Analysis of the requirement for β2-microglobulin for expression and formation of human CD1 antigens

Human CD1 form a group of nonpolymorphic leukocyte surface molecules with homology to major histocompatibility complex (MHC) proteins. Recent findings in human and in mouse demonstrate the capacity of CD1 molecules to present nonpeptide components like lipids or lipoglycans as well as peptides. We studied the involvement of β2-microglobulin (β2m) in expression of the classic human CD1 proteins CD1a, CD1b, and CD1c. The β2m-deficient human melanoma cell line FO-1 was transiently transfected with either CD1a, CD1b, or CD1c DNA alone, or in combination with β2m using the adenovirus-enhanced receptor-mediated transfer infection system. Only co-transfection of FO-1 cells with CD1 + β2m resulted in the detection of CD1 Ag by monoclonal antibodies (mAb). This indicated that CD1 mAb recognized determinants are dependent on β2m and raised the question whether β2m-free forms of CD1 can be expressed. Therefore, to visualize CD1 molecule expression independently of β2m, we expressed tagged recombinant forms. A full-length CD1b construct tagged at the very C terminus with a small peptide was transported to the plasma membrane only when β2m was co-transfected. β2m involvement in the transport of CD1 was confirmed by expression of soluble forms of CD1a, CD1b, and CD1c in three different cell types. Analogous to tagged full-length CD1b, secretion of the soluble CD1 constructs was strictly dependent on β2m. The soluble CD1 chimeras were secreted as complexes with endogenous β2m. Thus, similar to its role for MHC class I expression, β2m is essential for processing and surface transport of the classic human CD1 molecules CD1a, CD1b, and CD1c.     

肾癌细胞冻融抗原负载树突状细胞瘤苗的活化

目的: 探索体外诱导DCs活化的方法和制备肾癌树突状细胞(DCs)瘤苗。方法: 制备肾癌细胞冻融抗原；取健康人新鲜血分离外周血单个核细胞(PBMC),应用GM-CSF+IL-4刺激，诱导PBMC为iDCs,然后进行分组，采用不同因子刺激iDCs转化为mDCs,其中Ａ组:冻融抗原负载；Ｂ组: TNF-α+冻融抗原负载；Ｃ组: IL-1β+冻融抗原负载；Ｄ组: TNF-α+IL-1β+冻融抗原负载。 结果:各组均可诱导iDCs的成熟,并高表达CD86、CD80和HLA-DR；相对于其它组，D组DCs 更显著上调CD83和CD54表达(P<0.05)和IL-12分泌(P<0.01)，且D组mDCs更有效地刺激淋巴细胞增殖(P<0.05)。 结论: TNF-α+IL-1β与肾癌细胞冻融抗原协同可有效促进DCs成熟、增强诱导淋巴细胞活化的能力。     

B超结合血清CA－125测定诊断卵巢恶性肿瘤

１２１例卵巢肿瘤患者手术后经病理学诊断，本文对他们的术前Ｂ超结果和ＣＡ－１２５浓度进行分析评价。Ｂ超对卵巢恶性肿瘤的检测灵敏度为８５％，特异性８９％。ＣＡ－１２５阳性界值为４５Ｕ／ｍｌ时，诊断恶性卵巢肿瘤的灵敏度为９７％，特异性８２％，两者联合使用的特异性９８％，联合阳性预测值（ＰＰＶ）为９５％；当ＣＡ－１２５的阳性界值定为６５Ｕ／ｍｌ时，特异性为９９％，ＰＰＶ９８％。因此，Ｂ超和ＣＡ－１２５联合检测，为卵巢恶性肿瘤的诊断提供了一个有力的指标。     

CD40-CD40 ligand interactions stimulate B cell antigen processing

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II E^k molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.     

CD8 alpha- and Langerin-negative dendritic cells, but not Langerhans cells, act as principal antigen-presenting cells in leishmaniasis

In the early phase of leishmaniasis three types of potential antigen-presenting cells, including epidermal Langerhans cells (LC), dermal dendritic cells (DC) and inflammatory DC, are localized at the site of infection. Therefore, it has been a central question which cell type is responsible for the initiation of a protective immune response. In the early stage of an anti-Leishmania immune response, detectable Leishmania major antigen was localized in the paracortex of the draining lymph nodes (LN). Characterization of antigen-positive cells showed that L. major co-localized with DC of a CD11c(+) CD8 alpha(-) Langerin(-) phenotype. To determine the area of antigen uptake, dermis or epidermis, and to further define the type of antigen-transporting cells, L. major was inoculated subcutaneously and concurrently LC were mobilized with fluorescein isothiocyanate (FITC). After 3 days, DC carrying L. major antigen were always FITC(-), indicating a dermal and not an epidermal origin. Moreover, addition of L. major antigen to ex vivo isolated CD8 alpha(-) and CD8 alpha(+) DC from the draining LN of L. major-infected C57BL/6 mice demonstrated that both DC subpopulations were able to stimulate antigen-specific T cell proliferation in vitro. Without addition of exogenous antigen only the CD8 alpha(-) Langerin(-) DC were capable of stimulating antigen-specific T cell proliferation. Thus, we demonstrate that CD8 alpha(-) Langerin(-) DC and not LC are the basis of the protective immune response to intracellular L. major parasites in vivo.     

