Augmenting DAF levels in vivo ameliorates experimental autoimmune encephalomyelitis

Recent studies in experimental autoimmune encephalomyelitis (EAE) have found that CNS injury in Daf1^−/− mice is much greater than in wild types (WTs), suggesting that upregulating DAF levels in vivo might ameliorate disease. To test this, we generated a Daf1 transgenic (Tg) mouse which had elevated DAF levels on its cell surfaces. In by-stand C3b uptake assays, Daf1 Tg mouse erythrocytes took up less C3b on their surfaces than WT erythrocytes. When co-cultured with OT-II CD4⁺ T cells together with OVA_323–339 peptide, Daf1 Tg mouse bone marrow derived dendritic cells (BM-DCs) produced less C5a and C3a than WT BM-DCs and stimulated a lesser T cell response. In MOG_35–55 immunization induced EAE model, Daf1 Tg mice exhibited delayed disease onset and decreased clinical scores compared to WTs. Histological analyses showed that there were less inflammation and demyelination in spinal cords in Daf1 Tg mice than those in WTs. In accordance with these results, Daf1 Tg mice had decreased MOG_35–55 specific Th1 and Th17 responses. These data provide further evidence that DAF suppresses autoreactive T cell responses in EAE, and indicate that augmenting its expression levels could be effective therapeutically in treating multiple sclerosis as well as other T cell mediated diseases.     

Diversification and senescence of Foxp3+ regulatory T cells during experimental autoimmune encephalomyelitis

The fate of Foxp3⁺ regulatory T (Treg) cells responding during autoimmunity is not well defined. We observed a marked elevation in KLRG1⁺ (where KLRG1 stands for killer cell lectin‐like receptor G1) CNS‐infiltrating Treg cells in experimental autoimmune encephalomyelitis (EAE), and assessed their origin and properties. KLRG1⁺ Treg cells showed increased activation marker expression, Foxp3 and CD25 levels, and more rapid cell cycling than KLRG1^? cells. KLRG1^? Treg cells converted into KLRG1⁺ cells and this was increased in autoimmune inflammation. Conversion was unidirectional; KLRG1⁺ Treg cells did not revert to a KLRG1^? state. KLRG1⁺ but notKLRG1^?Treg cells survived poorly, indicative of terminal differentiation. This was associated with diminished BCL2 and increased apoptosis of isolated cells. KLRG1 was also upregulated on iTreg cells after transfer and EAE induction or on iTreg cells developing spontaneously during EAE. KLRG1⁺ Treg cells produced more IL‐10 and had altered effector cytokine production compared with their KLRG1^? counterparts. Despite their differences, KLRG1⁺ and KLRG1^? Treg cells proved similarly potent in suppressing EAE. KLRG1⁺ and KLRG1^? populations were phenotypically heterogeneous, with the extent and pattern of activation marker expression dependent both on cellular location and inflammation. Our results support an extensive diversification of Treg cells during EAE, and associate KLRG1 with altered Treg‐cell function and senescence.     

Vasoactive intestinal peptide induces regulatory T cells during experimental autoimmune encephalomyelitis

CD4(+)CD25(+) regulatory T cells (Treg) control the immune response to a variety of antigens, including self-antigens. Several models support the idea of the peripheral generation of CD4(+)CD25(+) Treg from CD4(+)CD25(-) T cells. Little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4(+)CD25(+) Treg. In this study we report on the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, to induce functional Treg in vivo during the development of experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. The administration of VIP to EAE mice results in the expansion of the CD4(+)CD25(+), Foxp3-expressing T cells in the periphery and the nervous system, which inhibit encephalitogenic T cell activation. In addition to the increase in the number of CD4(+)CD25(+) Treg, VIP induces more efficient suppressors on a per cell basis. The VIP-generated CD4(+)CD25(+) Treg transfer suppression and significantly ameliorate the progression of the disease.     

