Characterization of CD45 and CD45R monoclonal antibodies using transfected mouse cell lines that express individual human leukocyte common antigens

Five cell lines that express one of five isoforms of the human leukocyte common Ag (CD45) were established by transfecting a murine pre-B cell line with leukocyte common Ag cDNA constructs. Using these cell lines, the specificities of CD45 and CD45R mAb were examined. Of the 43 mAb tested, 25 antibodies recognized sequences common to all five isoforms, 16 antibodies recognized isoforms that include exon A encoded sequences, one antibody recognized isoforms that include exon B encoded sequences, and one antibody recognized only the isoform that does not include either exon A, B, or C encoded sequences.     

Biochemical nature and topographic localization of epitopes defining four distinct CD45 antigen specificities. Conventional CD45, CD45R, 180 kDa (UCHL1) and 220/205/190 kDa

The biochemical nature and relative topographic localization of Ag determinants recognized on CD45 molecular complex by mAb defining four distinct Ag specificities (conventional CD45, CD45R, 180 kDa and 220/205/190 kDa) have been investigated. These Ag specificities display a differential biochemical, cellular, and histochemical distributions and are important in the definition of CD4-positive complementary functional T cell subsets and/or distinct stages of thymic maturation. Protease treatment of either CD45-positive cells or purified CD45 molecules revealed that both conventional CD45 and 180-kDa (UCHL1 epitope) Ag specificities are defined by epitopes present on a protease-resistant domain which is internal to the protease-sensitive epitopes defining both CD45R and 220/205/190-kDa Ag specificities. In addition, it is shown that carbohydrate moieties are contributing to the epitopes recognized by both the anti-180-kDa UCHL1 and the anti-220/205/190-kDa mAb. Neuraminidase treatment, which cleaves sialic acids either from N- or O-linked oligosaccharides, abrogated the reactivity of both mAb. However, N-glycanase treatment, which selectively cleaves N-linked sugars, did not affect the recognition of these two epitopes. Thus, these results demonstrate that the Ag determinants recognized by the UCHL1 and the anti-220/205/190-kDa mAb, which are topographically unrelated, are associated with sialic acids from O-linked-type oligosaccharides, emphasizing the contribution of carbohydrates to the Ag heterogeneity of CD45 molecular complex.     

Comparative biochemical and tissue distribution study of four distinct CD45 antigen specificities

Studies on the antigenic structure of the human CD45 glycoproteins using mAb have revealed the existence of four reactivity patterns defined specifically by distinct biochemical, cellular, and histochemical distributions. In addition to the two well characterized Ag specificities, CD45 and CD45R, present on the four glycoproteins (220, 205, 190, and 180 kDa) and on the 220-kDa member, respectively, we have identified two novel specificities. These have been defined by two different mAb, UCHL1 and PD7/26/16, that recognize an epitope exclusively expressed on the 180-kDa lowest molecular sized polypeptide and an epitope shared by the three higher molecular sized polypeptides (220, 205, 190 kDa) of the complex, respectively. It has been demonstrated that they are also part of the CD45 complex by immunoprecipitation, immunoblotting, and binding assays with purified CD45 molecules. Comparative analysis by flow cytometry and immunoperoxidase techniques also showed a distinct pattern of cell distribution for each specificity. The 180-kDa specificity is present on a subset of T cells but absent on B lymphocytes, whereas the 220-kDa specificity is mainly expressed by B cells and a subpopulation of T lymphocytes. On the other hand, the cell distribution of the epitope common to the three higher members is slightly different to the conventional pan-leukocyte CD45 specificity. Thus, certain CD45+ cell types such as cutaneous Langerhans' cells, sinusoidal lymph node macrophages, and a small subset of T cells in both lymph node and tonsil did not express the 220/205/190-kDa specificity. These results further support that CD45 glycoproteins constitute a very heterogeneous molecular complex with epitopes that are selectively expressed by different cell types and by T cells at different stages of maturation.     

Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions

A chimeric human-mouse anti-T lymphocyte mAb (CHT2; SDZ 214-380) has been constructed by cloning the variable region exons of both the L and H chains from the murine hybridoma RFT2 which have CD7 specificity and reactivity with a 40-kDa Ag. The variable L chain exon was joined to the human C kappa, and the variable H chain exon was joined to the human IgG1 region exon encoding the human allotype nGlm(z), nGlm(a). The gene constructs were introduced by electroporation into SP2/0, a non-Ig-producing murine myeloma. The identical tissue reactivity of the newly made CHT2 and the original murine RFT2 mAb (CD7) was confirmed by blocking experiments as well as by immunohistology and flow cytometry. Because this new mAb may have clinical use, the CD7 Ag expression of T lineage cells has also been quantitated in double and triple immunofluorescence assays in combinations with mAb to restricted forms of leukocyte common Ag that designate unprimed (CD45R+) and primed T lymphocyte populations (UCHL1+). CHT2 shows very strong reactivity with large thymic blast cells and cortical thymocytes from which T-ALL originates. Strong staining is seen on CD45R+ unprimed "virgin" T lymphocytes, whereas the expression on UCHL1+ primed "memory" cell types is weaker unless these cells are reactivated by mitogens or Ag. Thus CHT2 may spare a substantial population of resting memory T cells which is relevant to its potential therapeutic use. In addition the chimeric antibody had a greater in vitro antibody dependent cytotoxicity and a prolonged half-life (4.2 to 5.0 days) in Rhesus monkeys.     

Phosphotyrosine-containing proteins in Dictyostelium discoideum.

Phosphotyrosine-containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and immunoprecipitation using a monoclonal anti-phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205-220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [gamma-32P]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205-220 kDa bands.     

Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors

The alternative splicing and variable expression of the exons near to the N-terminus of the leukocyte common antigen (L-CA, CD45) result in distinct extracellular isoforms expressed by cells with different functional and developmental properties. Here we report the tissue reactivity pattern and epitope specificity of a novel rat monoclonal antibody (IBL-8) against a restricted epitope of mouse CD45. We found that this mAb reacts with an epitope displayed by B cells and their precursors (both in newborn spleen and adult bone marrow). Moreover, peripheral CD8-positive T cells were also recognised at an intermediate intensity, whereas the CD4 T cell subset was weakly reactive. The epitope of this mAb was determined with M13 filamentous phages that display cysteine constrained nonapeptides on their coat proteins. The isolated bacteriophages expressing the putative epitope showed an isoform-specific inhibition of the binding of exon-specific mAbs. Deduced amino acid sequence data of these phages indicate that the epitope recognised by the IBL-8 mAb lies at the 136-144 region of the mouse CD45 molecule within its C exon, with a TAFP consensus sequence at its centre.     

Preliminary characterization of a soluble immunosuppressive molecule from DBA/2 spleen cells using monoclonal antibody immunoadsorbence

In a previous publication a monoclonal antibody (B16G) which appeared to recognize T suppressor cells and a T-suppressor factor (TsF) in the spleens of DBA/2 mice was described. B16G appears to be directed to a public specificity of DBA/2 TsF and therefore has been shown to inhibit a variety of immunological reactions. The present study involves preliminary characterization of the material with which B16G reacts. It was found that the B16G-reactive protein (putative TsF) could be absorbed and eluted specifically from a B16G immunoadsorbent column. Material eluting from the B16G column reacted with B16G in an ELISA and appeared to run as two or more bands of 40-45 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted material was biologically active (i.e., suppressive) in the standard assay (mixed leukocyte reaction of DBA/2 splenocytes with B10.BR targets), and its suppressive activity was abrogated by the addition of B16G to the mixed leukocyte reaction cultures. Sephadex G-150 chromatography of the B16G-reactive material showed that under these conditions, its native molecular mass was between 80-90 kDa, indicating that it might occur as a dimer under natural conditions.     

