Cytotoxic Cytokine Production Research Articles

Expression of CD39 is associated with T cell exhaustion in ovarian cancer and its blockade reverts T cell dysfunction

ABSTRACT Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

Abstract LB217: Ascorbic acid rescues antitumor immune response by reversing hypoxia-induced CD8+T cell dysfunction

Abstract Growing evidence suggests that hypoxia is one of the most important phenomena of solid tumors that supports tumor progression as well as therapeutic resistance. When T cells infiltrate the tumor microenvironment (TME), they encounter hypoxia and, in this condition, cells upregulate an evolutionary conserved Hypoxia-inducible factor 1 (HIF-1) transcription factor to adapt to the hypoxic stress. Hypoxia has been reported to alter specific aspects of CD8+ T cell biology through various mechanisms but in large part by the HIF1-α, leading to immune suppression. Therefore, identification and development of drugs that target hypoxia is critical to augment immune cells' function and enhance the anti-tumor response of immunotherapy. Over the past decades, several cellular and pre-clinical studies have shown that ascorbic acid (AA) possesses anti-tumor properties and acts as an adjuvant to various cancer therapies. AA, as a proven redox potential modulator, has proved to also be effective in targeting oxygen sensing regulator (reducing Hif1-α levels) and epigenetic reprogramming (increasing ten-eleven translocation (TET) methylcytosine dioxygenase enzymatic activity) in cancer cells. Most recently, immunomodulating potential of AA has been recognized as it enhanced the efficacy of checkpoint blockade by increasing immune cell infiltration within the TME. However, the exact mechanisms by which AA exerts immunotherapeutic effects in cancer have not been fully understood. Here, we hypothesized that AA exerts its immunotherapeutic effects by targeting tumor-induced hypoxic stress in CD8 T cells. Indeed, we found that AA induces a robust anti-tumor response by decreasing tumor volume and increasing mice survival. Immune profiling in the TME revealed that AA increases the infiltration of CD8+ T cells with enhanced cytotoxic cytokine production and proliferation. To further evaluate the underlying mechanism, we challenged CD8+ T cells with a cognate antigen ex vivo under the hypoxic/normoxic conditions. We found that hypoxia negatively affected the effector functions and proliferation of CD8 T cells. Interestingly, AA could reverse these effects, bringing the function and proliferation of cells back to the normoxic level by increasing the expression of Granzyme B, Interferon gamma and Ki-67. This reversal of hypoxia-induced adverse effects on CD8 T cells was achieved by AA-mediated decrease in the Hif1-α levels (a hypoxia mediator) and increase in TET-2 protein expression and function (characterized by increased 5-hydroxymethylcytosine (5-hmc) levels). Moreover, AA treatment could significantly enhance the metabolic fitness of CD8+ T cells (characterized by improved glycolytic ability and mitochondrial function) that were exposed to severe hypoxia. In conclusion, our data indicate that AA treatment shows enhanced anti-tumor effects via restoring cytotoxic T cell functions under hypoxic TME. These results also provide new insights into the immunomodulatory effects of AA via targeting hypoxia in CD8 T cells. Given that hypoxia in the TME is a barrier for generation of effective immune response, our results suggest that AA treatment is an effective strategy against cancer and may further enhance the anti-tumor effects of immunotherapeutic agents. Citation Format: Nazli Jafarzadeh, Pankaj Gaur, Dareen Sarhan, Nour Shobaki, Wael Traboulsi, Zainab Ramlaoui, Vivek Verma, Seema Gupta, Samir N. Khleif. Ascorbic acid rescues antitumor immune response by reversing hypoxia-induced CD8+T cell dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB217.

Abstract 6395: Butyrophilins 3A as an immune checkpoint and a therapeutic target in hematological malignancies

Abstract Introduction: Butyrophilins (BTN) are a family of immune regulators in the immunoglobin superfamily, structurally similar to the B7 family. BTN3A, highly expressed in a variety of human cancer cells, suppresses αβ T cell activation by preventing CD45 segregation and immune synapse formation. Human BTN3A has three isoforms with almost identical extracellular domains, but each has unique cytoplasmic tail. BTN3A is highly expressed in a variety of human cancer cells. Therefore, we hypothesize that blocking BTN3A can remove the immunosuppressive effects of cancer cells and therefore enhance the tumor killing effects of immune cells. Methods: The expression of BTN3A on primary human immune cells was examined by flow cytometry. Human BTN3A2-IgG Fc fusion protein was generated in the Drosophila expression system. BALB/c wild type mice were immunized with the fusion protein and hybridomas were generated. High-affinity monoclonal antibodies (mAb) were screened and purified. NIH 3T3 cells transfected with human BTN3A1, BTN3A2 or BTN3A3 were used to examine if the purified mAbs can recognize each isoform on cell surface. Artificial CHO antigen-presenting cells (APC CHO cells) were transfected with or without hBTN3A2, and then co-cultured with human primary T cells in the presence of mouse IgG isotype or anti-hBTN3A2 mAb. A mixed lymphocyte reaction with different donor dendritic cells and T cells was performed with isotype or anti-hBTN3A mAb. The hBTN3A2 mRNA expression in a variety of tumor cell lines was screened in a gene database, and the protein expression of selected tumor cell lines was examined by flow cytometry with anti-hBTN3A mAb. Findings: Multiple tumor cell lines, originating from hematologic malignancies, carcinomas, or sarcomas, showed high cell surface expression of hBTN3A by flow cytometry. Two clones of high-affinity, anti-hBTN3A mAb were generated and purified. Both are IgG1 isotypes. Anti-hBTN3A mAb bound to BTN3A1, BTN3A2 or BTN3A3 expressed on NIH 3T3 cells. The majority of human immune cells isolated from peripheral blood from healthy donors expressed hBTN3A, including CD4 and CD8 T cells, B cells, monocytes, natural killer cells and γδ T cells. BTN3A2 expressing APC CHO cells inhibited primary αβ T cell activation, and anti-BTN3A mAb restored T cell proliferation as well as stimulated cytotoxic cytokine production in some healthy donors. Conclusion: BTN3A is an immunosuppressive receptor that inhibits αβ T cell activation. Anti-hBTN3A mAb restores T cell activation, suggesting the potential for BTN3A to be a target for cancer immunotherapy. Citation Format: Yixian Li, Xiaoxin Ren, Yao Wei, Mou Peng, Hao Wang, Marc Christopher Pulanco, Christopher David Nishimura, Philip Galbo, Anne Madsen, R. Alejandro Sica, Xingxing Zang. Butyrophilins 3A as an immune checkpoint and a therapeutic target in hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6395.

