How Do Cytotoxic Lymphocytes Kill Cancer Cells?

In the past few years, cancer immunotherapy has emerged as a safe and effective alternative for treatment of cancers that do not respond to classical treatments, including those types with high aggressiveness. New immune modulators, such as cytokines, blockers of CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and PD-1(programmed cell death protein 1)/PD-L1 (programmed death-ligand 1), and interaction or adoptive cell therapy, have been developed and approved to treat solid and hematologic carcinomas. In these scenarios, cytotoxic lymphocytes (CL), mainly cytotoxic T cells (Tc) and natural killer (NK) cells, are ultimately responsible for killing the cancer cells and eradicating the tumor. Extensive studies have been conducted to assess how Tc and NK cells get activated and recognize the cancer cell. In contrast, few studies have focused on the effector molecules used by CLs to kill cancer cells during cancer immunosurveillance and immunotherapy. In this article, the two main pathways involved in CL-mediated tumor cell death, granule exocytosis (perforin and granzymes) and death ligands, are briefly introduced, followed by a critical discussion of the molecules involved in cell death during cancer immunosurveillance and immunotherapy. This discussion also covers unexpected consequences of proinflammatory and survival effects of granzymes and death ligands and recent experimental evidence indicating that perforin and granzymes of CLs can activate nonapoptotic pathways of cell death, overcoming apoptosis defects and chemoresistance. The consequences of apoptosis versus other modalities of cell death for an effective treatment of cancer by modulating the patient immune system are also briefly discussed. Clin Cancer Res; 21(22); 5047–56. ©2015 AACR

Lipid nanoparticles decorated with TNF-related aptosis-inducing ligand (TRAIL) are more cytotoxic than soluble recombinant TRAIL in sarcoma

Sarcomas are rare and heterogeneous cancers classically associated with a poor outcome. Sarcomas are 1% of the cancer but recent estimations indicate that sarcomas account for 2% of the estimated cancer-related deaths. Traditional treatment with surgery, radiotherapy, and chemotherapy has improved the outcome for some types of sarcomas. However, novel therapeutic strategies to treat sarcomas are necessary. TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand initially described as capable of inducing apoptosis on tumor cell while sparing normal cells. Only few clinical trials have used TRAIL-based treatments in sarcoma, but they show only low or moderate efficacy of TRAIL. Consequently, novel TRAIL formulations with an improved TRAIL bioactivity are necessary. Our group has developed a novel TRAIL formulation based on tethering this death ligand on a lipid nanoparticle surface (LUV-TRAIL) resembling the physiological secretion of TRAIL as a trasmembrane protein inserted into the membrane of exosomes. We have already demonstrated that LUV-TRAIL shows an improved cytotoxic activity when compared to soluble recombinant TRAIL both in hematological malignancies and epithelial-derived cancers. In the present study, we have tested LUV-TRAIL in several human sarcoma tumor cell lines with different sensitivity to soluble recombinant TRAIL, finding that LUV-TRAIL was more efficient than soluble recombinant TRAIL. Moreover, combined treatment of LUV-TRAIL with distinct drugs proved to be especially effective, sensitizing even more resistant cell lines to TRAIL

Selective IgA deficiency and presumptive polyclonal spike in the beta fraction in a dog with leishmaniosis

A 1-year-old, entire, crossbred, female dog examined for apathy, lethargy, unilateral nosebleed of 2-month duration and the presence of symmetrical ulcerative dermatitis (small ulcers covered by a haemorrhagic crust and surrounded by alopecia on the tip of the ear pinna). Laboratory alterations included anaemia and hyperglobulinemia with a presumptive polyclonal spike in the beta fraction. High antibody levels to Leishmania infantum were detected by enzyme-linked immunosorbent assay. An anti-Leishmania therapeutic protocol was established and a reduction of the anti-Leishmania antibodies was detected by an end point sera dilution enzyme-linked immunosorbent assay during the first weeks of therapeutic protocol, while C-reactive protein concentration was reduced during the period of time when meglumine antimoniate was administered. A good clinical response to the treatment was detected after initiating the anti-Leishmania treatment. Quantitative serology is useful in the short-term, because using a two-fold serial dilution technique correlates with the clinical response. © 2022 British Veterinary Association

Onto better TRAILs for cancer treatment

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-R-targeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists

In Vitro Genotoxicity Evaluation of an Antiseptic Formulation Containing Kaolin and Silver Nanoparticles

