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1 Switch Transcript and Rad51 Probes1
Departments of
*
Molecular Biophysics and Biochemistry and
Genetics, Yale University School of Medicine, New Haven, CT 06520
B cells are activated for switch recombination by signals from Th
cells, but the site at which this first occurs in vivo has yet to be
identified. By in situ hybridization of splenic sections using
riboprobes specific for the I
1 switch transcript and Rad51
mRNA, we have visualized B cells that are newly activated for
switch recombination and characterized the spatial and temporal
patterns of I1 and Rad51 mRNA expression. Within 2 days after
immunization with (4-hydroxy-3-nitrophenyl)acetyl-chicken
gamma-globulin, expression of I1 switch transcripts and Rad51
mRNA was evident and was localized to B220+ B cells
clustered within the T cell-rich periarteriolar lymphoid sheath (PALS)
and surrounding follicles. By Ab staining, we have shown previously
that cells switching from IgM to IgG expression can be visualized at 3
to 5 days postimmunization and colocalize to clusters of
Rad51+ cells. Hybridization of adjacent sections with
probes for Cµ and C1 mRNA now shows that switching from µ to
expression occurs within Rad51+I1+ regions
of the PALS and peaks between days 3 and 5. Colocalized expression of
I1 and Rad51 transcripts was observed from days 2 through 12 of the
immune response. I1 and Rad51 transcripts were down-regulated but
still detectable at 12 days postimmunization, when they were evident in
peanut agglutinin-positive germinal center B cells. Taken
together, these observations show that B cells are first activated for
switch recombination in the T cell-rich PALS.
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