Cell type-specific processing of the I-Ed-restricted hen egg lysozyme determinant 107–116

Class II major histocompatibility complex heterodimers present to T cells determinants as sets of antigen fragments with ragged N and C termini. It is not yet elucidated whether different types of antigen-presenting cells generate identical sets of peptides containing the same determinant. Taking advantage of recombinant I-E^d molecules produced by insect cells as empty heterodimers, a sensitive T cell stimulation assay was developed to analyze naturally processed hen egg lysozyme (HEL) peptides. I-E^d preparations were isolated from antigen-presenting cells cultured with HEL. Following acid treatment, peptides eluted from I-E^d were chromatographed and the fractions incubated at acidic pH with purified recombinant I-E^d molecules, conditions which favor peptide binding. The stimulatory capacity of the reconstituted peptide-I-E^d complexes adsorbed on the well surface of cell culture plates was then evaluated by measuring interleukin-2 secreted by an HEL 107–116-specific, I-E^d-restricted T cell hybridoma. We found that the B lymphoma A20 and an I-E^d-transfected fibroblast cell line generated distinct sets of peptides containing the HEL sequence 107–116. Our results suggest the possibility that presentation of one determinant by different types of antigen-presenting cells stimulates populations of T cells with distinct fine antigen specificities.     

Anaplastic large cell lymphoma,CD30/Ki-1 positive,expressing the CD15/Leu-M1 antigen

Summary In this report we analyze the morphological and immunohistochemical findings observed in 5 cases of CD30/Ki-1 positive anaplastic large cell lymphoma, a recently recognized neoplastic entity. In comparison with the Ki-1 lymphomas so far described, these cases showed a fairly large number of Reed-Sternberg-like cells, often admixed with small lymphocytes and occasional eosinophils. Moreover, in all our cases immunohistochemical reactions detected the CD15/ Leu-M1 antigen, together with markers of the T-lineage and of lymphoid activation. In previous studies the CD15/Leu-M1 antigen has been found in the majority of cases of Hodgkin's disease, but has been stated to be absent typically in Ki-1 lymphomas. Our results indicate that this antigen cannot be considered a reliable tool to distinguish between Ki-1 lymphomas and Hodgkin's disease. Furthermore, the morphological and immunohistochemical findings reported suggest that in some cases Ki-1 cell lymphoma and Hodgkin's disease may be closely related. They may represent different steps in the progression of the same lymphoproliferative disorder.This work has been partially supported by the Italian Association for Cancer Research, Milan Italy     

V(D)J recombination catalyzed by mutant RAG proteins lacking consensus DNA-PK phosphorylation sites

The process of antigen receptor gene rearrangement, V(D)J recombination, involves DNA cleavage by the RAG-1 and RAG-2 proteins. Cleavage generates covalently sealed (hairpin) DNA ends, termed coding ends, which must be opened by an endonuclease prior to joining. Resolution of these hairpin ends requires the activity of the DNA-dependent protein kinase (DNA-PK), a protein kinase whose specific role is yet undetermined. It has been suggested that phosphorylation of one or both RAG proteins by DNA-PK is required to activate or recruit the hairpin-opening nuclease. Furthermore, very recent work has shown that RAG proteins themselves can open hairpins. These data raise the possibility that DNA-PK-mediated phosphorylation of the RAG proteins could regulate the hairpin opening reaction. To test this hypothesis, we constructed mutant versions of RAG-1 and RAG-2 in which all four DNA-PK consensus phosphorylation sites were removed by site-directed mutagenesis. Our data provide conclusive evidence that phosphorylation of these conserved serine/threonine residues is not required for hairpin opening or joining of V(D)J recombination intermediates.     

噬菌体展示技术筛选bFGF抗原表位

目的：以抗—bFGF单克隆抗体GF22为目标，用噬菌体随机七肽库进行筛选。经三轮筛选后的噬菌体阳性克隆能特异地抑制bFGF与CF22结合。测序结果表明与CF22结合的序列高度保守，为LP(P／L)GH(F／1)K，LPPGHFK与bFGF分子(155氨基酸)上22—27位氨基酸一致，表明该序列在bFGF上是一个主要的抗原表位。用此序列的阳性噬菌体克隆免疫小鼠，发现此序列模拟肽能诱导抗bFGF抗体反应，且序列LPLGHIK诱导的抗体反应比IPPGHFK序列强三倍，序列IPLGHIK可能作为一种有价值的肽疫苗诱导抗bFGF抗体的产生。     

Use of the alpha-hemolysin secretion system of Escherichia coli for antigen delivery in the Salmonella typhi Ty21a vaccine strain

This study examined the suitability of the hemolysin secretion system of Escherichia coli for expression and delivery of alpha-hemolysin (HlyA) by the S. typhi Ty21a strain, the only live oral Salmonella vaccine strain licensed for human use, under in vitro and in vivo conditions. For this purpose, two plasmid vectors encoding either the whole alpha-hemolysin of E. coli (pANN202-812/pMOhly2) or the hemolysin secretion signal (pMOhly1) were transferred into S. typhi Ty21a. S. typhi Ty21a carrying pANN202-812/pMOhly2 revealed efficient secretion of hemolysin in vitro. After formulation according to a process suitable for commercial production of Salmonella-based live bacterial vaccines, plasmids were shown to be stable in Ty21a and hemolysin secretion was demonstrated even after storage of the strains under real-time and stress conditions. After intranasal immunization of mice with S. typhi Ty21a/pANN202-812 plasmids are stable in vivo, and immunization induced a profound immune response against the heterologous HlyA antigen. Therefore, the combination of the hemolysin secretion system and S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines.