Human CD4 internal antigen anti-idiotypic monoclonal antibody. Immunochemical and sequence analysis

The mouse mAb2 16D7 recognizes the paratope of the syngeneic anti-human CD4 mAb HP2/6 (mAb1 of our idiotypic cascade) and mimics CD4 in xenogeneic settings in humans. Immunochemical and sequence analyses were performed to define the minimum structural requirement for this mimicry. Binding assay of mAb1 with isolated naive 16D7 H and L chains showed that only the second reacted with mAb1. Specificity was indicated by the lack of reactivity of mAb1 with the L chain of mAb2 14D6, which also recognizes mAb1-paratope. It is likely that the 16D7-L mAb1-specific epitope is “sequence-dependent”, since fully denatured 16D7-L still reacted with mAb1. Sequence analysis of 16D7 and mAb1 showed a high degree of homology of their VH, as both were coded by the same gene family (V/II), whereas CDR3 showed the greatest diversity. Alignment of 16D7-H CDR3 with CD4, however, produced no similarity. In contrast, analyses of the 16D7 VL sequence (XX/V) defined a CDR3 6-mer peptide with a 50% identity (83% of similarity) to the CD4 stretch 218–223. This peptide seems a suitable replacement for 16D7 in active immunotherapy as it did not match any protein fragment retrieved from the n-r database (NCBI) and both the peptide and the corresponding CD4 amino acid stretch are surface accessible. Based on their immunochemical profiles and similarity to CD4, four additional 16D7-derived peptides were designed for synthesis. The data indicate that CD4 mimicry by mAb2 can be obtained at the level of primary structure and provide useful information for the synthesis of peptide(s) with bioactive potential. Received: 17 October 2000 / Accepted: 23 May 2001     

Convergence between CD98 and integrin-mediated T-lymphocyte co-stimulation

CD98 is a widely expressed cell surface heterodimeric glycoprotein, which is rapidly up-regulated upon activation of T lymphocytes. Monoclonal antibody (mAb) 80A10 recognizes an epitope on CD98 and in combination with CD3 antibody causes proliferation of peripheral blood T lymphocytes. CD98 co-stimulatory activity, mediated by either mAb 80A10 or 4F2, a well-characterized CD98-specific mAb, is blocked in the presence of the soluble beta1 integrin antibody 18D3. Previously we have reported that co-stimulatory activity of antibodies to integrins alpha4beta1, alpha5beta1, alphaLbeta2 and alpha4beta7 is inhibited by 18D3, whereas co-stimulation mediated by non-integrins was unaffected. Thus the non-integrin CD98 is uniquely sensitive to the inhibitory effects of beta1 integrin-blocking antibodies, which may reflect convergent signalling mechanisms between integrins and CD98. This is consistent with recent reports suggesting that CD98 may regulate integrin-mediated adhesive events.     

噬菌体表达短肽模拟乙脑病毒糖蛋白的研究

为研究日本脑炎病毒 (JEV )E蛋白模拟肽 ,将抗JEVE蛋白的mAb 2H4淘筛噬菌体 15肽库。经夹心ELISA、竞争ELISA鉴定后 ,随机挑取 10个阳性克隆 ,测序并与JEVE蛋白同源比较。将阳性噬菌体免疫小鼠 ,检测血清中特异性抗体。ELISA结果显示筛选到的噬菌体能特异地与mAb 2H4结合 ,并且这种结合可被JEV天然抗原所竞争抑制。 10个阳性克隆的氨基酸序列相同 :—RQDPQWPYANSTIAR— ,同源分析得到的序列STXAR可能为mAb 2H4识别的模拟表位。阳性噬菌体表达的 15肽能够刺激小鼠产生特异性抗体。该噬菌体表达短肽模拟JEVE蛋白的部分抗原性。     

Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody

Peptides based on the complementarity-determining region 1 (CDR1) and CDR3 of an anti-DNA monoclonal antibody (mAb) carrying the 16/6 idiotype (Id) were shown to induce experimental systemic lupus erythematosus (SLE) in susceptible mouse strains. In the present study, T-cell lines specific to the pCDR1 and pCDR3 peptides were established in BALB/c and in SJL mice, respectively. The T-cell lines were characterized and analysed for their pathogenicity upon administration to syngeneic mouse strains. Both T-cell lines expressed the alphabeta T-cell receptor (TCR) and the CD4+ CD8- phenotype. Additionally, both cell lines secreted interleukin (IL)-4 and IL-10 upon stimulation with their specific peptide, thus belonged to the T helper 2 (Th2) subset. Upon immunization, the pCDR3-specific T-cell line induced experimental SLE in SJL mice. The animals produced high levels of autoimmune anti-DNA and antinuclear protein antibodies, as well as anti-16/6 Id antibodies (Abs). Furthermore, the mice developed clinical manifestations, including leukopenia, proteinuria and accumulation of immune complex deposits in their kidneys. The pCDR1-specific T-cell line failed to induce SLE when injected into BALB/c mice. It is thus suggested that pCDR3 is an immunodominant epitope in experimental SLE and that pCDR3-specific T cells initiate autoimmunity, leading to SLE, probably via epitope spreading.     

Molecular mimicry rather than identity breaks T-cell tolerance in the CYP2D6 mouse model for human autoimmune hepatitis

In our novel mouse model for autoimmune hepatitis (AIH), wildtype FVB mice infected with an Adenovirus (Ad) expressing the major AIH autoantigen human cytochrome P450 2D6 (hCYP2D6) show persistent histological and immunological features associated with AIH, including the generation of anti-hCYP2D6 antibodies with an epitope specificity identical to LKM-1 autoantibodies in AIH-patients. Since FVB mice do not express hCYP2D6, the immune response was directed against mouse CYP (mCYP) homologues. Additional expression of hCYP2D6 in transgenic mice resulted in amelioration of the liver disease. In the present study we used the CYP2D6 model to assess why tolerance breakdown and induction of autoimmune liver disease is more efficient if the triggering antigen is similar but not identical to the target autoantigen. We found that in contrast to the specificity and magnitude of anti-hCYP2D6 antibody responses, T-cell responses differ profoundly between wildtype and transgenic mice. Detailed T-cell epitope mapping studies show a robust, antigen-specific T-cell reactivity in FVB mice largely directed against one CD4 and three CD8 epitopes, activating a total of approximately 1% CD4 and 10% CD8 T-cells, respectively, while infected hCYP2D6 mice generated almost no hCYP2D6-specific T-cells. The frequency of hCYP2D6-specific T-cells was approximately 3-fold higher in the liver compared with the spleen. Amino acid sequence comparison revealed that the immunodominant epitopes were located in hCYP2D6-segments of intermediate homology between hCYP2D6 and its mCYP homologues. Our data indicate that self/non-self molecular mimicry, rather than molecular identity, is a prerequisite for breaking T-cell tolerance in the liver.     

Close localization of mouse CD14 and CD32/16 in the cell surface of monocytic cell lines.

The binding of a rat anti-mouse CD14 monoclonal antibody (mAb) (rmC5-3) was inhibited by pretreatment of a mouse monocytic cell line WEHI-3 cells with anti-mouse CD32/16 mAb (2.4G2), whereas that of 2.4G2 was not inhibited by pretreatment of WEHI-3 cells with rmC5-3. An enzyme-linked immunosorbent assay showed that rmC5-3 detected peptide 9 corresponding to amino acid position 308-322 of CD14 but 2.4G2 did not. A Western blot analysis of sera revealed that rmC5-3 and 2.4G2 detected the bands thought to be soluble CD14 and CD32/16, respectively. rmC5-3 reacted with mouse CD14-transfected CHO cells, CD14-CHO-K1 cells, but 2.4G2 did not. Lipopolysaccharide-induced tumor necrosis factor (TNF)-alpha release was enhanced when a monocyte cell line (J774) was pretreated with rmC5-3. The enhancement was abolished by pretreatment with 2.4G2. The release of TNF-alpha was observed following treatment of J774 cells with 2.4G2 followed by anti-rat IgG F(ab')2.     

Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever

Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B-cell epitopes of nonstructural 1 (NS1) glycoprotein and anti-NS1 antibody response in DEN infection, a series of 15-mer synthetic peptides from the predicted B-cell linear epitopes of DEN-2 NS1 protein were prepared. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN-2 NS1, D2 NS1-P1 (amino acids 1–15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1-P1-specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1-P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti-D2 NS1-P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti-DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection. J. Med. Virol. 57:1–8, 1999. © 1999 Wiley-Liss, Inc.     

Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells

Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1 D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4- T cells, implying that the 1 D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1 D4 was completely abolished when cells were treated with neuraminidase, while them remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.     

Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies

Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well-established role of class II antigens in low-affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human class II-positive monocytic (I937) and T leukemic (HUT78) tumor cell lines and Epstein-Barr virus (EBV) transformed B-lymphoid cell lines (EBV-LCL). Class II-negative cell lines (U-937 and the EBV-LCL mutant line 616) were not induced to aggregate. An HLA-G-transfected EBV-LCL, 221-AGN, but not the class I-negative parental line, 221, showed homotypic aggregation in response to an HLA-G specific mAb (87G) and a broad reacting class I-specific mAb (IOT2). Both cell lines responded with aggregation to anti-class II mAb (TU35). The anti-class I mAb, W6/32, had no effect on all cell lines tested and two anti-beta 2-microglobulin mAb had variable, weak effects. The aggregation response was an active, temperature-sensitive process which was almost totally abrogated by azide and by cytochalasins B and E, but unaffected by colchicine, EDTA, aphidicolin, actinomycin D and protein tyrosine kinase inhibitors (genistein, herbimycin A). Serine/threonine protein kinase inhibitors (staurosporin, H7) partly inhibited the aggregation responses. There was no strict correlation between induction of aggregation and epitope density. FcR were not involved in the aggregation response, since F(ab')2 fragments of anti-DR mAb, L243, were as effective as the whole antibody. The aggregation was not influenced by mAb against accessory molecules previously shown to be involved directly or indirectly in homotypic aggregation [CD11a (LFA-1)/CD18/CD54 (ICAM-1), CD58 (LFA-3)/CD2, BB1/CD28, CD43, and CD44]. In conclusion, these data provide further evidence that HLA molecules are implicated in a novel, cellular aggregation phenomenon involving the cytoskeleton.     

A cloned gene of Cryptosporidium parvum encodes neutralization-sensitive epitopes

Two mAb, C6B6 and 7D10, each significantly reduced infection of mice by Cryptosporidium parvum and reacted with a 23-kDa glycoprotein (p23) of geographically disperse C. parvum isolates. The antibodies were used to identify plaques in a cDNA library prepared from C. parvum sporozoite mRNA. cDNA insert sequences from positive plaques were determined and used to isolate additional clones encoding p23 coding sequences. A consensus open reading frame of 333 base pairs, encoding 111 amino acids, was identified in this collection of cDNAs. The predicted amino acid sequence contained one N-glycosylation site, but lacked hydrophobic membrane spanning regions. Epitope mapping revealed that mAb 7D10 defines the linear epitope QDKPAD which occurs twice in the C terminal region of the peptide encoded by the ORF. This same C terminal peptide region contains a non-linear epitope bound by mAb C6B6. Serum from mice immunized with synthetic C terminal peptide reacted with sporozoite p23. The occurrence of neutralization-sensitive epitopes encoded by defined regions of the C. parvum genome suggests that recombinant proteins or synthetic peptides containing these epitopes may prove useful for inducing immune responses that diminish infection.     

Monoclonal antibodies to human neutrophil Fc gamma RIII (CD16) identify polypeptide epitopes.

Human neutrophils constitutively express two low-affinity Fc gamma R, Fc gamma RII (CD32) and Fc gamma RIII (CD16). Eleven monoclonal antibodies (mAb) to CD16 were used to identify antigenic differences among Fc gamma RIII-bearing cells, to define functional epitopes of Fc gamma RIII on neutrophils, and to characterize biochemically the epitopes identified by some of these mAb. Flow cytometry demonstrated that 9 of the 11 mAb reacted with neutrophils, 10 of the 11 reacted with natural killer cells, and 9 of 11 reacted with monocytes and monocyte-derived macrophages. These mAb reacted with CD16 positive cells with varying fluorescence intensities. The ability of anti-CD16 mAb to block the binding of 125I-labeled immune complexes to neutrophils was examined. Four monoclonal antibodies strongly inhibited (87-96%) the binding to neutrophils of 125I-labeled immune complexes. Competitive binding assays were performed to determine whether any other anti-CD16 mAb identify the epitope identified by mAb 3G8. Two other mAb, CLBFCGRAN 1 and CLBGRAN 11, blocked binding of 125I-3G8 IgG to neutrophils. Six of the anti-CD16 mAb efficiently immunoprecipitated polypeptides of broad mobility ranging from 45 to 84 kDa from 125I-labeled neutrophils. When Fc gamma RIII, a complex sialoglycoprotein consisting of almost 50% oligosaccharides, was immunoprecipitated from neutrophils with 3G8 Fab Sepharose and subsequently digested with N-glycanase, 5 of the 6 mAb were capable of immunoprecipitating a deglycosylated polypeptide migrating at 29 kDa. These results demonstrate that these 5 mAb identify polypeptide epitopes of Fc gamma RIII, whereas 1 mAb, YFC120.5, may react with a glycosyl moiety or a determinant whose conformation is dependent on the presence of oligosaccharides.     

