Innate immune response to encephalomyocarditis virus infection mediated by CD1d

CD1d-reactive natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and/or Th2 cytokines, can activate antigen-presenting cell (APC) interleukin-12 (IL-12) production, and are implicated in the regulation of adaptive immune responses. The role of the CD1d system was assessed during infection with encephalomyocarditis virus (EMCV-D), a picornavirus that causes acute diabetes, paralysis and myocarditis. EMCV-D resistance depends on IL-12-mediated interferon-gamma (IFN-gamma) production. CD1d-deficient mice, which also lack CD1d-reactive NKT cells, were substantially more sensitive to infection with EMCV-D. Infected CD1d knockout mice had decreased IL-12 levels in vitro and in vivo, and indeed were protected by treatment with exogenous IL-12. IFN-gamma production in CD1d knockout mice was decreased compared with that in wild-type (WT) mice in response to EMCV-D in vitro, although differences were not detected in vivo. Treatment with anti-asialo-GM1 antibody, to deplete NK cells, caused a marked increase in susceptibility of WT mice to EMCV-D infection, whereas CD1d knockout mice were little affected, suggesting that NK-cell-mediated protection is CD1d-dependent. Therefore, these data indicate that CD1d is essential for optimal responses to acute picornaviral infection. We propose that CD1d-reactive T cells respond to early immune signals and function in the innate immune response to a physiological viral infection by rapidly augmenting APC IL-12 production and activating NK cells.     

Comparison of the nucleotide sequences of diabetogenic and nondiabetogenic encephalomyocarditis virus

The nucleotide sequences of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) have been compared. These variants differ at eight sites. The poly(C) tract of EMC-B is three bases shorter than that of EMC-D. Of the seven sites at which nucleotide substitutions were confirmed using RNA templates, four resulted in amino acid changes in three different proteins, the leader peptide, 1B (VP2), and 1D (VP1). The biological significance of these differences can now be investigated.     

Genomic differences between the diabetogenic and nondiabetogenic variants of encephalomyocarditis virus

Plaque purification of the M variant of encephalomyocarditis (EMC-M) virus resulted in the isolation of two stable variants. One is a highly diabetogenic D variant (EMC-D) and the other is a nondiabetogenic B variant (EMC-B). The cDNA of EMC-D and EMC-B genomes were cloned and seven overlapping cDNA clones were selected to cover the entire genome except the 5'-end 310 bases which were determined by RNA-dependent DNA sequencing and enzymatic RNA sequencing. Each clone was restriction-mapped, subcloned, and sequenced. The genomes of EMC-D and EMC-B are composed of 7829 and 7825 bases, respectively. Both genomes contain a long open reading frame of 6876 nucleotides starting at position 830 on the consensus sequence, which encodes a polyprotein of 2292 amino acids. The sequences of EMC-D and EMC-B differ by two deletions, one insertion, and eight point mutations. The first deletion of 3 nucleotides is located in the 5' poly(C) tract where EMC-B has 127 nucleotides compared with 130 nucleotides in EMC-D. The second deletion in EMC-B involves 2 nucleotides at the 3'-end polyadenylation site. A single base insertion of U occurs at the 5' noncoding region of EMC-B. The eight point mutations are located in the polyprotein coding region. Two are silent and are each located in the structural gene 1B and in the nonstructural gene 2B. The remaining six mutations, one on the L gene and the other five on the 1D gene, introduce respective amino acid changes. It is concluded that the diabetogenic EMC-D viral genome (7829 bases) differs from the nondiabetogenic EMC-B viral genome (7825 bases) by 14 nucleotides out of 7829.     

The genomic RNA of diabetogenic encephalomyocarditis virus: characterization and molecular cloning

The RNA of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) which were derived from the same virus stock (M strain) have been compared. The size of both genomes is estimated at 7.7 kb. The poly(C) tract of EMC-D is estimated at 144 bases, whereas that of EMC-B is 141 bases in length. The untranslated 5' terminal 103 nucleotides are identical for B and D with preservation of a stable terminal hairpin structure. The entire open reading frame of both variants has been cloned and the restriction maps of 12 different enzymes are identical. These maps were compared to a computer-generated restriction map of another strain of EMC virus which has been cloned and sequenced by Palmenberg et al. (1984, Nucleic Acids Res. 12, 2969-2985). Approximately 50% of the restriction sites of the B and D variants had similar locations in this strain of EMC. We conclude that significant genetic variation exists between the M strain (B and D variants) and the Palmenberg strain of EMC virus. However, the B and D variants are very similar at the molecular level and comparison of their nucleotide sequences will be necessary to reveal the basis for their different biological properties.     

