CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape.

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma.

Cancer immunotherapy has made unprecedented breakthrough in the fields of chimeric antigen receptor-redirected T (CAR T) cell therapy and immune modulation. Combination of CAR modification and the disruption of endogenous inhibitory immune checkpoints on T cells represent a promising immunotherapeutic modality for cancer treatment. However, the potential for the treatment of hepatocellular carcinoma (HCC) has not been explored. In this study, the gene expressing the programmed death 1 receptor (PD-1) on the Glypican-3 (GPC3)-targeted second-generation CAR T cells employing CD28 as the co-stimulatory domain was disrupted using the CRISPR/Cas9 gene-editing system. It was found that, in vitro, the CAR T cells with the deficient PD-1 showed the stronger CAR-dependent anti-tumor activity against native programmed death 1 ligand 1-expressing HCC cell PLC/PRF/5 compared with the wild-type CAR T cells, and meanwhile, the CD4 and CD8 subsets, and activation status of CAR T cells were stable with the disruption of endogenous PD-1. Additionally, the disruption of PD-1 could protect the GPC3-CAR T cells from exhaustion when combating with native PD-L1-expressing HCC, as the levels of Akt phosphorylation and anti-apoptotic protein Bcl-xL expression in PD-1 deficient GPC3-CAR T cells were significantly higher than those in wild-type GPC3-CAR T cells after coculturing with PLC/PRF/5. Furthermore, the in vivo anti-tumor activity of the CAR T cells with the deficient PD-1 was investigated using the subcutaneous xenograft tumor model established by the injection of PLC/PRF/5 into NOD-scid-IL-2Rγ-/- (NSG) mice. The results indicated that the disruption of PD-1 enhanced the in vivo anti-tumor activity of CAR T cells against HCC, improved the persistence and infiltration of CAR T cells in the NSG mice bearing the tumor, and strengthened the inhibition of tumor-related genes expression in the xenograft tumors caused by the GPC3-CAR T cells. This study indicates the enhanced anti-tumor efficacy of PD-1-deficient CAR T cells against HCC and suggests the potential of precision gene editing on the immune checkpoints to enhance the CAR T cell therapies against HCC.

NprR-NprX Quorum-Sensing System Regulates the Algicidal Activity of Bacillus sp. Strain S51107 against Bloom-Forming Cyanobacterium Microcystis aeruginosa.

Harmful cyanobacterial blooms have severely impaired freshwater quality and threatened human health worldwide. Here, a Gram-positive bacterium, Bacillus sp. strain S51107, which exhibits strong algicidal activity against Microcystis aeruginosa, was isolated from Lake Taihu. We found that the algicidal activity of strain S51107 was regulated primarily by NprR-NprX quorum sensing (QS), in which the mature form of the signaling peptide NprX was identified as the SKPDIVG heptapeptide. Disruption of the nprR-nprX cassette markedly decreased the algicidal activity, and complemented strains showed significantly recovered algicidal activity. Strain S51107 produced low-molecular-weight algicidal compounds [indole-3-carboxaldehyde and cyclo(Pro-Phe)] and high-molecular-weight algicidal substance(s) (>3 kDa). Moreover, the production of high-molecular-weight algicidal substance(s) was regulated by NprR-NprX QS, but the production of low-molecular-weight algicidal compounds was not. High-molecular-weight algicidal substance(s) played a more important role than low-molecular-weight algicidal compounds in the algicidal activity of strain S51107. The results of this study could increase our knowledge about algicidal characteristics of a potential algicidal bacterium, Bacillus sp. strain S51107, and provide the first evidence that the algicidal activity of Gram-positive algicidal bacteria is regulated by QS, which will greatly enhance our understanding of the interactions between algae and indigenous algicidal bacteria, thereby providing aid in the design and optimization of strategies to control harmful algae blooms.

The algicidal activity of Aeromonas sp. strain GLY-2107 against bloom-forming Microcystis aeruginosa is regulated by N-acyl homoserine lactone-mediated quorum sensing.

Cyanobacterial blooms have disrupted the efficient utilization of freshwater worldwide. A new freshwater bacterial strain with strong algicidal activity, GLY-2107, was isolated from Lake Taihu and identified as Aeromonas sp. It produced two algicidal compounds: 2107-A (3-benzyl-piperazine-2,5-dione) and 2107-B (3-methylindole). Both compounds exhibited potent algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. The EC50 values (concentration for 50% maximal effect) of 3-benzyl-piperazine-2,5-dione and 3-methylindole were 4.72 and 1.10 µg ml-1 respectively. Based on a thin-layer chromatography biosensor assay and ultra-performance liquid chromatography-coupled high resolution-tandem mass spectrometry (UPLC-HRMS/MS), the N-acyl homoserine lactone (AHL) profile of strain GLY-2107 was identified as two short side-chain AHLs: N-butyryl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL). The production of the two algicidal compounds was controlled by AHL-mediated quorum sensing (QS), and C4-HSL was the key QS signal for the algicidal activity of the strain GLY-2107. Moreover, 3-methylindole was found to be positively regulated by C4-HSL-mediated QS, whereas 3-benzyl-piperazine-2,5-dione might be negatively controlled by C4-HSL-mediated QS. This study suggests that a QS-regulated algicidal system may have potential use for the development of a novel control strategy for harmful cyanobacterial blooms.