Characterization of a CD4(L3T4)-positive cytotoxic T cell clone that is restricted by class I major histocompatibility complex antigen on FBL-3 tumor cell

An L3T4(CD4)+ CTL clone specific for Friend virus-induced tumor FBL-3 was isolated, characterized and compared with a conventional Lyt-2(CD8)+ CTL clone. This clone L3.1 was obtained from the limiting dilution culture of splenic MLTC cells from a CB6F1 mouse whose CD8+ T cells had been suppressed by an in vivo injection of anti-Lyt-2.2 mAb. The phenotype of clone L3.1 was sIg-, Thy-1.2+, L3T4(CD4)+, Lyt-2 (CD8)-, and Ia- as determined by flow-cytometry. Northern blot analysis also confirmed that mRNA for L3T4(CD4), but not for Lyt-2 (CD8) was present in the total RNA of L3.1. The FBL-3-specific killing activity of L3.1 was inhibited by anti-H-2D6 mAb, and the tumor cells did not express class II MHC antigen, indicating that the recognition of tumor antigen by this CD4+ CTL clone was restricted by the class I MHC molecule on the tumor cells. Furthermore, the finding that anti-L3T4(CD4) mAb GK1.5 inhibited the specific and lectin-dependent non-specific cytotoxicity of L3.1 suggested that CD4 molecules on this CTL clone are not ligand (MHC class II)-binding proteins, but are involved in signal transduction.     

Human monocytes CD36 and CD16 are signaling molecules. Evidence from studies using antibody-induced chemiluminescence as a tool to probe signal transduction.

A panel of monoclonal antibodies (mAb) directed against human monocyte surface antigens was tested for their capacity to mediate signal transduction by measuring luminol-enhanced chemiluminescence (CL). The response patterns fell into three categories. The mAb Mo4, OKM3, OKM6 and antibodies specific for Fc receptor (FcR) type I and II did not mediate signal transduction directly or were weak triggers, but upon second-order cross-linking by goat anti-mouse immunoglobulin (Ig) F(ab')2 or rabbit anti-mouse Ig, a strong CL response was induced. The mAb recognizing different epitopes on CD11b (complement receptor type III alpha chain) were unique in their ability to induce a CL response either by direct stimulation or by second-order cross-linking. The mAb 3G8 recognizing FcR type III (FcRIII; CD16) on a monocyte subpopulation and CD36-specific monoclonals directly elicited a CL response. A response of similar magnitude was obtained with 3G8 F(ab')2 or with intact 3G8. Furthermore, elutriation-centrifugation-purified monocytes were stimulated by 3G8 to a similar extent as unseparated mononuclear cells, whereas lymphocytes did not respond. This suggests that a FcRIII-expressing monocyte subpopulation may mediate effector functions, including the generation of reactive oxygen species, via FcRIII triggering. Our finding that anti-CD36 F(ab')2 directly induces an oxidative burst is the first evidence that CD36 itself is a trigger molecule. CD36, which is thought to interact with erythrocytes infected with Plasmodium falciparum and with thrombospondin, may constitute a signal-transducing element and thus may have functions extending beyond mediation of adherence. The present CL system constitutes a simple assay for detecting mAb directed against monocyte surface signalling elements. Probing mAb for signal transduction requires suspended cells and antibodies in the fluid phase in order to avoid inadvertent FcR triggering, which may occur when cells are stimulated by surface-adherent whole antibodies.     

Assessment of Safety and the Immune Response to the CD4 “Internal Antigen” Mouse Anti-Idiotypic MAb 16D7 in Four Patients with SLE

