Two HLA-DQ-specific human-human hybridoma antibodies (TrG6;TrC5) define epitopes also expressed by a transcomplementing hybrid DQ molecule (DQw7 alpha/DQw4 beta)

We have generated two IgG human-human hybridoma Abs, TrG6 and TrC5, that define subsets of HLA-DQ. TrG6 combined selectively with lymphoblastoid cell lines that expressed DQw1 or DQw4. By sequential immunoprecipitation, competition with other mAbs of defined specificity for binding to antigen, and experiments where HLA antigens from cell lysates crosslinked pairs of mAbs, it was established that TrG6 bound a DQ-molecule. mAb TrC5 specifically recognized DQw2+DR3+ and DQw7+DR5+ cells. The reaction pattern of TrC5 with HLA-loss mutants indicated that TrC5 bound to DQw2 of the DQw2+DR3+ haplotype. Antigens in lysate from DQw7+DR5+ cells crosslinked TrC5 to the murine mAbs Tü22 (anti-DQ monomorphic) and IVD-12 (anti-DQw7 + DQw8 + DQw9), demonstrating that on these cells the TrC5 epitope is located on DQw7 molecules. Lysates from DQw7+DR5+/DQw4+DRw8+ heterozygous cells crosslinked TrG6 and TrC5, and available evidence indicated that the epitopes defined by these two mAbs were expressed by the transcomplementing DQ-molecule DQw7 alpha/DQw4 beta, where the DQw7 alpha chain specifies epitope TrC5 and the DQw4 beta chain specifies epitope TrG6. Taken together with published nucleotide sequences of DQ alpha and beta genes, our data are consistent with the conclusion that the amino acids at positions 69 and/or 75 of the DQ alpha chain of DQw2+DR3+ and DQw7+DR5+ haplotypes are critical for epitope TrC5. The previously reported human-human hybridoma Ab TrB12 reacts with DQw6, DQw8, and DQw9. The specificity of the murine mAb IIB3 is similar to that of TrB12, but, unlike TrB12, IIB3 also binds DQw4+ cells.     

Characterization and epitope mapping of four HLA class II reactive mouse monoclonal antibodies using transfected L cells and human cells transfected with mutants of DQB1*0302

Abstract: To study epitopes of HLA class II molecules, four mouse monoclonal antibodies (mAbs) 13B6, 17F8, 19A1 and 12G6 were made using HLA-DQ8, DP2 and DP4 expressing mouse transfectants for immunization. Three of the mAbs, 13B6, 17F8 and 19A1, bound to all DQ1, 4, 8 or 9 positive B-lymphoblastoid cell lines (B-LCLs) and transfectants tested, i.e. cells carrying the DQB1 genes 0302-3, 0401-2, 0501-3, 0601-4 and 0609 irrespective of the accompanying DQA1 gene. These DQB1 genes code for the shared amino acids (aa) GVY in position 45–47 of the DQ β chain. DQ1+4+8+9 specific (IIB3) and DQ3 specific (IVD12) reference mAbs inhibited binding of all three mAbs. Testing 13B6, 17F8 and 19A1 with cells made using aa substitutions in various positions of DQP1*0302 indicated involvement of aa 45 in the epitopes of all three mAbs. The last mAb (12G6) bound to all B–LCLs and all DP transfected cells. However, only some DR transfectants and a single DQ transfectant (carrying DQA1 *0201 and DQB1*0202) bound mAb 12G6. This reactivity pattern correlates with a shared sequence of aa (RFDSDVGE) in position 39–46 of DR-and DQ- and 37–44 of DP p chains.     

T cells from the small intestinal Mucosa of a DR4, DQ7/DR4. DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8

CD is an immunologic disease of the small intestine which is precipitated by ingestion of wheat gliadin. Most patients carry the HLA-DQ (1^*0501,β1^*0201) (DQ2) heterodimer. We recently reported that a prepoderance of gliadin-specific T cells from the small intestinal mucosa of DQ2-positive CD patients were restricted by this DQ heterodimer. However, a small percentage of CD patients do not carry this DQ heterodimer, and most of them instead carry DQ (1^*0301,β1^*0302) (DQ8). Here we report that a majority of gliadin-specific T cells from the small intestinal mucosa of a DR4,DQ7/DR4,DQ8 heterozygous CD patient are restricted by DQ8. Thus, preferential presentation of gliadin-derived peptides to T cells by the CD-associated DQ2 and DQ8 molecules may be an initial and important immunopathogenic step in CD. Human Immunology 41, 285–291 (1994)     

Interactions of peptide side chains with structurally complementary pockets in DQ molecules are critical for allele-specific peptide binding and T cell reactivity.