Defining antigen-dependent stages of T cell migration from the blood to the central nervous system parenchyma

In experimental autoimmune encephalomyelitis (EAE), intravenous transfer of activated CD4(+) myelin-specific T cells is sufficient to induce disease. Transferred T cells access the CNS parenchyma by trafficking across the blood brain barrier (BBB) vascular endothelium into the perivascular space, and then across the glial limitans that is made up of astrocytes and microglia. Flow cytometry analysis of cells isolated from CNS tissue does not distinguish between T cell populations at the various stages of migration. In this study, we have used GK1.5 (anti-CD4) treatment along with immunohistochemistry to distinguish between populations of T cells that are associated with the vasculature, T cells that have migrated into the perivascular space, and T cells in the parenchyma. We have also re-evaluated antigen specificity requirements of T cells as they are recruited to the CNS parenchyma. Activated myelin-specific T cells are restricted to the CNS vasculature for at least 24 h post transfer. MHC class II expression on the recipient is required for cells to traffic across the CNS vascular endothelium. Further, Con A-stimulated or non-CNS-specific (ovalbumin-specific) T cells fail to migrate into the perivascular space, and only enter the CNS parenchyma when co-transferred with myelin-specific T cells. Our results indicate that Th1 populations cannot accumulate in the perivascular (subarachnoid, Virchow-Robbins) space without a CNS antigen-specific signal.     

Blockade of tumour necrosis factor‐α in experimental autoimmune encephalomyelitis reveals differential effects on the antigen‐specific immune response and central nervous system histopathology

In various autoimmune diseases, anti‐tumour necrosis factor (TNF)‐α treatment has been shown to reduce both clinical disease severity and T helper type 1 (Th1)1/Th17 responses. In experimental autoimmune encephalomyelitis (EAE), however, the role of TNF‐α has remained unclear. Here, C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 and treated with anti‐TNF‐α, control antibody or vehicle. The clinical disease course, incidence and severity were assessed. On day 20 after immunization the antigen‐specific Th1/Th17 response was evaluated by enzyme‐linked immunospot (ELISPOT) in spleen and central nervous system (CNS). Also, the extent of spinal cord histopathology was analysed on semi‐ and ultrathin sections. Our results demonstrate that anti‐TNF‐α treatment reduced the incidence and delayed the onset of EAE, but had no effect on disease severity once EAE had been established. Whereas anti‐TNF‐α treatment induced an increase in splenic Th1/Th17 responses, there was no effect on the number of antigen‐specific Th1/Th17 cells in the spinal cord. Accordingly, the degree of CNS histopathology was comparable in control and anti‐TNF‐α‐treated mice. In conclusion, while the anti‐TNF‐α treatment had neither immunosuppressive effects on the Th1/Th17 response in the CNS nor histoprotective properties in EAE, it enhanced the myelin‐specific T cell response in the immune periphery.     

γδ T cells in EAE: Early trafficking events and cytokine requirements

We have previously shown that γδ T cells traffic to the CNS during EAE with concurrently increased expression of β₂‐integrins and production of IFN‐γ and TNF‐α. To extend these studies, we transferred bioluminescent γδ T cells to WT mice and followed their movement through the acute stages of disease. We found that γδ T cells rapidly migrated to the site of myelin oligodendrocyte glycoprotein peptide injection and underwent massive expansion. Within 6 days after EAE induction, bioluminescent γδ T cells were found in the spinal cord and brain, peaking in number between days 10 and 12 and then rapidly declining by day 15. Reconstitution of γδ T cell^?/? mice with γδ T cells derived from β₂‐integrin‐deficient mice (CD11a, ‐b or ‐c) demonstrated that γδ T‐cell trafficking to the CNS during EAE is independent of this family of adhesion molecules. We also examined the role of γδ T‐cell‐produced IFN‐γ and TNF‐α in EAE and found that production of both cytokines by γδ T cells was required for full development of EAE. These results indicate that γδ T cells are critical for the development of EAE and suggest a therapeutic target in demyelinating disease.     

