An improved method for growing and analysing human antigen-specific CD4+ T-cell clones

Diabetes Metab Res Rev. 2011 Nov;27(8):906-12. doi: 10.1002/dmrr.1271.

Abstract

Background: T-cell clones are valuable tools for investigating T-cell specificity in type 1 diabetes. Efficient methods for isolating T-cell clones have been developed, but growing enough cells to undertake a detailed analysis remains a challenge.

Methods: We optimized the conditions for isolating and growing antigen-specific human CD4+ effector T-cell clones. T-cell clones were isolated by FACS sorting antigen-responsive cells identified by carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution. The cloning efficiency was compared between T cells cloned in the presence of 21 different combinations of cytokines. Following cloning, the growth of cloned T cells in the presence of seven different combinations of cytokines was compared. Finally, we sought a quicker and more sensitive assay to measure cloned T cells' responses to antigen.

Results: IL-2+IL-4 were optimal for cloning antigen-specific CD4+ T cells. Following cloning, the most antigen-specific CD4+ T-cell clones grew in the presence of IL-15+IL-21. Antigen recognition by T cells cloned and grown under these conditions was readily detected by the increase in the expression of CD25. Induction of CD25 was a more sensitive measure of antigen recognition than 3H-thymidine incorporation assays. These findings were confirmed with two proinsulin-specific CD4+ T-cell clones isolated from an individual with type 1 diabetes.

Conclusion: The optimal cytokines for isolating, and growing, proinsulin-specific human, CD4+ T-cell clones are IL-2+IL-4 and IL-15+IL-21, respectively. Antigen recognition, by clones isolated and grown under these conditions is best detected by the induction of CD25.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD4-Positive T-Lymphocytes / drug effects
  • CD4-Positive T-Lymphocytes / immunology*
  • Cell Culture Techniques / methods*
  • Cell Separation
  • Clone Cells / metabolism
  • Flow Cytometry
  • Humans
  • Interleukin-15 / pharmacology
  • Interleukin-2 / pharmacology
  • Interleukin-2 Receptor alpha Subunit / biosynthesis
  • Interleukin-4 / pharmacology
  • Interleukins / pharmacology
  • Proinsulin / biosynthesis

Substances

  • IL2RA protein, human
  • Interleukin-15
  • Interleukin-2
  • Interleukin-2 Receptor alpha Subunit
  • Interleukins
  • Interleukin-4
  • Proinsulin
  • interleukin-21