Development Of Novel Immune Therapies Research Articles

TEMPORAL CHANGES IN INNATE AND ADAPTIVE IMMUNITY DURING SEPSIS AS DETERMINED BY ELISPOT.

Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The enzyme-linked immunospot (ELISpot) assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture. Blood and spleens were harvested serially, and ex vivo interferon γ and TNF-α production were compared by ELISpot and enzyme-linked immunosorbent assay. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example, dexamethasone, arginine, and IL-7, in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and enzyme-linked immunosorbent assay results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. Primary human macrophages activated by IFNγ (M[IFNγ]) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M[IFNγ]i) and phagocytic (M[IFNγ]p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Adjuvant therapy for stage II melanoma: the need for further studies

Immunotherapy with checkpoint inhibitors has revolutionised the outcomes for melanoma patients. In the metastatic setting, patients treated with nivolumab and ipilimumab have an expected 5-year survival of> 50%. For patients with resected high-risk stage III disease, adjuvant pembrolizumab, nivolumab or dabrafenib and trametinib are associated with a significant improvement in both relapse-free survival (RFS) and distant metastasis-free survival (DMFS). More recently neoadjuvant immunotherapy has shown very promising outcomes in patients with clinically detectable nodal disease and is likely to become a new standard of care. For stage IIB/C disease, two pivotal adjuvant trials of pembrolizumab and nivolumab have also reported a significant improvement in both RFS and DMFS. However, the absolute benefit is low and there are concerns about the risk of severe toxicities as well as long-term morbidity from endocrine toxicity. Ongoing registration phase III trials are currently evaluating newer immunotherapy combinations and the role of BRAF/MEK-directed targeted therapy for stage II melanoma. However, our ability to personalise therapy based on molecular risk stratification has lagged behind the development of novel immune therapies. There is a critical need to evaluate the use of tissue and blood-based biomarkers, to better select patients that will recur and avoid unnecessary treatment for the majority of patients cured by surgery alone.

CD56−CD16+ NK cells from HIV-infected individuals negatively regulate IFN-γ production by autologous CD8+ T cells

The percentage of human CD56- CD16+ NK cells increases during chronic infection with human HIV; however, the biologic role of CD56- CD16+ NK cells in HIV infection is unclear. Our results demonstrate that the percentage of CD56- CD16+ NK cells producing IL-10 and TGF-β was higher than CD56dim CD16+ NK cells. CD56- CD16+ NK cells could inhibit IFN-γ production by autologous CD8+ T cells, and this inhibition could be partially reversed by anti-IL-10, anti-TGF-β, or anti-PD-L1 mAbs. CD56- CD16+ NK cells are potential targets for the development of novel immune therapies against HIV infection.

The Immunobiology of Interleukin-35 and Its Regulation and Gene Expression.

Interleukin-35 (IL-35) is the latest addition to the IL-12 family of heterodimeric cytokines, consisting of IL-12 p35 subunit and IL-27β subunit Epstein-Barr virus induced 3 (EBI3). Since its discovery, IL-35 has been shown to exhibit immunosuppressive activities which are distinct from other members of IL-12 family. IL-35 is also unique in that it is expressed primarily by regulatory T-cells (Tregs) rather than by antigen-presenting cells (APCs). IL-35 can directly suppress effector T-cell proliferation and function and inhibit the differentiation of Th17 cells. It is also able to expand regulatory responses to promote tolerance to infections by generating a potent population of IL-35-producing inducible Tregs (iTr35). As the new cellular sources of IL-35 such as CD8+Tregs, Bregs, and tolerogenic dendritic cells DCs (tolDCs) are identified, more immunoregulatory functions of this cytokine are explored. IL-35 has been shown to be associated with a range of autoimmune diseases and cancer models. It appears to be a promising diagnostic biomarker. A greater understanding of the expression and regulatory mechanisms of IL-35 will be beneficial to the development of novel immune therapies.

The Different Immunoregulatory Functions on Dendritic Cells between Mesenchymal Stem Cells Derived from Bone Marrow of Patients with Low-Risk or High-Risk Myelodysplastic Syndromes

Myelodysplastic syndrome (MDS) is a group of progressive,clonal, neoplastic bone marrow disorders characterized by hematopoietic stem cell dysregulation and abnormalities in the immune system. Mesenchymal stem cells (MSC) appear to modulate the immune system at the very first step of the immune response through the inhibition of dendritic cells (DCs) differentiation and maturation. However, it is still unclear whether the effects of MSC on the development of DCs will be altered with disease state. In addition, it is not clear whether there are differences in the effects between low-risk and high-risk MDS-MSC on DCs development. In this study, our data confirm that MDS-MSC mediate a potent inhibition of DCs differentiation. Additionaly, MDS-MSC greatly alter DCs functions, including endocytosis, IL-12 secretion, their ability to inhibit T cell proliferation. Moreover, our results show that there are major differences in DCs development and function between low-risk and high-risk MDS-MSC. Compared to high-risk MDS-MSC, low-risk MDS-MSC is characterized by a poor ability to inhibit DCs differentiation and maturation; and correspondingly, less dysfunctional DC endocytosis, mildly decreased IL-12 secretion, and a reduction in DC-mediated inhibition of T cell proliferation. Finally, our results demonstrate that MDS-MSC derived TGF-β1 is largely responsible for the inhitory effects. These results elucidate the different immunoregulatory role of MSC in low-risk and high-risk MDS on DCs development, which may be important for understanding the pathogenesis of MDS and the development of novel immune therapies for the treatment of MDS.