Requirement of the Fc receptor common gamma-chain for gamma delta T cell-mediated promotion of murine experimental autoimmune encephalomyelitis

Immunoglobulin Fcgamma receptors (FcgammaR) are comprised of a ligand-binding alpha-chain that sometimes associates with a cell signaling common gamma-chain. These receptors comprise an important family of effector molecules that link humoral and cell-mediated adaptive immunity and regulate innate immunity. Recent animal studies suggest that FcgammaR in general, and FcR alpha-chains in particular, are required for full development of experimental autoimmune encephalomyelitis (EAE). We show here that deletion of the gamma-chain renders mice resistant to EAE, whereas deletion of the alpha-chains of FcgammaRI, FcgammaRIIB and FcgammaRIII has no protective effect. Susceptibility to EAE is fully restored in common gamma-chain-/- mice into which wild-type splenocytes are adoptively transferred, but EAE is not restored in common gamma-chain-/- mice given wild-type splenocytes depleted of gammadelta T cells. These data indicate that although the common gamma-chain is required for full development of EAE in mice, this requirement is likely FcgammaR alpha-chain-independent. Expression of the common gamma-chain by gammadelta T cells, probably in conjunction with the T cell receptor/CD3 complex, is likely the key requirement for full development of EAE.     

T-bet is essential for the progression of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T helper 1 (Th1) cells, while recovery from the disease is associated with the presence of Th2 cells. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of EAE. T-bet regulates the production of interferon-gamma (IFN-gamma) in CD4+ and natural killer cells, and CD4+ T cells from T-bet-deficient mice were unable to differentiate into a Th1 phenotype. Moreover BALB/c mice deficient in T-bet were resistant to the induction of EAE disease, with minimal inflammatory infiltrates in the central nervous system. These mice were resistant to EAE induction even when PLP(180-199) peptide specific effector T cells from BALB/c wild type were transferred to BALB/c T-bet-deficient mice. This resistance to EAE is may be caused by the production of the anti-inflammatory cytokine interleukin-10 (IL-10) from the spleen cells upon ex vivo stimulation with PLP(180-199) peptide and in vivo presence in the central nervous system. There was no difference in the recall responses in spleen cells from T-bet-deficient and wild type mice; however, less secretion of IFN-gamma was observed from primed splenocytes. The expression of IFN-gamma was less in the central nervous system of T-bet-deficient mice whereas IL-10 was significantly higher in T-bet-deficient as compared to wild type mice. These data indicate that T-bet genes play a critical role in maintaining the encephalitogenic nature of CD4+ T cells in autoimmune responses during EAE disease progression.     

Plasmacytoid DC promote priming of autoimmune Th17 cells and EAE

EAE, an animal model for MS, is a Th17 and Th1‐cell‐mediated autoimmune disease, but the mechanisms leading to priming of encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. To investigate the role of plasmacytoid DC (pDC) in the initiation of autoimmune Th17‐ and Th1‐cell responses and EAE, we depleted pDC with anti‐pDC Ag‐1 (anti‐PDCA1) mAb prior to immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG). pDC‐depleted mice developed less severe clinical and histopathological signs of EAE than control mice, which demonstrates a promoting role for pDC in the initiation of EAE. The levels of type I IFN were much lower in the sera from anti‐PDCA1‐treated mice. However, neutralization of type I IFN ameliorated the early phase of EAE but did not alter the severity of disease. Thus, only a minor part of the EAE‐promoting effect of pDC appears to be mediated by IFN‐α/β secretion. The numbers of MOG‐specific Th17 cells, but not Th1 cells, were lower in spleen from anti‐PDCA1‐treated mice compared with controls. In contrast, pDC depletion a week after MOG immunization resulted in more severe clinical signs of EAE. In conclusion, we demonstrate that pDC promote initiation of MOG‐induced Th17‐cell responses and EAE.     

Estrogen-induced protection against experimental autoimmune encephalomyelitis is abrogated in the absence of B cells

