Anti-PD1 does not improve pyroptosis induced by γδ T cells but promotes tumor regression in a pleural mesothelioma mouse model

IntroductionMesothelioma is an aggressive tumor in the pleural cavity that is difficult to treat. Diagnosis is usually late with minimal treatment options available for the patients and with unfavorable outcomes. However, recent advances in immunotherapy using γδ T cells may have potential against mesothelioma, given its ample tumoricidal and tumor-migratory properties could allow its infiltration to the widespread tumor mass. Thus, we hypothesize that Vδ2 T cells can perform cytotoxic activities against mesothelioma especially when combined with immune checkpoint blocker against PD-1.MethodsHuman Vδ2 T cells were expanded from peripheral blood mononuclear cells using Tetrakis‐pivaloyloxymethyl 2‐(thiazole‐2‐ylamino) ethylidene‐1,1‐bisphosphonate (PTA) plus IL-2 for 13 days, before used to test for cytotoxicity against mesothelioma cell lines. Mesothelioma-bearing mice was established by Intrapleural administration of mesothelioma cell lines to test for the efficacy of Vδ2 T cells plus anti-PD-1 antibody combination treatment. Pyroptosis was evaluated by cell morphology, western blot analysis, and ELISA experiments. Flow cytometry was used to examine expression of BTN2A1, BTN3A1, PD-L1, PD-L2 on mesothelioma cell lines. Immunofluorescence staining was performed to detect Vδ2 T cells post adoptive transfer and characteristics of pyroptosis in ex vivo mesothelioma tissue sections.ResultsIndeed, our data demonstrated that Vδ2 T cells killing mesothelioma can be enhanced by anti-PD-1 antibody in vitro, especially for high PD-1 expressing cells, and in vivo in the intrapleural mesothelioma mice model established by us. Adoptive transfer of Vδ2 T cells into these mice leads to tumor regression by 30-40% compared to control. Immunofluorescence of the tumor section confirmed infiltration of Vδ2 T cells into the tumor, especially to cells with BTN2A1 expression (a Vδ2 T cell activating molecule) despite PD-L1 co-localization. Interestingly, these cells co-expressed cleaved gasdermin D, suggesting that pyroptosis was induced by Vδ2 T cells. This was verified by Vδ2 T/mesothelioma co-culture experiments demonstrating membrane ballooning morphology, increased cleaved caspase-3 and gasdermin E, and upregulated IL-1β and IL-18.DiscussionVδ2 T cells plus anti-PD1 exhibited cytotoxicity against mesothelioma in vivo. However, we found no advantage for anti-PD-1 against PD-1 high expressing Vδ2 T cells in promoting pyroptosis. Taken together, our work demonstrated that Vδ2 T cells combined with anti-PD-1 antibody can be developed as a potential combination immunotherapy for mesothelioma

Piezoelectric thermoelastic dissipation research of piezoelectric harvester under different vibration

Piezoelectric materials are widely used to form piezoelectric energy harvesters. Also, the thermoelastic dissipation always influences the energy harvesting efficiency, during the energy harvest process. Therefore, in this paper, we discuss the effect of thermoelastic dissipation on the piezoelectric harvester through numerical calculation, simulation and experiment. The piezoelectric thermoelastic coupling governing equations under different vibration are derived, which are based on the Euler-Bernoulli beam theory, thermal conduction model and piezoelectric field model. Then, the structure frequency shift and thermoelastic damping are studied via numerical calculation and simulation. Meanwhile, we show the influence of the temperature field on the piezoelectric structure under different vibration modes. Furth more, we research the variations of piezoelectric structure thermoelastic dissipation characteristics under different structure geometry sizes. Based on these analyses, the effect of piezoelectric thermoelastic dissipation on the piezoelectric harvester is researche

Piezoelectric thermoelastic dissipation research of piezoelectric harvester under different vibration

Piezoelectric materials are widely used to form piezoelectric energy harvesters. Also, the thermoelastic dissipation always influences the energy harvesting efficiency, during the energy harvest process. Therefore, in this paper, we discuss the effect of thermoelastic dissipation on the piezoelectric harvester through numerical calculation, simulation and experiment. The piezoelectric thermoelastic coupling governing equations under different vibration are derived, which are based on the Euler-Bernoulli beam theory, thermal conduction model and piezoelectric field model. Then, the structure frequency shift and thermoelastic damping are studied via numerical calculation and simulation. Meanwhile, we show the influence of the temperature field on the piezoelectric structure under different vibration modes. Furth more, we research the variations of piezoelectric structure thermoelastic dissipation characteristics under different structure geometry sizes. Based on these analyses, the effect of piezoelectric thermoelastic dissipation on the piezoelectric harvester is researche