Amino acid differences in capsid protein, VP1, between diabetogenic and nondiabetogenic variants of encephalomyocarditis virus

The genes for the major capsid protein, VP1(1D), of both diabetogenic D variant (EMC-D) and nondiabetogenic B variant (EMC-B) of encephalomyocarditis virus were cloned by using two synthetic primers which are common to both EMC-D and EMC-B. The cloned genes were mapped for major restriction enzyme sites including AccI, BamHI, EcoRI, HincII, KpnI, PvuII, SstI, TaqI, and XbaI. Among those nine restriction enzyme sites, only the TaqI site distinguished EMC-D genome from the counterpart of EMC-B genome. The complete nucleotide sequences (831 bases) of the VP1 genes revealed five amino acid differences between the two variants. Three of the changes, at positions 41, 58, and 152, were Thr (EMC-B) to Ala (EMC-D). The additional two changes occurred at positions 63 [Gln (EMC-B) to Glu (EMC-D)] and 181 [Thr (EMC-B) to Ser (EMC-D)]. All of these amino acid changes were due to point mutations at the first base of each codon.     

Pathological anatomy of an experimental disease of small laboratory animals caused by encephalomyocarditis virus strain ÉMK-70

The results of a pathomorphological investigation of an experimental disease of small laboratory animals caused by strain ÉMK-70 of encephalomyocarditis virus isolated from monkeys are described. Regardless of the method of infection of the newborn and juvenile mice with the virus, they developed lesions of the brown fat, striated muscle, brain, and heart. The changes in guinea pigs were characterized by the development of severe myocarditis and encephalitis, accompanied by accumulation of virus antigen. The disease, caused by strain ÉMK-70 could not be differentiated from Coxsackie infection on the basis of the pathomorphological data. This fact must be taken into account when problems concerning the diagnosis of virus diseases at autopsy are examined.Institute of Experimental Pathology and Therapy, Academy of Medical Sciences of the USSR, Sukhumi. (Presented by Academician of the Academy of Medical Sciences of the USSR B. A. Lapin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84 No. 9, pp. 368–372, September, 1977.     

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of inflammatory bowel disease

4-1BB (CDw 137), a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB monoclonal antibody (mAb) enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of anti-4-1BB mAb reduced the incidence and severity of inflammatory bowel disease. In this study, we investigated the effects of anti-4-1BB mAb in a murine intestinal inflammation model, which induced by the hapten reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) and mimics immunologic characteristics of human Crohn's disease (CD). Colitis was induced by rectal administration of 2mg of TNBS in 35% ethanol using a vinyl catheter positioned 4cm from the anus. All mice were sacrificed 3 and 10 days after the TNBS administration. The disease activity index (DAI), histological changes of the colon and production of cytokines (IL-2, IL-4, IL-10 and IFN-gamma) were evaluated. The surface molecules of T cells in peripheral blood, spleen and mesenteric lymph nodes were analyzed by flow cytometry. When mice were treated with anti-4-1BB mAb, improvement in both wasting and histopathologic signs of colonic inflammation was observed. The increase a number of splenic CD4(+)CD25(+) T cells and decreased synthesis of the Th1 cytokine IL-2 also occurred. Interestingly, increased production of Th1 cytokine IFN-gamma and proportion of CD8(+) T cells were observed in mice treated with anti-4-1BB mAb in comparison to the colitic mice. These studies show, for the first time, that agonistic anti-4-1BB mAb can improve experimental colitis by reduction of IL-2 and augmentation of CD4(+)CD25(+) regulatory T cells. TNBS colitis is Th1-mediated and has similar histologic features and distribution of inflammation to CD. This study suggests that anti-4-1BB mAb therapy could be effective in the treatment of patients with CD.     

Inhibitory effects on HLA-DR1-specific T-cell activation by influenza virus haemagglutinin-derived peptides

