The first B cell clones produced following VDJ rearrangement express Igs with low affinity for antigens so may be less susceptible to tolerisaion but also less of a threat because they are low affinity.
antibodies produced after affinity maturation will be produced in the periphery and will be a threat if autoreactive so must be tolerised
Rearranged heavy and light chain transgenes encoding antibody specific for an antigen are inserted into a line of mice.
A separate line of mice is produced that are transgenic for the antigen that the antibody transgenes are specific for.
Cross the two lines x double transgenic mice that express both the transgenic self antigen and the transgenic antibody targeted against it.
Tolerance in these animals may be compared in mice that are only transgenic for the self antigen.
Two systems have been employed to this end: mice transgenic for HEL and its antibody and mice expressing various combinations of MHC haplotype and haplotype specific anti-MHC antibody.
Goodnow et al. produced transgenic mice carrying HEL under the control of the metallothionein or mouse albumen promoter.
For these mice HEL was self and consequently they were tolerant to it compared to non-transgenic littermates.
Mice were produced that were transgenic for an immunoglobulin that is specific for HEL.
To distinguish between transgenic Ig and endogenously rearranged Ig the heavy chain constant region Ig was derived from a different mouse strain so that cells could be fluorescently labelled for each allotype by monoclonal antibodies.
Fluorescence activated cell sorter analysis showed that 90% of peripheral B cells expressed only the transgenic Ig.
B cells that were transgenic for the Ig spontaneously differentiated into plasma cells and secreted IgM into the serum.
When the double transgenic mice were examined, however, they showed almost complete cessation of anti-HEL Ig secretion. FACS analysis of spleen and lymph node cells showed that anti-HEL B cells had not been deleted but had become anergic.
Nemazee and B rki introduced light and heavy chain genes encoding an antibody for H-2k MHC class I molecules into the germline of H-2d mice.
antibody not self reactive in the resulting animals and 50% of peripheral B cells expressed the transgene products with remainder expressing endogenous genes.
When these animals were crossed with non-transgenic H-2k/d heterozygous strain mice, anti H-2k antibody secretion was not observed in those progeny that expressed the H-2k MHC haplotype.
Unlike the case of the anti-HEL B cells mentioned above, no cells expressing the transgene could be detected, implying that in this case the autoreactive cells had been clonally deleted.
anergic cells observed in lysozyme mice expressed 90 to 98% less IgM on their surface – receptor down-regulation
may also have played a role in the clonal deletion in the H-2k transgenic mice since B cells in the bone marrow of these mice also showed reduced levels of surface IgM.
may be downregulation of IgM in the anti-MHC specific B cells prevented their homing to the peripheral lymphoid organs, accounting for their absence.
In the case of the anti-HEL B cells, they express IgD which could mediate this homing process. However despite IgD ligation being sufficient for activation, these cells are unresponsive and so a mechanism of tachyphylaxis whereby there are changes in the signal transduction across the membrane may be playing a part.- bit tenuous.