Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis.

The data presented in this article are related to the research article entitled "The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis" Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

The autoimmune risk gene ZMIZ1 is a vitamin D responsive marker of a molecular phenotype of multiple sclerosis.

Multiple Sclerosis (MS) is a neurological condition driven in part by immune cells from the peripheral circulation, the targets for current successful therapies. The autoimmune and MS risk gene ZMIZ1 is underexpressed in blood in people with MS. We show that, from three independent sets of transcriptomic data, expression of ZMIZ1 is tightly correlated with that of hundreds of other genes. Further we show expression is partially heritable (heritability 0.26), relatively stable over time, predominantly in plasmacytoid dendritic cells and non-classical monocytes, and that levels of ZMIZ1 protein expression are reduced in MS. ZMIZ1 gene expression is increased in response to calcipotriol (1,25 Vitamin D3) (p < 0.0003) and associated with Epstein Barr Virus (EBV) EBNA-1 antibody titre (p < 0.004). MS therapies fingolimod and dimethyl fumarate altered blood ZMIZ1 gene expression compared to untreated MS. The phenotype indicates susceptibility to MS, and may correspond with clinical response and represent a novel clinical target.

IFNL3/4 genotype is associated with altered immune cell populations in peripheral blood in chronic hepatitis C infection.

Single-nucleotide polymorphisms near the interferon lambda 3 (IFNL3) gene predict outcomes to infection and anti-viral treatment in hepatitis C virus (HCV) infection. To identify IFNL3 genotype effects on peripheral blood, we collected phenotype data on 400 patients with genotype 1 chronic hepatitis C (CHC). The IFNL3 responder genotype predicted significantly lower white blood cells (WBCs), as well as lower absolute numbers of monocytes, neutrophils and lymphocytes for both rs8099917 and rs12979860. We sought to define the WBC subsets driving this association using flow cytometry of 67 untreated CHC individuals. Genotype-associated differences were seen in the ratio of CD4CD45RO+ to CD4CD45RO-; CD8CD45RO+ to CD8CD45RO-, NK CD56 dim to bright and monocyte numbers and percentages. Whole blood expression levels of IFNL3, IFNLR1 (interferon lambda receptor 1), IFNLR1-mem (a membrane-associated receptor), IFNLR1-sol (a truncated soluble receptor), MxA and T- and NK (natural killer) cell transcription factors TBX21, GATA3, RORC, FOXP3 and EOMES in two subjects were also determined. CHC patients demonstrated endogenous IFN activation with higher levels of MxA, IFNLR1, IFNLR1-mem and IFNLR1-sol, and IFNL3 genotype-associated differences in transcription factors. Taken together, these data provide evidence of an IFNL3 genotype association with differences in monocyte, T- and NK cell levels in the peripheral blood of patients with CHC. This could underpin genotype associations with spontaneous and treatment-induced HCV clearance and hepatic necroinflammation.

Cistromic and genetic evidence that the vitamin D receptor mediates susceptibility to latitude-dependent autoimmune diseases.

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), P

Targeting fibroblast-like synovial cells at sites of inflammation with peptide targeted liposomes results in inhibition of experimental arthritis.

In this study we examined a synovium-specific targeted liposomal drug delivery system for its ability to localize and release its drug cargo to inflamed joints. Targeted liposomes were tested in vitro for binding to synovial fibroblast like (FLS) and endothelial cells using flow cytometry and in vivo for localization to joints using a rat model of adjuvant induced arthritis (AIA). Targeted liposomes were then loaded with anti-arthritic medications and examined for clinical efficacy in AIA. Targeted liposomes specifically bound to rabbit FLS and human FLS and showed a 7-10 fold increase in vivo localization in affected joints compared to unaffected joints. Histological sections from rats treated with prednisone and a new immunosuppressive peptide CP showed minimal inflammation. This report substantiates the ability of the novel FLS sequence to target liposomal drug delivery and offers an alternative therapeutic approach for the treatment of arthritis.

Haplotypes of the interleukin 7 receptor alpha gene are correlated with altered expression in whole blood cells in multiple sclerosis.

IL7 regulates T cell survival, differentiation and proliferation. The alpha chain of its receptor, CD127, is polymorphic, and its haplotypes are associated with recovery from transplantation and with the autoimmune disease multiple sclerosis (MS), especially primary progressive MS (PPMS). We demonstrate that two CD127 haplotypes are highly associated with the proportion of the mRNA encoding the soluble isoform of CD127 (P

HIV-Nef enhances interleukin-2 production and phosphatidylinositol 3-kinase activity in a human T cell line.

