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Background: The cGAS (GMP-AMP synthase)-triggered senescence-associated secretory phenotype (SASP) in promotion of cancer progression has been extensively documented. However, the role of cGAS-mediated DNA autophagy is little evaluated in cancer cells.Methods: Immunofluorescence, senescence associated-β-galactosidase staining (SA-β-gal) and Western blot were performed to detect gene expression, distribution and phenotypes. PCR, IP-PCR, FISH, BrdU, Comet assay, coimmunoprecipitation, sucrose density gradient centrifugation were carried out to detect possible mechanisms. Trypan blue exclusion, Live/dead staining and MTS assay were to measure the cell viability. All analyses were performed using GraphPad Prism 8. Relationships were analyzed using t-tests. A P-value of less than 0.05 was considered significant. All statistical tests and P values were 2-sided, and the level of significance was set at Results: Active DNA autophagy but not SASP activity could be detected in the BT-549 breast cancer cells with high micronucleus (MN). The selective autophagy of free genomic DNA in the cytoplasm is mediated by cGAS and usually coordinated with SQSTM1-mediated autophagy of ubiquitinated histones. Cytoplasmic DNA together with nuclear proteins derive from DNA replication-induced nuclear damage and MN collapse. The inhibition of DNA autophagy through either chemical inhibitors or genomic silencing of cGAS or SQSTM1 suppresses the growth and survival of cancer cells, while enhanced DNA damage increases the sensitivity to these inhibitors. Human cancer cells with either high DNA autophagy or enhancement of DNA damage are sensitive to inhibition of DNA autophagy.Conclusions: Our investigation revealed DNA autophagy in breast cancer cells with high MN formation. Autophagy of genomic DNA in the cytosol could be mediated by cGAS but is usually coordinated with other autophagic mediators. The selective autophagy mediated clearance of free genomic DNA protects of growth and survival cancer cells, and autophagic inhibition could be a potential therapeutic approach for cancer cells with high DNA autophagic activity.

Background: The cGAS (GMP-AMP synthase)-triggered senescence-associated secretory phenotype (SASP) in promotion of cancer progression has been extensively documented. However, the role of cGAS-mediated DNA autophagy is little evaluated in breast cancer cells.Methods: Immunofluorescence, senescence associated-β-galactosidase staining (SA-β-gal) and Western blot were performed to detect gene expression, distribution and phenotypes. PCR, IP-PCR, FISH, BrdU, Comet assay, coimmunoprecipitation, sucrose density gradient centrifugation were carried out to detect possible mechanisms. Trypan blue exclusion, Live/dead staining and MTS assay were to measure the cell viability. All analyses were performed using GraphPad Prism 8. Relationships were analyzed using t-tests. A P-value of less than 0.05 was considered significant. All statistical tests and P values were 2-sided, and the level of significance was set at Results: Active DNA autophagy but not SASP activity could be detected in breast cancer cells with high micronucleus (MN). The selective autophagy of free genomic DNA in the cytoplasm is mediated by cGAS and usually coordinated with SQSTM1-mediated autophagy of ubiquitinated histones in breast cancer cell lines with high frequency of MN formation. Cytoplasmic DNA together with nuclear proteins derive from DNA replication-induced nuclear damage and MN collapse in breast cell lines which with severe DNA damage. The inhibition of DNA autophagy through either chemical inhibitors or genomic silencing of cGAS or SQSTM1 suppresses the growth and survival of breast cancer cells, while enhanced DNA damage increases the sensitivity to these inhibitors for more cancer cells. Human cancer cells with either high DNA autophagy or enhancement of DNA damage are sensitive to inhibition of DNA autophagy.Conclusions: Our investigation revealed DNA autophagy in breast cancer cells with high MN formation. Autophagy of genomic DNA in the cytosol could be mediated by cGAS but is usually coordinated with other autophagic mediators. The selective autophagy mediated clearance of free genomic DNA protects of growth and survival in breast cancer cells even, and autophagic inhibition could be a potential therapeutic approach for cancer cells with high DNA autophagic activity.

The graft-versus-leukemia effect reminds us to observe the allogeneic cell elicited anti-tumor immune responses. Here we immunized recipient B6 mice with different types of allogenic leukocytes and found that vaccination with allogenic dendritic cells (alloDC) elicited the most efficient protection against broad-spectrum tumors. The recipient lymphocytes were analyzed and the data showed that CD8 T cells increased significantly after immunization and expressed effector memory T cell marker KLRG1. Functional evaluation demonstrated that these KLRG1+CD8 T cells could kill tumor cells in vitro and in vivo in Granzyme B- and Fas/FasL-dependent manners with no tumor antigen specificity, and tend to migrate into tumor sites by high expression of heparanase. Adoptive transfer of these cells could provide antitumor protection against tumors. AlloDC could also treat mice with residual tumors and combination of anti-PD1 antibody could enhance this effects. Together, our study showed that alloDC-immunization could induce potent antitumor effect through the expansion of KLRG1+CD8 T cells, which can work as both preventive and therapeutic tumor vaccines.

The graft-versus-leukemia effect reminds us to observe the allogeneic cell elicited anti-tumor immune responses. Here we immunized recipient B6 mice with different types of allogenic leukocytes and found that vaccination with allogenic dendritic cells (alloDC) elicited the most efficient protection against broad-spectrum tumors. The recipient lymphocytes were analyzed and the data showed that CD8 T cells increased significantly after immunization and expressed effector memory T cell marker KLRG1. Functional evaluation demonstrated that these KLRG1+CD8 T cells could kill tumor cells in vitro and in vivo in Granzyme B- and Fas/FasL-dependent manners with no tumor antigen specificity, and tend to migrate into tumor sites by high expression of heparanase. Adoptive transfer of these cells could provide antitumor protection against tumors. AlloDC could also treat mice with residual tumors and combination of anti-PD1 antibody could enhance this effects. Together, our study showed that alloDC-immunization could induce potent antitumor effect through the expansion of KLRG1+CD8 T cells, which can work as both preventive and therapeutic tumor vaccines.

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators' ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

CD1d-dependent NKT cells have been extensively studied; however, the function of CD8+NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8+NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8+NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8+NKT-like cell development is normal in CD1d−/− mice, which suggests that CD8+NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8+NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8+NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8+NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×10(4) PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.