In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides

Peggy A. Wentworth, Esteban Celis, Claire Crimi, Suzette Stitely, Laura Hale, Van Tsai, Horacio M. Serra, Marie France Del Guercio, Brian Livingston, David Alazard, John Fikes, Ralph T. Kubo, Howard M. Grey, Robert W. Chesnut, Francis V. Chisari, Alessandro Sette

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and β2-microglobulin (β2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4 + T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Original languageEnglish (US)
Pages (from-to)603-612
Number of pages10
JournalMolecular Immunology
Volume32
Issue number9
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

Fingerprint

Blood Cells
Antigens
Peptides
Staphylococcus aureus
Antigen-Presenting Cells
Interleukin-7
Histocompatibility Antigens Class I
Acids
In Vitro Techniques
Citric Acid
Epitopes
Buffers
Alleles
Phosphates
T-Lymphocytes

Keywords

  • APC
  • CTL
  • SAC-1 activated PBMCs
  • acid stripping
  • class I MHC
  • endogenous
  • peptide avidity

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

Cite this

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides. / Wentworth, Peggy A.; Celis, Esteban; Crimi, Claire; Stitely, Suzette; Hale, Laura; Tsai, Van; Serra, Horacio M.; Guercio, Marie France Del; Livingston, Brian; Alazard, David; Fikes, John; Kubo, Ralph T.; Grey, Howard M.; Chesnut, Robert W.; Chisari, Francis V.; Sette, Alessandro.

In: Molecular Immunology, Vol. 32, No. 9, 01.01.1995, p. 603-612.

Research output: Contribution to journalArticle

Wentworth, PA, Celis, E, Crimi, C, Stitely, S, Hale, L, Tsai, V, Serra, HM, Guercio, MFD, Livingston, B, Alazard, D, Fikes, J, Kubo, RT, Grey, HM, Chesnut, RW, Chisari, FV & Sette, A 1995, 'In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides', Molecular Immunology, vol. 32, no. 9, pp. 603-612. https://doi.org/10.1016/0161-5890(95)00037-F
Wentworth, Peggy A. ; Celis, Esteban ; Crimi, Claire ; Stitely, Suzette ; Hale, Laura ; Tsai, Van ; Serra, Horacio M. ; Guercio, Marie France Del ; Livingston, Brian ; Alazard, David ; Fikes, John ; Kubo, Ralph T. ; Grey, Howard M. ; Chesnut, Robert W. ; Chisari, Francis V. ; Sette, Alessandro. / In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides. In: Molecular Immunology. 1995 ; Vol. 32, No. 9. pp. 603-612.
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AU - Wentworth, Peggy A.

AU - Celis, Esteban

AU - Crimi, Claire

AU - Stitely, Suzette

AU - Hale, Laura

AU - Tsai, Van

AU - Serra, Horacio M.

AU - Guercio, Marie France Del

AU - Livingston, Brian

AU - Alazard, David

AU - Fikes, John

AU - Kubo, Ralph T.

AU - Grey, Howard M.

AU - Chesnut, Robert W.

AU - Chisari, Francis V.

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N2 - A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and β2-microglobulin (β2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4 + T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

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