Neoadjuvant PD-1 blockade induces T cell and cDC1 activation but fails to overcome the immunosuppressive tumor associated macrophages in recurrent glioblastoma

Primary brain tumors, such as glioblastoma (GBM), are remarkably resistant to immunotherapy, even though pre-clinical models suggest effectiveness. To understand this better in patients, here we take advantage of our recent neoadjuvant treatment paradigm to map the infiltrating immune cell landscape of GBM and how this is altered following PD-1 checkpoint blockade using high dimensional proteomics, single cell transcriptomics, and quantitative multiplex immunofluorescence. Neoadjuvant PD-1 blockade increases T cell infiltration and the proportion of a progenitor exhausted population of T cells found within the tumor. We identify an early activated and clonally expanded CD8+ T cell cluster whose TCR overlaps with a CD8+ PBMC population. Distinct changes are also observed in conventional type 1 dendritic cells that may facilitate T cell recruitment. Macrophages and monocytes still constitute the majority of infiltrating immune cells, even after anti-PD-1 therapy. Interferon-mediated changes in the myeloid population are consistently observed following PD-1 blockade; these also mediate an increase in chemotactic factors that recruit T cells. However, sustained high expression of T-cell-suppressive checkpoints in these myeloid cells continue to prevent the optimal activation of the tumor infiltrating T cells. Therefore, future immunotherapeutic strategies may need to incorporate the targeting of these cells for clinical benefit.

The Recruitment of Neutrophils to the Tumor Microenvironment Is Regulated by Multiple Mediators

Neutrophils sense and migrate towards chemotactic factors released at sites of infection/inflammation and contain the affected area using a variety of effector mechanisms. Aside from these established immune defense functions, neutrophils are emerging as one of the key tumor-infiltrating immune cells that influence cancer progression and metastasis. Neutrophil recruitment to the tumor microenvironment (TME) is mediated by multiple mediators including cytokines, chemokines, lipids, and growth factors that are secreted from cancer cells and cancer-associated stromal cells. However, the molecular mechanisms that underlie the expression and secretion of the different mediators from cancer cells and how neutrophils integrate these signals to reach and invade tumors remain unclear. Here, we discuss the possible role of the epithelial to mesenchymal transition (EMT) program, which is a well-established promoter of malignant potential in cancer, in regulating the expression and secretion of these key mediators. We also summarize and review our current understanding of the machineries that potentially control the secretion of the mediators from cancer cells, including the exocytic trafficking pathways, secretory autophagy, and extracellular vesicle-mediated secretion. We further reflect on possible mechanisms by which different mediators collaborate by integrating their signaling network, and particularly focus on TGF-β, a cytokine that is highly expressed in invasive tumors, and CXCR2 ligands, which are crucial neutrophil recruiting chemokines. Finally, we highlight gaps in the field and the need to expand current knowledge of the secretory machineries and cross-talks among mediators to develop novel neutrophil targeting strategies as effective therapeutic options in the treatment of cancer.

Role of Inflammatory Mediators, Macrophages, and Neutrophils in Glioma Maintenance and Progression: Mechanistic Understanding and Potential Therapeutic Applications

Gliomas are the most common, highly malignant, and deadliest forms of brain tumors. These intra-cranial solid tumors are comprised of both cancerous and non-cancerous cells, which contribute to tumor development, progression, and resistance to the therapeutic regime. A variety of soluble inflammatory mediators (e.g., cytokines, chemokines, and chemotactic factors) are secreted by these cells, which help in creating an inflammatory microenvironment and contribute to the various stages of cancer development, maintenance, and progression. The major tumor infiltrating immune cells of the tumor microenvironment include TAMs and TANs, which are either recruited peripherally or present as brain-resident macrophages (microglia) and support stroma for cancer cell expansion and invasion. These cells are highly plastic in nature and can be polarized into different phenotypes depending upon different types of stimuli. During neuroinflammation, glioma cells interact with TAMs and TANs, facilitating tumor cell proliferation, survival, and migration. Targeting inflammatory mediators along with the reprogramming of TAMs and TANs could be of great importance in glioma treatment and may delay disease progression. In addition, an inhibition of the key signaling pathways such as NF-κB, JAK/STAT, MAPK, PI3K/Akt/mTOR, and TLRs, which are activated during neuroinflammation and have an oncogenic role in glioblastoma (GBM), can exert more pronounced anti-glioma effects.

Neoadjuvant PD-1 blockade induces T cell and cDC1 activation but fails to overcome the immunosuppressive tumor associated macrophages in recurrent glioblastoma

Primary brain tumors, such as glioblastoma (GBM), have been remarkably resistant to immunotherapy, even though pre-clinical models suggest effectiveness. To understand this better in patients, we took advantage of our recent neoadjuvant treatment paradigm to map the infiltrating immune cell landscape of GBM and how this is altered following PD-1 checkpoint blockade using high dimensional proteomics, single cell transcriptomics, and quantitative multiplex immunofluorescence. Neoadjuvant PD-1 blockade increased T cell infiltration and proportion of a progenitor exhausted population of T cells found within the tumor. We identified an early activated and clonally expanded CD8+ T cell cluster whose TCR overlapped with a CD8+ PBMC population. Distinct changes were also observed in conventional type 1 dendritic cells that may facilitate T cell recruitment. Macrophages and monocytes still constituted the majority of infiltrating immune cells, even after anti-PD-1 therapy. Interferon-mediated changes in the myeloid population were consistently observed following PD-1 blockade; these also mediated an increase in chemotactic factors that recruit T cells. However, sustained high expression of T-cell-suppressive checkpoints in these myeloid cells continued to prevent the optimal activation of the tumor infiltrating T cells. Therefore, future immunotherapeutic strategies may need to incorporate the targeting of these cells for clinical benefit.