Collagen (CII) 263-272 peptide, an autoantigen in rheumatoid arthritis, is a specific human leukocyte antigen (HLA)-DR1/4-binding peptide recognized by T-cell receptors (TCR). The affinity of influenza virus haemagglutinin (HA) 306-318 peptide for the antigen-binding groove of HLA-DR1/4 molecules is higher than that of CII263-272. The HLA-DR1/4-binding residues of HA306-318 are located in the region 308-317. Altered HA308-317 peptides with substitutions of TCR-contact residues may inhibit HLA-DR1/4-specific T-cell activation by blocking the antigen-binding site of HLA-DR1/4 molecules. To evaluate the role of altered HA308-317 peptides in HLA-DR1-restricted T-cell activation, we synthesized three altered HA308-317 peptides. The specific binding of altered HA308-317 peptides to HLA-DR1 molecules was examined using flow cytometry. Effects of altered HA308-317 peptides on HLA-DR1-specific T-cell hybridoma were studied by measuring T-cell proliferation and surface expression of CD69 or CD25. The results showed that altered HA308-317 peptides were able to bind to HLA-DR1 molecules and competed with CII263-272 or wildtype HA308-317 peptide. Compared with wildtype CII263-272 or HA308-317, altered HA308-317 peptides did not stimulate significant T-cell proliferation and CD69 or CD25 expression. Furthermore, the altered HA308-317 peptides inhibited HLA-DR1-specific T-cell activation induced by CII263-272 or wildtype HA308-317 peptide, which may suggest an effective therapeutic strategy in inhibition of HLA-DR1-specific T-cell responses in autoimmunity.     

Paradoxical lessening of autoimmune processes in non-obese diabetic mice after infection with the diabetogenic variant of encephalomyocarditis virus

In order to gain insight into the interaction between autoimmunity and viral infection in the onset of insulin-dependent diabetes, non-obese diabetic (NOD) mice which spontaneously develop autoimmune diabetes were inoculated with the diabetogenic variant of the encephalomyocarditis virus (EMCV-D) before the onset of the disease. The pre-diabetic period was divided into two phases: the early phase (days 88 to 116) during which development of spontaneous diabetes is rare and the late phase (day 123 to 200) during which the incidence of spontaneous diabetes is high. As controls ICR mice of common ancestry were also inoculated. During the early phase diabetes was observed in 4/10 inoculated, 0/13 control NOD and 7/13 inoculated ICR males vs. 6/12 inoculated, 1/11 control NOD and 0/15 inoculated ICR females. However, in NOD female, virus-induced diabetes prevalence was variable from one experiment to another. In parallel the flow cytometric analysis showed a high percentage of L3T4+ T lymphocytes in the pancreas of inoculated female NOD mice 10 days after the infection. At this time a large proportion of both L3T4+ and Ly-2+ cells expressed the interleukin 2 receptor. During the late phase no new case of diabetes occurred in inoculated NOD mice but one case was observed in control NOD males and five in control NOD females. This prevention of autoimmune diabetes was constantly found in other experiments. Insulitis was milder in inoculated NOD mice of both sexes than in control NOD. Adoptive transfer of diabetes into irradiated 8-week-old males by splenocytes from 28-week-old females was successful in five out seven attempts with control splenocytes and in zero out of six attempts with splenocytes from inoculated mice. This immunosuppression was specific as the ability of lymphocytes to respond to soluble or allogeneic antigens was preserved. In the early phase EMCV-D precipitated the onset of diabetes in females NOD mice by amplifying L3T4+ T lymphocyte-mediated immune mechanisms. During the late phase viral infection had lessened immune processes in animals which had resisted or recovered from virus-induced diabetes.     

Aggregation of encephalomyocarditis virus induced by radio-iodination

Radio-iodination causes encephalomyocarditis virus to behave aberrantly when examined by affinity chromatography and to sediment rapidly during analysis on sucrose density gradients suggesting that aggregation had taken place. The change in physical properties of the virus occurred whether iodination was carried out with ¹²⁵I or ¹³¹I, with radio-iodine from two different sources, or using two different iodination procedures. The changes were not observed in virus subjected to an iodination procedure in the absence of radio-iodine suggesting that modification of tyrosine residues was involved rather than a side reaction such as amino acid oxidation. It is recommended that caution be exercised when following the fate of radio-iodinated virus in any particular study because its behaviour may not reflect that of normal, non-iodinated virus present.     

CD154-dependent priming of diabetogenic CD4(+) T cells dissociated from activation of antigen-presenting cells

We followed the fate of K(d)- or I-A(g7)-restricted beta cell-autoreactive T cells in monoclonal TCR-transgenic NOD mice expressing or lacking CD154. 8.3-NOD.RAG-2(-/-)/CD154(-/-) mice, which bear autoreactive CD8(+) T cells, developed diabetes with the same incidence and tempo as 8.3-NOD.RAG-2(-/-)/CD154(+) mice. Recruitment of CD154(-/-) 8.3-CD8(+) CTL was accelerated by CD154(+)CD4(+) T cells, by expression of a B7.1 transgene in beta cells or by treatment of the mice with CpG-DNA or an agonistic anti-CD40 antibody. In contrast, the autoreactive CD4(+) T cells maturing in 4.1-NOD.RAG-2(-/-) mice lost their diabetogenic potential if they lacked CD154, even in the presence of CD154(+)CD4(+) T cells, B7.1 molecules on beta cells, CpG-DNA treatment, or systemic CD40 ligation. These results demonstrate the existence of a novel, CD154-dependent pathway of CD4(+) T cell activation that is independent of CD40-mediated activation of APCs.     

