Development of a highly cytotoxic, clinical-grade virus-specific T cell product for adoptive T cell therapy.

At present, recipients of allogeneic hematopoietic stem-cells are still suffering from recurrent infections after transplantation. Infusion of virus-specific T cells (VST) post-transplant reportedly fights several viruses without increasing the risk of de novo graft-versus-host disease. This study targeted cytomegalovirus (CMV) for the development of an innovative approach for generating a very specific VST product following Good Manufacturing Practices (GMP) guidelines. We used a sterile disposable compartment named the Leukoreduction System Chamber (LRS-chamber) from the apheresis platelet donation kit as the starting material, which has demonstrated high levels of T cells. Using a combination of IL-2 and IL-7 we could improve expansion of CMV-specific T cells. Moreover, by developing and establishing a new product protocol, we were able to stimulate VST proliferation and favors T cell effector memory profile. The expanded VST were enriched in a closed automated system, creating a highly pure anti-CMV product, which was pre-clinically tested for specificity in vitro and for persistence, biodistribution, and toxicity in vivo using NOD scid mice. Our results demonstrated very specific VST, able to secrete high amounts of interferon only in the presence of cells infected by the human CMV strain (AD169), and innocuous to cells partially HLA compatible without viral infection.

Cytokines as an important player in the context of CAR-T cell therapy for cancer: Their role in tumor immunomodulation, manufacture, and clinical implications.

CAR-T cell therapies have been recognized as one of the most advanced and efficient strategies to treat patients with hematologic malignancies. However, similar results have not been observed for the treatment of solid tumors. One of the explanations is the fact that tumors have extremely hostile microenvironments for the infiltration and effector activity of T-cells, mainly due to the presence of highly suppressive cytokines, hypoxia, and reactive oxygen species. Taking advantage of cytokines functionally, new fourth-generation CAR constructs have been developed to target tumor cells and additionally release cytokines that can contribute to the cytotoxicity of T-cells. The manufacturing process, including the use of cytokines in the expansion and differentiation of T cells, is also discussed. Finally, the clinical aspects and the influence of cytokines on the clinical condition of patients, such as cytokine release syndrome, who receive treatment with CAR-T cells are addressed. Therefore, this review aims to highlight how important cytokines are as one of the major players of cell therapy.

Low RECK Expression Is Part of the Cervical Carcinogenesis Mechanisms.

Human papillomavirus (HPV)-induced carcinogenesis comprises alterations in the expression and activity of matrix metalloproteinases (MMP) and their regulators. Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK) inhibits the activation of specific metalloproteinases and its expression is frequently lost in human cancers. Here we analyzed the role of RECK in cervical carcinogenesis. Cervical cancer derived cell lines over expressing RECK were used to determine tumor kinetics as well as, cellular, immune and molecular properties in vivo. Besides, we analyzed RECK expression in cervical cancer samples. RECK over expression (RECK+) delayed tumor growth and increased overall survival in vivo. RECK+ tumors displayed an increase in lymphoid-like inflammatory infiltrating cells, reduced number and viability of tumor and endothelial cells and lower collagenase activity. RECK+ tumors exhibited an enrichment of cell adhesion processes both in the mouse model and cervical cancer clinical samples. Finally, we found that lower RECK mRNA levels were associated with cervical lesions progression and worse response to chemotherapy in cervical cancer patients. Altogether, we show that increased RECK expression reduced the tumorigenic potential of HPV-transformed cells both in vitro and in vivo, and that RECK down regulation is a consistent and clinically relevant event in the natural history of cervical cancer.

Swainsonine, an alpha-mannosidase inhibitor, may worsen cervical cancer progression through the increase in myeloid derived suppressor cells population.

Cervical cancer, caused by high oncogenic risk Human Papillomavirus (HPV) infection, continues to be a public health problem, mainly in developing countries. Using peptide phage display as a tool to identify potential molecular targets in HPV associated tumors, we identified α-mannosidase, among other enriched sequences. This enzyme is expressed in both tumor and inflammatory compartment of the tumor microenvironment. Several studies in experimental models have shown that its inhibition by swainsonine (SW) led to inhibition of tumor growth and metastasis directly and indirectly, through activation of macrophages and NK cells, promoting anti-tumor activity. Therefore, the aim of this work was to test if swainsonine treatment could modulate anti-tumor immune responses and therefore interfere in HPV associated tumor growth. Validation of our biopanning results showed that cervical tumors, both tumor cells and leukocytes, expressed α-mannosidase. Ex vivo experiments with tumor associated macrophages showed that SW could partially modulate macrophage phenotype, decreasing CCL2 secretion and impairing IL-10 and IL-6 upregulation, which prompted us to proceed to in vivo tests. However, in vivo, SW treatment increased tumor growth. Investigation of the mechanisms leading to this result showed that SW treatment significantly induced the accumulation of myeloid derived suppressor cells in the spleen of tumor bearing mice, which inhibited T cell activation. Our results suggested that SW contributes to cervical cancer progression by favoring proliferation and accumulation of myeloid cells in the spleen, thus exacerbating these tumors systemic effects on the immune system, therefore facilitating tumor growth.

Local and systemic immunomodulatory mechanisms triggered by Human Papillomavirus transformed cells: a potential role for G-CSF and neutrophils.

Cervical cancer is the last stage of a series of molecular and cellular alterations initiated with Human Papillomavirus (HPV) infection. The process involves immune responses and evasion mechanisms, which culminates with tolerance toward tumor antigens. Our objective was to understand local and systemic changes in the interactions between HPV associated cervical lesions and the immune system as lesions progress to cancer. Locally, we observed higher cervical leukocyte infiltrate, reflected by the increase in the frequency of T lymphocytes, neutrophils and M2 macrophages, in cancer patients. We observed a strong negative correlation between the frequency of neutrophils and T cells in precursor and cancer samples, but not cervicitis. In 3D tumor cell cultures, neutrophils inhibited T cell activity, displayed longer viability and longer CD16 expression half-life than neat neutrophil cultures. Systemically, we observed higher plasma G-CSF concentration, higher frequency of immature low density neutrophils, and tolerogenic monocyte derived dendritic cells, MoDCs, also in cancer patients. Interestingly, there was a negative correlation between T cell activation by MoDCs and G-CSF concentration in the plasma. Our results indicate that neutrophils and G-CSF may be part of the immune escape mechanisms triggered by cervical cancer cells, locally and systemically, respectively.

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

BACKGROUND: Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. METHODS: First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. RESULTS: Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. CONCLUSIONS: These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Abstract Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody a potential biomarker of Bothrops jararacussu venom against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.(AU)