title: An improved method for growing and analysing human antigen-specific CD4+ T-cell clones.
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.
  
  
  CONCLUSIONS
  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.
authors: Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.
authors: Joseph P
authors: JP
authors: Ciantar
authors: Stuart I
authors: SI
authors: Mannering
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology*; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D002999:Clone Cells / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D019409:Interleukin-15 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D053645:Interleukin-2 Receptor alpha Subunit / Q000096:biosynthesis; D015847:Interleukin-4 / Q000494:pharmacology; D007378:Interleukins / Q000494:pharmacology; D011384:Proinsulin / Q000096:biosynthesis; D000098546:Interleukin-21
chemical_list: C508017:IL2RA protein, human; D019409:Interleukin-15; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D007378:Interleukins; D015847:Interleukin-4; D011384:Proinsulin; D000098546:Interleukin-21