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Clinical and Vaccine Immunology, June 2009, p. 798-805, Vol. 16, No. 6
1071-412X/09/$08.00+0     doi:10.1128/CVI.00022-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Interleukin-12 Is the Optimum Cytokine To Expand Human Th17 Cells In Vitro{triangledown}

Soad Nady,{dagger} James Ignatz-Hoover, and Mohamed T. Shata*

The University of Cincinnati College of Medicine, Division of Digestive Diseases, Viral Immunology Laboratory, Cincinnati, Ohio

Received 16 January 2009/ Returned for modification 29 January 2009/ Accepted 13 April 2009

Recently, a new lineage of CD4+ T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4+ T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R+ cells from the CD4+ T cells. Functional studies revealed that CD4+ IL-23R+ cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-{gamma}). Furthermore, we expanded the CD4+ IL-23R+ cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4+ IL-23R+ cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN-{gamma} secreted by IL-23R+ CD4+ cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R+ CD4+ cells and the secretion of IFN-{gamma}, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.

* Corresponding author. Mailing address: Viral Immunology Laboratory, MSB 6360, Department of Internal Medicine, Division of Digestive Diseases, 231 Albert B. Sabin Way, University of Cincinnati Medical Center, P.O. Box 670595, Cincinnati, OH 45267-0595. Phone: (513) 558-6110. Fax: (513) 558-1744. E-mail: mohamed.shata{at}uc.edu

{triangledown} Published ahead of print on 22 April 2009.

{dagger} Present address: Zoology Department, Faculty of Science, Helwan University, Helwan, Egypt.

Clinical and Vaccine Immunology, June 2009, p. 798-805, Vol. 16, No. 6
1071-412X/09/$08.00+0     doi:10.1128/CVI.00022-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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