Proteome Based Construction of the Lymphocyte Function-Associated Antigen 1 (LFA-1) Interactome in Human Dendritic Cells

The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) plays an important role in the migration, adhesion and intercellular communication of dendritic cells (DCs). During the differentiation of human DCs from monocyte precursors, LFA-1 ligand binding capacity is completely lost, even though its expression levels were remained constant. Yet LFA-1-mediated adhesive capacity on DCs can be regained by exposing DCs to the chemokine CCL21, suggesting a high degree of regulation of LFA-1 activity during the course of DC differentiation. The molecular mechanisms underlying this regulation of LFA-1 function in DCs, however, remain elusive. To get more insight we attempted to identify specific LFA-1 binding partners that may play a role in regulating LFA-1 activity in DCs. We used highly sensitive label free quantitative mass-spectrometry to identify proteins co-immunoprecipitated (co-IP) with LFA-1 from ex vivo generated DCs. Among the potential binding partners we identified not only established components of integrin signalling pathways and cytoskeletal proteins, but also several novel LFA-1 binding partners including CD13, galectin-3, thrombospondin-1 and CD44. Further comparison to the LFA-1 interaction partners in monocytes indicated that DC differentiation was accompanied by an overall increase in LFA-1 associated proteins, in particular cytoskeletal, signalling and plasma membrane (PM) proteins. The here presented LFA-1 interactome composed of 78 proteins thus represents a valuable resource of potential regulators of LFA-1 function during the DC lifecycle.     

A Monoclonal Antibody Defining Human B Cell Differentiation Antigen (HLB-1 Antigen)

A new monoclonal antibody specific for human B cell differentiation antigen (HLB-1) is produced by a hybridoma established by fusion of splenocytes of mice immunized with the Epstein-Barr virus (EBV)-transformed peripheral B cell line, RPMI-8057. This monoclonal, antibody designated anti-HLB-1 monoclonal antibody (anti-HLB-1), reacted with surface immunoglobulin (sIg)-positive B cells of normal peripheral blood and lymphoid tissues and sIg-positive leukemic cells. The cells of T cell leukemia, non-T non-B acute lymphoblastic leukemia (ALL) and nonlymphoid leukemia were HLB-1 negative. These data were further confirmed by studying a panel of cultured human hematopoietic cell lines. Anti-HLB-1 reacted with B cell lines derived from pre-B, Burkitt's lymphoma, B cell type ALL and EBV-transformed peripheral B cells. Anti-HLB-1 was reactive with only one of three human myeloma cell lines, and with none of the T cell, myeloid and non-T non-B ALL cell lines. This newly defined HLB-1 antigen is different from other conventional human B cell markers such as sIg, Ia antigens, and receptors for the Fc portion of Ig and complement C3.     

Human and Murine Cytomegalovirus Evasion of Cytotoxic T Lymphocyte and Natural Killer Cell-Mediated Immune Responses

Herpesviruses, such as human and murine cytomegalovirus, possess an impressive array of genes believed to assist in virus survival against the host immune response. In this review, we cover the rapidly growing area of cytomegalovirus evasion of cellular immunity, specifically cytotoxic T lymphocytes and natural killer cells. The proposed mechanisms of action of viral proteins involved in blocking peptide presentation to CD8⁺T cells, namely, interference with peptide generation, inhibition of peptide assembly with class I MHC and retention/destabilization of class I MHC complexes, are described. In addition, recent evidence implicating the viral class I MHC-like proteins as inhibitors of natural killer cell-mediated clearance is reviewed.     

A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS³ Poisson detection mass spectrometry. Only one epitope (E7_11–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7_11–20), previously used as a vaccine candidate, was neither detected by MS³ on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.     

