Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors (original) (raw)
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Activation of T cells through a T cell-specific epitope of CD45
Cellular Immunology, 1992
The 180-and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the I90-kDa isoform of CD45 and to a minor extent, the 205-and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitopc was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the I90-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca"]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the I90-kDa CD45 isoform on human CD4 cells is heterogeneous and that the I90-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells. 8 1992 Academic PWSS, IK.
Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation
Proceedings of the National Academy of Sciences, 1989
Leukocytes express a family of plasma membrane proteins called CD45 or the leukocyte common antigen. Isoforms of various molecular masses, 180-240 kDa, are produced by alternative splicing and usage of three exons, named A, B, and C, that encode the N-terminal portion of the external domain. By using monoclonal antibode that precipitate B exon-dependent and B exon-independent isoforms we
Generating and characterizing the anti-human CD45 monoclonal antibody
Science and Technology Development Journal, 2020
Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis. Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity. Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen...
Blood, 1995
Lymphocyte interactions with other leukocytes and other cell types, as well as with components of the extracellular matrix, are one of the key steps in the immune response. Three novel monoclonal antibodies (MoAbs) have been produced and selected for their ability to induce intercellular adhesion in B cells. These three MoAbs immunoprecipitated a polypeptide of 220 kD, displaying specific phosphotyrosine phosphatase activity that has been identified as CD45. These MoAbs recognize epitopes located on the alternative spliced exon-A-encoded region of CD45. These epitopes are of polypeptidic nature, but they can be masked by addition of carbohydrate during CD45 biosynthesis. Interestingly enough, CD45 epitopes recognized by these MoAbs appeared to be selectively expressed on both peripheral blood and tonsillar B lymphocytes as well as on peripheral blood natural killer (NK) cells. CD45-mediated intercellular adhesion was abrogated upon incubation with anti-leukocyte function-associated ...
A new epitope on sheep CD45R molecule detected by a monoclonal antibody
Comparative Immunology Microbiology and Infectious Diseases, 1999
This paper describes the production and characterization of a monoclonal antibody (mAb), Co-46D5, which recognizes a new epitope on the isoform of the homologous sheep leukocyte common antigen (LCA) or CD45. This mAb was submitted to the 3rd workshop on ruminant leukocyte antigens and was assigned to a cluster reactive with B-and T-cells subsets. Co-46D recognizes a 220 kDa molecule on peripheral blood mononuclear cells (PBMC) and spleen cells but not on thymocytes. Flow cytometry (FCM) analysis shows that Co-46D5 reacted with 30% of PBMC and 50% of spleen cells and more than 95% of cells freshly isolated from lymphoid follicles of the ileal Peyer's patches (IPP) of young lambs. By immunohistochemistry, the antigen was detected mainly on B-cell areas of lymph nodes and spleen. It was also found on a subpopulation of medullar thymocytes. Based on these results, we assume that Co-46D5 recognizes a new epitope on the largest isoform of the sheep CD45 receptor, probably on the homologous to the human CD45RA isoform. #
Veterinary immunology …, 2005
Previous studies in our laboratory suggested that there was positive selection of B cells during early development in the appendix of normal and V H mutant (ali/ali) rabbits. Preferential expansion and survival of B lymphocytes was affected by the Ig V H frameworks 1 and 3 sequences expressed on the cell surface. We demonstrated a specific interaction between rabbit CD5 and the V region of rabbit heavy chains and suggested that CD5 is a potential selecting ligand for B-cell surface immunoglobulin framework region sequences. To further investigate the role of CD5 in rabbit B-cell selection and survival we prepared recombinant constructs and obtained stable expression of the three scavenger receptor cysteine-rich (SRCR) extracellular domains of rabbit CD5. Here we describe the production and purification of this expressed recombinant CD5 protein, polyclonal antibody obtained by immunization of a goat and initial production and characterization of specific mAbs against peptides selected from each sequenced SRCR domain. Published by Elsevier B.V.
Immunological evaluation of predicted linear B-cell epitopes of human CD20 antigen
Biotechnology and Applied Biochemistry, 2012
The importance of B-lymphocyte-restricted differentiation antigen Bp35 (CD20) as a target for immunotherapeutic depletion of B cells is irrefutable. Several anti-human CD20 (anti-hCD20) monoclonal antibodies are expressed at different stages of development. However, resistance to anti-CD20 therapy has made the search for new alternatives imperative. Identification of B-cell epitopes within hCD20 using in silico tools can provide new opportunities to develop monoclonal antibodies with different binding sites. Furthermore, identification of the relationship between amino acid sequences of predicted B-cell epitopes and immune responses facilitates the determination of immunogenic regions of proteins by using their primary structure. Experimental evaluation of predicted linear B-cell epitopes as candidate peptides and bioinformatics allows us to explore this relationship. In this study, we selected three candidate epitopes within the extra membrane loop of hCD20 with the aid of five immunoinformatics predictor web servers and evaluated mouse humoral response to keyhole-limpet-hemocyaninconjugated peptides, and P4 and P5 peptides (the extracellular loop of hCD20 without and with a disulfide bond, respectively). Injection of the peptides yielded results that confirmed the prediction and selection of candidates. ELISA and flow cytometry corroborated the in silico selections. The B-cell epitopes P1, P2, and P3 were effective for immunization of mice.
Immunology, 1996
In vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp 120 from human immunodeficiency virus type 1 (HIV-1)] and a T-helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro immune response demonstrated an antigen-specific isotype switch, which was dependent on the presence of antigen-specific T-helper cells, CD40 ligation and antigen. Antibody libraries were constructed from cells derived directly from the naive donors, or from primary or secondary in vitro immunized B cells. Five libraries were displayed on filamentous phage and selected for anti-V3-specific Fab fragments, using a selection approach that linked recognition and phage replication. A panel of 19 recombinant antigen-specific Fab, representing different phases of the humoral in vitro immune response, were sequenced, expressed and analysed using a biosensor. Recombinant Fab fragments derived from cultures on day 12 exhibited an increase in affinity of close to two orders of magnitude compared to those obtained from cells primary immunized for 7 days. This study provides the first evidence that an antigen-specific in vitro immune response can yield high-affinity immunoglobulinG antibodies.
Blood, 2009
Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL–derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL–derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL–isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting wi...