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[Promiscuous T cell hybridoma derived from NOD mouse]
1Section of Pathology, Hokkaido University, Sapporo, Japan.
In previous studies of this laboratory, epitope (site contacts with TCR) and agretope (site contacts with MHC molecule) were determined on p43-58 peptide composed of residues 43 to 58 of pigeon cytochrome c. Position 50 was shown to be a major epitopic site. Positions 46 and 54 were agretopic sites and we could determine specific amino acids on the agretopic positions which bound to each relevant I-A or I-E molecule. Using NOD mice, amino acids on the agretopic positions bound to I-Ag7 molecules were analyzed. Arginine (R) at position 46 and alanine (A) at position 54 were shown to be an agretopic motif bound to I-Ag7 molecules. I then established hybridomas specific for a p43-58 analogue, 46R50E54A, which contains R, glutamic acid (E) and A at positions 46, 50 and 54, respectively. Among the 46R50E54A-specific T cell hybridomas, a highly promiscuous hybridoma, NOE33-1-2, which recognized 46R50E54A with a variety of I-A molecules (I-Ad,s,u,v) as well as with I-Ag7 was obtained. Interestingly, NOE33-1-2 cells exhibited almost same responding pattern to various 46R50E54A analogue peptides as bulk lymph node T cells from each I-A bearing mice immunized with a 46R50E54A analogue did. Thus, the responding pattern of NOE33-1-2 appeared to be simply reflected by the binding affinity of the 46R50E54A analogues to the relevant I-A molecule. However, subsequent analysis with I-Ad and mutant I-Ad expressing antigen presenting cells (APC) showed that the floor part of beta chain of the I-Ad molecule was profoundly involved in interaction among TCR of NOE33-1-2, 46R50E54A and the I-Ad molecule. On the other hand, this part appeared not to be important in responses of all other 46R50E54A-specific and I-Ad restricted T cell hybridomas derived from I-Ad or I-Ab mice. No difference was shown in the expression of accessory molecules which might interpret the queer interaction between TCR of NOE33-1-2, antigenic peptide and MHC molecule. When NOE33-1-2 cells were competed with T cell hybridomas derived from I-Ad mice for I-Ad plus peptide Ag, the TCR of NOE33-1-2 cells showed higher affinity to the stimulatory complexes than that of latter hybridoma cells. These findings suggest that NOE33-1-2 hybridoma cells are of peculiar characteristics which may be attributable to negative or positive selection under influence of I-Ag7 molecules.
In previous studies of this laboratory, epitope (site contacts with TCR) and agretope (site contacts with MHC molecule) were determined on p43-58 peptide composed of residues 43 to 58 of pigeon cytochrome c. Position 50 was shown to be a major epitopic site. Positions 46 and 54 were agretopic sites and we could determine specific amino acids on the agretopic positions which bound to each relevant I-A or I-E molecule. Using NOD mice, amino acids on the agretopic positions bound to I-Ag7 molecules were analyzed. Arginine (R) at position 46 and alanine (A) at position 54 were shown to be an agretopic motif bound to I-Ag7 molecules. I then established hybridomas specific for a p43-58 analogue, 46R50E54A, which contains R, glutamic acid (E) and A at positions 46, 50 and 54, respectively. Among the 46R50E54A-specific T cell hybridomas, a highly promiscuous hybridoma, NOE33-1-2, which recognized 46R50E54A with a variety of I-A molecules (I-Ad,s,u,v) as well as with I-Ag7 was obtained. Interestingly, NOE33-1-2 cells exhibited almost same responding pattern to various 46R50E54A analogue peptides as bulk lymph node T cells from each I-A bearing mice immunized with a 46R50E54A analogue did. Thus, the responding pattern of NOE33-1-2 appeared to be simply reflected by the binding affinity of the 46R50E54A analogues to the relevant I-A molecule. However, subsequent analysis with I-Ad and mutant I-Ad expressing antigen presenting cells (APC) showed that the floor part of beta chain of the I-Ad molecule was profoundly involved in interaction among TCR of NOE33-1-2, 46R50E54A and the I-Ad molecule. On the other hand, this part appeared not to be important in responses of all other 46R50E54A-specific and I-Ad restricted T cell hybridomas derived from I-Ad or I-Ab mice. No difference was shown in the expression of accessory molecules which might interpret the queer interaction between TCR of NOE33-1-2, antigenic peptide and MHC molecule. When NOE33-1-2 cells were competed with T cell hybridomas derived from I-Ad mice for I-Ad plus peptide Ag, the TCR of NOE33-1-2 cells showed higher affinity to the stimulatory complexes than that of latter hybridoma cells. These findings suggest that NOE33-1-2 hybridoma cells are of peculiar characteristics which may be attributable to negative or positive selection under influence of I-Ag7 molecules.