Worldwide antimicrobial resistance is partly caused by the overuse of antibiotics as growth promoters. Based on the known bactericidal effect of silver, a new material containing silver in a clay base was developed to be used as feed additive. An in vitro genotoxicity evaluation of this silver-kaolin clay formulation was conducted, which included the mouse lymphoma assay in L5178Y TK+/- cells and the micronucleus test in TK6 cells, following the principles of the OECD guidelines 490 and 487, respectively. As a complement, the standard and Fpg-modified comet assays for the evaluation of strand breaks, alkali labile sites and oxidative DNA damage were also performed in TK6 cells. The formulation was tested without metabolic activation after an exposure of 3 h and 24 h; its corresponding release in medium, after the continuous agitation of the silver-kaolin for 24 h was also evaluated. Under the conditions tested, the test compound did not produce gene mutations, chromosomal aberrations or DNA damage (i.e., strand breaks, alkali labile sites or oxidized bases). Considering the results obtained in the present study, the formulation seems to be a promising material to be used as antimicrobial in animal feed. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

In vivo potential of recombinant granulysin against human melanoma

9-kDa granulysin is a protein expressed into the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It has been shown to exert cytolysis on microbes and tumors. We showed previously that 9-kDa granulysin exerted cell death by apoptosis in vitro on hematological tumor cell lines and also on cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. In addition, we have shown the anti-tumor efficiency of granulysin as a single agent in two in vivo models of human tumor development in athymic mice, the MDA-MB-231 mammary adenocarcinoma and the NCI-H929 multiple myeloma, without signs of overt secondary effects by itself. In this work, we have tested recombinant 9-kDa granulysin in an in vivo and especially aggressive model of melanoma development, xenografted UACC62 cells in athymic mice. Recombinant granulysin was administered once UACC62-derived tumors were detectable and it substantially retarded the in vivo development of this aggressive tumor. We could also detect apoptosis induction and increased NK cell infiltration inside granulysin-treated tumor tissues. These observations are especially interesting given the possibility of treating melanoma by intra-tumor injection

Conjugation of the 9-kDa isoform of Granulysin with liposomes potentiates its cytotoxicity

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni2+ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-xL. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway

Comparative proteomics of exosomes secreted by tumoral jurkat t cells and normal human t cell blasts unravels a potential tumorigenic role for valosin-containing protein

We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion

The Multifaceted Function of Granzymes in Sepsis: Some Facts and a Lot to Discover

Sepsis is a serious global health problem. In addition to a high incidence, this syndrome has a high mortality and is responsible for huge health expenditure. The pathophysiology of sepsis is very complex and it is not well-understood yet. However, it is widely accepted that the initial phase of sepsis is characterized by a hyperinflammatory response while the late phase is characterized by immunosuppression and immune anergy, increasing the risk of secondary infections. Granzymes (Gzms) are a family of serine proteases classified according to their cleavage specificity. Traditionally, it was assumed that all Gzms acted as cytotoxic proteases. However, recent evidence suggests that GzmB is the one with the greatest cytotoxic capacity, while the cytotoxicity of others such as GzmA and GzmK is not clear. Recent studies have found that GzmA, GzmB, GzmK, and GzmM act as pro-inflammatory mediators. Specially, solid evidences show that GzmA and GzmK function as extracellular proteases that regulate the inflammatory response irrespectively of its ability to induce cell death. Indeed, studies in animal models indicate that GzmA is involved in the cytokine release syndrome characteristic of sepsis. Moreover, the GZM family also could regulate other biological processes involved in sepsis pathophysiology like the coagulation cascade, platelet function, endothelial barrier permeability, and, in addition, could be involved in the immunosuppressive stage of sepsis. In this review, we provide a comprehensive overview on the contribution of these novel functions of Gzms to sepsis and the new therapeutic opportunities emerging from targeting these proteases for the treatment of this serious health problem

High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer

During the last years, a great effort has been invested into developing new TRAIL formulations with increased bioactivity, trying to overcome the resistance to conventional soluble TRAIL (sTRAIL) exhibited by many primary tumours. In our group, we have generated artificial lipid nanoparticles decorated with sTRAIL (LUV-TRAIL), emulating the physiological TRAIL-containing exosomes by which T-cells release TRAIL upon activation. We already demonstrated that LUV-TRAIL has greater cytotoxicity against both chemoresistant haematologic tumour cells and epithelial carcinoma cells compared to a form of sTRAIL similar to that used in clinical trials. In this study we have tested LUV-TRAIL in several human colon cancer cell lines with different sensitivity to sTRAIL. LUV-TRAIL significantly improved sTRAIL cytotoxicity in all colon cancer cell lines tested. Trying to ascertain the molecular mechanism by which LUV-TRAIL exhibited improved cytotoxicity, we demonstrated that TRAIL-coated lipid nanoparticles were able to activate DR5 more efficiently than sTRAIL, and this relied on LUV-TRAIL ability to promote DR5 clustering on the cell surface. Moreover, we show that TRAIL molecules are arranged in higher order oligomers only in LUV-TRAIL, which may explain their enhanced DR5 clustering ability. Finally, LUV-TRAIL showed significantly better antitumour activity than sTRAIL in an in vivo model using HCT-116 xenograft tumours in nude mice, validating its potential clinical application