Increased remissions in multiple sclerosis (MS) during pregnancy suggest that elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2=17β-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Studies demonstrate that depletion of B cells prior to induction of EAE exacerbates disease severity, implicating regulatory B cells. We thus evaluated pathogenic and E2-induced protective mechanisms in B-cell-deficient (μMT(-/-)) mice. EAE-protective effects of E2 were abrogated in μMT(-/-) mice, with no reduction in disease severity, cellular infiltration or pro-inflammatory factors in the central nervous system compared to untreated controls. E2 treatment of WT mice selectively upregulated expression of PD-L1 on B cells and increased the percentage of IL-10-producing CD1d(high) CD5(+) regulatory B cells. Upregulation of PD-L1 was critical for E2-mediated protection since E2 did not inhibit EAE in PD-L1(-/-) mice. Direct treatment of B cells with E2 significantly reduced proliferation of MOG(35-55)-specific T cells that required estrogen receptor-α (ERα). These results demonstrate, for the first time, a requirement for B cells in E2-mediated protection against EAE involving direct E2 effects on regulatory B cells mediated through ERα and the PD-1/PD-L1 negative co-stimulatory pathway. E2-primed B cells may represent an important regulatory mechanism in MS and have strong implications for women receiving current MS therapies that cause B-cell depletion.     

The Peyer's patch is a critical immunoregulatory site for mucosal tolerance in experimental autoimmune encephalomylelitis (EAE)

Expression of MCP-1 in the central nervous system (CNS) is associated with various neuroinflammatory diseases, including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we found that MCP-1 was decreased in the CNS but increased in the gut following oral administration of myelin basic protein (MBP) correlating with protection from EAE. To study the trafficking and the fate of T cells during oral tolerance, MBP-specific TCR transgenic (Tg) CD4(+) T cells were labeled using 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE) and transferred intravenously to syngeneic B10.PL recipients before feeding with either MBP or PBS. We observed that the CFSE-labeled T cells traffic to the peripheral lymphoid tissue and the Peyer's patches (PP). The labeled T cells proliferate in vivo in both the lymph node and the PP 48h after MBP feeding, but the cells are maintained in the PP longer than in the LN. CFSE-labeled cells in the PP have high levels of CD69 and Fas expression which is accompanied by increased apoptosis after MBP feeding. Our observations suggest that oral administration of autoantigen induces an elevation of MCP-1 in the gut, early T cell trafficking and activation in the periphery and the PP, followed by deletion of autoreactive T cells in the PP.     

Modulation of the severity of experimental autoimmune encephalomyelitis by gammadelta T lymphocytes activated by mycobacterial antigens

Immunity to mycobacterial antigens may contribute to the maintenance of self-tolerance. Exposure of the immune system to mycobacterial antigen might well stimulate the immune system to exert control over unwanted self-reactive clones. We demonstrated that in vivo administration of Mycobacterium tuberculosis, PPD, and PPD peptide (180-196) prior to immunization with Myelin Basic Protein (MBP)led to a moderate increase of γδ T cells, suppression of the immune response, and reduction in the severity of Experimental Autoimmune Encephalomyelitis. The immunosuppression observed is due, at least in part, to the production of Transforming growth factor-β (TGFβ) by the γδ T lymphocytes.     

Dissimilar background genes control susceptibility to autoimmune disease in the context of different MHC haplotypes: NOD.H-2(s) congenic mice are relatively resistant to both experimental autoimmune encephalomyelitis and type I diabetes

Nonobese diabetic (NOD) mice develop multi-organ autoimmune diseases, including type 1 diabetes. We hypothesized that backcrossing the MHC region from SJL (H-2(s)) mice, which have an endogenous PLP(139-151)-reactive repertoire, onto the background of autoimmune-prone NOD mice would result in a mouse strain that is highly susceptible to experimental autoimmune encephalomyelitis (EAE). Unexpectedly, although we detected an endogenous PLP(139-151) repertoire in the NOD.S mice, they did not develop spontaneous EAE and were relatively resistant to PLP(139-151)-induced EAE when compared to SJL mice. This resistance was associated with lower production of proinflammatory cytokines and a decreased expansion of PLP(139-151)-specific CD4(+) T cells after immunization and restimulation with PLP peptide in vitro. V(beta) chain usage among PLP(139-151)-reactive T cells differed between SJL and NOD.S mice. Furthermore, NOD.S mice were resistant to the development of insulitis and cyclophosphamide-induced diabetes, but not sialadenitis. Altogether, even though NOD mice develop spontaneous autoimmune diseases, they become relatively resistant to induction of EAE even when they express the EAE-permissive class II molecule I-A(s). Our data show that certain combinations of otherwise susceptibility-conferring MHC and non-MHC genes can mediate autoimmune-disease resistance when they are paired together. These findings do not support the "shared autoimmune gene" hypothesis.     

