B. Thomas Bäckström

Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation.

Treatment with ex vivo–generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)35–55/IAb (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer–reactive, Foxp3+ T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3+ T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4+Foxp3+ T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.

Schistosomiasis Decreases Central Nervous System Inflammation and Alters the Progression of Experimental Autoimmune Encephalomyelitis

ABSTRACT A preestablished infection with the parasitic helminth, Schistosoma mansoni, significantly reduced the incidence and delayed the onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. The altered disease progression was not solely due to the induction of a strong Th2 response, since intraperitoneal injection of schistosome eggs did not affect disease development. MOG-specific gamma interferon (IFN-γ), nitric oxide, and tumor necrosis factor alpha production by splenocytes was significantly reduced in schistosome-infected mice compared to uninfected mice. However, similar levels of interleukin-10 (IL-10) were produced in an antigen-specific manner, suggesting that the induction of antigen-specific responses was not inhibited. Analysis of in vivo cytokine production by real-time PCR indicated that IL-12p40, but not IFN-γ, transcript levels were dramatically reduced in the spinal cords of schistosome-infected, MOG-immunized mice. Furthermore, analysis of the cellular composition of the spinal cords and brains revealed that a preestablished infection with S. mansoni decreased central nervous system (CNS) inflammation, particularly of macrophages and CD4 T cells. These results suggest that schistosomiasis may negatively regulate the onset of EAE by downregulating the production of proinflammatory cytokines and altering CNS inflammation.

Depletion of Langerhans Cells in Human Papillomavirus Type 16-Infected Skin Is Associated with E6-Mediated Down Regulation of E-Cadherin

ABSTRACT Human papillomavirus type 16 (HPV16) is an oncogenic virus that causes persistent infections in cervical epithelium. The chronic nature of HPV16 infections suggests that this virus actively evades the host immune response. Intraepithelial Langerhans cells (LC) are antigen-presenting cells that are critical in T-cell priming in response to viral infections of the skin. Here we show that HPV16 infection is directly associated with a reduction in the numbers of LC in infected epidermis. Adhesion between keratinocytes (KC) and LC, mediated by E-cadherin, is important in the retention of LC in the skin. Cell surface E-cadherin is reduced on HPV16-infected basal KC, and this is directly associated with the reduction in numbers of LC in infected epidermis. Expression of a single viral early protein, HPV16 E6, in KC reduces levels of cell surface E-cadherin thereby interfering with E-cadherin-mediated adhesion. Through this pathway, E6 expression in HPV16-infected KC may limit presentation of viral antigens by LC to the immune system, thus preventing the initiation of a cell-mediated immune response and promoting survival of the virus.

Experimental autoimmune encephalomyelitis induction in naive mice by dendritic cells presenting a self-peptide.

Self‐reactive T cells escape deletion in the thymus and are found in the peripheral repertoire. Because bone‐marrow‐derived dendritic cells (BM‐DC) are potent activators of antigen‐specific T cells, these cells could theoretically activate self‐reactive T cells leading to autoimmunity. We investigated whether BM‐DC could induce the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our results show that transfer of BM‐DC presenting a self‐peptide from the myelin oligodendrocyte glycoprotein (MOG35–55) into naive mice induced EAE 7–14 days later. MOG35–55‐specific T cells of the Th1 phenotype were present in the lymph nodes and spleens of mice that received live peptide‐pulsed BM‐DC. Heat‐killed or formaldehyde‐fixed BM‐DC presenting MOG35–55 could induce neither clinical signs of EAE nor a measurable T‐cell response in vitro. These data show that live BM‐DC presenting a self‐antigen can induce the organ‐specific autoimmune disorder EAE in a non‐transgenic system. Therefore, this new EAE model could be used as a more clinically relevant model for the human disease multiple sclerosis. These findings could also have implications for the use of DC immunotherapy in a clinical setting.

Recombinant Escherichia coli produces tailor-made biopolyester granules for applications in fluorescence activated cell sorting: functional display of the mouse interleukin-2 and myelin oligodendrocyte glycoprotein

