Solid-phase selection of human T lymphocyte subpopulations using monoclonal antibodies

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, a-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a >1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescenceactivated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation ofendothelial cells but also as a tool for the identification and pharmacological study of ACE.Angiotensin-converting enzyme (ACE), or kininase II, which cleaves the terminal dipeptide from angiotensin I to form vasoactive angiotensin II and which is active as a dipeptidyl hydrolase in its action on bradykinin and other small peptides, serves as a useful marker for the identification ofendothelial cells from capillaries, veins, and arteries (1-4). Because the enzyme is associated with the cell surface (5), antibodies directed against ACE can serve not only to identify endothelial cells but also can mark them for analysis and isolation without loss of viability.Our interest in the growth and development of murine endothelial cells prompted us to generate monoclonal reagents directed at strongly cross-species reactive ACE. Tests carried out with conventionally generated rabbit anti-rat ACE (6) pointed to rat lung ACE as a suitable immunogen; therefore, we used a rat lung ACE preparation to induce an immune response in mice. Spleens from these mice then were fused by hybridization to nonsecreting mouse myeloma cells to permit isolation and characterization of hybrids (hybridomas) that produced monoclonal antibodies to ACE.We now report on the properties of one such hybridomaits cell-specific and enzyme-directed binding properties and its use as a reagent for identifying and isolating murine, bovine, and human endothelial cells.MATERIALS AND METHODS ACE Preparation. ACE was obtained by following the methods of Lanzillo and Fanberg (6, 7). In brief, rat lungs were dissected after lavage, lightly homogenized in 0.02 M potassium phosphate buffer (pH 8.3), and centrifuged (250 x g; 10 min) to remove cells and debris. The supernatant was recentrifuged (54,000 X g; 60 min) and the pellets then were rehomogenized to yield the "crude enzyme preparation." For further purification, sodium deoxycholate was added; this was followed by centrifugation and dialysis, filtration through Whatman no. 1 paper, and fractionation on DEAE-cellulose columns ...

show abstract

“…Selective adhesion to antibody-coated dishes (panning; ref. 19) or alternative solid-phase selection methods (20) (23).…”

mentioning

confidence: 99%

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

Auerbach¹,

Alby²,

Grieves³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This method is similar to that of other investigators except that they used E-resetting (T) cells as starting material instead of PBL or they used EDTA to remove adherent cells (7,8,19).…”

Section: Discussionmentioning

confidence: 99%

Panning Separation for Monoclonal Antibody‐Specific T‐Cell Subsets: Effects of Enzymes and Metabolic Inhibitors

Goto

Bluestein

Zvaifler

1984

Microbiology and Immunology

View full text Add to dashboard Cite

The mechanisms of “panning,” a simple positive cell separation technique, were examined. Using monoclonal antibodies to “pan” for T cells (T101+, T4+, and T8+), we obtained enriched populations with 90% purity and viability from unfractionated human peripheral blood lymphocytes (PBL). We found that the “panning” method reflects an active process. It is sodium azide inhibitable and independent of the divalent cation concentration. Effective panning does not require capping, patch formation, or DNA synthesis. The cell yields are unaffected by mitomycin‐C or microtubule and microfilament blocking reagents such as concanavalin A, colchicine, and cytochalasin B. This economical technique provides large numbers of functionally intact monoclonal antibody‐specific cells within a relatively short time for further functional and biochemical characterization.

show abstract

“…The second method for positive selection of T cells was a modification of the technique recently described by Fong et al (34). In these experiments, cells were treated with FITCconjugated anti-Thy-l.2 antibody for 30 min on ice, washed, and added to plates coated with affinity-purified goat anti-FITC antibody, a gift from Dr. James Monthony, Bio-Rad Laboratories, Richmond, CA.…”

Section: Preparation Of Immune Complexes Tepc-15 or Mcpc-603 Myelomamentioning

confidence: 99%

Induction of idiotype-specific suppressor T cells with antigen/antibody complexes.

Caulfield¹,

Luce²,

Proffitt³

et al. 1983

The Journal of Experimental Medicine

View full text Add to dashboard Cite

The effects of immune complexes on the antibody response of BALB/c mice to Streptococcus pneumoniae R36a (Pn) were investigated. The cell wall polysaccharide (PnC) extracted from Pn was used to form complexes with TEPC-15, a myeloma protein that binds to phosphorylcholine determinants on the PnC. Complexes formed at equivalence were cultured with splenic T cells from BALB/c mice for 2 d, and then the cells were added to fresh BALB/c spleen cell cultures to test their effect on the antibody response to Pn, a response dominated by the T15 idiotype family. The results indicate that TEPC-15/PnC complexes induced potent suppressor T cells (Ts) whereas cells cultured with free antigen or free antibody alone had no effect on the plaque-forming cell response to Pn. The suppression was specific since the response to control antigens such as sheep erythrocytes was unaffected. The suppression appears to be idiotype-specific since the Ts had a relatively weak (and in some cases no) effect on the anti-Pn response of BALB/c mice that had been suppressed for T15 idiotopes by neonatal injection of a monoclonal anti-T15 antibody, MaId 5-4. Furthermore, cells cultured with TEPC-15/PnC complexes were shown to express specific receptors for TEPC-15 idiotopes. The results indicate that antigen/antibody complexes may have important immunoregulatory effects because they are potent inducers of idiotype-specific Ts.

show abstract

Solid-phase selection of human T lymphocyte subpopulations using monoclonal antibodies

Cited by 17 publications

References 15 publications

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

Panning Separation for Monoclonal Antibody‐Specific T‐Cell Subsets: Effects of Enzymes and Metabolic Inhibitors

Induction of idiotype-specific suppressor T cells with antigen/antibody complexes.

Contact Info

Product

Resources

About