This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Brain derived neurotrophic factor (BDNF) acting through the high affinity tropomyosin‐related kinase receptor B (TrkB) plays an important role in synaptic plasticity and function at the neuromuscular junction (NMJ) as well as maintenance of neuromuscular transmission. Our previous studies showed that susceptibility to age‐related changes in diaphragm muscle (DIAm) NMJ morphology and denervation depends on trophic influences exerted by BDNF/TrkB signaling. Indeed, TrkB kinase activity is necessary to stabilize DIAm NMJ pre‐synaptic terminals in 18 month old mice (representing early old age; ~90% survival). However, by 24 months of age (~75% survival), BDNF/TrkB signaling has no effect on DIAm neuromuscular transmission, suggesting reduced TrkB kinase activity in the older age groups. We hypothesized that the NMJs that are resilient to old age do not depend on TrkB kinase activity for stabilization of pre‐synaptic terminals. Male and female 24 month old TrkB F616A mice were used to examine the structural properties of NMJs across various muscles with varying susceptibility to age‐related effects (DIAm, EDLm, and SOLm). Confocal microscopy was used to compare pre‐ and post‐synaptic morphology and denervation following a 7 day treatment with the phosphoprotein phosphatase‐1 derivative 1NMPP1, which inhibits TrkB kinase activity in TrkB F616A mice vs. vehicle treatment. Inhibition of TrkB kinase activity had no effect on pre‐synaptic or post‐synaptic terminal volume in DIAm, EDLm, and SOLm. Similarly, the overlap of pre‐ and post‐synaptic structures and the proportion of denervated NMJs did not change with 1NMPP1 treatment in all muscle types. The average percent of denervated NMJs was 8, 22, and 7% in DIAm, EDLm, and SOLm, respectively. Collectively these results suggest that the requirement of TrkB kinase activity for stabilization of NMJs in DIAm, EDLm and SOLm wanes in old age. Support or Funding Information Supported by R01‐AG057052. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

In skeletal muscles, force gradation depends on the level of motor unit (MU) activation. MU recruitment generally follows a specific order, with MUs comprising type I or type IIa fibers more active than type IIx and/or IIb. Direct evidence of MU recruitment across motor behaviors is limited to samples of relatively few MUs within a pool. The purpose of this study was to determine the extent of diaphragm MU recruitment across different motor behaviors. We hypothesized that in vivo labeling of individual neuromuscular junctions (NMJs) by FM4‐64 reflects the activation of MUs . Adult rats were exposed to eupneic (E), hypoxic/hypercarbic (H) conditions or supramaximal phrenic nerve stimulation (S). Individual diaphragm NMJs were type‐identified by myosin heavy chain expression. Average FM4‐64 labeling for NMJs at type I or IIa fibers vs. type IIx and/or IIb fibers increased progressively from E to H to S. The proportion of labeled NMJs of each type also increased significantly. At type I or IIa fibers, 40%, 59% and 90% of NMJs were labeled during E, H and S, respectively. At type IIx and/or IIb fibers, 21%, 32% and 82% of NMJs were labeled during E, H and S, respectively. Thus, using an in vivo labeling method there is direct evidence of progressive MU recruitment across different ventilatory and non‐ventilatory behaviors in the rat diaphragm. Supported by NIH grants HL37680 and AR51173, and the Mayo Clinic.

TAR DNA‐binding protein of 43 kDa (TDP‐43) is a nuclear protein involved in transcriptional repression and alternative splicing. It has been shown to be a component of ubiquitin‐positive, tau‐ and á‐synuclein‐negative inclusions in neuronal soma and nuclei as well as neurites of frontotemporal lobar degeneration (FTLD‐U) and in motor neurons of amyotrophic lateral sclerosis (ALS). Recently, it has been co‐localized with Lewy bodies in Lewy body disease (LBD). In this study we use immunogold electron microscopy to study the ultrastructural features of TDP‐43‐labeled structures. The results showed that in all FTLD‐U, ALS and LBD, TDP‐43 was localized to filaments coated with granular material. Both coated and uncoated regions of the filaments were labeled. In FTLD‐U a subset of neurites filled with densely packed filaments were also labeled. The filaments have a diameter of 10–17 nm. Neurofilament and glial filaments were not labeled by TDP‐43. The data suggests that TDP‐43 is a constituent protein in filamentous inclusions in neurodegenerative diseases. Supported by NIH grant AG17216.