A Biased Competition Theory of Cytotoxic T Lymphocyte Interaction with Tumor Nodules

The dynamics of the interaction between Cytotoxic T Lymphocytes (CTL) and tumor cells has been addressed in depth, in particular using numerical simulations. However, stochastic mathematical models that take into account the competitive interaction between CTL and tumors undergoing immunoediting, a process of tumor cell escape from immunesurveillance, are presently missing. Here, we introduce a stochastic dynamical particle interaction model based on experimentally measured parameters that allows to describe CTL function during immunoediting. The model describes the competitive interaction between CTL and melanoma cell nodules and allows temporal and two-dimensional spatial progression. The model is designed to provide probabilistic estimates of tumor eradication through numerical simulations in which tunable parameters influencing CTL efficacy against a tumor nodule undergoing immunoediting are tested. Our model shows that the rate of CTL/tumor nodule productive collisions during the initial time of interaction determines the success of CTL in tumor eradication. It allows efficient cytotoxic function before the tumor cells acquire a substantial resistance to CTL attack, due to mutations stochastically occurring during cell division. Interestingly, a bias in CTL motility inducing a progressive attraction towards a few scout CTL, which have detected the nodule enhances early productive collisions and tumor eradication. Taken together, our results are compatible with a biased competition theory of CTL function in which CTL efficacy against a tumor nodule undergoing immunoediting is strongly dependent on guidance of CTL trajectories by scout siblings. They highlight unprecedented aspects of immune cell behavior that might inspire new CTL-based therapeutic strategies against tumors.     

Specific Lymphocyte Sensitization in Cancer: Is There a Common Antigen in Human Malignant Neoplasia?

From human cancer tissue a basic protein can be extracted by the method which yields encephalitogenic factor when applied to human brain. This tumour basic protein (obtained from several different neoplasms) acts as an antigen in the cytopherometric test for malignant neoplasia and in general gives higher results than does brain basic protein. The reverse is true when degenerative disease of the nervous system is studied. The basic protein extractable from brain and from tumours thus has some degree of specificity probably referable to its amino-acid sequence.     

Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain

The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.     

Specific Lymphocyte Stimulation by Purified,Heat-inactivated Hepatitis-B Antigen

The ability of purified, heat-inactivated hepatitis-B antigen (HBAg) to stimulate sensitized lymphocytes in vitro was investigated with the lymphocyte stimulation test on lymphocytes from three groups of individuals. Stimulation was minimal in the lymphocytes of two out of 15 normal controls, whereas lymphocytes from nine out of 12 patients who had recovered from hepatitis B showed stimulation, as did lymphocytes from five out of 12 laboratory technicians who had been regularly exposed to HBAg but who had no history of hepatitis or signs of it in the previous two years. No differences were observed in the responses to phytohaemagglutinin of lymphocytes from persons in the three groups. HBAg and HBAg inactivated by heat were shown in immunodiffusion to be immunologically identical. Inactivated HBAg stimulated antibody production in guinea-pigs. These findings suggest that not only humoral but also cell-mediated immunity might be induced by vaccination with purified, heat-inactivated HBAg.     

人前列腺特异抗原分离纯化

本文报道了一种从人精液中提纯前列腺特异抗原的方法,本方法仅需Affigel-Blue gel柱和凝胶HPLC二步层析,即可得到均一的PSA纯品。这是一个快速、有效的提纯方法。PSA是前列腺癌的特异肿瘤标志物。     

Cross-Allele Cytotoxic T Lymphocyte Responses against 2009 Pandemic H1N1 Influenza A Virus among HLA-A24 and HLA-A3 Supertype-Positive Individuals

Lack of a universal vaccine against all serotypes of influenza A viruses and recent progress on T cell-related vaccines against influenza A virus illuminate the important role of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) in anti-influenza virus immunity. However, the diverse HLA alleles among humans complicate virus-specific cellular immunity research, and elucidation of cross-HLA allele T cell responses to influenza virus specificity requires further detailed work. An ideal CTL epitope-based vaccine would cover a broad spectrum of epitope antigens presented by most, if not all, of the HLAs. Here, we evaluated the 2009 pandemic influenza A (H1N1) virus-specific T cell responses among the HLA-A24⁺ population using a rationally designed peptide pool during the 2009 pandemic. Unexpectedly, cross-HLA allele T cell responses against the influenza A virus peptides were detected among both HLA-A11⁺ and HLA-A24⁺ donors. Furthermore, we found cross-responses in the entire HLA-A3 supertype population (including HLA-A11, -A31, -A33, and -A30). The cross-allele antigenic peptides within the peptide pool were identified and characterized, and the crystal structures of the major histocompatibility complex (MHC)-peptide complexes were determined. The subsequent HLA-A24-defined cross-allele peptides recognized by the HLA-A11⁺ population were shown to mildly bind to the HLA-A*1101 molecule. Together with the structural models, these results partially explain the cross-allele responses. Our findings elucidate the promiscuity of the cross-allele T cell responses against influenza A viruses and are beneficial for the development of a T cell epitope-based vaccine applied in a broader population.     