New kid on the block: does histamine get along with inflammation in amyotrophic lateral sclerosis?

Results from amyotrophic lateral sclerosis (ALS) patients and pre-clinical studies strongly suggest that systemic and CNS-intrinsic immune activation plays a central role in ALS pathogenesis. Microglial cells are emerging in this context as master regulators with a bi-functional role in the progression of the pathological response. They foster a pro-inflammatory setting through the production of cytotoxic cytokines and chemokines (M1 phenotype), after an aborted effort to sustain an anti-inflammatory environment for motor neurons through the release of beneficial cytokines and growth factors (M2 phenotype). In this review, we gather information meant to propose that histamine and ATP, which are released from mast cells, microglia and damaged neurons at sites of injury where they function as transmitters, have to be considered as new players in the ALS neuroinflammatory arena. After all, abnormal histamine and ATP signalling in the brain are already documented in neurodegenerative/neuroinflammatory conditions such as multiple sclerosis, Alzheimer and Parkinson's disease and, at present, histamine- as well as ATP-related compounds are in clinical trial for these same pathologies. Concerning ALS, while emerging data are now available about purinergic mechanisms, the involvement of histamine is basically unexplored. The circumstantial evidence that we present here thus constitutes a solid background for formulating novel hypotheses, stimulating a scientific debate and, most of all, inspiring future research. We deem that a new potential role of histamine in the setting of ALS neuroinflammation might find a fertile ground where to thrive. ALS is still a disease without a cure: why not to play with a new kid on the block?

Fluoride toxicity and new-onset diabetes in Finland: a hypothesis

The incidence of type 1 diabetes (T1D) has increased substantially in Finland, but the exact trigger for the onset of T1D is still unknown. We know that use of amoxicillin and anti-cariogenic fluoride tablets is a common practice for children in Finland. It seems that beta-cell destruction is initiated by modification of the proinsulin by combined effects of fluoride (F2) and amoxicillin. Amoxicillin especially when used together with clavulanic acid results in an acid environment around the beta-cells that promotes the conversion of F2 to hydrogen fluoride (HF). Unlike F2, HF can diffuse easily into the beta-cell cytosol. Because the cytosol has a neutral pH, virtually all HF reverts to F2 in the cytosol and F2 cannot easily diffuse out of the cell. Exposure to excess F2 promotes proinsulin covalent dimerization and simultaneously hyperexpression of MHC Class I molecules. Proinsulin dimers then migrate to the cell membrane with MHC class I molecules, accumulate at the beta-cell membrane and produces a powerful immunogenic stimulus for the cytotoxic T-cells. Production of cytotoxic cytokines from the infiltrating T-cells initiates the destruction of beta-cells. In Finnish children, this might be helped along by a higher beta-cell activity and by a reactive thymus-dependent immune system induced by higher levels of thyroid hormones and calcitonin respectively. After repeated similar attacks, more and more effector T-cells are raised and more and more beta-cells are destroyed, and clinical diabetes occurs.

Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction

Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains. The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice. The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-β and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion. The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.

Hereditary hemochromatosis: An opportunity for gene therapy

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen: iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.

Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine production in human mononuclear cells.

Enzymatically depolymerized guluronate and mannuronate oligomers were prepared from polyuronates with an alginate lyase from a Pseudoalteromonas sp., and their effects on mononuclear cells from human peripheral blood were examined. Conditioned medium prepared by the incubation of cells with an untreated polyuronate had little effect on growth of human leukemic U937 cells, but a medium prepared with depolymerized uronate oligomers inhibited their growth. Inhibition was greater in a medium prepared with guluronate oligomer than one prepared with mannuronate oligomer. The cytotoxic activity of the medium was heat-labile and nondialyzable. Apoptotic nuclear morphological changes and increased caspase-3-like activity were found in U937 cells treated with a medium prepared with depolymerized uronates. The medium prepared with purified tetra-guluronate and tetra-mannuronate also was cytotoxic; these effects were inhibited by antibodies to tumor necrosis factor-alpha. Our results suggested that enzymatically depolymerized guluronate and mannuronate oligomers induced the production of cytotoxic cytokines in human mononuclear cells, although the uronate polymers before depolymerization had no such activity.