Cross-linking CD28 leads to activation of 70-kDa S6 kinase

Proliferation of T lymphocytes in response to antigen/MHC complexes is dependent upon the presence of a co-stimulatory signal; in its absence, T cells are rendered unresponsive to specific antigen. CD28 is a T cell surface glycoprotein that acts as a co-stimulatory molecule when combined with signals initiated by the T cell receptor-CD3 complex. While the biochemical signaling events following CD28 stimulation are still poorly defined, monoclonal antibodies (mAb) directed against CD28 have been shown to transduce a variety of early signals that are different in the presence of cross-linking antibody or the presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). Stimulation of human T cells with cross-linked anti-CD28 mAb alone resulted in the activation of 70-kDa (p70) S6 kinase, a rapamycin-sensitive serine/threonine kinase that is believed to be important for cell cycle progression. Activation of p70 S6 kinase through CD28 was inhibited by rapamycin. Activation of p70 S6 kinase also increased in response to cross-linked CD3, but followed a more rapid time course than activation via CD28. Cyclosporin A and FK506 had no effect on p70 S6 kinase activity initiated via either pathway. The combination of cross-linked CD28 and cross-linked CD3 had no more than an additive effect on the induction of p70 S6 kinase activity. Thus, recruitment of p70 S6 kinase activity appears to represent a common signal transduction event shared by both the CD28 and CD3 pathways of T cell activation.     

Immunological dysfunction in HIV-1-infected individuals caused by impairment of adenosine deaminase-induced costimulation of T-cell activation

The cell surface association between CD26 and adenosine deaminase (ADA) has a costimulatory function during T-cell activation. Several studies have revealed correlations among CD4⁺ CD26⁺ T-cell depletion, increased serum levels of ADA, and the evolution of human immunodeficiency virus (HIV) infection, implicating CD26 and ADA in HIV disease progression. In this context, we aimed to determine whether ADA costimulation could be altered during HIV infection. ADA costimulation was investigated in cells from HIV-infected patients (n = 36) in terms of proliferation and cytokine secretion. An effect of ADA on T-cell proliferation was found in HIV-1-infected patients and correlated positively with the CD4⁺ percentage and the nadir CD4 count and negatively with viral load, demonstrating that the response depends on the immunological status of the patient. The robust ADA-induced increase in cytokine production [interferon (IFN)-γ, interleukin (IL)-6 and IL-10] was markedly reduced in T cells from HIV-1-infected subjects. To eliminate some of the variables associated with immunological defects in HIV-1-infected patients, anti-CD3 plus ADA assays with T cells from healthy volunteers were performed in the presence of recombinant glycoprotein 120 (gp120). It was found that gp120 was responsible for the impairment of the ADA–CD26 interaction and consequently of the ADA-induced effect on both costimulation and cytokine production. The gp120-mediated disruption of the CD26–ADA interaction is a novel mechanism that might explain, at least in part, the altered immunological features observed in HIV-1-infected patients and may have significant relevance in AIDS pathogenesis.     

负载灭活口蹄疫病毒树突状细胞对CD8+T细胞的旁位活化效应

目的:探讨在体外条件下负载灭活口蹄疫病毒(FMDV)树突状细胞活化CD8+T细胞的机制.方法:用重组粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和白介素-4(rmIL-4) 将小鼠外周血单核细胞诱导分化成树突状细胞(MoDCs),以信号阻断法从淋巴结T细胞制备CD8+T细胞,利用负载灭活FMDV的MoDCs与CD8+T细胞共培养,以FMDV+MoDCs+淋巴结T细胞作为对照,用ELISA检测不同时间培养上清液中IFN-γ的含量.用蛋白酶体抑制剂和溶酶体抑制剂处理MoDCs,2小时后再负载灭活FMDV,并与CD8+T细胞共培养,对照组则用FMDV+MoDCs+CD8+T细胞和FMDV+MoDCs,用ELISA检测不同时间培养上清液中IFN-γ的含量.结果:在共培养9小时,CD8+T细胞的IFN-γ释放量约为淋巴结T细胞IFN-γ释放量的1/6,随后,淋巴结T细胞和CD8+T细胞均产生少量IFN-γ.用灭活FMDV负载被lactacystin和氯喹处理的DCs与CD8+ T细胞共培养后9～12小时,CD8+T细胞产生IFN-γ显著减少,在共培养24小时,IFN-γ释放显著增多,并在36小时达到峰值,与对照组相比,差异有统计学意义.结论:负载灭活FMDV的MoDCs可以活化淋巴结T细胞,而且,还可通过非MHCⅠ和非MHCⅡ类分子依赖途径激活CD8+T细胞,此即旁位活化效应.     

