Meike Mitsdoerffer

A Functional Role of HLA-G Expression in Human Gliomas: An Alternative Strategy of Immune Escape

HLA-G is a nonclassical MHC molecule with highly limited tissue distribution that has been attributed chiefly immune regulatory functions. Glioblastoma is paradigmatic for the capability of human cancers to paralyze the immune system. To delineate the potential role of HLA-G in glioblastoma immunobiology, expression patterns and functional relevance of this MHC class Ib molecule were investigated in glioma cells and brain tissues. HLA-G mRNA expression was detected in six of 12 glioma cell lines in the absence of IFN-γ and in 10 of 12 cell lines in the presence of IFN-γ. HLA-G protein was detected in four of 12 cell lines in the absence of IFN-γ and in eight of 12 cell lines in the presence of IFN-γ. Immunohistochemical analysis of human brain tumors revealed expression of HLA-G in four of five tissue samples. Functional studies on the role of HLA-G in glioma cells were conducted with alloreactive PBMCs, NK cells, and T cell subpopulations. Expression of membrane-bound HLA-G1 and soluble HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG) rendered cells highly resistant to direct alloreactive lysis, inhibited the alloproliferative response, and prevented efficient priming of cytotoxic T cells. The inhibitory effects of HLA-G were directed against CD8 and CD4 T cells, but appeared to be NK cell independent. Interestingly, few HLA-G-positive cells within a population of HLA-G-negative tumor cells exerted significant immune inhibitory effects. We conclude that the aberrant expression of HLA-G may contribute to immune escape in human glioblastoma.

Interferon-β enhances monocyte and dendritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis

Antigen-presenting cells (APC) are considered to play a critical role in promoting the (re)activation of potentially autoreactive T cells in multiple sclerosis (MS), an inflammatory demyelinating disorder of the central nervous system (CNS). B7-H1 (PD-L1) is a novel member of the B7 family proteins which exert costimulatory and immune regulatory functions. Here we characterize the expression and functional activity of B7-H1 expressed on monocytes and dendritic cells (DC) of healthy donors and MS patients. B7-H1 is constitutively expressed on monocytes and differentially matured DC, but not on B cells. IFN-beta, the principle immune modulatory agent used for the treatment of MS, strongly enhances B7-H1 expression on monocytes and semi-matured DC, but not B cells, in vitro. Importantly, B7-H1 expressed on APC strongly inhibits autologous CD4 T-cell activation. Neutralization of B7-H1 on monocytes or differentially matured monocyte-derived DC markedly increases the secretion of the pro-inflammatory cytokines, IFN-gamma and IL-2, T-cell proliferation, and the expression of T-cell activation markers. B7-H1 exhibits strong inhibitory effects when expressed on monocytes, immature or semi-mature DC, but less so when expressed on fully matured DC. B7-H1-dependent immune inhibition is in part mediated by CD4/CD25+ regulatory T cells. There is no difference in the baseline expression levels of monocytic B7-H1 between untreated MS patients and healthy donors. However, both groups show a significant concentration-dependent up-regulation of B7-H1 mRNA and protein in response to IFN-beta in vitro. Serial measurements of B7-H1 mRNA in MS patients before and 6 months after initiation of IFN-beta therapy corroborated the relevance of these results in vivo: Nine of nine patients showed a significant increase in B7-H1 mRNA levels after 6 months of IFN-beta therapy (median 1.04 vs. 8.78; p<0.05, two-sided t-test). Accordingly, protein expression of B7-H1 on monocytes was up-regulated after 24 h of IFN-beta application. In summary, B7-H1 expressed on APC acts as a strong inhibitor of autologous CD4 T-cell activation and may thus contribute to the maintenance of peripheral immune tolerance. IFN-beta up-regulates B7-H1 in vitro and in MS patients in vivo and might represent a novel mechanism how IFN-beta acts as a negative modulator on APC T-cell interactions in the periphery.

Human muscle cells express a B7-related molecule, B7-H1, with strong negative immune regulatory potential: a novel mechanism of counterbalancing the immune attack in idiopathic inflammatory myopathies

B7‐H1 is a novel B7 family protein attributed to costimulatory and immune regulatory functions. Here we report that human myoblasts cultured from control subjects and patients with inflammatory myopathies as well as TE671 muscle rhabdomyosarcoma cells express high levels of B7‐H1 after stimulation with the inflammatory cytokine IFN‐γ. Coculture experiments of MHC class I/II‐positive myoblasts with CD4 and CD8 T cells in the presence of antigen demonstrated the functional consequences of muscle‐related B7‐H1 expression: production of inflammatory cytokines, IFN‐γ and IL‐2, by CD4 as well CD8 T cells was markedly enhanced in the presence of a neutralizing anti‐B7‐H1 antibody. This observation was paralleled by an augmented expression of the T cell activation markers CD25, ICOS, and CD69, thus showing B7‐H1‐mediated inhibition of T cell activation. Further, we investigated 23 muscle biopsy specimens from patients with polymyositis (PM), inclusion body myositis (IBM), dermatomyositis (DM), and nonmyopathic controls for B7‐H1 expression by immunohistochemistry: B7‐H1 was expressed in PM, IBM, and DM specimens but not in noninflammatory and nonmyopathic controls. Staining was predominantly localized to areas of strong inflammation and to muscle cells as well as mononuclear cells. These data highlight the immune regulatory properties of muscle cells and suggest that B7‐H1 expression represents an inhibitory mechanism induced upon inflammatory stimuli and aimed at protecting muscle fibers from immune aggression.