Characterization of the CD45 molecule on murine intestinal intraepithelial lymphocytes

The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.     

Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells

A group of mAb recognizing the 200- and/or 220-kDa determinants (CD45R) of the leukocyte common Ag such as 2H4, WR16, MD4.3, and SN130 cross-block each other showing that they recognize a closely related epitope. The antibody UCHL1 reacts with a 180-kDa determinant of the leukocyte common Ag and exhibits a reciprocal T subset distribution pattern to the CD45R group. Peripheral blood T cells were 40% positive for UCHL1 and 58 to 65% positive for the CD45R antibodies; less than 1% of cells stained for both. On activation of CD45R+,UCHL1- T cells by PHA, up to 40% of cells became positive for both CD45R and UCHL1 by day 3. By day 7, CD45R+,UCHL1- cells fell from 90 to less than 21% whereas UCHL1+,CD45R- cells rose from 2 to 93%. Conversely, PHA-stimulated UCHL1+,CD45R- cells remained UCHL1+,CD45R- during the 7 days in culture showing that phenotypic change was unidirectional from CD45R+ to UCHL1+. In primary allogeneic mixed lymphocyte reactions, activated CD45R+ T cells also showed a change to UCHL1+. When these cells were rechallenged by the original alloantigen, the UCHL1+ cells showed 7- to 20-fold greater proliferation than the CD45R+ cells on day 3 after rechallenge. The recovery of virtually all alloantigen induced secondary proliferative response in the UCHL1+,CD45R- T cell population suggests that UCHL1 identifies a primed population of T cells which may include memory cells.     

Analysis of stage-specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and Western blotting

e carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.     

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.     

The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses.

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3.Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.     

C1q receptor on murine cells

Different cells and cell lines of murine origin were tested for their capacity to bind the C subcomponent C1q by using biotinylated human C1q and streptavidin-FITC. Cytofluorometric analysis of splenocytes and thymocytes shows that the majority of C1q-reactive cells reside in the population of B cells and macrophages. There is a significant difference in the C1q-binding capacity of in vitro activated cells; although more than half of the B cell blasts bind the C subcomponent, T cell blasts are virtually negative. It is shown that pre-B lymphomas and cell lines of myeloid origin bind C1q strongly (90 to 98%), whereas in the case of mature B cell lymphomas, plasmocytomas, and the tested T cell lines, the percentage of C1q binding cells varies from 0 to 56. C1q affinity chromatography of the detergent extracts from P388D1 and WEHI-3 cells followed by SDS-PAGE of the eluted proteins under reducing conditions reveals a band at approximately 80 kDa. Analysis of splenocytes shows two additional minor C1q-binding molecules with apparent molecular masses of 50 and 45 kDa, whereas in the case of B cell blasts three bands of similar density are seen at approximately 95, 50 and 45 kDa. C1q-receptors of murine cells are shown to be antigenically related to their human counterpart, because a polyclonal antibody (266A) raised against the human C1q receptor reacts with them.     

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 microM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein.     

Extraction of membrane antigens from Brucella ovis and an assessment of their serological activity by immunoblotting

