GM-CSF-mediated epithelial-immune cell crosstalk orchestrates pulmonary immunity to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening mold pneumonia in immune compromised patients, particularly in those with quantitative or qualitative defects in neutrophils. While innate immune cell crosstalk licenses neutrophil antifungal activity in the lung, the role of epithelial cells in this process is unknown. Here, we find that that surfactant protein C (SPC)-expressing lung epithelial cells integrate infection-induced IL-1 and type III interferon signaling to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) preferentially at local sites of fungal infection and neutrophil influx. Using in vivo models that distinguish the role of GM-CSF during acute infection from its homeostatic function in alveolar macrophage survival and surfactant catabolism, we demonstrate that epithelial-derived GM-CSF increases the accumulation and fungicidal activity of GM-CSF-responsive neutrophils, with the latter being essential for host survival. Our findings establish SPC + epithelial cells as a central player in regulating the quality and strength of neutrophil-dependent immunity against inhaled mold pathogens. HIGHLIGHTS: GM-CSF is essential for host defense against A. fumigatus in the lung IL-1 and IFN-λ promote GM-CSF production by lung epithelial cells in parallelEpithelial cell-derived GM-CSF increases neutrophil accumulation and fungal killing capacityEpithelial cells preferentially upregulate GM-CSF in local sites of inflammation.

IL-12 sensing in neurons induces neuroprotective CNS tissue adaptation and attenuates neuroinflammation in mice.

Interleukin-12 (IL-12) is a potent driver of type 1 immunity. Paradoxically, in autoimmune conditions, including of the CNS, IL-12 reduces inflammation. The underlying mechanism behind these opposing properties and the involved cellular players remain elusive. Here we map IL-12 receptor (IL-12R) expression to NK and T cells as well as neurons and oligodendrocytes. Conditionally ablating the IL-12R across these cell types in adult mice and assessing their susceptibility to experimental autoimmune encephalomyelitis revealed that the neuroprotective role of IL-12 is mediated by neuroectoderm-derived cells, specifically neurons, and not immune cells. In human brain tissue from donors with multiple sclerosis, we observe an IL-12R distribution comparable to mice, suggesting similar mechanisms in mice and humans. Combining flow cytometry, bulk and single-nucleus RNA sequencing, we reveal an IL-12-induced neuroprotective tissue adaption preventing early neurodegeneration and sustaining trophic factor release during neuroinflammation, thereby maintaining CNS integrity in mice.

Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors.

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

Age-induced alterations of granulopoiesis generate atypical neutrophils that aggravate stroke pathology.

Aging accounts for increased risk and dismal outcome of ischemic stroke. Here, we investigated the impact of age-related changes in the immune system on stroke. Upon experimental stroke, compared with young mice, aged mice had increased neutrophil clogging of the ischemic brain microcirculation, leading to worse no-reflow and outcomes. Aged mice showed an enhanced granulopoietic response to stroke that led to the accumulation of CD101+CD62Llo mature and CD177hiCD101loCD62Llo and CD177loCD101loCD62Lhi immature atypical neutrophils in the blood, endowed with increased oxidative stress, phagocytosis and procoagulant features. Production of CXCL3 by CD62Llo neutrophils of the aged had a key role in the development and pathogenicity of aging-associated neutrophils. Hematopoietic stem cell rejuvenation reverted aging-associated neutropoiesis and improved stroke outcome. In elderly patients with ischemic stroke, single-cell proteome profile of blood leukocytes identified CD62Llo neutrophil subsets associated with worse reperfusion and outcome. Our results unveil how stroke in aging leads to a dysregulated emergency granulopoiesis impacting neurological outcome.

CD4+ T cells produce GM-CSF and drive immune-mediated glomerular disease by licensing monocyte-derived cells to produce MMP12.

GM-CSF in glomerulonephritisDespite glomerulonephritis being an immune-mediated disease, the contributions of individual immune cell types are not clear. To address this gap in knowledge, Paust et al. characterized pathological immune cells in samples from patients with glomerulonephritis and in samples from mice with the disease. The authors found that CD4+ T cells producing granulocyte-macrophage colony-stimulating factor (GM-CSF) licensed monocytes to promote disease by producing matrix metalloproteinase 12 and disrupting the glomerular basement membrane. Targeting GM-CSF to inhibit this axis reduced disease severity in mice, implicating this cytokine as a potential therapeutic target for patients with glomerulonephritis. -CM.

Preexisting comorbidities shape the immune response associated with severe COVID-19.