Roles of CCL2-CCR2 Axis in the Tumor Microenvironment

Chemokines are a small family of cytokines that were first discovered as chemotactic factors in leukocytes during inflammation, and reports on the relationship between chemokines and cancer progression have recently been increasing. The CCL2-CCR2 axis is one of the major chemokine signaling pathways, and has various functions in tumor progression, such as increasing tumor cell proliferation and invasiveness, and creating a tumor microenvironment through increased angiogenesis and recruitment of immunosuppressive cells. This review discusses the roles of the CCL2-CCR2 axis and the tumor microenvironment in cancer progression and their future roles in cancer therapy.

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.

Malnutrition Aggravates Alterations Observed in the Gut Structure and Immune Response of Mice Infected with Leishmania infantum

Malnutrition is a risk factor for developing visceral leishmaniasis and its severe forms. Our group demonstrated that malnourished animals infected with Leishmania infantum had severe atrophies in lymphoid organs and T cell subpopulations as well as altered levels of thymic and splenic chemotactic factors, all of which resulted in dysfunctional lymphoid microenvironments that promoted parasite proliferation. Here, we hypothesize that malnutrition preceding parasite infection leads to structural and immunological changes in the gut mucosae, resulting in a failure in the immune response sensed in the intestine. To evaluate this, we analyzed the immunopathological events resulting from protein malnutrition in the guts of BALB/c mice infected with L. infantum. We observed lymphocytic/lymphoplasmacytic inflammatory infiltrates and lymphoid hyperplasia in the duodenum of well-nourished-infected mice; such alterations were worsened when malnutrition preceded infection. Parasite infection induced a significant increase of duodenal immunoglobulin A (IgA) of well-nourished animals, but those levels were significantly decreased in malnourished-infected mice. In addition, increased levels of Th17-related cytokines in duodenums of malnourished animals supported local inflammation. Together, our results suggest that the gut plays a potential role in responses to L. infantum infection—and that such responses are impaired in malnourished individuals.

Complement Proteins as Soluble Pattern Recognition Receptors for Pathogenic Viruses

The complement system represents a crucial part of innate immunity. It contains a diverse range of soluble activators, membrane-bound receptors, and regulators. Its principal function is to eliminate pathogens via activation of three distinct pathways: classical, alternative, and lectin. In the case of viruses, the complement activation results in effector functions such as virion opsonisation by complement components, phagocytosis induction, virolysis by the membrane attack complex, and promotion of immune responses through anaphylatoxins and chemotactic factors. Recent studies have shown that the addition of individual complement components can neutralise viruses without requiring the activation of the complement cascade. While the complement-mediated effector functions can neutralise a diverse range of viruses, numerous viruses have evolved mechanisms to subvert complement recognition/activation by encoding several proteins that inhibit the complement system, contributing to viral survival and pathogenesis. This review focuses on these complement-dependent and -independent interactions of complement components (especially C1q, C4b-binding protein, properdin, factor H, Mannose-binding lectin, and Ficolins) with several viruses and their consequences.

Novel method to analyze cell kinetics for the rapid diagnosis and determination of the causative agent in allergy

Drug-induced allergy (DIA), an unexpectedly triggered side effect of drugs used for therapeutic purposes, is a serious clinical issue that needs to be resolved because it interrupts the treatment of the primary disease. Since conventional allergy testing is insufficient to accurately predict the occurrence of DIA or to determine the drugs causing it, the development of diagnostic and predictive tools for allergic reactions is important. We demonstrated a novel method, termed high-sensitive allergy test (HiSAT), for the rapid diagnosis of allergy (within 1 hr; with true-positive diagnosis rates of 89% and 9% for patients with and without allergy-like symptoms, respectively). HiSAT analyzes the cell kinetics as an index against chemotactic factors in a patient’s serum, as different from the diagnosis using conventional methods. Once allergy has occurred, HiSAT can be used to determine the causative medicine using culture supernatants incubated with the subject’s lymphocytes and the test allergen. This test is more efficient (60%) than the lymphocyte transformation test (20%). Furthermore, in HiSAT, cell mobility significantly increases in a dose-dependent manner against supernatant incubated with lymphocytes from a subject with pollinosis collected at a time when the subject is without allergic symptoms and the antigen. The result demonstraed that HiSAT might be a promising method to rapidly diagnose DIA or to determine with high accuracy the antigen causing allergy.

Intrapulmonary vaccination with delta-inulin adjuvant stimulates non-polarised chemotactic signalling and diverse cellular interaction

There is an urgent need for novel vaccination strategies to combat respiratory pathogens. Mucosal vaccine delivery is an attractive option as it directly targets the site of infection; however, preclinical development has been hindered by a lack of suitable mucosal adjuvants and a limited understanding of their immune effects in the lung environment. Herein, we define the early immune events following the intrapulmonary delivery of a vaccine incorporating the adjuvant delta-inulin. Analysis of the early inflammatory response showed vaccine-induced innate cell recruitment to lungs and local lymph nodes (LN) was transient and non-polarised, correlating with an increase in pulmonary chemotactic factors. Use of fluorescently labelled adjuvant revealed widespread tissue dissemination of adjuvant particles, coupled with broad cellular uptake and transit to the lung-draining LN by a range of innate immune cells. Mass cytometric analysis revealed extensive phenotypic changes in innate and adaptive cell subsets induced by vaccination; this included identification of unconventional lymphocytes such as γδ-T cells and MAIT cells that increased following vaccination and displayed an activated phenotype. This study details a comprehensive view of the immune response to intrapulmonary adjuvant administration and provides pre-clinical evidence to support delta-inulin as a suitable adjuvant for pulmonary vaccines.