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Multifocal inflammatory lesions featuring destruction of lipid-rich myelin are pathologic hallmarks of multiple sclerosis. Lesion activity is assessed by the extent and composition of myelin uptake by myeloid cells present in such lesions. In the inflamed CNS, myeloid cells are comprised of brain-resident microglia, an endogenous cell population, and monocyte-derived macrophages, which infiltrate from the systemic compartment. Using microglia isolated from the adult human brain, we demonstrate that myelin phagocytosis is dependent on the polarization state of the cells. Myelin ingestion is significantly enhanced in cells exposed to TGF-β compared with resting basal conditions and markedly reduced in classically activated polarized cells. Transcriptional analysis indicated that TGF-β-treated microglia closely resembled M0 cells. The tyrosine kinase phagocytic receptor MerTK was one of the most upregulated among a select number of differentially expressed genes in TGF-β-treated microglia. In contrast, MerTK and its known ligands, growth arrest-specific 6 and Protein S, were downregulated in classically activated cells. MerTK expression and myelin phagocytosis were higher in CNS-derived microglia than observed in monocyte-derived macrophages, both basally and under all tested polarization conditions. Specific MerTK inhibitors reduced myelin phagocytosis and the resultant anti-inflammatory biased cytokine responses for both cell types. Defining and modulating the mechanisms that regulate myelin phagocytosis has the potential to impact lesion and disease evolution in multiple sclerosis. Relevant effects would include enhancing myelin clearance, increasing anti-inflammatory molecule production by myeloid cells, and thereby permitting subsequent tissue repair., (Copyright © 2016 by The American Association of Immunologists, Inc.)

In multiple sclerosis, successful remyelination within the injured CNS is largely dependent on the survival and differentiation of oligodendrocyte progenitor cells. During inflammatory injury, oligodendrocytes and oligodendrocyte progenitor cells within lesion sites are exposed to secreted products derived from both infiltrating immune cell subsets and CNS-resident cells. Such products may be considered either proinflammatory or anti-inflammatory and have the potential to contribute to both injury and repair processes. Within the CNS, astrocytes also contribute significantly to oligodendrocyte biology during development and following inflammatory injury. The overall objective of the current study was to determine how functionally distinct proinflammatory and anti-inflammatory human immune cell subsets, implicated in multiple sclerosis, can directly and/or indirectly (via astrocytes) impact human oligodendrocyte progenitor cell survival and differentiation. Proinflammatory T cell (Th1/Th17) and M1-polarized myeloid cell supernatants had a direct cytotoxic effect on human A2B5(+) neural progenitors, resulting in decreased O4(+) and GalC(+) oligodendrocyte lineage cells. Astrocyte-conditioned media collected from astrocytes pre-exposed to the same proinflammatory supernatants also resulted in decreased oligodendrocyte progenitor cell differentiation without an apparent increase in cell death and was mediated through astrocyte-derived CXCL10, yet this decrease in differentiation was not observed in the more differentiated oligodendrocytes. Th2 and M2 macrophage or microglia supernatants had neither a direct nor an indirect impact on oligodendrocyte progenitor cell differentiation. We conclude that proinflammatory immune cell responses can directly and indirectly (through astrocytes) impact the fate of immature oligodendrocyte-lineage cells, with oligodendrocyte progenitor cells more vulnerable to injury compared with mature oligodendrocytes., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The mechanisms whereby immune cells infiltrating the CNS in multiple sclerosis patients contribute to tissue injury remain to be defined. CD4 T cells are key players of this inflammatory response. Myelin-specific CD4 T cells expressing CD56, a surrogate marker of NK cells, were shown to be cytotoxic to human oligodendrocytes. Our aim was to identify NK-associated molecules expressed by human CD4 T cells that confer this oligodendrocyte-directed cytotoxicity. We observed that myelin-reactive CD4 T cell lines, as well as short-term PHA-activated CD4 T cells, can express NKG2C, the activating receptor interacting with HLA-E, a nonclassical MHC class I molecule. These cells coexpress CD56 and NKG2D, have elevated levels of cytotoxic molecules FasL, granzyme B, and perforin compared with their NKG2C-negative counterparts, and mediate significant in vitro cytotoxicity toward human oligodendrocytes, which upregulated HLA-E upon inflammatory cytokine treatment. A significantly elevated proportion of ex vivo peripheral blood CD4 T cells, but not CD8 T cells or NK cells, from multiple sclerosis patients express NKG2C compared with controls. In addition, immunohistochemical analyses showed that multiple sclerosis brain tissues display HLA-E(+) oligodendrocytes and NKG2C(+) CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4 T cells contribute to tissue injury in multiple sclerosis.

