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Therapeutics against coronavirus disease 2019 (COVID-19) are urgently needed. Granulocyte–macrophage colony-stimulating factor (GM-CSF), a myelopoietic growth factor and pro-inflammatory cytokine, plays a critical role in alveolar macrophage homeostasis, lung inflammation and immunological disease. Both administration and inhibition of GM-CSF are currently being therapeutically tested in COVID-19 clinical trials. This Perspective discusses the pleiotropic biology of GM-CSF and the scientific merits behind these contrasting approaches.Recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) as well as antibodies targeted at GM-CSF or its receptor are being tested in clinical trials for coronavirus disease 2019 (COVID-19). This Perspective introduces the pleiotropic functions of GM-CSF and explores the rationale behind these different approaches.

Key Points Alzheimer disease (AD), like other proteinopathic neurodegenerative disorders, is characterized by the accumulation of amyloidogenic proteins A neuroinflammatory component in AD has been known for more than a decade; however, the importance of the contribution of inflammation in the pathogenesis of AD has been appreciated only recently. Genetic and bioinformatic data from individuals with AD and insights from preclinical models now substantiate the present view that inflammation participates in and exacerbates AD pathology. Neuroinflammation in AD is primarily driven by the brain's intrinsic myeloid cells (known as microglia) and escalates with disease progression; thus AD-associated neuroinflammation contrasts with traditionally defined neuroinflammatory diseases such as multiple sclerosis and encephalitides, which are mainly driven by blood-derived leukocytes and B and T lymphocytes, invading the CNS. Manipulation of some of the molecules of the innate immune system or their respective pathways in animal models of AD has resulted in substantial alteration of disease pathology, indicating the potential to ameliorate the disease course through targeting components of the immune system. The immune system thus appears to provide exciting novel and accessible targets for the diagnosis, control and treatment of AD; however, precise knowledge about specific and defined immune events, which may change during the disease course or differ among individuals with AD, is required. Diagnostics research needs to develop sensitive methods to detect immune alterations prior to the onset of AD to identify those patients at risk who may benefit most from specific, tailored anti-inflammatory interventions. It is now emerging that the neuroinflammation that is associated with Alzheimer disease may have a key role in driving this disease. In this Review, Heppner, Ransohoff and Becher examine the contribution of the immune system to the pathogenesis of this disorder. The past two decades of research into the pathogenesis of Alzheimer disease (AD) have been driven largely by the amyloid hypothesis; the neuroinflammation that is associated with AD has been assumed to be merely a response to pathophysiological events. However, new data from preclinical and clinical studies have established that immune system-mediated actions in fact contribute to and drive AD pathogenesis. These insights have suggested both novel and well-defined potential therapeutic targets for AD, including microglia and several cytokines. In addition, as inflammation in AD primarily concerns the innate immune system — unlike in 'typical' neuroinflammatory diseases such as multiple sclerosis and encephalitides — the concept of neuroinflammation in AD may need refinement.

The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis. This protocol describes the design, application and computational analysis of high-dimensional fluorescent antibody panels for flow cytometry. Up to 28 colors can be characterized to study complex cellular populations such as the immune system.

The invasion of immune cells into the central nervous system (CNS) is a hallmark of the process we call neuroinflammation. Diseases such as encephalitides or multiple sclerosis (MS) are characterised by the dramatic influx of T lymphocytes and monocytes. The communication between inflammatory infiltrates and CNS resident cells is primarily mediated through cytokines. Over the years, numerous cytokine networks have been assessed to better understand the development of immunopathology in neuroinflammation. In MS for instance, many studies have shown that CD4 + T cells infiltrate the CNS and subsequently lead to immunopathology. Inflammatory CD4 + T cells, such as T H 1, T H 17, GM-CSF-producing helper T cells are big players in chronic neuroinflammation. Conversely, encephalitogenic or meningeal regulatory T cells (T REGs ) and T H 2 cells have been shown to drive a decrease in inflammatory functions in microglial cells and thus promote a neuroprotective microenvironment. Recent studies report overlapping as well as differential roles of these cells in tissue inflammation. Taken together, this suggests a more complex relationship between effector T cell subsets in neuroinflammation than has hitherto been established. In this overview, we review the interplay between helper T cell subsets infiltrating the CNS and how they actively contribute to neuroinflammation and degeneration. Importantly, in this context, we will especially focus on the current knowledge regarding the contribution of various helper cell subsets to neuroinflammation by referring to their helper T cell profile in the context of their target cell.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4 + T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM. Glioblastoma multiform (GBM) is a common and aggressive type of primary brain cancer that currently has no effective therapy. Here, the authors show, using a mouse GBM model and EGFRvIII-targeting chimeric antigen receptor (CAR)-T cells, that Intratumoral injection of interleukin-12 helps condition the microenvironment and promote anti-tumor immunity.

Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy. Tumor-draining lymph nodes are often the first site of metastasis in breast cancer patients. Here, the authors show that metastatic lymph nodes are characterized by the accumulation of suppressive regulatory T cells with a distinct phenotype compared to matched non-invaded lymph nodes and tumors.

Among many populations of blood cells, high dimensional analysis using mass cytometry reveals classical monocyte frequency as strong predictors of response to PD-1 blockade therapy of melanoma. Immune-checkpoint blockade has revolutionized cancer therapy. In particular, inhibition of programmed cell death protein 1 (PD-1) has been found to be effective for the treatment of metastatic melanoma and other cancers. Despite a dramatic increase in progression-free survival, a large proportion of patients do not show durable responses. Therefore, predictive biomarkers of a clinical response are urgently needed. Here we used high-dimensional single-cell mass cytometry and a bioinformatics pipeline for the in-depth characterization of the immune cell subsets in the peripheral blood of patients with stage IV melanoma before and after 12 weeks of anti-PD-1 immunotherapy. During therapy, we observed a clear response to immunotherapy in the T cell compartment. However, before commencing therapy, a strong predictor of progression-free and overall survival in response to anti-PD-1 immunotherapy was the frequency of CD14 + CD16 − HLA-DR hi monocytes. We confirmed this by conventional flow cytometry in an independent, blinded validation cohort, and we propose that the frequency of monocytes in PBMCs may serve in clinical decision support.

When helper T (T H ) cell polarization was initially described three decades ago, the T H cell universe grew dramatically. New subsets were described based on their expression of few specific cytokines. Beyond T H 1 and T H 2 cells, this led to the coining of various T H 17 and regulatory (T reg ) cell subsets as well as T H 22, T H 25, follicular helper (T FH ), T H 3, T H 5 and T H 9 cells. High-dimensional single-cell analysis revealed that a categorization of T H cells into a single-cytokine-based nomenclature fails to capture the complexity and diversity of T H cells. Similar to the simple nomenclature used to describe innate lymphoid cells (ILCs), we propose that T H cell polarization should be categorized in terms of the help they provide to phagocytes (type 1), to B cells, eosinophils and mast cells (type 2) and to non-immune tissue cells, including the stroma and epithelium (type 3). Studying T H cells based on their helper function and the cells they help, rather than phenotypic features such as individual analyzed cytokines or transcription factors, better captures T H cell plasticity and conversion as well as the breadth of immune responses in vivo. The T helper subset paradigm has been instrumental in informing our understanding of T cell diversity; however, modern single-cell analyses have revealed the limits of the concept. In their Perspective, Becher and colleagues propose an alternative framework in which to understand T helper diversity, based not on transcription factors and cytokines but rather physiological functionality.

Almost every disorder of the CNS is said to have an inflammatory component, but the precise nature of inflammation in the CNS is often imprecisely defined, and the role of CNS-resident cells is uncertain compared with that of cells that invade the tissue from the systemic immune compartment. To understand inflammation in the CNS, the term must be better defined, and the response of tissue to disturbances in homoeostasis (eg, neurodegenerative processes) should be distinguished from disorders in which aberrant immune responses lead to CNS dysfunction and tissue destruction (eg, autoimmunity). Whether the inflammatory tissue response to injury is reparative or degenerative seems to be dependent on context and timing, as are the windows of opportunity for therapeutic intervention in inflammatory CNS diseases.

Extracorporeal photopheresis has been an effective treatment for graft-versus-host disease. In this case, it was used to treat a patient with severe autoimmune colitis induced by checkpoint-inhibitor therapy for melanoma. The extracorporeal photopheresis resulted in an increased number of immunoregulatory natural killer cells.