Intratumor heterogeneity and T cell exhaustion in primary CNS lymphoma.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) is a rare lymphoma of the central nervous system, usually of diffuse large B cell phenotype. Stereotactic biopsy followed by histopathology is the diagnostic standard. However, limited material is available from CNS biopsies, thus impeding an in-depth characterization of PCNSL. METHODS: We performed flow cytometry, single-cell RNA sequencing, and B cell receptor sequencing of PCNSL cells released from biopsy material, blood, and cerebrospinal fluid (CSF), and spatial transcriptomics of biopsy samples. RESULTS: PCNSL-released cells were predominantly activated CD19+CD20+CD38+CD27+ B cells. In single-cell RNA sequencing, PCNSL cells were transcriptionally heterogeneous, forming multiple malignant B cell clusters. Hyperexpanded B cell clones were shared between biopsy- and CSF- but not blood-derived cells. T cells in the tumor microenvironment upregulated immune checkpoint molecules, thereby recognizing immune evasion signals from PCNSL cells. Spatial transcriptomics revealed heterogeneous spatial organization of malignant B cell clusters, mirroring their transcriptional heterogeneity across patients, and pronounced expression of T cell exhaustion markers, co-localizing with a highly malignant B cell cluster. CONCLUSIONS: Malignant B cells in PCNSL show transcriptional and spatial intratumor heterogeneity. T cell exhaustion is frequent in the PCNSL microenvironment, co-localizes with malignant cells, and highlights the potential of personalized treatments.

Stroke induces disease-specific myeloid cells in the brain parenchyma and pia.

Inflammation triggers secondary brain damage after stroke. The meninges and other CNS border compartments serve as invasion sites for leukocyte influx into the brain thus promoting tissue damage after stroke. However, the post-ischemic immune response of border compartments compared to brain parenchyma remains poorly characterized. Here, we deeply characterize tissue-resident leukocytes in meninges and brain parenchyma and discover that leukocytes respond differently to stroke depending on their site of residence. We thereby discover a unique phenotype of myeloid cells exclusive to the brain after stroke. These stroke-associated myeloid cells partially resemble neurodegenerative disease-associated microglia. They are mainly of resident microglial origin, partially conserved in humans and exhibit a lipid-phagocytosing phenotype. Blocking markers specific for these cells partially ameliorates stroke outcome thus providing a potential therapeutic target. The injury-response of myeloid cells in the CNS is thus compartmentalized, adjusted to the type of injury and may represent a therapeutic target.

Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges.

The CNS is ensheathed by the meninges and cerebrospinal fluid, and recent findings suggest that these CNS-associated border tissues have complex immunological functions. Unlike myeloid lineage cells, lymphocytes in border compartments have yet to be thoroughly characterized. Based on single-cell transcriptomics, we here identified a highly location-specific composition and expression profile of tissue-resident leukocytes in CNS parenchyma, pia-enriched subdural meninges, dura mater, choroid plexus and cerebrospinal fluid. The dura layer of the meninges contained a large population of B cells under homeostatic conditions in mice and rats. Murine dura B cells exhibited slow turnover and long-term tissue residency, and they matured in experimental neuroinflammation. The dura also contained B lineage progenitors at the pro-B cell stage typically not found outside of bone marrow, without direct influx from the periphery or the skull bone marrow. This identified the dura as an unexpected site of B cell residence and potentially of development in both homeostasis and neuroinflammation.

Redefining the heterogeneity of peripheral nerve cells in health and autoimmunity.

Peripheral nerves contain axons and their enwrapping glia cells named Schwann cells (SCs) that are either myelinating (mySCs) or nonmyelinating (nmSCs). Our understanding of other cells in the peripheral nervous system (PNS) remains limited. Here, we provide an unbiased single cell transcriptomic characterization of the nondiseased rodent PNS. We identified and independently confirmed markers of previously underappreciated nmSCs and nerve-associated fibroblasts. We also found and characterized two distinct populations of nerve-resident homeostatic myeloid cells that transcriptionally differed from central nervous system microglia. In a model of chronic autoimmune neuritis, homeostatic myeloid cells were outnumbered by infiltrating lymphocytes which modulated the local cell-cell interactome and induced a specific transcriptional response in glia cells. This response was partially shared between the peripheral and central nervous system glia, indicating common immunological features across different parts of the nervous system. Our study thus identifies subtypes and cell-type markers of PNS cells and a partially conserved autoimmunity module induced in glia cells.

Deciphering immune mechanisms in chronic inflammatory demyelinating polyneuropathies.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune disease of the peripheral nerves that presents with either chronic progression or relapsing disease. Recent studies in samples from patients with CIDP and mouse models have delineated how defects in central (thymic) and peripheral (extrathymic) immune tolerance mechanisms can cause PNS autoimmunity. Notably, nerve parenchymal cells actively contribute to local autoimmunity and also control disease outcome. Here, we outline how emerging technologies increasingly enable an integrated view of how immune cells and PNS parenchymal cells communicate in CIDP. We also relate the known heterogeneity of clinical presentation with specific underlying mechanisms. For example, a severe subtype of CIDP with tremor is associated with pathogenic IgG4 autoantibodies against nodal and paranodal proteins. An improved understanding of pathogenic mechanisms in CIDP will form the basis for more effective mechanism-based therapies.