FTY720 (fingolimod) treatment of multiple sclerosis (MS) results in lymphopenia due to increased recruitment into and decreased egress from secondary lymphoid organs of CCR7(+) lymphocytes. Although absolute numbers of NK lymphocytes were reported as being unaltered in FTY720-treated MS patients (MS-FTY), such analyses did not detect a change in a minor subset. Because expression of CCR7 has been described on CD56(bright) NK cells, a minority population of NK cells, we investigated the effect of FTY720 treatment on the phenotype and function of human NK cells in the peripheral circulation of MS patients. MS-FTY patients displayed a decreased proportion of peripheral CD56(bright)CD62L(+)CCR7(+) NK cells compared with untreated MS and healthy donors. In vitro treatment with FTY720-P increased migration of untreated donor NK cells to CXCL12 while reducing the response to CX3CL1 with similar migration responses seen in NK cells from MS-FTY patients. FTY720-P inhibited sphingosine 1-phosphate-directed migration of CD56(bright) and CD56(dim) NK cells subsets from untreated healthy donors. IL-12- and IL-15-stimulated NK cells from MS-FTY patients displayed similar capacity to produce IFN-γ, TNF, IL-10, and MIP-1α cytokines/chemokines compared with NK cells from untreated healthy donors and displayed comparable levels of degranulation in response to K562 tumor cells compared with untreated donors. Subset alterations and function of NK cell populations will need to be considered as part of assessing overall immunosurveillance capacity of patients with MS who will receive sustained FTY720 therapy.

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.

Microglia are resident cells of the CNS that belong to the myeloid cell lineage. In experimental models of neuroinflammation, they have limited capacity to function as APCs when compared with dendritic cells (DCs). Human peripheral blood monocytes have the plasticity to differentiate into mature DCs when exposed to GM-CSF and IL-4 followed by LPS. In this study we addressed the potential of human microglia to acquire phenotypic and functional properties of mature DCs under similar inducing conditions. Treated adult and fetal microglia became CD14(low) and acquired limited expression of CD209 (DC-SIGN); they remained CD1a(-) and CD83(-), and decreased MHCII expression, suggesting that they had not achieved a complete DC phenotype. The monocyte-derived DCs efficiently promoted CD4 T cell proliferation in an allogeneic MLR, whereas differentiated adult microglia had a decreased ability to stimulate CD4 T cell proliferation compared with their untreated counterparts. Differentiated fetal microglia did support CD4 T cell proliferation, whereas untreated cells could not. Fetal and adult microglia produced significant amounts of IL-10 following differentiation but no detectable IL-12 p70, in contrast to differentiated monocytes that produced IL-12 p70. Our data indicate that neither adult nor fetal microglia acquired the full characteristic phenotype of mature stimulatory DCs when treated with DC-inducing cytokines in vitro. Moreover, such treatment, especially of adult microglia, induces functional responses that could promote an antiinflammatory environment in the CNS.

Autoantibody neuromyelitis optica-IgG (NMO-IgG) recognizing aquaporin-4 (AQP4) is implicated as playing a central role in the physiopathology of NMO. The aim of this in vitro-based study was to characterize functional consequences of interaction between NMO-IgG and cells of the neurovascular unit (astrocytes and brain endothelium) that would provide insight into recognized features of NMO, namely altered blood-brain barrier (BBB) permeability and granulocyte recruitment. We used sera from NMO and longitudinally extensive transverse myelitis cases shown to bind in a characteristic perivascular pattern to primate cerebellar slices. Using flow cytometry, we found that sera from NMO-IgG-positive patients reacted with CNS-derived human fetal astrocytes, whereas sera from multiple sclerosis patients did not. We demonstrated that NMO-IgG binding to astrocytes alters aquaporin-4 polarized expression and increases permeability of a human BBB endothelium/astrocyte barrier. We further demonstrated that NMO-IgG binding to human fetal astrocytes can result in NK cell degranulation, astrocyte killing by Ab-dependent cellular cytotoxicity and complement-dependent granulocyte attraction through the BBB model. Our study highlights important functional roles for NMO-IgG that could account for pathological lesions and BBB dysfunction observed in NMO.