OBJECTIVE: The Nef protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examine the effects of Nef on T lymphocyte activation and associated signalling events. DESIGN: A recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, and either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates signal transduction pathways and results in IL-2 production and IL-2 receptor alpha-chain (CD25) expression. Cellular responses were examined in cells expressing either Nef or an irrelevant control protein. METHODS: Activation of signalling was assessed by immunoblot analysis, or by in-vitro phosphatidylinositol 3-kinase (PI3K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expression was examined using flow cytometry. RESULTS: Infection of cells with recombinant vaccinia expressing HIV-nef resulted in a marked increase in the production of IL-2 when cells were activated. The enhanced IL-2 response was accompanied by an increase in the level of PI3K activity. IL-2 production remained sensitive to inhibition with the PI3K competitive inhibitor Ly294002, and to the fungal macrolide, rapamycin. In contrast, CD25 expression was not affected, and there were no measurable changes to nuclear factor kappaB (NFkappaB) activation pathways. CONCLUSION: Enhanced IL-2 production in stimulated T cells expressing HIV-Nef is associated with increased activation of PI3K-dependent signalling pathways. The results support a model in which Nef affects HIV disease progression by distorting T cell responses.

Augmentation of lymphocyte responses by monoclonal antibodies to the gangliosides GD3 and GD2: the role of protein kinase C, cyclic nucleotides, and intracellular calcium.

Previous studies have shown that MAb's against the gangliosides GD3 and GD2 may augment T cell responses to a variety of stimuli. We present evidence that antiganglioside MAb's, like PHA, increase intracellular cGMP and protein kinase C yet have no effect on intracellular Ca2+. Stimulation of T cells with MAb's to GD3 was associated with increased cGMP levels, particularly in the CD8+ T cell subset which showed the highest degree of potentiation by the MAb's. Augmentation of T cell responses by the MAb's to GD3 and GD2 was also mimicked by activation of PKC with phorbol esters but both agents together produced marked synergistic effects on cell division, suggesting they had different but complementary modes of action. Furthermore, use of neomycin to inhibit PKC activation only partially reversed the augmentation of proliferative responses by the antiganglioside MAb's. It did however inhibit the MAb-induced increase in IL2 production and IL2 receptor (Tac) expression. These studies suggest therefore that the potentiation of IL2 production by the MAb's against GD2 and GD3 was due to enhanced activation of PKC whereas their augmentation of proliferative responses appeared to be due to effects on late events in T cell activation and was associated with both increased cGMP levels and activation of PKC.

Potentiation of interleukin-2 production and its binding by monoclonal antibodies to the gangliosides GD3 and GD2.

Previous studies have shown that monoclonal antibodies (mAbs) against certain gangliosides, which induced remissions in patients with melanoma, also potentiated the response of lymphocytes to a variety of stimuli, including lectins, interleukin-2 (IL-2) and antigens. The present studies have investigated the mechanism of these effects on lymphocytes. Although the mAbs potentiated phytohemagglutinin(PHA)-induced IL-2 production at high concentrations of mAbs and of PHA, this did not appear to explain their potentiation of the proliferative responses of lymphocytes. Hence, although IL-2 production was minimal or absent from the CD8+ subset the latter showed the highest degree of augmentation. Furthermore, addition of IL-2 to PHA-stimulated cultures did not produce similar augmentation of mitogenic responses to that produced by the mAb to GD3 or GD2. The augmented and normal mitogenic responses were, however, dependent on IL-2, as shown by their inhibition with mAbs against IL-2. The antiganglioside mAbs did not have significant effects on IL-2 receptor expression measured by mAbs to Tac. However, the mAbs appeared to increase the affinity of binding of radiolabelled IL-2 to IL-2 receptor and increased internalization of the latter. These results suggest that the effects of the mAbs on IL-2 production may be distinguished from their effects on the proliferative responses of T cells and that the latter were associated with changes in affinity and internalization of 125I-IL-2. Whether the latter is a direct cause of the increased proliferative response remains unknown. The ability of mAbs to GD2 and GD3 to increase IL-2 production and to "enhance" IL-2-dependent proliferative responses suggests the may have valuable clinical roles as immunopotentiating agents.

Suppression of natural killer cell activity in humans by radiation from solarium lamps depleted of UVB.

Previous studies have shown that ultraviolet radiation (UVR) from solarium lamps suppressed natural killer (NK) cell activity in the blood and that sunscreen lotions offered no protection against this effect. In the present study we tried to determine whether the effects on NK cell activity were caused by the UVB or the UVA components of radiation from solarium lamps by filtering out UVB with Mylar sheeting. Groups of 10 normal subjects were either left untreated or exposed for 30 min on 12 consecutive days to radiation that was filtered or not filtered through a 0.1 mm thick Mylar sheeting. NK cell activity was depressed in the group exposed to solarium radiation and this was not prevented by filtration through Mylar. The latter procedure, however, appeared to prevent changes in blood lymphocyte subsets that are induced by solarium radiation as well as the reduction in Langerhans cell numbers in skin biopsies taken after exposure to solarium radiation. Suppression of NK cell activity was evident up to 14 days after cessation of UVR exposure. This would be consistent with the replacement of NK cells from bone marrow that had been damaged as a result of direct effects of UVA on NK cells in the microcirculation of the skin or else indicate functional suppression of NK cells by suppressor cells induced by UVR as postulated for UVR-induced suppression of delayed hypersensitivity responses in murine models. These studies suggest that UVA may be important in the induction of certain effects on the immune system in human subjects. Further studies are required to assess the implications of these findings with respect to induction of neoplasia and the design of sunscreens effective against UVA.