Peptides derived from glutamate decarboxylase, λ-repressor and VP-16 of HSV-2 were used to study peptide binding to different DQ3 alleles. Four residues at relative positions n, n+3, n+5 and n+8 of the peptide were found to be important for binding to the DQ3.1 and DQ3.2 molecules, presumably by anchoring into pockets designated 1, 4, 6 and 9 of the DQ molecules. Preferential binding of peptides with specific amino acid substitutions defined distinct binding motifs for each allelic molecule. The DQ3.1 molecule differs from DQ3.2 in its motif for position 4 and position 9, and both of these differences in the binding motif correlate with the allelic polymorphisms between the DQ3.1 and DQ3.2 β chains polymorphisms in pocket 4 and 9 of the class II peptide binding groove. The polymorphism at residue 57 of DQ was of particular interest: single amino acid substitutions at position 57 of the DQ3.2 molecule or at position n+8 of the peptide reciprocally regulated DQ-peptide complex formation. These binding studies were paralleled by specific T cell recognition, in which the substituted peptide abolished T cell reactivity which was directed to the DQ3.2-peptide complex, while the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57.     

Monoclonal antibody to β2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca mobilization in the mouse diaphragm muscle

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca²⁺ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for 1 subunit of muscle nAChR) depressed contractile Ca²⁺ transients without affecting non-contractile Ca²⁺ transients. mAb 270 (specific for β2 subunit of neuronal nAChR) depressed only non-contractile Ca²⁺ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-β2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca²⁺ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.     

Functional roles of human class II antigens in T cell proliferative response. Comparison of the DR and DQ molecules using mouse monoclonal antibodies

The monoclonal antibody (mAb) NC1 recognizing a monomorphic DR determinant and the anti-DQ mAbs PLM2, PLM9, and PLM12 recognizing polymorphic DQ determinants were produced. NC1 reacted with the H1L1 (DR) and H1L2(DRw52, DRw53 and allelic products) complexes of a DR family, while the specificity of PLM12 was DQw3 (previously called TB21). Both PLM2 and PLM9 reacted with the allodeterminant of TA10 (a subtype of DQw3). These three mAbs immunoprecipitated the same H2L3 complex of a DQ family. With the aid of these mAbs, DR molecules but not DQw3 molecules were detected on monocytes in significant quantities. The functional roles of these class II molecules in the T cell proliferative responses to three soluble antigens (PPD, mite, Candida) and alloantigens were examined by blocking assays in vitro. NC1 almost completely inhibited T cell proliferative responses against both soluble antigens and allogeneic antigens. In contrast, PLM2, PLM9 and PLM12 showed no significant inhibitory effects at all. These results indicate that DR and DQ antigens are different in their functional roles as well as their serological and immunochemical characteristics and tissue distributions; these three soluble antigens and the alloantigen were presented in association with determinants residing predominantly on two forms of NC1-reactive molecule, namely the H1L1 and/or H1L2 complex of a DR family but not in association with the DQw3 allodeterminant on the H2L3 complex of a DQ family.     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

A human—human hybridoma (Tr7E2) producing cytotoxic antibody to HLA-DQw1

A human—human IgM (λ) hybridoma antibody (called Tr7E2) was constructed by fusing Epstein-Barr virus-transformed cells from a multiparous woman with the human fusion partner KR12. By eosin exclusion microcytotoxicity the monoclonal antibody killed 12 of 13 human leukocyte antigen DQw1-bearing lymphoblastoid cells. No reaction was seen with any of 19 DQw1-negative cells. The single DQw1⁺ cell line that was not killed by Tr7E2 was the homozygous cell called 9WS 806 TAB (DR8,8; DQw1,1) of Japanese origin. The radioimmunoassay indicated that this result was probably not because of a decreased expression by this cell of DQ antigens, and these cells were killed by the mouse monoclonal antibody Genox3.53G2a5, reported to be specific for DQw1. Thus, the Tr7E2⁻ cell line TAB probably expresses a novel structural DQw1 variant. Of 213 Norwegians, 107 were Tr7E2⁺ Genox⁺ ; none expressed only one of these epitopes. The putative split is, therefore, probably very rare in this population. Monodisperse magnetic polymer beads coated with Tr7E2 formed rosettes selectively with peripheral blood mononuclear cells from DQw1-positive individuals, suggesting a new approach to typing for class II antigens.     