The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1-9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-alpha, IL-6, CXCL-10, and IL-10, and the expression of IFN-beta. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-beta, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.

Glatiramer acetate (GA) therapy of patients with multiple sclerosis (MS) represents a unique setting in which in vivo Th2 deviation of T cells is consistently observed and associated with clinical benefit in a human autoimmune disease. We postulated that APCs are important targets of GA therapy and demonstrate that treatment of MS patients with GA reciprocally regulates the IL-10/IL-12 cytokine network of monocytes in vivo. We further show that Th1- or Th2-polarized GA-reactive T cells isolated from untreated or treated MS patients mediate type 1 and 2 APC differentiation of human monocytes, based on their ability to efficiently induce subsequent Th1 and Th2 deviation of naive T cells, respectively. These observations are extended to human microglia, providing the first demonstration of type 2 differentiation of CNS-derived APCs. Finally, we confirm that the fundamental capacity of polarized T cells to reciprocally modulate APC function is not restricted to GA-reactive T cells, thereby defining a novel and dynamic positive feedback loop between human T cell and APC responses. In the context of MS, we propose that GA therapy results in the generation of type 2 APCs, contributing to Th2 deviation both in the periphery and in the CNS of MS patients. In addition to extending insights into the therapeutic mode of action of GA, our findings revisit the concept of bystander suppression and underscore the potential of APCs as attractive targets for therapeutic immune modulation.

Axonal and neuronal loss occurs in inflammatory diseases of the CNS such as multiple sclerosis. The cause of the loss remains unclear. We report that polyclonally activated T cells align along axons and soma of cultured human neurons leading to substantial neuronal death. This occurs in an allogeneic and syngeneic manner in the absence of added Ag, requires T cells to be activated, and is mediated through cell contact-dependent mechanisms involving FasL, LFA-1, and CD40 but not MHC class I. Activated CD4(+) and CD8(+) T cell subsets are equally neuronal cytotoxic. In contrast to neurons, other CNS cell types (oligodendrocytes and astrocytes) are not killed by T cells. These results demonstrate for the first time the high and selective vulnerability of human neurons to T cells, and suggest that when enough activated T cells accumulate in the CNS, neuronal cytotoxicity can result through Ag-independent non-MHC class I mechanisms.

Circulating B cells enter the CNS as part of normal immune surveillance and in pathologic states, including the common and disabling illness multiple sclerosis. However, little is known about the molecular mechanisms that mediate human B cell interaction with the specialized brain endothelial cells comprising the blood-brain barrier (BBB). We studied the molecular mechanisms that regulate the migration of normal human B cells purified ex vivo, across human adult brain-derived endothelial cells (HBECs). We found that B cells migrated across HBECs more efficiently than T cells from the same individuals. B cell migration was significantly inhibited by blocking Abs to the adhesion molecules ICAM-1 and VLA-4, but not VCAM-1, similar to the results previously reported for T cells. Blockade of the chemokines monocyte chemoattractant protein-1 and IL-8, but not RANTES or IFN-gamma-inducible protein-10, significantly inhibited B cell migration, and these results were correlated with the chemokine receptor expression of B cells measured by flow cytometry and by RNase protection assay. Tissue inhibitor of metalloproteinase-1, a natural inhibitor of matrix metalloproteinases, significantly decreased B cell migration across the HBECs. A comprehensive RT-PCR comparative analysis of all known matrix metalloproteinases and tissue inhibitors of metalloproteinases in human B and T cells revealed distinct profiles of expression of these molecules in the different cell subsets. Our results provide insights into the molecular mechanisms that underlie human B cell migration across the BBB. Furthermore, they identify potential common, and unique, therapeutic targets for limiting CNS B cell infiltration and predict how therapies currently developed to target T cell migration, such as anti-VLA-4 Abs, may impact on B cell trafficking.

