Raising allo-restricted cytotoxic T lymphocytes by co-culture of murine splenocytes with autologous macrophage bearing the peptide/allo-major histococompatibility complex.
Generation and adoptive transfusion of alloreactive cytotoxic T lymphocytes (CTLs) specific for tumor are expected to circumvent tumor tolerance. Here we describe a novel protocol to raise allo-restricted, peptide-specific CTLs by co-culture of murine splenocytes and autologous macrophage bearing an allogeneic H-2K molecule associated with its restricted peptide (peptide/allo-MHC). The extracellular domains of H-2K(d) were fused with constant domains of murine IgG2a heavy chain to generate a fusion protein (peptide/H-2K(d)/IgG2aFc, the dimer) consisting of divalent TCR-ligands and an IgG Fc receptor type I (FcgammaRI)-reactive moiety. The dimer is able to bind to macrophage (Mvarphi) of H-2K(k) via the interaction of the Fc part with FcgammaRI, and cause the H-2K(k) Mvarphi to be coated with the peptide/H-2K(d) complex. The results show that proliferation of CD8+ cells is enhanced and that the specific-tetramer stained CD8+ cells appear more frequently by co-culture of H-2K(k) splenocytes with the autologous Mvarphi loaded with the dimer. Furthermore, the CD8+ T cells from the co-cultural bulk exhibit an elevated cytotoxicity against a specific target (H-2K(d)-restricted, peptide-specific cytotoxicity), compared with that against the irrelevant targets. This study provides a strategy for preparation of allo-restricted, peptide-specific CTLs, which may add to our arsenal for adoptive immunotherapy to eliminate chronic virally infected or tumor cells.
Chen
Xueling
X
Department of Immunology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China.
Yan
Yongxiang
Y
Lu
Shengjun
S
Weng
Xiufang
X
Liang
Zhihui
Z
Li
Jianan
J
Zhong
Maohua
M
Tang
Jie
J
Xiao
Wei
W
Sun
Wei
W
Shen
Guanxin
G
Wu
Xiongwen
X
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D005260:Female; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology*; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000166:cytology* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D013154:Spleen / Q000166:cytology*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000276:immunology*
D006183:H-2 Antigens; D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments