Targeting the GM-CSF receptor for the treatment of CNS autoimmunity.

In multiple sclerosis (MS), there is a growing interest in inhibiting the pro-inflammatory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to evaluate the therapeutic potential and underlying mechanisms of GM-CSF receptor alpha (Rα) blockade in animal models of MS. We show that GM-CSF signaling inhibition at peak of chronic experimental autoimmune encephalomyelitis (EAE) results in amelioration of disease progression. Similarly, GM-CSF Rα blockade in relapsing-remitting (RR)-EAE model prevented disease relapses and inhibited T cell responses specific for both the inducing and spread myelin peptides, while reducing activation of mDCs and inflammatory monocytes. In situ immunostaining of lesions from human secondary progressive MS (SPMS), but not primary progressive MS patients shows extensive recruitment of GM-CSF Rα+ myeloid cells. Collectively, this study reveals a pivotal role of GM-CSF in disease relapses and the benefit of GM-CSF Rα blockade as a potential novel therapeutic approach for treatment of RRMS and SPMS.

Contribution of astrocyte-derived IL-15 to CD8 T cell effector functions in multiple sclerosis.

The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.

The majority of infiltrating CD8 T lymphocytes in multiple sclerosis lesions is insensitive to enhanced PD-L1 levels on CNS cells.

Central nervous system (CNS) cells locally modulate immune responses using numerous molecules that are not fully elucidated. Engagement of programmed death-1 (PD-1), expressed on activated T cells, by its ligands (PD-L1 or PD-L2) suppresses T-cell responses. Enhanced CNS PD-1 and PD-L1 expression has been documented in inflammatory murine models; however, human CNS data are still incomplete. We determined that human primary cultures of astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA led to significantly increased CD8 T-cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of postmortem brain tissues demonstrated a significantly greater PD-L1 expression in multiple sclerosis (MS) lesions compared with control tissues, which colocalized with astrocyte or microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Thus, our results demonstrate that inflamed human CNS cells such as in MS lesions express significantly elevated PD-L1, providing a means to reduce CD8 T cell responses, but most of these infiltrating immune cells are devoid of PD-1 and thus insensitive to PD-L1/L2. Strategies aimed at inducing PD-1 on deleterious activated human CD8 T cells that are devoid of this receptor could provide therapeutic benefits since PD-L1 is already increased in the target organ.

Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), is characterized by blood-brain barrier (BBB) disruption and massive infiltration of activated immune cells. Engagement of programmed cell death-1 (PD-1) expressed on activated T cells with its ligands (PD-L1 and PD-L2) suppresses T cell responses. We recently demonstrated in MS lesions elevated PD-L1 expression by glial cells and absence of PD-1 on many infiltrating CD8 T cells. We have now investigated whether human brain endothelial cells (HBECs), which maintain the BBB, can express PD-L1 or PD-L2 and thereby modulate T cells. METHODS: We used primary cultures of HBECs isolated from non-tumoral CNS tissue either under basal or inflamed conditions. We assessed the expression of PD-L1 and PD-L2 using qPCR and flow cytometry. Human CD8 T cells were isolated from peripheral blood of healthy donors and co-cultured with HBECs. Following co-culture with HBECs, proliferation and cytokine production by human CD8 T cells were measured by flow cytometry whereas transmigration was determined using a well established in vitro model of the BBB. The functional impact of PD-L1 and PD-L2 provided by HBECs was determined using blocking antibodies. We performed immunohistochemistry for the detection of PD-L1 or PD-L2 concurrently with caveolin-1 (a cell specific marker for endothelial cells) on post-mortem human brain tissues obtained from MS patients and normal controls. RESULTS: Under basal culture conditions, PD-L2 is expressed on HBECs, whilst PD-L1 is not detected. Both ligands are up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 leads to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increases CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions does not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues are PD-L2-positive, while only about 50% express PD-L2 in MS lesions. CONCLUSIONS: Our observations suggest that brain endothelial cells contribute to control T cell transmigration into the CNS and immune responses via PD-L2 expression. However, such impact is impaired in MS lesions due to downregulation of endothelium PD-L2 levels.

Humanized mouse model of Rasmussen's encephalitis supports the immune-mediated hypothesis.

Rasmussen's encephalitis (RE) is a chronic inflammatory brain disorder that causes frequent seizures and unilateral hemispheric atrophy with progressive neurological deficits. Hemispherectomy remains the only treatment that leads to seizure freedom for this refractory epileptic syndrome. The absence of an animal model of disease has been a major obstacle hampering the development of effective therapies. Here, we describe an experimental mouse model that shares several clinical and pathological features with the human disease. Immunodeficient mice injected with peripheral blood mononuclear cells from RE patients and monitored by video electroencephalography developed severe seizures of cortical origin and showed intense astrogliosis and accumulation of human IFN-γ- and granzyme B-expressing T lymphocytes in the brain compared with mice injected with immune cells from control subjects. We also provide evidence for the efficacy of α4 integrin blockade, an approved therapy for the treatment of multiple sclerosis and Crohn's disease, in reducing inflammatory markers associated with RE in the CNS. This model holds promise as a valuable tool for understanding the pathology of RE and for developing patient-tailored experimental therapeutics.

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry.

BACKGROUND: Flow cytometry is an efficient and powerful technique to characterize and quantify numerous cells. However, the strengths of this technique have not been widely harnessed in neurosciences due to the critical step of CNS tissue preparation into a single cell suspension. Previous reports assessed either neural cells or infiltrating leukocytes but simultaneous detection has not been extensively implemented. We optimized CNS tissue preparation for flow cytometry analysis. NEW METHOD: We subjected CNS tissue from individual adult mice to different digestion protocols and Percoll™ methods. We quantified and characterized by flow cytometry neural cells (neurons, oligodendrocytes, microglia) and leukocytes (macrophages, T lymphocytes). RESULTS: The one step Percoll™ method significantly increased cell yield compared to the gradient Percoll™ method. The collagenase D+DNase I digestion led to the maximal cell number recovery while preserving cell marker (O4, NeuN, CD45, CD11b, CD3, CD4, CD8) integrity compared to papain, trypsin digestion, and no digestion. The combination of collagenase D+DNase I digestion and one step Percoll™ method was optimal for the recovery and analysis of cells from the CNS of naïve and experimental autoimmune encephalomyelitis (multiple sclerosis model) mice. COMPARISON WITH EXISTING METHOD(S): Although flow cytometry does not reveal CNS localization, this technique allows concurrent quantification of multiple parameters. In contrast to other protocols, our novel method simultaneously analyzes neural and immune cells in individual mice in healthy and pathological conditions. CONCLUSIONS: We strongly believe that the field of neurosciences will benefit from an optimal use of flow cytometry to elucidate physiological and pathological processes.