Th1-polarized CD4+ T cells are considered central to the development of a number of target-directed autoimmune disorders including multiple sclerosis. The APC-derived cytokine IL-12 is a potent inducer of Th1 polarization in T cells. Inhibition of IL-12 in vivo blocks the development of experimental allergic encephalomyelitis, the animal model for multiple sclerosis. Based on previous work that suggests that the production of IL-12 by activated human central nervous system-derived microglia is regulated by autocrine TNF-alpha, we wanted to determine whether inhibition of TNF could induce a reduction of Th1 responses by its impact on systemic APCs. We found that soluble TNFR p75-IgG fusion protein (TNFR:Fc) inhibited production of IFN-gamma by allo-Ag-activated blood-derived human CD4 T cells. We documented reduced IL-12 p70 production by APCs in the MLR. By adding back recombinant IL-12, we could rescue IFN-gamma production, indicating that TNFR:Fc acts on APC-derived IL-12. Consistent with an inhibition of the Th1 polarization, we found a decreased expression of IL-12R-beta2 subunit on the T cells. Furthermore, the capacity of T cells to secrete IFN-gamma upon restimulation when previously treated with TNFR:Fc is impaired, whereas IL-2 secretion was not altered. Our results define a TNF-dependent cytokine network that favors development of Th1 immune responses.

Multiple sclerosis (MS) is considered to be an autoimmune disease that is directed either at myelin or at its cell of origin, the oligodendrocytes (OL). The inflammatory lesions in the central nervous system contain multiple myelin Ag-restricted and nonrestricted cell populations with the potential to mediate tissue injury. Previous studies indicate that it is possible to generate MHC class I-restricted myelin peptide-specific cytotoxic CD8 T cells, and that human adult OLs express MHC class I molecules in vitro. The purpose of this study was to demonstrate that myelin basic protein peptide-specific CD8 T cells could induce OL injury. We generated CD8 T cell lines from six healthy donors and five MS patients, and all cell lines were HLA-A2 positive. The obtained CD8 cell lines induced lysis of HLA-A2- but not HLA-A3-transfected HMy2.C1R cells in the presence of myelin basic protein peptide 110-118. In the absence of exogenous peptide, the CD8 T cell lines were cytotoxic to HLA-A2 but not to non-HLA-A2 OLs. Cytotoxicity was blocked with anti-MHC class I-blocking Ab. These results support the postulate that autoreactive CD8 cytotoxic T cells can contribute to the tissue injury in MS.

Oligodendrocytes and their myelin membranes are the apparent target of the autoimmune response that characterizes the human adult central nervous system-demyelinating disease multiple sclerosis. Human oligodendrocytes do not express MHC class II molecules, a requirement for MHC-restricted injury mediated by myelin-reactive CD4+ T cells, the cell type implicated in initiating the disease process. In this study we observed that human adult central nervous system-derived oligodendrocytes can be susceptible to non-MHC-restricted lysis mediated by myelin basic protein-reactive CD4+ T cell lines. Cytotoxicity was significantly greater (37 +/- 4 vs 7 +/- 3%) with cell lines in which a high proportion of cells (mean, 28 +/- 6%) expressed CD56 compared with cytotoxicity mediated by low CD56 cell lines (8 +/- 2%). High CD56 cell lines, when rested in IL-2, lost cytotoxic activity and had reduced expression of CD56 (mean, 5 +/- 2%). CD4+ T cells isolated from short term (3-day) anti-CD3/IL-2-activated mononuclear cell cultures did not express CD56 and were not cytotoxic to oligodendrocytes unless lectin was added. In contrast, an enriched population of non-T cells extracted from the same activated MNC cultures expressed CD56 as well as other NK cell-associated surface molecules and was cytotoxic. These results indicate the potential susceptibility of human oligodendrocytes to non-MHC-restricted injury mediated by both Ag-reactive and nonspecific cellular immune effector cells, with CD56 expression being a common feature of the effector cells.

Cell-mediated immune mechanisms contribute to tissue injury within the central nervous system (CNS) in a number of experimental diseases, including experimental allergic encephalomyelitis and some viral infections, and may mediate lesion formation in multiple sclerosis. We investigated the conditions under which murine astrocytes can become susceptible targets of cytotoxic T cells. We demonstrate that mouse astrocytes in vitro can be susceptible targets of class I major histocompatibility complex (MHC)-specific cytotoxicity mediated by L3 cytotoxic T lymphocytes (CTL). Expression of appropriate class I MHC antigen on the astrocytes is a requirement, because only cells bearing the H-2d phenotype are susceptible to lysis by L3 cells. BALB/c-H-2dm2 astrocytes lacking the specific determinant recognized by L3 cells are not susceptible to lysis. Astrocyte lysis can, however, occur under culture conditions in which MHC antigen expression is immunocytochemically low or undetectable. Cytolysis can be inhibited by pretreatment of the effector L3 cells with either anti-Lyt-2 monoclonal antibody (mAb) or anti-clonotypic mAb and by preincubation of the glial target cells with an appropriate anti-H-2 antibody (anti-H-2Ld). mAb to lymphocyte function-associated antigen does not inhibit cytotoxicity of the L3 clone against glial cells. Knowledge regarding the role of CTL within the CNS, including the surface molecules involved in glial cell lysis, could further the development of immunotherapies designed to effect immune reactivity within the CNS.