The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells

Abstract Background Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive. Results Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein–protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles. Conclusions These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically)

Additional file 3 of The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells

Additional file 3: Figure S1. V1 induces the accumulation of CapZ on early endosomes. HeLa cells were transiently transfected with CapZβ-mCherry, RAB5A-BFP, and Rabaptin-5-GFP (A) or Rabex5-GFP (B), and treated with or without V1 (1 μM). The colocalization coefficients (MCCs) of CapZ/RAB5, CapZ/ Rabaptin-5, CapZ/Rabex-5, Rabex-5/RAB5, or Rabaptin-5/RAB5 were quantified. The images represent data from at least three independent experiments. The difference between two groups was calculated using an unpaired Student’s t-test. Differences were considered statistically significant when P < 0.05, *** P < 0.001. Figure S2. The role of CapZ in the early-to-late endosome transition. (A) CapZβ-GFP/RFP-FYVE-expressing (a PI(3)P sensor)/CapZβ-mCherry-expressing HeLa cells were subjected to confocal imaging, and the colocalization coefficients (MCCs) of mRFP-FYVE/CapZβ-EGFP were quantified. The scale bar is 5 μm. The images represent data from at least three independent experiments. (B) Coomassie staining of recombinant CapZa-CapZb complex purified from bacterial culture. The difference between the two groups was calculated using an unpaired Student’s t-test. Differences were considered statistically significant when P < 0.05, *** P < 0.001. Figure S3. CapZ participates in endosomal maturation. (A) HeLa cells were transiently transfected with FRB-RAB5 and FKBP-CapZβ, and then incubated with rapamycin (1 mM) for 12 h to induce an interaction between RAB5 and CapZ. (B) HeLa cells were transiently transfected with FRB-RAB5 and FKBP-CapZβ, and then they were incubated with rapamycin (1 mM) for 12 h, followed by anti-Lamp1 immunostaining and confocal imaging. (C) Control or CapZb knockout cells were labeled with lysosensor-Green DND-189, followed by confocal image and quantification. The scale bar is 5 μm. The colocalization coefficients (MCC) of RAB5A, CapZ, or Lamp1 were quantified. The difference between the two groups was calculated using an unpaired Student’s t-test. Differences were considered statistically significant when P < 0.05, *** P < 0.001. The images represent data from at least three independent experiments. Figure S4. The stabilization of CapZ on early endosomes inhibits endocytosis and cell migration. (A, B) Control or FRB-RAB5/FKBP-CapZβ expressing HeLa cells were incubated with or without rapamycin (1 mM) for 12 h, followed by incubation with transferin-594 on ice for 1.5 h. The cells were fixed at the time points indicated and processed for confocal imaging (A) and quantification (B). (C) Control or FRB-RAB5/FKBP-CapZβ expressing HeLa cells were placed into the upper chamber of a transwell plate in the absence or presence of rapamycin (1 mM). After 18 h, the cells in the lower chamber were stained with crystal violet and quantified. The difference between the two groups was calculated using the ANOVA test. Differences were considered statistically significant when P < 0.05, *** P < 0.001. The images and graphs represent data from at least three independent experiments. Figure S5. V1, chloroquine, and bafilomycin A1 inhibit ZIKV or MHV infection in host cells. (A, B) A549 cells were pretreated with V1, bafilomycin A1, and chloroquine for 3 h, and then infected with ~ 1 MOI of ZIKV for 24 h, followed by ZIKV-E and GAPDH immunoblot analysis (A) or the measurement of viral titers by TCID50 assay (B). (C) 17Cl-1 cells were pretreated with V1, bafilomycin A1 (50 nM), and chloroquine (25 mM) for 3 h, and then infected with ~ 1 MOI of MHV for 24 h, followed by the qRT-PCR analysis of MHV nsp9 mRNA expression. The blots, images, and graphs represent data from at least three independent experiments. The difference between the two groups was calculated using the ANOVA test. Differences were considered statistically significant when P < 0.05, *** P < 0.001. The blots and graphs represent data from at least three independent experiment

Vacuolin-1 inhibits endosomal trafficking and metastasis via CapZ?

10.1038/s41388-021-01662-3Oncogene40101775-179