a b s t r a c t "Inverse vaccination" refers to antigen-specific tolerogenic immunization treatments that are capable of inhibiting autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), initial trials using purified myelin antigens required repeated injections because of the rapid clearance of the antigens. This problem has been overcome by DNA-based vaccines encoding for myelin autoantigens alone or in combination with "adjuvant" molecules, such as interleukin (IL)-4 or IL-10, that support regulatory immune responses. Phase I and II clinical trials with myelin basic protein (MBP)-based DNA vaccines showed positive results in reducing magnetic resonance imaging (MRI)-measured lesions and inducing tolerance to myelin antigens in subsets of MS patients. However, DNA vaccination has potential risks that limit its use in humans. An alternative approach could be the use of protein-based inverse vaccines loaded in polymeric biodegradable lactic-glycolic acid (PLGA) nano/microparticles (NP) to obtain the sustained release of antigens and regulatory adjuvants. The aim of this work was to test the effectiveness of PLGA-NP loaded with the myelin oligodendrocyte glycoprotein (MOG) autoantigen and recombinant (r) IL-10 to inverse vaccinate mice with EAE. In vitro experiments showed that upon encapsulation in PLGA-NP, both MOG 35-55 and rIL-10 were released for several weeks into the supernatant. PLGA-NP did not display cytotoxic or proinflammatory activity and were partially endocytosed by phagocytes. In vivo experiments showed that subcutaneous prophylactic and therapeutic inverse vaccination with PLGA-NP loaded with MOG 35-55 and rIL-10 significantly ameliorated the course of EAE induced with MOG 35-55 in C57BL/6 mice. Moreover, they decreased the histopathologic lesions in the central nervous tissue and the secretion of IL-17 and interferon (IFN)-␥ induced by MOG in splenic T cells in vitro. These data suggest that subcutaneous PLGA-NP-based inverse vaccination may be an effective tool to treat autoimmune diseases.

Mutations decreasing function of the Fas death receptor cause the autoimmune lymphoproliferative syndrome (ALPS) with autoimmune manifestations, spleen/ lymph node enlargement, and expansion of CD4/CD8-negative T cells. Dianzani Autoimmune Lymphoproliferative Disease (DALD) is a variant lacking this expansion. Perforin is involved in cell-mediated cytotoxicity and its biallelic mutations cause familial hemophagocytic lymphohistiocytosis (HLH). We previously described an ALPS patient carrying heterozygous mutations of the Fas and perforin genes and suggested that they concurred in ALPS. This work extends the analysis to 14 ALPS, 28 DALD, and 816 controls, and detects an N252S amino acid substitution in 2 ALPS, and an A91V amino acid substitution in 6 DALD. N252S conferred an OR ‫؍‬ 62.7 (P ‫؍‬ .0016) for ALPS and A91V conferred an OR ‫؍‬ 3 (P ‫؍‬ .016) for DALD. Copresence of A91V and variations of the osteopontin gene previously associated with DALD conferred an OR ‫؍‬ 17 (P ‫؍‬ .0007) for DALD.In one N252S patient, NK activity was strikingly defective in early childhood, but became normal in late childhood. A91V patients displayed lower NK activity than controls. These data suggest that perforin variations are a susceptibility factor for ALPS/DALD development in subjects with defective Fas function and may influence disease expression. IntroductionFas is a death receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily and induces cell death upon triggering by FasL. [1][2][3] In the immune response, it is highly expressed by activated effector lymphocytes and is involved in switching off the immune response, limiting clonal expansion of lymphocytes, and favoring peripheral tolerance. Moreover, FasL is expressed by cytotoxic T cells and NK cells and is involved in killing of target cells expressing Fas. Fas induces cell apoptosis by triggering a cascade of caspases through 2 partly interconnected pathways: the extrinsic pathway involves caspase-8-mediated direct activation of the cascade, whereas the intrinsic pathway proceeds through mitochondrial release of cytochrome c and activation of caspase-9. Both pathways converge in the activation of effector caspases, such as caspase-3, -6, and -7. [1][2][3] Defective Fas function leads to the unwanted accumulation of lymphocytes and favors autoimmunity possibly by impairing the switching off of autoreactive lymphocytes. This has been shown in the autoimmune lymphoproliferative syndrome (ALPS), an inherited disease characterized by (1) defective function of Fas, (2) autoimmune manifestations that predominantly involve blood cells, (3) polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and (4) expansion of TCR␣␤ ϩ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Moreover, ALPS patients are predisposed to develop lymphomas in adulthood. 3-11 ALPS is generally due to deleterious mutations of the Fas gene (TNFRSF6) and is classified as ALPS type-Ia, but rare mutations of other ge...

Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of β-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2Rγnull mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h−ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10–metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response.

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P ¼ 0.0166; odds ratio (OR) ¼ 2.06, 95% confidence interval (CI): 1.13-3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P ¼ 0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P ¼ 0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR ¼ 1.38 (95% CI ¼ 1.10-1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

Osteopontin is a proinflammatory molecule, modulating TH1 and TH17 responses. Several reports suggest its involvement in multiple sclerosis (MS) pathogenesis. We previously reported that OPN gene variations at the 3′ end are a predisposing factor for MS development and evolution. In this paper, we extended our analysis to a gene variation at the 5′ end on the −156G > GG single nucleotide polymorphism (SNP) and replicated our previous findings at the 3′ end on the +1239A > C SNP. We found that only +1239A > C SNP displayed a statistically significant association with MS development, but both +1239A > C and −156G > GG had an influence on MS progression, since patients homozygous for both +1239A and −156GG alleles displayed slower progression of disability and slower switch to secondary progression than those carrying +1239C and/or −156G and those homozygous for +1239A only. Moreover, patients homozygous for +1239A also displayed a significantly lower relapse rate than those carrying +1239C, which is in line with the established role of OPN in MS relapses.

1 The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 mg ml À1 phytohemagglutinin plus 2 U ml À1 interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 mg ml À1 ; 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining).3 The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC 50 values were: 3.2 Â 10 À7 M for (1S,3R)-ACPD; 4.5 Â 10 À8 M for quisqualate; 1.0 Â 10 À6 M for (S)-3,5-DHPG; 2.0 Â 10 À5 M for CHPG. 4 Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 Â 10 À6 M. The IC 50 values calculated were: 8.7 Â 10 À5 , 4.3 Â 10 À6 and 6.3 Â 10 À7 M for AIDA, LY 367385 and MPEP, respectively. 5 L-Glutamate (1 Â 10 À6 M; 18 h) significantly (Pp0.05) inhibited FasL expression (40.8711.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6 Expression of both mGlu 1 and mGlu 5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase-PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. 7 These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms.

Osteopontin (OPN) is highly expressed in demyelinating lesions in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). OPN is cleaved by thrombin into N- (OPN-N) and C-terminal (OPN-C) fragments with different ligands and functions. In EAE, administering recombinant OPN induces relapses, whereas treatment with anti-OPN antibodies ameliorates the disease. Anti-OPN autoantibodies (autoAbs) are spontaneously produced during EAE but have never been detected in MS. The aim of the study was to evaluate anti-OPN autoAbs in the serum of MS patients, correlate them with disease course, and recapitulate the human findings in EAE. We performed ELISA in the serum of 122 patients collected cross-sectionally, and 50 patients with relapsing–remitting (RR) disease collected at diagnosis and followed longitudinally for 10 years. In the cross-sectional patients, the autoAb levels were higher in the RR patients than in the primary- and secondary-progressive MS and healthy control groups, and they were highest in the initial stages of the disease. In the longitudinal group, the levels at diagnosis directly correlated with the number of relapses during the following 10 years. Moreover, in patients with active disease, who underwent disease-modifying treatments, autoAbs were higher than in untreated patients and were associated with low MS severity score. The autoAb displayed neutralizing activity and mainly recognized OPN-C rather than OPN-N. To confirm the clinical effect of these autoAbs in vivo, EAE was induced using myelin oligodendrocyte glycoprotein MOG35–55 in C57BL/6 mice pre-vaccinated with ovalbumin (OVA)-linked OPN or OVA alone. We then evaluated the titer of antibodies to OPN, the clinical scores and in vitro cytokine secretion by spleen lymphocytes. Vaccination significantly induced antibodies against OPN during EAE, decreased disease severity, and the protective effect was correlated with decreased T cell secretion of interleukin 17 and interferon-γ ex vivo. The best effect was obtained with OPN-C, which induced significantly faster and more complete remission than other OPN vaccines. In conclusion, these data suggest that production of anti-OPN autoAbs may favor remission in both MS and EAE. Novel strategies boosting their levels, such as vaccination or passive immunization, may be proposed as a future strategy in personalized MS therapy.

Key Points In vitro, IL-17 inhibits Fas-induced cell death and IL-17 neutralization improves lymphocyte apoptosis in patients with ALPS and DALD. Treatment of MRLlpr/lpr mice with anti–IL-17A antibodies decreases the severity of autoimmune/lymphoproliferative disease.