Retrograde transport of fluorescent dyes permits imaging of nearly the entire pool of motoneurons when administered in the target muscle or axon shaft, but the extent of dendritic labeling is usually limited. We used an intracellular injection of Lucifer Yellow CH (LY) into retrogradely labeled phrenic motoneurons (PMNs) to enhance dendritic structure. PMNs were specifically labeled by Alexa Fluor 488‐conjugated cholera toxin fragment B (CTB) injected into the pleural space of 18‐days old rats. After a 72‐hr survival period, animals were transcardially perfused with 4% paraformaldehyde and 4% sucrose in 0.1 M phosphate buffer. Cervical spinal cords were sectioned in coronal plane. Individual CTB‐labeled PMNs were intracellularly injected with LY under direct visual control and subsequently imaged with an Olympus FluoView 200 laser‐scanning confocal system. Image processing and analyses were performed using Metamorph 7.4 software. PMNs were extensively labeled and reconstructed in 3D. The range of visible primary, secondary and tertiary processes was 3 ‐ 7, 2 ‐ 4 and 0 ‐ 2 per PMN, respectively. Average length of a single dendritic tree was 218.0±20.1 μm and surface area was 1295±199 μm 2 . The described technique provides 3D quantitative morphometry of a large sample of retrogradely‐labeled PMNs in great detail. Supported by NIH grants HL37680 and AR51173.

Unilateral denervation (DNV) of rat diaphragm muscle (DIAm) increases protein synthesis and degradation, with net protein breakdown by 14 days after DNV. We hypothesized that neural influences such as the nerve‐derived trophic factor neuregulin (NRG‐1) are essential in maintaining DIAm protein balance. NRG activates receptor tyrosine kinases of the ErbB family, with the ErbB2‐ErbB3 co‐receptor showing highest affinity for NRG‐1 and strong phosphoinositol‐3‐kinase (PI3K) activation. Both protein synthesis (via Akt phosphorylation) and degradation (via the transcription factor FoxO3A) are regulated by PI3K activity. We used Western blot analyses to assess NRG‐1, ErbB2, ErbB3 and FoxO3A protein expression at 1, 3, 7, and 14 days after DNV. By 1 day after DNV, NRG‐1 expression decreased, whereas ErbB2 and ErbB3 expression increased. Consistent with reduced NRG/ErbB signaling, association of the PI3K regulatory subunit p85 with ErbB3 decreased at 14 days after DNV. FoxO3A expression increased at 7 and 14 days after DNV, in agreement with delayed net protein breakdown. Importantly, DIAm inactivity induced by spinal hemisection had no effect on ErbB2, ErbB3, or FoxO3A expression. Taken together, these results indicate that DNV‐induced skeletal muscle adaptations result from removal of a trophic influence such as NRG‐1 rather than inactivity itself. Supported by NIH grant AR51173.

Muscle protein content reflects a balance between protein synthesis and degradation, which are likely regulated by neuromuscular activity and/or neurotrophin influence. In previous studies, we found that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) protein content after approximately 7 days. However, we did not determine whether the decrease in DIAm MHC protein content was the result of a decrease in protein synthesis, an increase in protein degradation, or both. We hypothesize that protein degradation plays a significant role in the decrease in DIAm protein content following DNV. In this study, we analyzed the ubiquitination state of cellular proteins as a marker of protein degradation. Conjugation of ubiquitin to cellular proteins targets proteins for degradation by proteosomes. We analyzed DIAm from adult male Sprague‐Dawley rats 3, 7 and 14 days after DNV. The DIAm was homogenized and protein ubiquitination was analyzed by Western analysis. No difference in ubiquitination was detected between control (CTL), sham surgery‐treated (sham), and 3‐day DNV animals. However, following 7 and 14 days of DNV, there was an approximate five‐fold increase in protein ubiquitination in DIAm compared to CTL and sham groups. These results demonstrate that DIAm protein ubiquitination increases between 3 and 7 days following DNV, and support the hypothesis that protein degradation contributes to the DNV‐induced decrease in DIAm MHC protein content. Supported by NIH grants AR51173 and HL37680.