The efficacy and selectivity of chaotropic and phase-partitioning procedures for the extraction of membrane proteins from Brucella ovis were compared with a standard Sarkosyl method. Major group 1, 2 and 3 outer-membrane proteins (OMPs) of B. ovis stained by Coomassie blue in SDS-PAGE gels had, respectively, apparent molecular masses of 81/82 kDa, 39-41 kDa and 30-32 kDa. The presence of these bands in the Sarkosyl extract of total membrane vesicles (TMVs) indicate that the procedure failed to selectively solubilize only inner-membrane proteins (IMPs). SDS-PAGE analyses also revealed the presence of OMPs and other additional bands following extraction of B. ovis TMVs by butanol phase-partitioning or with extraction solutions based on the chaotropic reagents potassium thiocyanate (KSCN), sodium salicylate (SSC) and lithium acetate (LAE). OMPs are therefore not selectively extracted by any one of these procedures. Based on the number and staining intensity of extracted membrane-associated polypeptides, the efficacy of different extraction procedures could be graded in decreasing order as follows: KSCN, SSC, butanol and LAE. Both butanol and SSC were particularly effective in extracting group 3 OMPs. Sera from chronic excretor rams were used to identify zones of seroreactivity in immunoblots. Essentially, two reactivity patterns were seen: strong antibody binding against polypeptides in zones A (46-85 kDa), C (28-32 kDa) and D (18-22 kDa) in one, and additional reactivity against zones B (34-44 kDa) and E (13-18 kDa) polypeptides in the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human leukocytes contain a large pool of free forms of CD18

Monoclonal antibodies to CD18, the common chain of leukocyte integrins, recognize in various types of human lymphoid and myeloid cells under the conditions of nonreducing Western blotting three species of CD18 of mol. wt. 96, 87, and 78 kDa, respectively. Using a unique monoclonal antibody MEM-148 reacts exclusively with free CD18 molecules, but not with leukocyte integrin heterodimers. We demonstrate that only the upper one (96 kDa) is present on the cell surface within the CD11/CD18 integrin heterodimers, while the lower ones (87 and 78 kDa) are found intracellularly as free molecules unassociated with CD11 chains or other molecules. These intracellular free CD18 chains may in part represent biosynthetic precursors; alternatively, these species may represent an intracellular source of the recently observed free, proteolytically truncated CD18 chains expressed on the surface of activated myeloid cells.     

Immunological detection of G-protein alpha-subunits in Dictyostelium discoideum

Putative G-protein alpha-subunits in Dictyostelium discoideum were detected on western blots using the antiserum A-569, raised against a peptide whose sequence is found in alpha-subunits of all known GTP-binding signal transducing proteins. Two bands with a MW of 40 kDa and 52 kDa were specifically recognized by the common peptide antiserum; the staining of both bands was strongly reduced when the antiserum was preincubated with the peptide that was used for antibody production. D.discoideum mutant HC213 (fgd A) lacks staining of the 40 kDa band, while the 52 kDa band is still present. This mutant is severely defective in cAMP receptor-G-protein interaction. We concluded that the primitive eukaryote D.discoideum contains proteins which show functional and physical similarity with the alpha-subunits of vertebrate G-proteins.     

Expression of myoblast and myocyte antigens in relation to differentiation and the cell cycle

Cell cycle parameters and expression of myoblast and myocyte antigens were investigated during exponential growth and during the differentiation phase of rat L8( E63 ) myoblasts by an integrated approach involving microspectrophotometry with DNA fluorochromes, [3H]thymidine autoradiography, and immunofluorescent staining with monoclonal antibodies. In addition to the majority of cells which are recruited into myotubes, two distinct populations of mononucleate cells were resolved in cultures of rat myoblasts undergoing differentiation. These mononucleate cells consist of (1) a population of proliferating cells with a prolonged G1 transit time; (2) a population of non-proliferating cells which remain arrested in G1 for more than 72 h. The latter group was examined with respect to the expression of two marker antigens recognized by two monoclonal antibodies: antibody B58 reacts with a macromolecular component present in undifferentiated myoblasts but not in mature myotubes, and antibody XMlb reacts with a muscle-specific isoform of myosin. All four possible combinations of expression of these antigens by single cells were found: B58 +XM1b -, B58 +XM1b +, B58 - XM1b -, and B58 - XMlb +. The implication of these findings with respect to the transition from the proliferative to the differentiative phase of myogenesis is discussed.     

Antibody 3G8, specific for the human neutrophil Fc receptor, reacts with natural killer cells

Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.     

The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans

The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.