We have analyzed the immune response elicited with the human CD4 internal antigen anti-idiotypic monoclonal antibody 16D7 in four patients with active systemic lupus erythematosus and assessed the safety of the treatment. Patients 1 and 2 received three 2-mg 16D7 subcutaneous (SC) injections at 3-week intervals and mainly developed IgG1, whereas IgG1, IgG3, and IgG4 were detected in the sera of the other two patients (3 and 4) who received the same amount of 16D7 on days 0, 28, and 70. 16D7-F(ab`)<sub>2</sub> isotypic determinant-specific antibodies levels, evaluated by sera reactivity with the 16D7-isotype matched anti-idiotypic monoclonal antibody 14D6-F(ab`)₂, were low or undetectable in patient 1 and became detectable following the first and second boosters in patient 3 and patients 2 and 4, respectively. The highest level was seen in patients 3 and 4. The focusing pattern (spectrotype) of 16D7 idiotypic-specific antibodies suggested that multiple V genes are involved or many somatic mutations occur. Once established, each patients spectrotype remained stable. Although spectrotype were individually distinct, all four patients produced CD4-specific antibodies, indicating that this response crosses the genetic barrier. Disease relapsed after 11 (patient 2), 40 (patients 3 and 4), and 125 (patient 1) weeks. The lack of side effects and the prolonged periods of disease control (33 and 103 weeks after the last booster) warrants an extension of this study.     

Signal transduction via a protein associated with a glycosylphosphatidylinositol-anchored protein, decay-accelerating factor (DAF/CD55)

Decay-accelerating factor (DAF/CD55) is a glycosylphosphatidylinositol- anchored protein which is known to have signal transducing capacity and to be associated with several proteins. To determine the signal transducer in the DAF-forming complex, we purified DAF-associated proteins from Raji B cells using an anti-DAF mAb (1C6)-bound affinity column and established five mAb against them. Among these, mAb 2E12- G7(IgM/kappa) reacted with a variety of intact cells, including peripheral blood mononuclear cells (PBMC), as well as cells from T and B cell lines, as shown by cytofluorimetric analyses. The Mr of 2E12-G7 antigen was estimated to be 43 kDa by surface biotinylation and immunoblotting analysis. This antigen was demonstrated in 1C6 immunoprecipitates, but not in anti-CD59 (another GPI-anchored complement regulatory factor)-immunoprecipitates. Sequential treatment with 1C6 F(ab')2 and then with anti-mouse Ig F(ab')2 stimulated PBMC to induce tyrosine phosphorylation on proteins of 45, 72, 78 and approximately 100 kDa. Also, mAb cross-linked to 2E12-G7 stimulated PBMC to induce tyrosine phosphorylation on proteins of 72, 78 and approximately 100 kDa. Furthermore, when 2E12-G7 and 1C6 immunoprecipitates were incubated with [gamma-32P]ATP, the main constituents detected in both were phosphorylated proteins of 26, 32 and 62 kDa. Thus, DAF-associated 2E12-G7 antigen transduces a signal, similar to the DAF molecule.      

Characterisation of a monoclonal antibody recognising specifically the HSP70 from Leishmania

Heat-shock protein 70 (HSP70) is ubiquitously distributed along the evolutionary scale and has such an amino acid sequence conservation that it is considered the most evolutionarily conserved protein. In order to obtain immunological tools specific against Leishmania infantum HSP70, hybridomas were established that secreted monoclonal antibodies (mAbs) against recombinant L. infantum HSP70. One of them, named mAb 2B8D2, specifically reacted with the Leishmania protein and did not recognise HSP70 from the related kinetoplastid Trypanosoma cruzi. The use of synthetic peptides allowed us to determine the B-cell epitope recognised by this mAb, an epitope located in the divergent C-terminal domain of the protein. Remarkably, the mAb possesses the capacity to immunoprecipate HSP70 from promastigote extracts of L. infantum. The fact that human HSP70 is not recognised by this mAb assures the usefulness of this antibody for diagnostic purposes and studies involving Leishmania infection of macrophages.     

Promotion of natural killer cell growth in vitro by bispecific (anti-CD3 x anti-CD16) antibodies.

Bispecific heteroconjugated F(ab')2 fragments were prepared from pepsin-digested monoclonal OKT3 (anti-CD3) and 3G8 (anti-CD16) antibodies with 5,5'-dithiobis- (2-nitrobenzoic acid). When these bispecific antibodies (BsA) were added to peripheral blood lymphocyte (PBL) cultures with 100 U/ml human recombinant interleukin-2 (rIL-2), preferable growth of natural killer cells occurred. After 3 weeks the frequencies of CD56+ and CD56+3- cells in cultures with BsA were 74 +/- 7% and 65 +/- 7%, respectively, compared with 48 +/- 6% and 29 +/- 7% in control cultures. The frequencies of CD3+ lymphocytes in the presence of BsA, cells from 1-day cultures were labelled with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4 and CD8 monoclonal antibodies (mAb) and propidium iodide which stains dead cells. Flow cytometry revealed that more than 95% of the dead cells in cultures with BsA were CD3+. Thirty-seven per cent of CD3+, 43% of CD4+ and 17% of CD8+ cells were dead on day 1, and after 3 days the CD4+/CD8+ ratio among viable lymphocytes was 1.6 in the control and 0.5 in BsA cultures. Taken together, these results show that bispecific (anti-CD3 x anti-CD16) F(ab')2 fragments are strongly immunomodulatory by inducing the killing of T cells by CD16+ cells.     