Development of novel immune therapies for solid tumors: phase I clinical trials in a single institute

Development of novel immune therapies for solid tumors: phase I clinical trials in a single institute

Role of immune responses in the pathogenesis of low‐risk MDS and high‐risk MDS: implications for immunotherapy

The myelodysplastic syndromes (MDS) constitute a group of heterogeneous clonal haemopoietic stem cell disorders, characterized by ineffective and dysplastic haematopoiesis with varying degrees of peripheral cytopenia. Low-risk MDS is characterized by increased apoptosis in the bone marrow (BM) with autoimmune characteristics whereas the advanced or high-risk stages involve immune evasion and secondary DNA damage, giving cells growth potential to progress into acute myeloid leukaemia (AML). Nevertheless, the causes of MDS remain poorly defined and it is not clear how the disease progresses from an early stage to advanced MDS and AML. Although there are clear indications for a role of the immune system, the exact mechanism by which the immune response contributes to the progression is not yet clear. New insights into the pathophysiology of MDS with regard to the immune system will be instrumental for the development of novel patient-oriented therapies. This review is focused on the role of immune responses in MDS and the implications for the development of novel immune therapies.

Anti-GD2 Antibody Therapy for GD2-Expressing Tumors

In the development of novel immune therapies for high-risk cancers, one goal is to find tumor targets that are not widely shared by normal cells. One such target is the surface disialoganglioside GD2. This antigen is expressed on the surface of a variety of tumors for which no curative therapies exist for patients with advanced disease. In childhood, the most common GD2-expressing tumor is neuroblastoma. GD2 is also expressed on several other high-risk tumors, including those of neuroectodermal or epithelial origin, virtually all melanomas, and approximately 50% of tumor samples from osteosarcoma and soft-tissue sarcomas. Because of the tumor-selective expression of this molecule, it is an attractive target for tumor-specific therapies such as antibody therapy. Over the last 2 decades, several anti-GD2 antibodies have been developed. To reduce both the toxicity of the antibody and the development of human anti-mouse antibodies (HAMA), research efforts have primarily focused on exploring anti-GD2 antibodies that have progressively more human elements while at the same time reducing the mouse components. This review will examine antibodies currently undergoing clinical testing as well as the most recent advances to improve antibody therapy for patients with GD2-expressing tumors.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4–16 h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3 -CD16 +CD56 +). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target ( E: T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Programmed death 1: a critical regulator of T-cell function and a strong target for immunotherapies for chronic viral infections

The intricate balance between positive and negative signals delivered by accessory molecules is crucial to generate efficient immune responses while maintaining tolerance and preventing autoimmunity. Of these molecules, programmed death 1 has been described as a negative regulator of T-cell activation. This review will focus on current knowledge about PD-1 regulation in different diseases and discuss its potential benefits for the development of novel immune therapies. PD-1 has recently been shown to be upregulated on HIV-specific CD8 T cells, whereas the PD-1 expression level was significantly correlated with viral load. Blockade of the PD-1/PD-L1 interaction enhanced the capacity of HIV-specific CD8 and CD4 T cells to proliferate or secrete cytokines and cytotoxic molecules. Future manipulations of this pathway could rescue the function of exhausted CD8 and CD4 T cells. The engagement of PD-1 with its ligands induces inhibitory signals as it blocks T-cell receptor-induced T-cell proliferation and cytokine production. The PD-1 pathway plays a crucial role in the maintenance of peripheral tolerance and the pathogenesis of cancer and chronic viral infections. Understanding the mechanisms by which PD-1 interferes with T-cell functions will pave the way for novel therapeutic immune interventions to treat these diseases.

Drug resistance mutations and the cellular immune response: a valuable synergy for the development of novel immune therapies

The escape of HIV-1 is a cardinal feature of the virus and a major hindrance to the development of effective therapeutic strategies. In highly active antiretroviral therapy-treated patients, the virus is subjected to selective pressures from cellular immune response directed against the viral proteome and antiretroviral treatment targetting a few genes of the HIV-1 genome. This review will focus on the relationship between these two pressures and its potential advantage in the development of novel immune therapies. Recent studies have investigated the conflicting selective forces between viral fitness and escape to immunological and therapeutic pressures in natural HIV infection and the SIV model. Simultaneous pressures driven by cytotoxic T lymphocytes and highly active antiretroviral therapy could potentially reduce viral fitness, leading to better control of the viral load. Two studies have described a potential therapeutic vaccine strategy against viral escape mutant epitopes from reverse transcriptase inhibitors. The emergence of multidrug-resistant viruses is associated with enhanced T-cell-mediated immune response as a possible consequence of reduced viral fitness. Amino acid substitutions generate potential cytotoxic T-lymphocyte epitopes that may elicit new reactivities against mutated viruses. Both could significantly enhance the immune response through direct and indirect mechanisms.