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa.

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

Effect of the termini of RNase Hs from Chlamydophila pneumoniae on enzymatic biochemical characterization.

A difference between prokaryotic RNase HII and HIII, which both belong to type 2 RNase H, is a long N-terminal extension of HIII; however, the main-fold structures of HII and HIII known as RNase H-fold are similar. To further understand the structure-function relationship of RNase HII and RNase HIII, biochemical analyses were carried out using N-terminal truncations of RNase HIII (IIIN56(Δ), IIIN81(Δ), and IIIN88(Δ)) and C-terminal truncation (IIC19(Δ)) of RNase HII from Chlamydophila pneumoniae. Compared with wild-type CpRNase HII/III, IIIN56(Δ) had no obvious variation on the cleavage site and efficiency of DNA-rN(1)-DNA/DNA (DR(1)D) and DNA-rN(4)-DNA/DNA (DR(4)D) substrates. IIC19(Δ) and IIIN81(Δ) both showed decreased activities, and IIIN88(Δ) exhibited little cleavage on these substrates. However, IIIN81(Δ) showed very different activities toward different substrates (20% for DR(1)D and 85% for DR(4)D). Moreover, IIC19(Δ)IIIN(82-88) mutant, prepared through adding N-terminal 82nd to 88th residues locating at the bound region of N- and C-terminal domains of CpRNase HIII to N-terminus of IIC19(Δ), cleaved DR(4)D substrate more efficiently and preferentially at the cleavage sites of CpRNase HIII but not those of CpRNase HII. These results indicated that C-termini of CpRNase HII, N-termini of CpRNase HIII, and bound region of N- and C-terminal domain are all important for enzymatic activities. Moreover, the 82nd to 88th residues of N-terminus of CpRNase HII are related with enzyme cleavage site specificity. These results will help to understand the importance of C-termini of CpRNase HII and N-termini of CpRNase HIII to the enzyme activities for DR(1)D and DR(4)D substrate.

[Effects of heavy metals pollution on soil microbial communities metabolism and soil enzyme activities in coal mining area of Tongchuan, Shaanxi Province of Northwest China].

This paper studied the metabolism of soil microbes, functions of soil microbial communities, and activities of soil enzymes in a coal mining area of Tongchuan. In the coal mining area, the concentrations of soil Cu, Zn, Cd, and Pb were significantly higher than those in the non-mining area, of which, Cd contributed most to the heavy metals pollution. By adopting Biolog method combining with principal component analysis (PCA) and cluster analysis, it was found that the metabolic characteristics of different soil microbial communities varied significantly with increasing soil heavy metals pollution, and the variation was mainly manifested in the metabolic patterns of carbon sources such as saccharides and amino acids. In slightly and moderately polluted soils, the utilization of carbon sources by soil microbial communities was activated; while in heavily polluted soils, the carbon sources utilization was inhibited. The activities of soil urease, protease, alkaline phosphatase, and catalase all tended to decline with intensifying soil heavy metals pollution. The soil urease, protease, alkaline phosphatase, and catalase activities in the coal mining area were 50.5%-65.1%, 19.1%-57.1%, 87.2%-97.5%, and 77.3%-86.0% higher than those in the non-mining area, respectively. The activities of soil sucrase and cellulase were activated in slightly and moderately polluted soils, but inhibited in heavily polluted soils.

Effects of Cu on metabolisms and enzyme activities of microbial communities in the process of composting.

With the compost matrix of pig manure, wheat straw, and spent mushroom substrate, and then inoculated with the Compound Microbe Preparation, the study investigated the effects of the heavy metal Cu on the process of composting. Biolog EcoPlate™ test revealed that at a low content, Cu could improve the capacities of microbial communities to transform and exploit carbon sources in the form of polymer, thus speeding up the decomposition of agricultural wastes, and at a high content, Cu presented inhibiting effect on microbial communities to exploit complex macromolecular carbon sources, thus extending the decomposition of agricultural wastes. Enzyme activity testing showed that at a low content, Cu presented enzyme activity-activating effect at the early period of composting and inhibiting effect in the late period of composting, and at a high content, Cu presented enzyme activity-inhibiting effects through the process of composting.