Concanavalin A (Con A)-induced suppressor activity has previously been shown to be reduced in multiple sclerosis (MS) patients with active clinical disease. In this study, we demonstrate that OKT3, as well as Con A induced suppressor activity mediated by unfractionated peripheral blood mononuclear cells is reduced in patients with the progressive form of MS. By performing reconstitution experiments involving E+, T4+, or T8+ cells derived from either MS patients or controls, and normal allogeneic macrophages or E- cells, we sought to define the cellular basis for this suppressor defect. In both MS and control groups, E+ cells were required to obtain measurable levels of suppression. Suppressor levels induced by Con A-activated cultures containing E+ cells from MS patients were lower than those induced by those containing control donor E+ cells. Suppression mediated by T8+ cells from MS patients was also lower than for controls. In the control group, suppression mediated by T8+ cells exceeded that mediated by T4+ cells; such differences were not apparent in the MS group. These results suggest that although Con A-induced suppression can be mediated by a number of T and non-T cell subsets, the functional suppressor defect measured in the MS population does involve the T8+ cell subset.

We measured the pokeweed mitogen (PWM)-induced secretion of IgG by the unfractionated mononuclear cells (MNC) of young adult donors, and correlated the results with the functional activity of cell suspensions enriched for T helper (T4+) and T suppressor/cytotoxic (T8+) cells. The distribution of IgG levels secreted by MNC differs from a Gaussian curve, implying that the group is composed of distinct heterogeneous populations. When donors were compared who were judged to be very low responders or very high responders on the basis of IgG secretion levels by MNC (less than 700 ng/ml or greater than 2500 ng/ml), no differences were found in the capacity of T4+-enriched cells to support PWM-driven IgG secretion by a common B cell pool. In contrast, the addition of 0.2 X 10(5) T8+ cells from these low responders to PWM-stimulated cultures of 0.5 X 10(5) T4+ cells plus 0.5 X 10(5) B cells resulted in significantly less IgG secretion (389 +/- 121 ng/ml) than did the addition of the same number of T8+ cells from the high responders (2241 +/- 548 ng/ml, p less than 0.01). Normalized percent suppression by T8+ cells was higher in low responders than in high responders (77.0 +/- 9.9% vs 33.0 +/- 8.5%, p less than 0.01). Both high and low responders markedly suppressed IgG secretion when 0.5 X 10(5) T8+ cells were added. No correlation was found either between proportion of T3+, T8+, T4+, or M1+ cells within the MNC population and levels of IgG secretion by MNC or between T8+ numbers and levels of suppression induced by a constant number of T8+-enriched cells. Our data indicate that differences in the functional activity of T8+ cells, rather than quantitative differences, account for the wide range of PWM-induced IgG secretion by MNC.

In order to examine questions concerning immunologic privilege of the central nervous system, we placed neocortical transplants into cerebral ventricles of mice. We compared the fates of transplants between fully H-2 compatible (isografts) and H-2 incompatible (allografts) animals. Histologic evaluation comparing animals from iso- and allograft groups revealed significant differences in the number of inflammatory cells and in the degree of necrosis within the grafts. Response to allografted tissue within the brain mimics that seen in several immune-mediated diseases of the nervous system in that neurons appear to be selectively spared. Only upon subsequent stimulation of the host's immune system with an orthotopic skin graft bearing the major histocompatibility complex antigens of the neural graft are neurons destroyed. Immunohistochemical evaluation revealed that the inflammatory cell infiltrates in and around the allografts were composed of Lyt-2+, L3T4+, and Mac-1+ cells. In addition, Ia+ endothelial cells as well as Ia+ parenchymal CNS cells were found in both donor and host tissue of allografted animals. Hence, H-2 incompatible neural tissue transplanted to the CNS is recognized and rejected by the immune system of the recipient animal. The cellular infiltrates seen within the first weeks to months following transplantation of allogeneic CNS tissue resemble those seen in other allografts undergoing rejection. We conclude that the CNS is not unconditionally privileged as either a transplant site or as a source of transplanted tissue.