Modulation by α-2 Macroglobulin of Human Lymphocyte Proliferation in Response to Mitogens and Antigen

The effect was studied of purified alpha-2 macroglobulin (a potent and polyvalent protease inhibitor) on normal human lymphocytes stimulated by phytohaemagglutinin, pokeweed mitogen, or by purified protein derivative as an antigen. At physiological concentration alpha 2 M has been found to inhibit mitogenic as well as antigenic stimulation. However at lower dilutions alpha 2 M acts in synergy either with the mitogen or with the antigen. At these dilutions thymidine incorporation is enhanced. alpha 2 M has no toxic or mitogenic effect on normal human lymphocytes. These data might indicate that two proteases may play a role in the proliferation of normal human lymphocytes.     

Design Parameters for Granzyme-Mediated Cytotoxic Lymphocyte Target-Cell Killing and Specificity

Cytotoxic lymphocytes are key elements of the immune system that are primarily responsible for targeting cells infected with intracellular pathogens, or cells that have become malignantly transformed. Target cells are killed mainly via lymphocyte exocytosis of specialized lysosomes containing perforin, a pore-forming protein, and granzymes, which are proteases that induce apoptosis. Due to its central role in lymphocyte biology, as well as its implication in a host of pathologies from cancer to autoimmunity, the granzyme-perforin pathway has been the subject of extensive investigation. Nevertheless, the details of exactly how granzyme and perforin cooperate to induce target-cell death remain controversial. To further investigate this system, we developed a biophysical model of the immunological synapse between a cytotoxic lymphocyte and a target cell using a spatial stochastic simulation algorithm. We used this model to calculate the spatiotemporal evolution of granzyme B and perforin from the time of their exocytosis to granzyme internalization by the target cell. We used a metric of granzyme internalization to delineate which biological processes were critical for successful target-cell lysis. We found that the high aspect ratio of the immunological synapse was insufficient in this regard, and that molecular crowding within the synapse is critical to preserve sufficient concentrations of perforin and granzyme for consistent pore formation and granzyme transfer to target cells. However, even when pore formation occurs in our model, a large amount of both granzyme and perforin still escape from the synapse. We argue that a tight seal between the cytotoxic lymphocyte and its target cell is not required to avoid bystander killing. Instead, we propose that the requirement for spatiotemporal colocalization of granzyme and perforin acts as an effective bimolecular filter to ensure target specificity.     

Tubulation of Endosomal Structures in Human Dendritic Cells by Toll-like Receptor Ligation and Lymphocyte Contact Accompanies Antigen Cross-presentation

Mouse dendritic cells (DCs) can rapidly extend their Class II MHC-positive late endosomal compartments into tubular structures, induced by Toll-like receptor (TLR) triggering. Within antigen-presenting DCs, tubular endosomes polarize toward antigen-specific CD4⁺ T cells, which are considered beneficial for their activation. Here we describe that also in human DCs, TLR triggering induces tubular late endosomes, labeled by fluorescent LDL. TLR triggering was insufficient for induced tubulation of transferrin-positive endosomal recycling compartments (ERCs) in human monocyte-derived DCs. We studied endosomal remodeling in human DCs in co-cultures of DCs with CD8⁺ T cells. Tubulation of ERCs within human DCs requires antigen-specific CD8⁺ T cell interaction. Tubular remodeling of endosomes occurs within 30 min of T cell contact and involves ligation of HLA-A2 and ICAM-1 by T cell-expressed T cell receptor and LFA-1, respectively. Disintegration of microtubules or inhibition of endosomal recycling abolished tubular ERCs, which coincided with reduced antigen-dependent CD8⁺ T cell activation. Based on these data, we propose that remodeling of transferrin-positive ERCs in human DCs involves both innate and T cell-derived signals.     