Vstm3 is a member of the CD28 family and an important modulator of T-cell function

Members of the CD28 family play important roles in regulating T-cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA-4-CD80-CD86 network. In the same way that soluble CTLA-4 can be used to block T-cell responses, we show that soluble Vstm3 attenuates T-cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T-cell responses.     

How myeloid cells shape experimental autoimmune encephalomyelitis: At the crossroads of outside-in immunity

Experimental autoimmune encephalomyelitis (EAE) is an animal model of central nervous system (CNS) autoimmunity. It is most commonly used to mimic aspects of multiple sclerosis (MS), a demyelinating disorder of the human brain and spinal cord. The innate immune response displays one of the core pathophysiological features linked to both the acute and chronic stages of MS. Hence, understanding and targeting the innate immune response is essential. Microglia and other CNS resident MUs, as well as infiltrating myeloid cells, diverge substantially in terms of both their biology and their roles in EAE. Recent advances in the field show that antigen presentation, as well as disease-propagating and regulatory interactions with lymphocytes, can be attributed to specific myeloid cell types and cell states in EAE lesions, following a distinct temporal pattern during disease initiation, propagation and recovery. Furthermore, single-cell techniques enable the assessment of characteristic proinflammatory as well as beneficial cell states, and identification of potential treatment targets. Here, we discuss the principles of EAE induction and protocols for varying experimental paradigms, the composition of the myeloid compartment of the CNS during health and disease, and systematically review effects on myeloid cells for therapeutic approaches in EAE.     

Identification of T cell epitopes on human proteolipid protein and induction of experimental autoimmune encephalomyelitis in HLA class II-transgenic mice

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS that is associated with HLA class II molecules HLA-DR2, -DR3 and -DR4. Previously, it has been difficult to analyze the role of individual HLA molecules in disease pathogenesis due to heterogeneity of MHC genes, linkage disequilibrium, influence of non-MHC genes and contribution of environment. To overcome some of these problems, we have generated HLA-transgenic (tg) mice to investigate function and interaction of these molecules in disease pathogenesis. To investigate the role of individual HLA class II genes in immune responses to human proteolipid protein (PLP), a candidate autoantigen in MS, mice expressing HLA genes DR2, DR3, DR4 (DRB1*0401 and DRB1*0402), DQ6 and DQ8, lacking endogenous class II molecules were immunized with overlapping peptides of PLP. In all tg mice, the majority of the dominant T cell epitopes were clustered mainly to three region; amino acids 31-70, 91-120 and 178-228, of the PLP molecules. We also identified an encephalitogenic epitope PLP(91-110) that induced clinical EAE in HLA-DR3 tg mice. These tg mice had inflammatory infiltrates classically associated with EAE and showed a Th1 cytokine profile. This humanized mouse model of MS will be valuable in deciphering the role of HLA molecules and autoantigens in MS.     

MicroRNA let‐7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis

MicroRNAs (miRNAs) play important roles in the regulation of immune responses. There is evidence that miRNAs also participate in the pathogenesis of multiple sclerosis (MS), but how the miRNAs regulate the pathogenesis of MS is still under investigation. The identification of new members of the miRNA family associated with the pathogenesis of MS could facilitate early diagnosis and treatment. Here, we show that the level of miRNA let‐7e is significantly upregulated in EAE, an animal model of MS using miRNA array and quantitative real‐time PCR. The expression of let‐7e was mainly in CD4⁺ T cells and infiltrated mononuclear cells of CNS, and highly correlated with the development of EAE. We found that let‐7e silencing in vivo inhibited encephalitogenic Th1 and Th17 cells and attenuated EAE, with reciprocal increase of Th2 cells; overexpression of let‐7e enhanced Th1 and Th17 cells and aggravated EAE. We also identified IL‐10 as one of the functional targets of let‐7e. Together, we propose that let‐7e is a new miRNA involved in the regulation of encephalitogenic T‐cell differentiation and the pathogenesis of EAE.