Analysis of the effect of a sunscreen agent on the suppression of natural killer cell activity induced in human subjects by radiation from solarium lamps.

Previous studies in rodents have shown that ultraviolet radiation (UVR) may have direct effects on the immune system in the skin and at higher doses may induce systemic suppression of immune responses. We have previously shown that UVR from sun or solarium beds may induce systemic effects in human subjects. The purpose of the present study was to examine whether these systemic effects in human subjects could be prevented by use of commercially available sunscreen agents. Groups of 12 normal subjects were exposed to radiation from solarium lamps after application of a sunscreen agent or the base used in its preparation. Twelve half-hourly exposures induced a depression of natural killer (NK) cell activity against a melanoma and the K562 target cell which was not prevented by use of the sunscreen agent. Changes in functional activity were accompanied by a reduction in NK cell numbers assessed by Leu-11 monoclonal antibodies against the labile Fc receptor. Application of the sunscreen agent also did not protect against effects of solarium exposure on recall antigen skin tests and immunoglobulin production in vitro in pokeweed mitogen-stimulated cultures of B and T cells. These results suggest that further evaluation of the wave-length spectrum of UVR and the effectiveness of sunscreen agents in prevention of UVR-induced effects on the immune system is needed.

Potentiation of lymphocyte responses by monoclonal antibodies to the ganglioside GD3.

Previous studies have shown that the ganglioside GD3 is expressed in high concentrations on melanoma cells and that monoclonal antibodies (MAbs) against GD3 may induce partial remissions in tumor growth in patients with melanoma. The present studies indicated that incubation of interleukin 2 (IL2) dependent murine natural killer cells with several MAbs against the ganglioside GD3 potentiated the proliferative response to IL2. There was no effect on cell division in the absence of added IL2. Similarly the mitogenic response of human blood lymphocytes to phytohemagglutinin (PHA) or the T3 antigen was enhanced by coculture with these MAbs. This was noted when the MAbs to GD3 were added either before or up to 48 h after addition of PHA or MAb to T3. Kinetic analysis revealed that the potentiation of the PHA induced mitogenic response followed the expected response to PHA suggesting that the MAbs amplified normal activation pathways. These effects were also seen wih MAb to GD3 and the T10 structure on T-cells but not with MAbs to the transferrin receptor or isotype MAb controls. Studies on T-cell subsets suggested that the enhanced PHA responses were confined mainly to the T8 subset and responses of the T4 subset were not enhanced. Flow cytometric analysis revealed low levels of GD3 expression on blood lymphocytes which increased during culture with PHA. IL2 receptor (Tac epitope) expression did not show close correlation with the enhanced lymphocyte responses and the mechanism of the potentiation remains uncertain. These studies raise questions concerning the role of gangliosides in T-cell activation and whether these in vitro effects of MAbs to GD3 may account in part for their antitumor effects in patients with melanoma.

Clonal analysis of cytotoxic T lymphocytes (CTL) against autologous melanoma. Classification based on phenotype, specificity and inhibition by monoclonal antibodies to T cell structures.

This study investigates the nature and specificity of cytotoxic T lymphocytes (CTL) in patients with melanoma which are able to kill autologous melanoma cells. Interleukin 2 (IL2)-dependent T cell clones from two melanoma patients and a normal subject were generated in mixed lymphocyte cultures (MLC) or mixed lymphocyte tumor cell cultures (MLTC) and propagated for prolonged periods in tissue culture. Analysis of their phenotype by a wide range of monoclonal antibodies (M.Abs) revealed two main phenotypes which depended on whether they expressed Fc receptors detected by Leu 11 M.Abs or not. Leu 11- T cells (referred to as Type 1) were inhibited by M.Abs to T3, T8, and a common HLA, ABC antigen. Conversely Leu 11+ T cells (referred to as Type 2) were inhibited by M.Ab to Leu 11 but not by M.Ab to T3, T8 and the HLA, ABC antigen. Subtypes among Type 1 cells were recognized which depended on their specificity. The most restricted were CTL [Type 1(a)] clones generated only in MLTC which recognized the autologous melanoma cell plus 1 of 11 other melanoma target cells. Type 1(b) CTL clones recognized a larger proportion (approximately 50%) of the melanoma cells. A third category [Type 1(c)] recognized antigens on melanoma cells shared with that on the EBV-transformed B cells used as stimulators in the MLC. Type 2 CTL clones had broad specificity to melanoma and nonmelanoma cells, characteristic of that described for lymphokine activated killer (LAK) cells. The latter were MHC unrestricted but further studies are required to clarify whether the Type 1 CTL clones are MHC restricted or not. The CTL activity of all clones was inhibited by M.Ab to the sheep red blood cell receptor and to the T10 antigens. It is suggested that recognition of these different types of CTL clones may assist future studies on the immune response against melanoma and the nature of antigens recognized by CTL.