Ginsenoside Rg1 improves anti-tumor efficacy of adoptive cell therapy by enhancing T cell effector functions.

Adoptive cell therapy (ACT) has emerged with remarkable efficacies for tumor immunotherapy. Chimeric antigen receptor (CAR) T cell therapy, as one of most promising ACTs, has achieved prominent effects in treating malignant hematological tumors. However, the insufficient killing activity and limited persistence of T cells in the immunosuppressive tumor microenvironment limit the further application of ACTs for cancer patients. Many studies have focused on improving cytotoxicity and persistence of T cells to achieve improved therapeutic effects. In this study, we explored the potential function in ACT of ginsenoside Rg1, the main pharmacologically active component of ginseng. We introduced Rg1 during the in vitro activation and expansion phase of T cells, and found that Rg1 treatment upregulated two T cell activation markers, CD69 and CD25, while promoting T cell differentiation towards a mature state. Transcriptome sequencing revealed that Rg1 influenced T cell metabolic reprogramming by strengthening mitochondrial biosynthesis. When co-cultured with tumor cells, Rg1-treated T cells showed stronger cytotoxicity than untreated cells. Moreover, adding Rg1 to the culture endowed CAR-T cells with enhanced anti-tumor efficacy. This study suggests that ginsenoside Rg1 provides a potential approach for improving the anti-tumor efficacy of ACT by enhancing T cell effector functions.

Strategies to enhance CAR-T persistence.

Chimeric antigen receptor T (CAR-T) cell therapy has significantly improved the life expectancy for patients with refractory or relapse B cell lymphoma. As for B cell acute lymphoblastic leukemia (B-ALL), although the primary response rate is promising, the high incidence of early relapse has caused modest long-term survival with CAR-T cell alone. One of the main challenges is the limited persistence of CAR-T cells. To further optimize the clinical effects of CAR-T cells, many studies have focused on modifying the CAR structure and regulating CAR-T cell differentiation. In this review, we focus on CAR-T cell persistence and summarize the latest progress and strategies adopted during the in vitro culture stage to optimize CAR-T immunotherapy by improving long-term persistence. Such strategies include choosing a suitable cell source, improving culture conditions, combining CAR-T cells with conventional drugs, and applying genetic manipulations, all of which may improve the survival of patients with hematologic malignancies by reducing the probability of recurrence after CAR-T cell infusion and provide clues for solid tumor CAR-T cell therapy development.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Synthesis, secretion, and antifungal mechanism of a phosphatidylethanolamine-binding protein from the silk gland of the silkworm Bombyx mori.

A silkworm cocoon contains several antimicrobial proteins such as protease inhibitors and seroins to provide protection for the enclosed pupa. In this study, we identified a new Bombyx mori phosphatidylethanolamine-binding protein (BmPEBP) with antimicrobial activity in the cocoon silk using semi-quantitative and quantitative RT-PCR, western blotting, and immunofluorescence. The results indicated that BmPEBP was synthesized in the middle silk gland and secreted into the sericin layer of the cocoon silk. Functional analysis showed that BmPEBP could inhibit the spore growth of four types of fungi, Candida albicans, Saccharomyces cerevisiae, Beauveriabassiana, and Aspergillus fumigates, by binding to the fungal cell membrane. Investigation of the interaction of BmPEBP with membrane phospholipids revealed that the protein showed a strong binding affinity to phosphatidylethanolamine, weak affinity to phosphatidylinositol, and no affinity to phosphatidylserine or phosphatidylcholine. Circular dichroism spectroscopy showed that binding to phosphatidylethanolamine caused conformational changes in the BmPEBP molecule by reducing ß-sheet formation and inducing the appearance of an α-helix motif. We speculate that BmPEBP performs antifungal function in the cocoon silk through interaction with phosphatidylethanolamine in the fungal membrane.

Identification of Bombyx mori sericin 4 protein as a new biological adhesive.

Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.

Functional analysis and characterization of antimicrobial phosphatidylethanolamine-binding protein BmPEBP in the silkworm Bombyx mori.

Phosphatidylethanolamine-binding proteins (PEBPs) are a class of highly conserved, biologically diverse proteins, which are widely distributed in plants, insects, and mammals. In this study, a Bombyx mori PEBP (BmPEBP) gene was reported, which encodes a protein composed of 209 amino acid residues. BmPEBP includes a predicted signal peptide, indicating that it is an extracellular protein, which differs from the cytoplasmic PEBPs of plants and mammals. Recombinant soluble BmPEBP was successfully synthesized using a prokaryotic expression system and was then purified effectively by Ni2+-NTA affinity chromatography and gel filtration. Far-ultraviolet circular dichroism spectra indicated that BmPEBP had a well-defined ß-sheet structure, with the ß-sheet content accounting for about 41% of the protein. BmPEBP had a relatively stable structure at temperatures ranging from 15 °C to 57.5 °C. The Tm, ΔH, and ΔS of BmPEBP were 62.27 °C ±â€¯0.14 °C, 570.10 ±â€¯0.17 kJ/mol, and 1.70 ±â€¯0.03 KJ/(mol·K), respectively. Homology modeling analysis suggested that the active sites of BmPEBP were conserved, comprising Pro96, His111, and His143. Quantitative real-time PCR showed that BmPEBP was highly expressed in the silk gland and had very low expression in other tissues. However, BmPEBP expression was significantly upregulated in the larval fat body after infection with two kinds of fungi, Beauveria bassiana and Candida albicans. Moreover, in vitro fungal inhibition tests showed that BmPEBP could significantly inhibit the sporular growth of Saccharomyces cerevisiae, C. albicans, B. bassiana, and Aspergillus fumigatus. To our knowledge, this is the first report to reveal the antifungal role of a PEBP in insects.