Monocyte-derived HLA-G acts as a strong inhibitor of autologous CD4 T cell activation and is upregulated by interferon-β in vitro and in vivo: rationale for the therapy of multiple sclerosis

Peripheral antigen presenting cells (APCs) contribute to the maintenance of immune tolerance and are considered to play a critical role in promoting the (re)activation of autoreactive T cells in multiple sclerosis (MS). Interferon-beta (IFN-beta) is the principle immune-modulatory agent used in the treatment of MS, but its mechanism of action remains elusive. HLA-G is a non-classical MHC molecule (MHC class Ib) attributed chiefly immune-regulatory functions. We here investigated the role of monocyte-derived HLA-G in the immune-regulatory processes of MS and its implications for current immune-modulatory therapies. Monocytes constitutively express cell surface HLA-G1 and soluble HLA-G5. Comparison of monocytic HLA-G expression between patients with relapsing-remitting MS (n=17) and healthy donors (n=20) revealed significantly lower levels of HLA-G1 protein in MS patients. However, both groups showed a significant upregulation of HLA-G in response to IFN-beta in vitro. Serial measurements of HLA-G mRNA levels in MS patients before and during IFN-beta therapy corroborated the relevance of these results in vivo: 1 month after initiation of IFN-beta1b therapy (n=9), HLA-G1 and HLA-G5 were significantly increased compared to baseline levels and remained elevated during treatment for 6 months (n=3). Importantly, functional experiments demonstrated that monocyte-derived HLA-G inhibits both Th1 (IFN-gamma, IL-2) and Th2 (IL-10) cytokine production by antigen-stimulated autologous CD4 T cells. Soluble HLA-G added to antigen-specific T cell lines (TCLs) has similar effects on the release of cytokines and reduces T cell proliferation. Although both IFN-beta and IFN-gamma strongly enhance HLA-G1 and HLA-G5 expression by monocytes in vitro, IFN-beta leads to a stronger relative upregulation of HLA-G compared to classical MHC class I molecules than stimulation with IFN-gamma. Taken together, monocyte-derived HLA-G mediates the inhibition of autologous CD4 T cell activation and might be involved in immune-regulatory pathways in the pathogenesis of MS. We conclude that some desirable immune-modulatory effects of INF-beta might be accomplished via the upregulation of the immune-tolerogenic molecule HLA-G.

Hide-and-seek in the brain: a role for HLA-G mediating immune privilege for glioma cells

This review summarizes the current knowledge on the expression and functional role of HLA-G in normal CNS cells and brain tumor cells in vitro and in vivo. The CNS has classically been viewed as an immune-privileged organ. Here we discuss some of the particularities of anti-tumoral responses within this compartment. Special emphasis is dedicated to the possible role of the non-classical MHC molecule HLA-G as an alternative mechanism of immune escape. We review the mechanisms how glioma cell-derived HLA-G may paralyze the immune system and which cellular subsets of the immune system are affected. Possible therapeutic implications derived from these observations include the targeting of HLA-G expression within the framework of inducing glioma-specific immunity.

Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo.

Human glioblastoma is a highly lethal tumor known for its capability of interfering with effective antitumor immune responses. Costimulatory signals are of critical relevance in both the inductive and effector phases of immune responses. Inducible costimulator‐ligand (ICOSL), a member of the B7 family of costimulatory molecules related to CD80/CD86, regulates CD4 as well as CD8 T‐cell responses via interaction with its receptor, ICOS, on activated T cells. We report the expression of ICOSL by glioma cells in vitro and in vivo. In contrast to CD80 (B7.1) and CD86 (B7.2), ICOSL protein and mRNA was expressed in 7 of 12 glioma cell lines. ICOSL expression is upregulated by the inflammatory cytokine, tumor necrosis factor‐α (TNF‐α), whereas interferon‐γ (IFN‐γ) has no such effect. Further, immunohistochemical analysis of human brain tumors demonstrates the expression of ICOSL in three of four tissue samples. ICOSL expression is functional in that a neutralizing ICOSL antibody (HIL‐131) reduces Th1 and Th2 cytokine levels in cocultures of peripheral blood lymphocytes or T‐cell subsets (CD4 and CD8) with glioma cells. However, ICOSL gene transfer into glioma cells does not alter their immunogenicity under primary or secondary alloreactive coculture assays.

The CD28 related molecule ICOS: T cell modulation in the presence and absence of B7.1/2 and regulational expression in multiple sclerosis

Costimulatory signals play a key role in regulating T cell activation and are believed to have decisive influence in the inciting and perpetuating cellular effector mechanisms in autoimmune diseases such as multiple sclerosis (MS). Inducible costimulator protein (ICOS), a recently identified member of the CD28-family, presumably affects the differentiation of Th1/Th2 cells after primary activation and modulates the immune response of effector/memory T cells. This study examines the expression and functional role of ICOS costimulation in healthy donors and patients with MS. After nonspecific or antigen-specific stimulation, ICOS is preferentially expressed on CD4 Th2-T cells. ICOS-costimulation affects the production of Th1 and Th2 cytokines both in the absence and presence of B7/CD28 costimulation, thus suggesting that ICOS costimulation can modulate cytokine secretion also in a CD28-independent manner. Levels of constitutive and inducible ICOS expression on human T cell subsets from peripheral blood were quantified in healthy donors and patients with MS. Constitutive expression of ICOS on T cells varies between 0.1% and 42.3%. There were no significant differences between both groups in the baseline expression or inducibility of ICOS on CD4 or CD8 T cells. ICOS expression could be demonstrated on CSF T lymphocytes in patients with acute MS relapses but was not elevated compared with peripheral blood. In essence we show that ICOS is upregulated on human T cells after stimulation and can modulate both Th1 and Th2 cytokine production in the absence and presence of B7-costimulation. In MS patients we demonstrate the functionality of the ICOS costimulatory pathway. Potential implications of ICOSL/ICOS interactions for MS immunopathogenesis are discussed.