Accuri C6 Flow Cytometer System
Originally published online as doi:10.1189/jlb.0408242 on December 26, 2008

Published online before print December 26, 2008
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(Journal of Leukocyte Biology. 2009;85:574-581.)
© 2009 by Society for Leukocyte Biology

Allo-restricted CTLs generated by coculturing of PBLs and autologous monocytes loaded with allogeneic peptide/HLA/IgG1-Fc fusion protein

Xiufang Weng*,{dagger},1, Shengjun Lu*,1, Maohua Zhong*, Zhihui Liang*, Guanxin Shen*, Jianguo Chen{dagger} and Xiongwen Wu*,2

Departments of
* Immunology and
{dagger} Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

2 Correspondence: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. E-mail: xiongwenwu{at}hotmail.com

ABSTRACT

The graft-versus-leukemia effect of allogeneic marrow transplantation suggests the dramatic effect of the allogeneic T cell to eradicate malignant disease. Preparation and adoptive transfusion of tumor-specific T cells from HLA-mismatched donors might be expected to circumvent CTL tolerance to the tumor. In this study, a soluble, divalent HLA-A2 molecule was constructed with the Fc part of human IgG1 and was pulsed with a peptide related to melanoma tyrosinase 368–376 [Tyr368–376 (Tyr)] to form the Tyr/HLA-A2 dimer, which allowed loading onto monocytes via interaction of the Fc and FcR. The HLA-A2-negative (HLA-A2-ve) monocytes loaded with the Tyr/HLA-A2 dimer acted as allo-APC with copies of a single allogeneic epitope. After coculture of the HLA-A2-ve PBLs and autologous monocytes loaded with the dimer, CD8+ cells in the coculture show an obvious proliferation and increased frequency of Tyr/HLA-A2 tetramer-stained cells. The sorted Tyr/HLA-A2 tetramer-positive CD8+ cells display an elevated cytotoxic activity against HLA-A2-positive melanoma cells expressing tyrosinase endogenously (i.e., SK-Mel-5) but little against tyrosinase-negative melanoma cells (i.e., A375). The coculture of PBLs and autologous monocytes loaded with allogeneic peptide/HLA complexes offers a novel approach to expand allo-restricted, peptide-specific CTLs, which might be a potential arsenal for treatment of patients with malignant disease, if the tumor-related epitope were defined.

Key Words: HLA-A2 dimer • allo-antigen-presenting cells • allo-restriction • T cells