Development of Human Recombinant Antibodies Against ROR1 Tumor Antigen

Background:Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal antigen expressed on many types of cancer cells, but not normal adult cells. ROR1 antigen contributes to cancer development and progression by several signaling pathways. ROR1 expression has been associated with tumor growth, survival, and metastasis. In this study specific human recombinant antibodies were selected against ROR1 antigen for their use in cancer immunotherapy.Methods:Phage display technology was used to produce phage antibody from a human scFv library. Phage concentration was determined to confirm the phage rescue process. Panning procedure was performed to isolate specific scFv clones against ROR1 epitope. Phage ELISA was done to evaluate the reactivity of the selected scFvs.Results:Two specific human scFvs with frequencies of 20% and 25% were selected against ROR1 peptide. The antibodies showed specific reaction to the corresponding epitopes in phage ELISA.DiscussionCancer targeted therapy using human specific antibodies is a new strategy, which is used in cancer therapy. The selected specific scFvs that target ROR1 epitope are human antibodies that originated from a human library and have the potential to be used in clinic in cancer immunotherapy of ROR1 positive tumors without induction of human anti mouse antibody (HAMA) response.Key Words: ROR1, Phage display, scFV library, Cancer     

荧光素标记检测乙型肝炎病毒表面抗原特异性T淋巴细胞方法的探讨

用绿色荧光前体化合物Calcein AM标记靶细胞,经过与杀伤性T淋巴细胞(CTL)效应细胞数小时的孵育,通过分析培养上清中的荧光强度检测CTL效应.通过对一系列标记条件的研究:不同浓度Calcein AM标记靶细胞、不同洗涤缓冲液、不同标记细胞浓度、不同洗涤次数、不同裂解液配方,确定CalceinAM荧光素标记法的最佳条件.用该方法检测乙型肝炎病毒表面抗原的DNA疫苗免疫Balb/c小鼠对P815S细胞的CTL效应,E/T比为10时,杀伤效率接近饱和,达到65%.通过荧光显微镜直接观察杀伤后的P815S细胞,细胞破裂程度与计算出的杀伤效率有一定相关性.     

Proceedings of the Second International Bovine Lymphocyte Antigen (BoLA) Workshop

The results of the second International BoLA Workshop, held in Wageningen, Netherlands in July 1980, are reported. These results arise from a comparison of 362 alloantisera to bovine lymphocytes, originating from 9 laboratories. The an-tisera were tested against a selected panel of 144 lymphocyte samples, originating from 7 laboratories. Some of the antisera and lymphocytes had also been tested during the First International BoLA Workshop in 1978.
Ten of the eleven specificities defined at the first workshop were confirmed, and an additional six new specificities were designated. Two of the additional specificities are subgroups of the w6 specificity. The data from this workshop are consistent with the hypothesis that BoLA antigens are controlled by a series of codominant alleles at a single autosomal locus.     

Development of a Novel In Silico Docking Simulation Model for the Fine HIV-1 Cytotoxic T Lymphocyte Epitope Mapping

Introduction

Class I HLA''s polymorphism has hampered CTL epitope mapping with laborious experiments. Objectives are 1) to evaluate the novel in silico model in predicting previously reported epitopes in comparison with existing program, and 2) to apply the model to predict optimal epitopes with HLA using experimental results.

Materials and Methods

We have developed a novel in silico epitope prediction method, based on HLA crystal structure and a peptide docking simulation model, calculating the peptide-HLA binding affinity at four amino acid residues in each terminal. It was applied to predict 52 HIV best–defined CTL epitopes from 15-mer overlapping peptides, and its predictive ability was compared with the HLA binding motif-based program of HLArestrictor. It was then used to predict HIV-1 Gag optimal epitopes from previous ELISpot results.

Results

43/52 (82.7%) epitopes were detected by the novel model, whereas 37 (71.2%) by HLArestrictor. We also found a significant reduction in epitope detection rates for longer epitopes in HLArestrictor (p = 0.027), but not in the novel model. Improved epitope prediction was also found by introducing both models, especially in specificity (p<0.001). Eight peptides were predicted as novel, immunodominant epitopes in both models.

Discussion

This novel model can predict optimal CTL epitopes, which were not detected by an existing program. This model is potentially useful not only for narrowing down optimal epitopes, but predicting rare HLA alleles with less information. By introducing different principal models, epitope prediction will be more precise.