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Lymphocyte display: a novel antibody selection platform based on T cell activation.

Alonso-Camino V, Sánchez-Martín D, Compte M, Sanz L, Alvarez-Vallina L - PLoS ONE (2009)

Bottom Line: Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens.Furthermore, these platforms are not well suited for in vivo selections.Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain.

ABSTRACT
Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

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Selection of CIR-activated T cells.JurkatαCEA-CIR-EGFP effector (E) cells and CIR-positive JurkatαNIP-CIR competitor (C) cells at a E∶C mixing ratio 1∶1000 were stimulated with CEA-positive target cells for 16 hours and further sorted on the basis of EGFP and CD69 expression. After a period of cell expansion the activation/selection cycle was repeated.
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pone-0007174-g007: Selection of CIR-activated T cells.JurkatαCEA-CIR-EGFP effector (E) cells and CIR-positive JurkatαNIP-CIR competitor (C) cells at a E∶C mixing ratio 1∶1000 were stimulated with CEA-positive target cells for 16 hours and further sorted on the basis of EGFP and CD69 expression. After a period of cell expansion the activation/selection cycle was repeated.

Mentions: To determine whether T cells expressing CIRs on their surfaces could serve as a platform for screening and isolating binders to tumor specific cell surface antigen, JurkatαCEA-CIR-EGFP cells and CIR-negative non-transduced Jurkat cells (Fig. 6) or CIR-positive transfected Jurkat cells (JurkatαNIP-CIR) (Fig. 7) were mixed at decreasing concentrations of the former, with a total number of 3×107 and incubated overnight with confluent monolayers of HeLaCEA cells, and the sensitivity of the isolation and enrichment process evaluated. Mixed Jurkat T cells were recovered from the tumor cell monolayer by EDTA treatment, ficoll purified, washed twice with medium and incubated with anti-CD69 PE mAb. At a mixing ratio 1∶1000 a single round of selection by direct FACS sorting of EGFP+CD69+ cells, resulted in a two-log enrichment of anti-CEA CIR expressing JurkatαCEA-CIR-EGFP cells, from the background CIR-negative Jurkat cell population (Fig. 6) or CIR-positive JurkatαNIP-CIR cells. The sorted JurkatαCEA-CIR-EGFP/1S population was propagated and submitted for an additional round of activation/selection on HeLaCEA cell monolayers. After staining with anti-CD69 PE mAb and FACS sorting nearly 100% of the cells expressed EGFP (JurkatαCEA-CIR-EGFP/2S). Overall, CIR-mediated activation of T cells combined with FACS sorting of CD69-expressing activated T cells resulted in a 103 fold-enrichment in a tandem two-step round of selection.


Lymphocyte display: a novel antibody selection platform based on T cell activation.

Alonso-Camino V, Sánchez-Martín D, Compte M, Sanz L, Alvarez-Vallina L - PLoS ONE (2009)

Selection of CIR-activated T cells.JurkatαCEA-CIR-EGFP effector (E) cells and CIR-positive JurkatαNIP-CIR competitor (C) cells at a E∶C mixing ratio 1∶1000 were stimulated with CEA-positive target cells for 16 hours and further sorted on the basis of EGFP and CD69 expression. After a period of cell expansion the activation/selection cycle was repeated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747005&req=5

pone-0007174-g007: Selection of CIR-activated T cells.JurkatαCEA-CIR-EGFP effector (E) cells and CIR-positive JurkatαNIP-CIR competitor (C) cells at a E∶C mixing ratio 1∶1000 were stimulated with CEA-positive target cells for 16 hours and further sorted on the basis of EGFP and CD69 expression. After a period of cell expansion the activation/selection cycle was repeated.
Mentions: To determine whether T cells expressing CIRs on their surfaces could serve as a platform for screening and isolating binders to tumor specific cell surface antigen, JurkatαCEA-CIR-EGFP cells and CIR-negative non-transduced Jurkat cells (Fig. 6) or CIR-positive transfected Jurkat cells (JurkatαNIP-CIR) (Fig. 7) were mixed at decreasing concentrations of the former, with a total number of 3×107 and incubated overnight with confluent monolayers of HeLaCEA cells, and the sensitivity of the isolation and enrichment process evaluated. Mixed Jurkat T cells were recovered from the tumor cell monolayer by EDTA treatment, ficoll purified, washed twice with medium and incubated with anti-CD69 PE mAb. At a mixing ratio 1∶1000 a single round of selection by direct FACS sorting of EGFP+CD69+ cells, resulted in a two-log enrichment of anti-CEA CIR expressing JurkatαCEA-CIR-EGFP cells, from the background CIR-negative Jurkat cell population (Fig. 6) or CIR-positive JurkatαNIP-CIR cells. The sorted JurkatαCEA-CIR-EGFP/1S population was propagated and submitted for an additional round of activation/selection on HeLaCEA cell monolayers. After staining with anti-CD69 PE mAb and FACS sorting nearly 100% of the cells expressed EGFP (JurkatαCEA-CIR-EGFP/2S). Overall, CIR-mediated activation of T cells combined with FACS sorting of CD69-expressing activated T cells resulted in a 103 fold-enrichment in a tandem two-step round of selection.

Bottom Line: Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens.Furthermore, these platforms are not well suited for in vivo selections.Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, Madrid, Spain.

ABSTRACT
Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Show MeSH
Related in: MedlinePlus