Production of IL-27 in multiple sclerosis lesions by astrocytes and myeloid cells: Modulation of local immune responses.

The mechanisms whereby human glial cells modulate local immune responses are not fully understood. Interleukin-27 (IL-27), a pleiotropic cytokine, has been shown to dampen the severity of experimental autoimmune encephalomyelitis, but it is still unresolved whether IL-27 plays a role in the human disease multiple sclerosis (MS). IL-27 contribution to local modulation of immune responses in the brain of MS patients was investigated. The expression of IL-27 subunits (EBI3 and p28) and its cognate receptor IL-27R (the gp130 and TCCR chains) was elevated within post-mortem MS brain lesions compared with normal control brains. Moreover, astrocytes (GFAP(+) cells) as well as microglia and macrophages (Iba1(+) cells) were important sources of IL-27. Brain-infiltrating CD4 and CD8 T lymphocytes expressed the IL-27R specific chain (TCCR) implying that these cells could respond to local IL-27 sources. In primary cultures of human astrocytes inflammatory cytokines increased IL-27 production, whereas myeloid cell inflammatory M1 polarization and inflammatory cytokines enhanced IL-27 expression in microglia and macrophages. Astrocytes in postmortem tissues and in vitro expressed IL-27R. Moreover, IL-27 triggered the phosphorylation of the transcription regulator STAT1, but not STAT3 in human astrocytes; indeed IL-27 up-regulated MHC class I expression on astrocytes in a STAT1-dependent manner. These findings demonstrated that IL-27 and its receptor were elevated in MS lesions and that local IL-27 can modulate immune properties of astrocytes and infiltrating immune cells. Thus, therapeutic strategies targeting IL-27 may influence not only peripheral but also local inflammatory responses within the brain of MS patients.

Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis.

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.

B cell-derived IL-15 enhances CD8 T cell cytotoxicity and is increased in multiple sclerosis patients.

Multiple lines of evidence suggest that CD8 T cells contribute to the pathogenesis of multiple sclerosis (MS). However, the sources and involvement of cytokines such as IL-15 in activating these cells is still unresolved. To investigate the role of IL-15 in enhancing the activation of CD8 T cells in the context of MS, we determined cell types expressing the bioactive surface IL-15 in the peripheral blood of patients and evaluated the impact of this cytokine on CD8 T cell cytotoxicity and migration. Flow cytometric analysis showed a significantly greater proportion of B cells and monocytes from MS patients expressing IL-15 relative to controls. We established that CD40L activation of B cells from healthy donors increased their IL-15 levels, reaching those of MS patients. We also demonstrated an enhanced cytotoxic profile in CD8 T cells from MS patients upon stimulation with IL-15. Furthermore, we showed that IL-15 expressed by B cells and monocytes is sufficient and functional, enhancing granzyme B production by CD8 T cells upon coculture. Exposure of CD8 T cells to this cytokine enhanced their ability to kill glial cells as well as to migrate across an in vitro inflamed human blood-brain barrier. The elevated levels of IL-15 in patients relative to controls, the greater susceptibility of CD8 T cells from patients to IL-15, in addition to the enhanced cytotoxic responses by IL-15-exposed CD8 T cells, stresses the potential of therapeutic strategies to reduce peripheral sources of IL-15 in MS.

Melanoma cell adhesion molecule identifies encephalitogenic T lymphocytes and promotes their recruitment to the central nervous system.

In multiple sclerosis, encephalitogenic CD4(+) lymphocytes require adhesion molecules to accumulate into central nervous system inflammatory lesions. Using proteomic techniques, we identified expression of melanoma cell adhesion molecule (MCAM) on a subset of human effector memory CD4(+) lymphocytes and on human blood-brain barrier endothelium. Herein, we demonstrate that MCAM is a stable surface marker that refines the identification of interleukin 17(+), interleukin 22(+), RAR-related orphan receptor γ and interleukin 23 receptor(+) cells within the CD161(+)CCR6(+) subset of memory CD4(+) lymphocytes. We also show that MCAM(+) lymphocytes express significantly more granulocyte/macrophage colony stimulating factor and granzyme B than MCAM(-) lymphocytes. Furthermore, the proportion of MCAM(+) CD4(+) lymphocytes is significantly increased in the blood and in the central nervous system of patients with multiple sclerosis and experimental autoimmune encephalomyelitis animals compared with healthy controls or other neurological diseases, and MCAM expression is upregulated at the blood-brain barrier within inflammatory lesions. Moreover, blockade of MCAM or depletion of MCAM(+) CD4(+) T lymphocytes both restrict the migration of T(H)17 lymphocytes across blood-brain barrier endothelial cells and decrease the severity of experimental autoimmune encephalomyelitis. Our findings indicate that MCAM could serve as a potential biomarker for multiple sclerosis and represents a valuable target for the treatment of neuroinflammatory conditions.

IL-27 increases the proliferation and effector functions of human naïve CD8+ T lymphocytes and promotes their development into Tc1 cells.

IL-27 has been shown to exhibit both pro- and anti-inflammatory properties; it favors mouse naïve CD4(+) T-cell differentiation into Th1 cells to the detriment of Th17 and Th2 skewing and regulates IL-10 and IL-17 production by human CD4(+) T cells. Moreover, IL-27 promotes proliferation and cytotoxic functions of mouse CD8(+) T lymphocytes, but no data are available on human CD8(+) T cells. We investigated the impact of IL-27 on human CD8(+) T cells. In contrast to mouse T cells, the IL-27 receptor (IL-27R), composed of T cell cytokine receptor (TCCR) and gp130, was detected on a greater percentage of human CD8(+) than CD4(+) T cells and these proportions increased upon polyclonal activation. IL-27 induced rapid STAT1 and STAT3 signaling, enhanced STAT1 protein levels, and induced SOCS1 and SOCS3 expression in a STAT1-dependent manner by human CD8(+) T cells. Addition of IL-27 to α-CD3-activated naïve CD8(+) T cells significantly increased T-box transcription factor expression levels, cell proliferation, and IFN-γ and granzyme B production leading to increased CD8(+) T-cell-mediated cytotoxicity. These results demonstrate that IL-27, a rapidly produced cytokine by activated APC, has a profound impact on human naïve CD8(+) T cells, driving them to become highly efficient Tc1 cells.

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.

Prostate-derived Ets transcription factor overexpression is associated with nodal metastasis and hormone receptor positivity in invasive breast cancer.

Prostate-derived Ets transcription factor (PDEF) has recently been associated with invasive breast cancer, but no expression profile has been defined in clinical specimens. We undertook a comprehensive PDEF transcriptional expression study of 86 breast cancer clinical specimens, several cell lines, and normal tissues. PDEF expression profile was analyzed according to standard clinicopathologic parameters and compared with hormonal receptor and HER-2/neu status and to the expression of the new tumor biomarker Dikkopf-1 (DKK1). Wide ranging PDEF overexpression was observed in 74% of tested tumors, at higher levels than the average expression found in normal breasts. High PDEF expression was associated with hormone receptor positivity (P < .001), moderate to good differentiation (less than grade III, P = .01), and dissemination to axillary lymph nodes (P = .002). PDEF was an independent risk factor for nodal involvement (multivariate analysis, odds ratio 1.250, P = .002). It was expressed in a different subgroup compared to DKK1-expressing tumors (P < .001). Our data imply that PDEF mRNA expression could be useful in breast cancer molecular staging. Further insights into PDEF functions at the protein level, and possible links with hormone receptors biology, bear great potential for new therapeutic avenues.

Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells.

The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.