BACKGROUND: Comorbidities are risk factors for development of severe coronavirus disease 2019 (COVID-19). However, the extent to which an underlying comorbidity influences the immune response to severe acute respiratory syndrome coronavirus 2 remains unknown. OBJECTIVE: Our aim was to investigate the complex interrelations of comorbidities, the immune response, and patient outcome in COVID-19. METHODS: We used high-throughput, high-dimensional, single-cell mapping of peripheral blood leukocytes and algorithm-guided analysis. RESULTS: We discovered characteristic immune signatures associated not only with severe COVID-19 but also with the underlying medical condition. Different factors of the metabolic syndrome (obesity, hypertension, and diabetes) affected distinct immune populations, thereby additively increasing the immunodysregulatory effect when present in a single patient. Patients with disorders affecting the lung or heart, together with factors of metabolic syndrome, were clustered together, whereas immune disorder and chronic kidney disease displayed a distinct immune profile in COVID-19. In particular, severe acute respiratory syndrome coronavirus 2-infected patients with preexisting chronic kidney disease were characterized by the highest number of altered immune signatures of both lymphoid and myeloid immune branches. This overall major immune dysregulation could be the underlying mechanism for the estimated odds ratio of 16.3 for development of severe COVID-19 in this burdened cohort. CONCLUSION: The combinatorial systematic analysis of the immune signatures, comorbidities, and outcomes of patients with COVID-19 has provided the mechanistic immunologic underpinnings of comorbidity-driven patient risk and uncovered comorbidity-driven immune signatures.

IFNγ and GM-CSF control complementary differentiation programs in the monocyte-to-phagocyte transition during neuroinflammation.

During inflammation, Ly6Chi monocytes are rapidly mobilized from the bone marrow (BM) and are recruited into inflamed tissues, where they undergo monocyte-to-phagocyte transition (MTPT). The in vivo developmental trajectories of the MTPT and the contribution of individual cytokines to this process remain unclear. Here, we used a murine model of neuroinflammation to investigate how granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFNγ), two type 1 cytokines, controlled MTPT. Using genetic fate mapping, gene targeting and high-dimensional single-cell multiomics analyses, we found that IFNγ was essential for the gradual acquisition of a mature inflammatory phagocyte phenotype in Ly6Chi monocytes, while GM-CSF was required to license interleukin-1ß (IL-1ß) production, phagocytosis and oxidative burst. These results suggest that the proinflammatory cytokine environment guided MTPT trajectories in the inflamed central nervous system (CNS) and indicated that GM-CSF was the most prominent target for the disarming of monocyte progenies during neuroinflammation.

Twin study reveals non-heritable immune perturbations in multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system underpinned by partially understood genetic risk factors and environmental triggers and their undefined interactions1,2. Here we investigated the peripheral immune signatures of 61 monozygotic twin pairs discordant for MS to dissect the influence of genetic predisposition and environmental factors. Using complementary multimodal high-throughput and high-dimensional single-cell technologies in conjunction with data-driven computational tools, we identified an inflammatory shift in a monocyte cluster of twins with MS, coupled with the emergence of a population of IL-2 hyper-responsive transitional naive helper T cells as MS-related immune alterations. By integrating data on the immune profiles of healthy monozygotic and dizygotic twin pairs, we estimated the variance in CD25 expression by helper T cells displaying a naive phenotype to be largely driven by genetic and shared early environmental influences. Nonetheless, the expanding helper T cells of twins with MS, which were also elevated in non-twin patients with MS, emerged independent of the individual genetic makeup. These cells expressed central nervous system-homing receptors, exhibited a dysregulated CD25-IL-2 axis, and their proliferative capacity positively correlated with MS severity. Together, our matched-pair analysis of the extended twin approach allowed us to discern genetically and environmentally determined features of an MS-associated immune signature.

Single-cell profiling of immune system alterations in lymphoid, barrier and solid tissues in aged mice.

Aging exerts profound and paradoxical effects on the immune system, at once impairing proliferation, cytotoxicity and phagocytosis, and inducing chronic inflammation. Previous studies have focused on individual tissues or cell types, while a comprehensive multisystem study of tissue-resident and circulating immune populations during aging is lacking. Here we reveal an atlas of age-related changes in the abundance and phenotype of immune cell populations across 12 mouse tissues. Using cytometry-based high parametric analysis of 37 mass-cytometry and 55 spectral flow-cytometry parameters, mapping samples from young and aged animals revealed conserved and tissue-type-specific patterns of both immune atrophy and expansion. We uncovered clear phenotypic changes in both lymphoid and myeloid lineages in aged mice, and in particular a contraction in natural killer cells and plasmacytoid dendritic cells. These changes correlated with a skewing towards myelopoiesis at the expense of early lymphocyte genesis in aged mice. Taken together, this atlas represents a comprehensive, systematic and thorough resource of the age-dependent alterations of the mammalian immune system in lymphoid, barrier and solid tissues.