BackgroundFluorescence activated cell sorting (FACS) is a powerful technique for the qualitative and quantitative detection of biomolecules used widely in both basic research and clinical diagnostic applications. Beads displaying a specific antigen are used to bind antibodies which are then fluorescently labelled using secondary antibodies. As the individual suspension bead passes through the sensing region of the FACS machine, fluorescent signals are acquired and analysed. Currently, antigens are tediously purified and chemically cross-linked to preformed beads. Purification and coupling of proteins often renders them inactive and they will not be displayed in its native configuration. As an alternative, we genetically engineered Escherichia coli to produce biopolyester (polyhdroxyalkanoate=PHA) granules displaying diagnostically relevant antigens in their native conformation and suitable for FACS analysis.ResultsHybrid genes were constructed, which encode either the mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG) fused via an enterokinase site providing linker region to the C terminus of the PHA granule associated protein PhaP, respectively. The hybrid genes were expressed in PHA-accumulating recombinant E. coli. MOG and IL2 fusion proteins were abundantly attached to PHA granules and were identified by MALDI-TOF/MS analysis and N terminal sequencing. A more abundant second fusion protein of either MOG or IL2 resulted from an additional N terminal fusion, which did surprisingly not interfere with attachment to PHA granule. PHA granules displaying either IL2 or MOG were used for FACS using monoclonal anti-IL2 or anti-MOG antibodies conjugated to a fluorescent dye. FACS analysis showed significant and specific binding of respective antibodies. Enterokinase treatment of IL2 displaying PHA granules enabled removal of IL2 as monitored by FACS analysis. Mice were immunized with either MOG or OVA (ovalbumin) and the respective sera were analysed using MOG-displaying PHA granules and FACS analysis showing a specific and sensitive detection of antigen-specific antibodies within a wide dynamic range.ConclusionE. coli can be genetically engineered to produce PHA granules displaying correctly folded eukaryotic proteins and which can be applied as beads in FACS based diagnostics. Since PHA granule formation and protein attachment occurs in one step already inside the bacterial cell, microbial production could be a cheap and efficient alternative to commercial beads.

Transplantation of Bone Marrow Transduced to Express Self-Antigen Establishes Deletional Tolerance and Permanently Remits Autoimmune Disease

Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-γ, and GM-CSF in response to MOG35–55 peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4+ MOG35–55-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.

Naïve blood monocytes suppress T-cell function. A possible mechanism for protection from autoimmunity.

In certain disease context, cells of the monocyte/macrophage lineage are known to exhibit T‐cell suppressor function. However, whether naïve monocytes are also able to suppress T‐cell responses has not been previously investigated. In this study, we have discovered that CD11b+Ly6G− mononuclear cells in the blood of naïve mice are potent suppressors of T‐cell proliferation in vitro. The suppression of T‐cell proliferation requires cell‐cell contact and is partially dependent on nitric oxide production. Following the induction of experimental autoimmune encephalomyelitis in mice, the suppressor function of this blood CD11b+Ly6G− cell population is impaired. Therefore, blood CD11b+Ly6G− cells appear to be intrinsically suppressive and may have a key role in maintaining immune homoeostasis. Loss of this suppressive function may contribute to development of autoimmunity.

Targeting Antigen to MHC Class II Molecules Promotes Efficient Cross-Presentation and Enhances Immunotherapy

An efficient pathway of cross-presentation common to a range of dendritic cell (DC) populations was identified by targeting Ag to MHC class II molecules. This finding was achieved by conjugating Ag to M1, which is a modified version of the superantigen streptococcal mitogenic exotoxin Z-2 that binds to MHC class II molecules but cannot directly stimulate T cells. M1 conjugates were efficiently presented to CD4+ and CD8+ T cells by bone marrow-derived DC and Langerhans cells in vitro. Whereas nonconjugated Ag was preferentially cross-presented by splenic CD8α+ DC in vivo, M1-conjugated Ag was cross-presented by all dendritic subtypes assessed. Potent effector T cell responses with antitumor activity were elicited when M1 conjugates were injected together with an adjuvant. This method of Ag delivery has significant potential in therapeutic applications.

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H‐2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG38–51 peptide important for peptide binding to I‐Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I‐Ab. In addition, G38 andW39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I‐Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC‐binding residue of the MOG38–51 peptide and most likely occupies the p1 pocket of I‐Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG38–51 peptide to study their therapeutic potential in the EAE model.

The phenotype of type 1 and type 2 CD8+ T cells activated in vitro is affected by culture conditions and correlates with effector activity

We used various culture conditions to generate type 1 (Tc1) or type 2 (Tc2) cytotoxic T cells in vitro. T‐cell receptor (TCR) transgenic T cells were cultured with antigen and spleen cells, or antigen and dendritic cells (DC), or anti‐CD3 and anti‐CD28. Tc1 cultures contained interleukin (IL)‐2 and IL‐6, and Tc2 cultures contained IL‐2, IL‐6 and IL‐4. Tc2 cells generated in each culture condition acquired a CD62Llow CD44high phenotype, had high cytotoxic activity, and secreted IL‐4, IL‐5 and moderate amounts of interferon‐γ (IFN‐γ). In contrast, the phenotype and function of Tc1 cells varied depending on culture conditions. Tc1 cells from anti‐CD3 and anti‐CD28 cultures had high cytotoxic activity and were CD62Llow CD44high, while Tc1 cells from antigen and spleen cell cultures had low cytotoxic activity and were CD62Lhigh CD44low. Tc1 cells from antigen and DC cultures had an intermediate phenotype. All Tc1 cells secreted high amounts of IFN‐γ, but only Tc1 from anti‐CD3 and anti‐CD28 cultures had antitumour activity in vivo. Differences were not caused by suboptimal culture conditions, as Tc1 cells divided at a similar rate whether cultured with antigen and spleen cells or with anti‐CD3 and anti‐CD28. We conclude that IL‐4 not only induces ‘type 2’ cytokine secretion in CD8+ T cells, but also affects their expression of surface markers and cytotoxic activity.