Role of the CD28 receptor in T-cell activation

Antigen-specific T-cell activation is initiated through the T-cell receptor. Recent evidence has shown that a number of additional T-cell surface receptors serve to regulate the responses of antigen-activated T cells. One such molecule, CD28, is a member of a heterophilic cell adhesion complex, and is the receptor for the B-cell-restricted B7/BB-1 antigen. As Carl June, Jeffrey Ledbetter, Peter Linsley and Craig Thompson review here, CD28 serves as the surface component of a novel signal transduction pathway that modulates T-cell lymphokine production and increases the resistance of T-cell responses to various immunosuppressive agents.     

T cell activation induced by cross-linking CD3 and CD28 leads to silencing of Epstein-Barr virus/C3d receptor (CR2/CD21) gene and protein expression

Complement receptor II (CR2) also known as CD21 is the receptor for C3d on immune complexes. In humans it serves as a receptor for the Epstein-Barr virus (EBV). CR2 is expressed on B cells and in low density in the T cell lineage. EBV can infect T cells and EBV-positive T lymphomas have been described. Although CR2 mRNA is readily detectable in T cells, the function of CR2 in human T lymphocytes remains elusive. Here we have analyzed the expression of CR2 in normal and activated T cells. PCR analyses and immunofluorescence/confocal microscopy of peripheral blood T cells and of activated T cells shows considerable reduction in CR2 mRNA and protein expression upon activation. The downregulation of CR2 expression may modulate life span or immunological reactivity of T cells and the susceptibility of cells to infection by lymphotropic viruses.     

Differential T-cell activation by B7-1 expression

T-cell receptor-mediated T-cell activation requires cosimulation signal, which can be provided by B7-1 molecule. Our previous study demonstrated that the coexpression of a covalent peptide/major histocompatibility complex class II molecule complex and costimulatory molecule B7-1 by recombinant adenovirus leads to synergy in peptide-specific T-cell activation. However, the viral antigen-specific T-cell activation is not enhanced by B7-1 expressed by the adenovirus. To verify the differential T cell activation by B7-1 and investigate its underlying mechanisms, we constructed an adenovirus coexpressing a covalent complex of hen egg lysozyme peptide/I-Ak (HEL46-61/I-Ak) and B7-1 in the present study. In vivo studies revealed that HEL46-61-specific T-cell response, but not viral antigen-specific T-cell response, was enhanced by B7-1 expression mediated by the adenovirus, suggesting that exogenous B7-1 expression may regulate T-cell response to these two different antigens through distinct mechanisms. Furthermore, our results revealed that antigen-presenting cells were not susceptible to adenovirus infection in vivo. Based on these findings, the possible mechanism of differential B7-1 costimulation on peptide-specific and viral antigen-specific T-cell activation is discussed.     

Human Soluble CD80 is generated by alternative splicing, and recombinant soluble CD80 binds to CD28 and CD152 influencing T-cell activation

CD80 is a costimulatory factor mainly expressed on the surface of activated monocytes, B cells and dendritic cells. In this study, we demonstrate that 24% of healthy individuals have soluble forms of CD80, sCD80, in their serum. The concentration of sCD80 ranged from 0 to 1 mg/l. At the mRNA level, we detected a spliced form s1CD80 (771 bp), in unstimulated monocytes and B cells, while another form named s2CD80 (489 bp) was expressed in activated T cells as well as in freshly isolated and activated monocytes. s1CD80 lacks the transmembrane domain, and the IgC-like domain plus the transmembrane domain are spliced out of s2CD80. We also present data demonstrating that recombinant s1CD80 binds to recombinant CD152-Ig and CD28-Ig. It can also bind to T cells, preferentially to activated T cells. Recombinant sCD80 had immunomodulatory effects shown by its inhibition of the mixed lymphocyte reaction and inhibition of T-cell proliferation. sCD80 in human serum adds a new member to the family of soluble receptors, implying a network of soluble costimulatory factors with functional relevance. The inhibitory effect of the recombinant protein on T-cell activation makes it a possible candidate for treatment of diseases associated with hyperactivated T cells.