Production of epitope specific monoclonal IgG antibodies to HLA class II molecules by combining in vivo and in vitro immunization.

To study the expression of HLA-DQ beta chain alleles associated with type 1 diabetes, mAbs were generated from mice immunized with synthetic peptides representing allelic HLA-DQw7 and HLA-DQw8 beta chain sequences. The splenocytes from immunized mice were fused with myeloma cells, either immediately after or following additional in vitro boosting with peptide. Peptide-specific mAbs, predominantly of the IgG isotype, were isolated only from in vitro boosted splenocytes. Immunoblot analysis showed that several of the mAbs cross-reacted with DQ beta chain molecules. One mAb to a peptide representing DQw8 beta position [49-60] specifically recognised the DQw8 beta chain. Three mAbs to a peptide representing DQw8 beta position [39-52] specifically recognised an epitope consisting of Gly-Val-Tyr in position 45-47, i.e., all DQ beta alleles except DQw7 beta (position 45-47: Glu-Val-Tyr) and DQw2 beta (position 45-47; Gly-Glu-Phe). In FACS analysis these mAbs bound lymphocytes with the same specificity as found by immunoblotting analysis. Thus, by combining in vivo and in vitro immunization we have generated a number of epitope specific monoclonal IgG antibodies that distinguish closely related HLA-DQ beta chain alleles in predetermined positions.     

Polymorphisms within the HLA-DRw6 haplotype. III. DQ alpha and DQ beta polymorphism associated with HLA-D

We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQ alpha and DQ beta polymorphism. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQ alpha and beta chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.     

The natural history of a microsatellite located in the HLA DQ region

Microsatellite sequences are very polymorphic stretches of di or more nucleotide repeats that are intensively used in gene mapping studies. In this study, we have characterized the polymorphism of a CA repeat sequence (DQCAR) located between DQA1 and DQB1. This allowed us to study the value of microsatellite typing for association studies and to speculate on the evolution and mutagenicity of dinucleotide sequences in the human genome.

The linkage disequilibrium pattern of 14 DQCAR alleles was established using 941 samples oligotyped at the DRB1, DQA1 and DQB1 level. Samples included unrelated subjects from Japan, Papua New Guinea, African American and Caucasians, 10th WS cell lines and patients with various Class II associated diseases. DQCAR typing was performed as described previously (Human Immunol, 42(3); 209-220, 1995). We also PCR amplified, cloned and sequenced various DQCAR size alleles in a subset of samples.

The most striking finding of this study was the discovery that DQCAR variation differed greatly between DQ1 and non DQ1 haplotypes. Almost all DQ1 samples shared a single DQCAR allele (DQCAR 103). In contrast, non DQ1 haplotypes displayed a large amount of additional DQCAR variation, with for example up to 8 alleles in association with DQB1^*0301. We also explored the value of DQCAR typing in a few selected autoimmune disorders but could not detect a tighter association than with HLA class II typing alone.

DQCAR sequencing results were very informative to explain the lack of DQCAR polymorphism in DQCAR103-DQ1 haplotypes. In the low variation DQ1 haplotypes, the stretch of CA was interrupted by a C to A mutation, thus leading to short CA repeat stretches flanking a triple A sequence. In contrast, all DQCAR mutating alleles had relatively longer perfect CA repeat sequences. Other sequence changes were also observed in the flanking area of the microsatellite where CA to GA conversions were frequently observed. These results suggest that sequence polymorphisms within individual microsatellite alleles may drastically influence the mutation rate of each allele. This may have consequences for disease association studies using microsatellite markers.     

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5− DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.     

不同刺激剂和培养环境对CD4+、CD8+T细胞内细胞因子表达的影响

目的探讨不同的刺激剂和不同的培养条件对CD4 和CD8 T细胞内细胞因子表达的影响。方法分离正常人的外周血单个核细胞(PBMC),分别加入3种不同的刺激剂(PHA,抗CD3和抗CD28mAb,PMA和离子霉素),置于4种不同的培养环境下(室温,37℃水浴,37℃培养箱,37℃50mL/LCO2培养箱)培养4·5~5h。收集细胞,以荧光素-mAb标记后,用流式细胞术分析CD4 、CD8 T细胞内IL-2、IFN-γ和TNF-α的表达。结果CD4 和CD8 T细胞内细胞因子的表达,随着刺激剂的不同而有所差别,且在上述4种培养环境中,PMA的刺激效果最强,抗CD3mAb次之,PHA的刺激效应最弱。以上述3种刺激剂刺激后,不同培养条件对T细胞内细胞因子的表达有一定的影响。室温培养时几乎检测不到细胞因子的表达,而在37℃水浴、37℃培养箱和37℃50mL/LCO2培养箱中培养时,表达细胞因子的CD4 和CD8 T细胞的百分率差异无统计学意义(P>0.05)。结论不同刺激剂体外刺激T细胞表达细胞因子的效应不同,依次为PMA和离子霉素>抗CD3和抗CD28mAb>PHA。体外刺激培养的T细胞活化过程中,温度是重要的条件,CO2无明显影响。     

抗艰难梭菌A毒素单克隆抗体的制备及特性分析

目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具     

A preliminary analysis of the 11th International Histocompatibility Workshop monoclonal antibodies

In the 11th International Histocompatibility Workshop prescreening study, HLA Class I and Class II monoclonal antibodies (mAb) were tested on panels of peripheral T and B cells and homozygous typing cell (HTC) lymphoblastoid lines. The 102 Class I mAb, of which 64 were new, were screened on 63 HTC lines and 20 peripheral T cells. The 144 Class II mAb were tested on 63 HTC lines, 20 peripheral B and 10 peripheral T cells. For Class I, 45 of the 102 mAbs tested proved to have clear and identifiable reaction patterns on both PBL and HTC lines and included several interesting new HLA-A and -C locus antibodies. Of the 144 Class II mAb, of which 99 were new, 18 DR, 22 DQ and 7 DP antibodies were judged excellent. The DR antibodies included some which were antigen-specific but the majority were complex antibodies, while the DQ antibodies, as previously, identified the majority of DQ antigens both singly and in combinations. The encouraging number of DP antibodies was interesting in the limited range of sites they appear to detect.     

The presence of gliadin specific, HLA DQ2 restricted T cells in the small intestinal mucosa is a common phenomenon of celiac disease.

The gastrointestinal disorder celiac disease is strongly associated with genes encoding the HLA-DQ2 heterodimer, DQ(1^*0501,β1^*02). We have isolated activated lamina propria derived lymphocytes from small intestinal biopsies of 20 DR3-DQ2+ celiac disease patients (17 treated and 3 untreated) by immunomagnetic positive selection of CD25+ cells after in vitro challenge of biopsies with wheat flour gliadin protein. Gliadin-specific, polyclonal T cell lines were established from all of the 20 patients. Inhibition studies of T cell lines with anti-HLA monoclonal antibodies indicated predominant antigen presentation by DQ2 in patients. Furthermore we generated 9 gliadin-specific T cell clones from 5 patients which all were DQ2 restricted. These data confirm our previous reports in a more limited material of preferential DQ2-restriction of gliadin-specific T cells from the small intestine. In conclusion, our findings strongly indicate that gliadin-specific T cells is a common phenomenon in the small intestinal mucosa of celiac disease patients and they support the notion that most of them recognize gliadin-peptide when presented by the disease associated HLA-DQ heterodimer (1^*0501,β1^*02).     

