Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that ...cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term 'intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.
Abstract
Local intratumor delivery with electroporation of low levels of plasmids encoding molecules, such as IL-12, IL-15, induces an antitumor effect without causing systemic toxicity. However, ...previous studies have predominately focused on the function of the delivered molecule encoded within the plasmid, and ignored the plasmid backbone. In this study, we found backbones pUMVC3 and pVax1 induced up-regulation of MHC class I (MHC-I), PD-L1, CD95 (Fas) and CD155 on tumor cell surface. These molecules participate in a considerable number of immunoregulatory functions through their interactions with activating and inhibitory immune cell receptors. MHC molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells and tumor cells. Increased PD-L1 expression on tumor cells is an important monitor of tumor growth and effectiveness of immune inhibitor therapy. During cancer progression CD95 is frequently downregulated, raising the possibility that loss of CD95 is part of a mechanism for tumor evasion. Due to its prominent endogenous and immune functions, CD155 has been gaining interest as a therapeutic target in the field of tumor immunology. Results from flow cytometry confirmed increased expression of MHC-I and PDL-1 on B16F10, 4T1 and KPC tumor cell lines. Preliminary animal data from tumor-bearing models, B16F10 melanoma, 4T1 breast cancer and KPC pancreatic cancer mouse models showed that tumor growth was attenuated after pUMVC3 intratumoral electroporation. Our data also documented pSTAT1-NF-κB signaling pathway might be associated with plasmid backbones’ function of up-regulating MHC-I, PD-L1, CD95 (Fas) and CD155 on tumor cells.
R01 CA186730
Despite improvements in disease diagnosis, treatment, and prognosis, breast cancer is still a leading cause of cancer death for women. Compelling evidence suggests that targeting cancer stem cells ...(CSCs) have a crucial impact on overcoming the current shortcomings of chemotherapy and radiotherapy. In the present study, we aimed to study the effects of T cells and a critical anti-tumor cytokine, interferon-gamma (IFN-γ), on breast cancer stem cells.
BALB/c mice and BALB/c nude mice were subcutaneously injected with 4T1 tumor cells. Tumor growth and pulmonary metastasis were assessed. ALDEFLOUR™ assays were performed to identify aldehyde dehydrogenasebright (ALDHbr) tumor cells. ALDHbr cells as well as T cells from tumor-bearing BALB/c mice were analyzed using flow cytometry. The effects of CD8+ T cells on ALDHbr tumor cells were assessed in vitro and in vivo. The expression profiles of ALDHbr and ALDHdim 4T1 tumor cells were determined. The levels of plasma IFN-γ were measured by enzyme-linked immunosorbent assay, and their associations with the percentages of ALDHbr tumor cells were evaluated. The effects of IFN-γ on ALDH expression and the malignancy of 4T1 tumor cells were analyzed in vitro.
There were fewer metastatic nodules in tumor-bearing BALB/c mice than those in tumor-bearing BALB/c nude mice (25.40 vs. 54.67, P < 0.050). CD8+ T cells decreased the percentages of ALDHbr 4T1 tumor cells in vitro (control vs. effector to target ratio of 1:1, 10.15% vs. 5.76%, P < 0.050) and in vivo (control vs. CD8+ T cell depletion, 10.15% vs. 21.75%, P < 0.001). The functions of upregulated genes in ALDHbr 4T1 tumor cells were enriched in the pathway of response to IFN-γ. The levels of plasma IFN-γ decreased gradually in tumor-bearing BALB/c mice, while the percentages of ALDHbr tumor cells in primary tumors increased. IFN-γ at a concentration of 26.68 ng/mL decreased the percentages of ALDHbr 4T1 tumor cells (22.88% vs. 9.88%, P < 0.050) and the protein levels of aldehyde dehydrogenase 1 family member A1 in 4T1 tumor cells (0.86 vs. 0.49, P < 0.050) and inhibited the abilities of sphere formation (sphere diameter <200 μm, 159.50 vs. 72.0; ≥200 μm, 127.0 vs. 59.0; both P < 0.050) and invasion (89.67 vs. 67.67, P < 0.001) of 4T1 tumor cells.
CD8+ T cells and IFN-γ decreased CSC numbers in a 4T1 mouse model of breast cancer. The application of IFN-γ may be a potential strategy for reducing CSCs in breast cancer.
Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are ...still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell composition after intratumoral injection of pIL-12 GET. The number of immune memory cells was markedly increased in pIL-12 GET melanoma groups compared to control group. This was validated using flow cytometry to analyze peripheral blood mononuclear cells as well as delineating immune cell content using immunohistochemistry. Significant differences in multiple cell types were observed, including CD8⁺ T cells, regulatory T cells and myeloid cells, which were induced to mount a CD8⁺PD1
T cells immune response. Taken together, these findings suggest a basic understanding of the sequence of immune activity following pIL-12 GET and also illuminates that adjuvant immunotherapy can have a positive influence on the host immune response to cancer.
Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide ...application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8
and CD4
T cells. Additionally, the combination therapy induced higher MHC-I surface expression on tumor cells to hamper tumor cells escaping from immune recognition. Furthermore, we found that activating T cells by exposure to IL-12 resulted in tumors sensitized to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.
Pediatric-onset systemic lupus erythematosus arises in humans and mice lacking the endonuclease Dnase1L3. When Dnase1L3 is absent, DNA from circulating apoptotic bodies is not cleared, leading to ...anti-DNA antibody production. Compared to early anti-DNA and anti-chromatin responses, other autoantibody responses and general immune activation in Dnase1L3
mice are greatly delayed. We investigated the possibility that immune activation, specifically inflammasome activation, is regulated by Dnase1L3. Here, we report that Dnase1L3 inhibition blocked both NLR family, pyrin domain containing 3 (NLRP3) and NLRC4 inflammasome-mediated release of high-mobility group box 1 protein and IL-1β. In contrast to IL-1β release, Dnase1L3 inhibition only mildly impaired NLRP3-dependent pyroptosis, as measured by propidium iodide uptake or LDH release. Mechanistically, we found that Dnase1L3 was needed to promote apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) nuclear export and speck formation. Our results demonstrate that Dnase1L3 inhibition separates cytokine secretion from pyroptosis by targeting ASC. These findings suggest that Dnase1L3 is necessary for cytokine secretion following inflammasome activation.
Abstract
Background
Immune checkpoint inhibition (ICI) has led to impressive gains in survival for many patients. However, many cancer patients treated with ICI experience intrinsic or acquired ...resistance which directly affects efficacy. Decreased levels of MHC class-I molecules on cancer cells and tumor infiltrating lymphocytes (TILs) have been associated with poor clinical outcomes.
Methods
Treatment of breast cancer cell lines with a plasmid encoding IL-12 (pIL-12) and vector pUMVC3 were analyzed using flow cytometry, western blotting and then validated functionally. The 4T1 breast cancer animal model was used to study T cell infiltration into the tumor as well as response following local delivery of pIL-12 with electrotransfer.
Results
Enhanced MHC class-I expression in 4T1 tumor cells, and elevated CD8+ T-cell infiltration into 4T1 tumor tissue were observed after pIL-12 gene electrotransfer. Furthermore, increased capacity to lyse 4T1 cells was observed with cultured cytotoxic T lymphocytes (CTLs) from long-term surviving mice compared to control group. A direct translation was observed in tumor-bearing mice, including attenuating tumor growth and improved tumor-free survival in mice. An immune memory response which protected against rechallenge was induced also.
Conclusion
Our findings suggest that the capacity of an induced endogenous immune response with pIL12 via the recruitment of CD8+ T cells and increased MHC class-I expression in tumor cells results in attenuating the resistance of anti-PD1 and leads to eradication of established, aggressive tumors and generates systemic tumor-specific protective immunity. Combinations of anti-PD-1 with pIL-12 gene therapy may be an effective future therapeutic strategy.
Supported by NCI R01 CA186730
Gene electrotransfer (GET) is a reliable and effective physical method for in vivo delivery of plasmid DNA (pDNA). Several preclinical and clinical studies have utilized GET to deliver plasmids ...encoding immune stimulating genes for treatment of melanoma and other tumor types. Intratumor delivery of plasmids encoding cytokines directly to tumors can induce not only a local immune response, but a systemic one as well. To obtain an effective immune response, it is critical to achieve the appropriate expression pattern of the delivered transgene. Expression pattern (levels and kinetics) can be modified by manipulating the electrotransfer parameters. These parameters include the tissue target and the electric pulse parameters of pulse width, electric field, and pulse number. We have found that to induce a robust immune response, we needed only low to moderately elevated expression levels compared to controls. When developing a therapeutic protocol, it is important to establish what expression profile will enable the appropriate response. In this chapter we describe how to determine the appropriate GET protocol to achieve the expression profile that can result in the desired clinical response.
Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and ...virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.
Local intratumor delivery with electroporation of low levels of plasmids encoding molecules, induces an antitumor effect without causing systemic toxicity. However, previous studies have ...predominately focused on the function of the delivered molecule encoded within the plasmid, and ignored the plasmid vector. In this study, we found vectors pUMVC3 and pVax1 induced upregulation of MHC class I (MHC-I) and PD-L1 on tumor cell surface. These molecules participate in a considerable number of immunoregulatory functions through their interactions with and activating inhibitory immune cell receptors. MHC molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells and tumor cells. Increased PD-L1 expression on tumor cells is an important monitor of tumor growth and the effectiveness of immune inhibitor therapy. Results from flow cytometry confirmed increased expression of MHC-I and PDL-1 on B16F10, 4T1, and KPC tumor cell lines. Preliminary animal data from tumor-bearing models, B16F10 melanoma, 4T1 breast cancer and KPC pancreatic cancer mouse models showed that tumor growth was attenuated after pUMVC3 intratumoral electroporation. Our data also documented that pSTAT1 signaling pathway might not be associated with plasmid vectors' function of upregulating MHC-I, PD-L1 on tumor cells.
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