Integrated single cell analysis of blood and cerebrospinal fluid leukocytes in multiple sclerosis.

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) - an autoimmune disease of the CNS - increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.

Tumor infiltrating lymphocytes expanded from pediatric neuroblastoma display heterogeneity of phenotype and function.

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically.

Immune Cell Profiling of the Cerebrospinal Fluid Provides Pathogenetic Insights Into Inflammatory Neuropathies.

Objective: Utilize immune cell profiles in the cerebrospinal fluid (CSF) to advance the understanding and potentially support the diagnosis of inflammatory neuropathies. Methods: We analyzed CSF cell flow cytometry data of patients with definite Guillain-Barré syndrome (GBS, n = 26) and chronic inflammatory demyelinating polyneuropathy (CIDP, n = 32) based on established diagnostic criteria in comparison to controls with relapsing-remitting multiple sclerosis (RRMS, n = 49) and idiopathic intracranial hypertension (IIH, n = 63). Results: Flow cytometry revealed disease-specific changes of CSF cell composition with a significant increase of NKT cells and CD8+ T cells in CIDP, NK cells in GBS, and B cells and plasma cells in MS in comparison to IIH controls. Principal component analysis demonstrated distinct CSF immune cells pattern in inflammatory neuropathies vs. RRMS. Systematic receiver operator curve (ROC) analysis identified NKT cells as the best parameter to distinguish GBS from CIDP. Composite scores combing several of the CSF parameters differentiated inflammatory neuropathies from IIH and GBS from CIDP with high confidence. Applying a novel dimension reduction technique, we observed an intra-disease heterogeneity of inflammatory neuropathies. Conclusion: Inflammatory neuropathies display disease- and subtype-specific alterations of CSF cell composition. The increase of NKT cells and CD8+ T cells in CIDP and NK cells in GBS, suggests a central role of cytotoxic cell types in inflammatory neuropathies varying between acute and chronic subtypes. Composite scores constructed from multi-dimensional CSF parameters establish potential novel diagnostic tools. Intra-disease heterogeneity suggests distinct disease mechanisms in subgroups of inflammatory neuropathies.

Cerebrospinal Fluid Concentrations of Neuronal Proteins Are Reduced in Primary Angiitis of the Central Nervous System.

Primary angiitis of the central nervous system (PACNS) is a rare autoimmune vasculitis limited to the CNS often causing substantial disability. Understanding of this disease is impaired by the lack of available biomaterial. Here, we collected cerebrospinal fluid (CSF) from patients with PACNS and matched controls and performed unbiased proteomics profiling using ion mobility mass spectrometry to identify novel disease mechanisms and candidate biomarkers. We identified 14 candidate proteins, including amyloid-beta A4 protein (APP), with reduced abundance in the CSF of PACNS patients and validated APP by Enzyme-linked Immunosorbent Assay (ELISA) in an extended cohort of patients with PACNS. Subsequent functional annotation surprisingly suggested neuronal pathology rather than immune activation in PACNS. Our study is the first to employ mass spectrometry to local immune reactions in PACNS and it identifies candidates such as APP with pathogenic relevance in PACNS to improve patient care in the future.

CD11b+Gr-1+ myeloid-derived suppressor cells reduce atherosclerotic lesion development in LDLr deficient mice.

AIMS: Myeloid-derived suppressor cells (MDSCs) form a heterogeneous population of cells composed of early myeloid progenitor cells and immature myeloid cells, which strongly suppress pro-inflammatory immune cells in inflammatory diseases. Currently, it is unknown whether MDSCs contribute to atherosclerosis, a chronic inflammatory disease in which accumulation of lipoproteins in the arterial wall activates the immune system causing abnormal vascular remodelling and vessel occlusion. Here, we investigated whether and how MDSCs contribute to the development of atherosclerosis. METHODS AND RESULTS: We show that MDSCs arise in the bone marrow of LDLr(-/-) mice during atherosclerosis and strongly suppress proliferation of T cells. Adoptive transfer of MDSCs into both female and male LDLr(-/-) mice fed a Western-type diet (WTD) ameliorates atherosclerosis with 35%. We observed a 54% reduction in adventitial T cells, and more specifically, MDSCs suppress Th1 and Th17 cells. In addition, treatment with MDSCs reduces circulating pro-atherogenic B2 cells. We found two subsets of MDSCs in the bone marrow of hypercholesterolemic mice, monocytic and granulocytic MDSCs (mo- and gr-MDSCs, respectively), of which the percentage of mo-MDSCs significantly increased during WTD feeding. Moreover, mo-MDSCs completely abolished splenocyte proliferation, whereas gr-MDSCs were unable to suppress proliferation. Mechanistically, we show that MDSCs from atherosclerotic mice suppress T cells in an IFN-γ- and nitric oxide-dependent manner, which is associated with the action of mo-MDSCs. CONCLUSION: This study demonstrates that MDSCs develop during atherosclerosis and reduce atherosclerosis via suppression of pro-inflammatory immune responses.