Equine class II MHC antigens: identification of two sets of epitopes using anti-human monoclonal antibodies

Six mouse and 13 rat monoclonal antibodies (mAb) recognizing HLA-DR, DQ and DP antigens were used for the detection of cell surface class II MHC antigens of equine lymphocytes. The monoclonal antibodies were tested against peripheral blood lymphocytes (PBL) from a panel of thoroughbred horses, using two-color fluorescence flow cytometry. Seven of these mAbs reacted with both surface immunoglobulin positive (sIg+) and surface immunoglobulin negative (sIg-) lymphocytes. sIg+ cells stained consistently brighter than sIg- cells. The fluorescence pattern did not vary from donor to donor for each of the mAbs tested, except for SFR1-MI.2, which reacted with a variable intensity with cells from 47 of 53 horses tested. Immunoprecipitation with mAb SFR1-MI.2 and analysis by two-dimensional electrophoresis demonstrated the presence of light and heavy chains equivalent to HLA class II alpha and beta chains. Antibody N297 (DQ specific), previously shown to react with an epitope expressed on human B cells but not on mitogen-induced T cells, reacted only with sIg+ cells in 42 of 53 horses tested. The lack of staining of horses sIg- cells with N297 may be due to a low or lack of expression of this determinant on these cells or to a weak cross-reactivity of this antibody with equine antigens.     

Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells

ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GFP-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the ability of cytohesin-1 mAbs to immunoprecipitate cytohesin-1. Among the hybridomas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-1. Furthermore, mAb 2E11 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditions. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected both cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not GFP-cytohesin-1 under native conditions. Another antibody, mAb 10A12, also selectively immunoprecipitated GFP-ARNO under native conditions, but the epitope recognized by this mAb is unlikely to be linear as no signal was obtained by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal antibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin-1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in levels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulocytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be useful as a potential marker for granulocytic differentiation.     

鼠抗人CD28分子单克隆抗体的研制及生物学特性研究

目的 制备鼠抗人CD28分子的单克隆抗体（mAb),研究其在T细胞的活化、增殖及信号传导中的作用。方法 以小鼠淋巴瘤细胞转染人CD28基因的细胞株（CD28-T）为免疫原,采用B淋巴细胞杂交瘤技术,获取分泌特异性mAb的杂交瘤细胞株,以体内诱生法生产腹水,并以免疫亲和层析法对其纯化,以快速定性试纸法鉴定mAb的Ig亚类,竞争抑制法分析mAb识别的抗原表位,3H-TdR掺入法研究mAb对T细胞的刺激效应,结果 成功地获得了5株分泌特异性抗入CD28mAb的杂交瘤细胞株,鉴定的1株（克隆18G8）属IgG2a,腹水效价（流式细胞仪分析）达1：2400以上,该mAb能60%阻断标准鼠抗入CD28抗体与相应抗原的结合,提示其识别的抗原表位与标准mAb不完全相同,mAb18G8可取代B7-1分子介导的协同刺激信号,促进人外周血T细胞增殖（ST=7）。结论 18G8是12株功能性mAb,具有重要的研究和应用价值。     

Evidence that HLA-DQ9 confers risk to celiac disease by presence of DQ9-restricted gluten-specific T cells

We describe the gluten T-cell response of a DR7DQ2/DR9DQ9 heterozygous celiac disease patient (CD555). Interestingly, this patient had T cells recognizing gluten in the context of human leukocyte antigen (HLA) molecules of both haplotypes. For the DR9DQ9 haplotype, DQ9 was identified as the antigen-presenting molecule. As DQ9 carries aspartate at DQ β57 but is otherwise identical to DQ8 and not considered associated with celiac disease, we aimed to characterize this DQ9-restricted T-cell response in detail. By fractionation of pepsin-trypsin digested gliadin we identified an epitope stimulatory for several T-cell clones. This epitope was identical to an epitope (DQ8-glut-1) previously identified in DQ8 patients. In CD555, this was the dominant DQ9-restricted epitope, whereas no T-cell response was found toward two other DQ8-restricted epitopes. These findings correlated with peptide binding data demonstrating that this epitope bound better to DQ9 than the two other DQ8-restricted epitopes. Although glutamine to glutamate exchange at P9 improved binding of all three epitopes to DQ8, no such effect was observed for DQ9. The differential ability of DQ8 and DQ9 to harness a negatively charged anchor at P9 may result in fewer potential gluten epitopes in DQ9 patients. Our data further indicate that DQ9 is a susceptibility factor for celiac disease.