Responses of coxsackievirus B4-specific T-cell lines to 2C protein-characterization of epitopes with special reference to the GAD65 homology region

Coxsackie B viruses (CBV) have been indicated as environmental triggers initiating autoimmune destruction of insulin-producing pancreatic beta-cells, and molecular mimicry might be the mechanism. A prime candidate for inducing cross-reactive immune responses is a homology sequence, PEVKEK, found both in CBV4 2C protein and in GAD65. To characterize the CBV4-specific T-cell epitopes, overlapping peptides covering the 2C protein were synthesized and CBV4-specific T-cell lines were established from healthy and diabetic subjects. The T-cell epitopes were dependent on the HLA-DR genotype of the T-cell donor, but no difference between diabetic and healthy subjects could be detected. Peptide p4, which included the PEVKEK sequence, contained an HLA-DR1-restricted T-cell epitope. Three randomly selected CBV4-specific T-cell lines, which responded to peptide p4, failed to recognize GAD65 protein or GAD65 peptides containing the PEVKEK sequence. We conclude that the CBV4 2C protein is strongly immunogenic for T-cells and PEVKEK is included in a T-cell epitope. However, presentation of this epitope in the context of neutral HLA-DR1 allele does not support its role in pathogenesis of type 1 diabetes.     

Multiple pathways to induction of virus-induced autoimmune demyelination: lessons from Theiler's virus infection

Infection of SJL mice with wild-type BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) leads to CD4(+)T cell-mediated CNS demyelination characterized by the development of anti-myelin epitope autoimmune responses via epitope spreading during the chronic stage of disease. To exmine the feasibility of virus-encoded mimic epitopes to initiate CNS autoimmunity, we recently developed a molecular mimicry model of virus-induced demyelinating disease wherein a non-pathogenic variant strain of TMEV was engineered to encode a 30-mer peptide encompassing the immunodominant myelin proteolipid protein, PLP139-151, epitope. SJL mice infected intracerebrally with TMEV encoding either the native PLP139-151 determinant or various peptide mimics of the epitope develop an early onset demyelinating disease mediated by activated PLP139-151-specific Th1 cells. The autoimmune nature of this early-onset demyelinating disease is shown by the fact that induction of tolerance to the PLP139-151 peptide prevents clinical disease and associated PLP139-151-specific T cell responses without affecting T cell reactivity to virus epitopes. Most significantly, TMEV encoding a molecular mimic peptide derived from the Haemophilus influenzae bacteria, homologous at only six out of thirteen of the core amino acids, led to CNS disease. These studies provide conclusive evidence that virus-induced myelin-specific autoreactive T cells can be induced by molecular mimicry and provide a useful model to study the disease inducing ability of viruses encoding human-disease-related mimicry peptides.     

Epitope mapping of anti-rat CD53 monoclonal antibodies. Implications for the membrane orientation of the Transmembrane 4 Superfamily

CD53 is a pan-leukocyte glycoprotein which is a member of the recently described Transmembrane 4 Superfamily (TM4SF) of membrane proteins that are predicted to span the lipid bilayer four times. The major hydrophilic region of murine CD53 was expressed as a glutathione-S-transferase fusion protein, and the epitopes of four mouse anti-rat CD53 monoclonal antibodies (mAb) (OX-44, 2D1, 6E2 and 7D2) were mapped to this region using mouse/rat chimeric fusion proteins. The epitopes of OX-44,6E2 and 7D2 are restored by the substitution of a single isoleucine residue for threonine at position 154 in the mouse protein. The 2D 1 epitope is non-linear and appears to require the juxtaposition of isoleucine at position 154 with one or more of the amino acids arginine (132), methionine (133) and serine (140). All of these epitopes are shown to be sensitive to reduction, thus indicating the importance of disulfide bonding in the correct folding of the CD53 hydrophilic domain. Moreover, as these four mAb recognize CD53 at the cell surface, the data provide direct molecular evidence for the proposed membrane orientation of the TM4SF.