Enumeration of human alloreactive helper T lymphocyte precursor frequencies by limiting dilution analysis of interleukin-2 production

Neil Thomas Young, D L Roelen, M J Dallman, K J Wood, P J Morris, K I Welsh

Research output: Contribution to journalArticle

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Abstract

We describe a limiting dilution assay for the enumeration of alloreactive interleukin-2 (IL-2) producing helper T lymphocyte precursors (HTLp). In place of the commonly used CTLL cell line, we have employed concanvalin A (ConA) stimulated rat thymocytes as IL-2 responsive indicator cells in a proliferation assay to detect IL-2 levels in limiting dilution microculture supernatants. The proliferation of ConA stimulated thymocytes induced by either recombinant IL-2 or culture supernatants could be blocked by co-incubation with a monoclonal antibody against the rat IL-2 receptor alpha chain, demonstrating the specificity of the response. Our investigations of alloantigen-induced IL-2 production show that (i) a minimum stimulator cell irradiation dose of 50-60 Gy is required to prevent backstimulation of microcultures; (ii) frequencies of alloreactive HTLp are significantly associated with HLA-DR antigen matching between responder and stimulator; (iii) HTLp frequencies detected in assays using B lymphoblastoid cell line stimulators are significantly higher than in assays employing peripheral blood lymphocyte stimulators but possibly reflect a degree of non-specific activation; and (iv) allosensitized responders exhibit altered kinetics of IL-2 production which may permit discrimination between sensitized and naive individuals. Our results both confirm and extend previous reports concerning such features of the alloresponse in humans and demonstrate that ConA stimulated thymocytes are a suitable alternative to CTLL as IL-2 responsive indicator cells in limiting dilution assays for HTLp analysis.
Original languageEnglish
Pages (from-to)33-41
Number of pages9
JournalJournal of Immunological Methods
Volume195
Issue number1-2
DOIs
Publication statusPublished - 9 Sep 1996

Fingerprint

Helper-Inducer T-Lymphocytes
Interleukin-2
Thymocytes
Interleukin-2 Receptor alpha Subunit
Cell Line
Isoantigens
HLA-DR Antigens
Monoclonal Antibodies
Lymphocytes

Keywords

  • Animals
  • Cell Count
  • Cell Differentiation
  • Cell Division
  • HLA-DR Antigens
  • Hematopoietic Stem Cells
  • Humans
  • Interleukin-2
  • Rats
  • T-Lymphocytes, Helper-Inducer

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Enumeration of human alloreactive helper T lymphocyte precursor frequencies by limiting dilution analysis of interleukin-2 production. / Young, Neil Thomas; Roelen, D L; Dallman, M J; Wood, K J; Morris, P J; Welsh, K I.

In: Journal of Immunological Methods, Vol. 195, No. 1-2, 09.09.1996, p. 33-41.

Research output: Contribution to journalArticle

Young, Neil Thomas ; Roelen, D L ; Dallman, M J ; Wood, K J ; Morris, P J ; Welsh, K I. / Enumeration of human alloreactive helper T lymphocyte precursor frequencies by limiting dilution analysis of interleukin-2 production. In: Journal of Immunological Methods. 1996 ; Vol. 195, No. 1-2. pp. 33-41.
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AB - We describe a limiting dilution assay for the enumeration of alloreactive interleukin-2 (IL-2) producing helper T lymphocyte precursors (HTLp). In place of the commonly used CTLL cell line, we have employed concanvalin A (ConA) stimulated rat thymocytes as IL-2 responsive indicator cells in a proliferation assay to detect IL-2 levels in limiting dilution microculture supernatants. The proliferation of ConA stimulated thymocytes induced by either recombinant IL-2 or culture supernatants could be blocked by co-incubation with a monoclonal antibody against the rat IL-2 receptor alpha chain, demonstrating the specificity of the response. Our investigations of alloantigen-induced IL-2 production show that (i) a minimum stimulator cell irradiation dose of 50-60 Gy is required to prevent backstimulation of microcultures; (ii) frequencies of alloreactive HTLp are significantly associated with HLA-DR antigen matching between responder and stimulator; (iii) HTLp frequencies detected in assays using B lymphoblastoid cell line stimulators are significantly higher than in assays employing peripheral blood lymphocyte stimulators but possibly reflect a degree of non-specific activation; and (iv) allosensitized responders exhibit altered kinetics of IL-2 production which may permit discrimination between sensitized and naive individuals. Our results both confirm and extend previous reports concerning such features of the alloresponse in humans and demonstrate that ConA stimulated thymocytes are a suitable alternative to CTLL as IL-2 responsive indicator cells in limiting dilution assays for HTLp analysis.

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