GM-CSF: Master regulator of the T cell-phagocyte interface during inflammation.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) was sequentially redefined during the past decades. Originally described as a hematopoietic growth factor for myelopoiesis, GM-CSF was recognized as a central mediator of inflammation bridging the innate and adaptive arms of the immune system. Phagocytes sensing GM-CSF adapt an inflammatory phenotype and facilitate pathogen clearance. However, in the context of chronic tissue inflammation, GM-CSF secreted by tissue-invading lymphocytes has detrimental effects by licensing tissue damage and hyperinflammation. Accordingly, therapeutic intervention at the T cell-phagocyte interface represents an attractive target to ameliorate disease progression and immunopathology. Although GM-CSF is largely dispensable for steady state myelopoiesis, dysregulation, as seen in chronic inflammatory diseases, may however lead to disrupted haematopoiesis and long-term effects on bone marrow output. Here, we will survey the role of GM-CSF during inflammation, discuss the extent to which GM-CSF-secreting T cells, debate their introduction as a separate T cell lineage and explore current and future clinical implications of GM-CSF in human disease settings.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

Single-cell profiling of myasthenia gravis identifies a pathogenic T cell signature.

Myasthenia gravis (MG) is an autoimmune disease characterized by impaired neuromuscular signaling due to autoantibodies targeting the acetylcholine receptor. Although its auto-antigens and effector mechanisms are well defined, the cellular and molecular drivers underpinning MG remain elusive. Here, we employed high-dimensional single-cell mass and spectral cytometry of blood and thymus samples from MG patients in combination with supervised and unsupervised machine-learning tools to gain insight into the immune dysregulation underlying MG. By creating a comprehensive immune map, we identified two dysregulated subsets of inflammatory circulating memory T helper (Th) cells. These signature ThCD103 and ThGM cells populated the diseased thymus, were reduced in the blood of MG patients, and were inversely correlated with disease severity. Both signature Th subsets rebounded in the blood of MG patients after surgical thymus removal, indicative of their role as cellular markers of disease activity. Together, this in-depth analysis of the immune landscape of MG provides valuable insight into disease pathogenesis, suggests novel biomarkers and identifies new potential therapeutic targets for treatment.

Intratumoral IL-12 delivery empowers CAR-T cell immunotherapy in a pre-clinical model of glioblastoma.

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.

Oncogenic KrasG12D causes myeloproliferation via NLRP3 inflammasome activation.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1ß axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1ß axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies.

Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas.

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and Treg expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB.

Graft-versus-host disease, but not graft-versus-leukemia immunity, is mediated by GM-CSF-licensed myeloid cells.

Allogeneic hematopoietic cell transplantation (allo-HCT) not only is an effective treatment for several hematologic malignancies but can also result in potentially life-threatening graft-versus-host disease (GvHD). GvHD is caused by T cells within the allograft attacking nonmalignant host tissues; however, these same T cells mediate the therapeutic graft-versus-leukemia (GvL) response. Thus, there is an urgent need to understand how to mechanistically uncouple GvL from GvHD. Using preclinical models of full and partial MHC-mismatched HCT, we here show that the granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by allogeneic T cells distinguishes between the two processes. GM-CSF drives GvHD pathology by licensing donor-derived phagocytes to produce inflammatory mediators such as interleukin-1ß and reactive oxygen species. In contrast, GM-CSF did not affect allogeneic T cells or their capacity to eliminate leukemic cells, retaining undiminished GvL responses. Last, tissue biopsies and peripheral blood mononuclear cells from patients with grade IV GvHD showed an elevation of GM-CSF-producing T cells, suggesting that GM-CSF neutralization has translational potential in allo-HCT.

Assessing the role of innovative therapeutic paradigm on multiple sclerosis treatment response.

OBJECTIVE: Within the last decade, many changes have been made to the management of patients with multiple sclerosis (MS). The aim of our study was to investigate the global impact of all these changes on the disease's course. MATERIALS AND METHODS: This single-centre study was carried out on patients with multiple sclerosis (pwMS) who started treatment with first-line disease-modifying therapies. We have compared three large cohorts of patients with MS diagnosis, for three consecutive periods within July 2001, August 2001-December 2005, and January 2006-September 2011. RESULTS: A total of 1068 relapsing-remitting pwMS cases were included. Patients in the last cohort began treatment earlier (P < 0.0001), started more frequent treatment with high-dose interferon beta or glatiramer acetate (P < 0.0001), and had experienced a more frequent treatment escalation strategy (P = 0.004) than patients in other cohorts. The multivariate analysis adjusted for baseline characteristics showed that pwMS of the last cohort had a high probability of showing no evidence of disease activity (NEDA3) at 4 years (OR 3.22, 95% CIs 1.89-5.47; P < 0.0001). These results were confirmed in a propensity score analysis. CONCLUSIONS: Our study showed an improvement over the last 15 years in the treatment response; this observation can be associated to a paradigm shift in MS treatment strategies.