Redirected cytotoxic effector function. Requirements for expression of chimeric single chain high affinity immunoglobulin e receptors.
The aim of this study was to construct a single chain chimeric FcepsilonRIalpha receptor capable of effector function in leukocytes, including cytotoxic lymphocytes. To determine the most effective single chain FcepsilonRIalpha receptor with respect to IgE binding and signaling function, a variety of chimeric gene constructs were transiently transfected into COS-7 cells. The most effective chimera consisted of four parts including: wild-type or mutated extracellular domains (Trp130 --> Ala130, W130A) of FcepsilonRIalpha, membrane proximal and transmembrane regions of FcgammaRIIa, and intracellular CD3zeta (epsilonIIaIIazeta). Scatchard analysis indicated that these FcepsilonRIalpha chimeric receptor bound ligand with an affinity of 0.9 to 2.2 x 10(9) -1. Ligand binding capacity was dramatically reduced with the deletion of 11 membrane proximal amino acids of FcepsilonRIalpha; however, function was restored by substitution with the equivalent region of FcgammaRIIa, suggesting a crucial requirement for a "spacer" segment between the transmembrane and extracellular ligand binding domain. Chimeras that bound IgE effectively also mediated phagocytosis. Chimeric receptors that contained transmembrane zeta were expressed as multimers and consequently did not bind IgE effectively; however, cotransfection of these chimeras with gamma-chain largely reconstituted IgE-mediated phagocytosis. The mouse cytotoxic T lymphocyte cell line, CTLLR8 was stably transfected with epsilonIIaIIazeta, and cloned transfectants were demonstrated to lyse target cells in an anti-FcepsilonRIalpha or IgE antibody-dependent manner. Therefore, functional single chain chimeric FcepsilonRIalpha receptors were expressed in the absence or presence of associated zeta or gamma molecules and were used to redirect killer lymphocytes to target cells.
Kershaw
M H
MH
Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, 3084, Victoria, Australia.
Darcy
P K
PK
Hulett
M D
MD
Hogarth
P M
PM
Trapani
J A
JA
Smyth
M J
MJ
D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D000882:Haplorhini; D051379:Mice; D008969:Molecular Sequence Data; D010587:Phagocytosis; D017455:Receptors, IgE / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012397:Rosette Formation; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D017931:DNA Primers; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins