Biomedical Engineering Reference
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FIGURE 36.3 CD32B CD79B DART 1 mediated negative signaling. In B-lymphocytes,
recognition of an antigen by the B-cell receptor (BCR) induces a signal that can direct clonal
expansion. CD79B of the BCR contains an immunoreceptor tyrosine-based activation motif
(ITAM) in the cytoplasmic domain. Recruitment of the inhibitory receptor Fc g RII (CD32B) to
the BCR signaling complex by antigen-bound antibodies results in phosphorylation of tyrosine
residue (Y292) of the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CD32B and
activation of the inhibitory pathway. Following this paradigm, the CD32B CD79B DART 1
inhibits B-cell activation by directly cross-linking CD32B to the CD79B (Ig b ) subunit of
the BCR.
using FL-labeled alkaline phosphatase. 4420-hCB3.1
DART 1 was readily detected under these conditions, while
the negative control, 4420
binding of a fluoresceinated mAb at the site of activation
was required for DART 1 inhibition.
Interestingly,
the
2B6 U-DART, was not captured
4420
hCB3.1 U-DART also mediated B-cell inhibition
when combined with a fluoresceinated anti-CD22 mAb
demonstrating that alternative inhibitory receptors
expressed on human B cells can be utilized as long as
they are appropriately recruited to the activation complex.
CD22 has multiple ITIMs and has been shown to regulate
B-cell activation; its biological role, however, is less well
understood than that of CD32B. Similar inhibition of B-cell
proliferation was observed when the 4420
and gave no signal.
Activation of human B cells isolated from PBMC by
negative selection was initiated using anti- m mAb cross-
linked with F(ab)'2 of goat anti mouse IgG or using goat
anti- m polyclonal antibody. Activation was monitored using
incorporation of 3H-thymidine, calcium flux, and IgG secre-
tion. The results show that U-DART molecules in combina-
tion with the appropriate fluoresceinated partners were able
to effectively inhibit proliferation in a dose-dependent man-
ner (Figure 36.4C, D). Specifically, inclusion of the 4420
2B6 U-DART
was used in combination with hCB3.1-agly-FITC
(Figure 36.4D). The level of B-cell inhibition mediated
by U-DART 1 approach was also consistent with the
level mediated by the CD32BxCD79B I-DART 1 molecule
itself supporting the predictive value of the U-DART
approach (Figure 36.4D—upper panels). Recapitulation
of the U-DART 1 activity in the I-DART 1 format was
also reflected in the comparable inhibition of IgG secretion
mediated by the combination of 4420
hCB3.1 DART 1 in combination with fluoresceinated
CD32B mAb resulted in an
70% decrease in the amount
of 3H-thymidine incorporation (Figure 36.4C). The aglyco-
sylated N297Q form of the antibody was utilized in order to
avoid FcRn engagement of antibody. Neither an irrelevant
fluoresceinated mAb nor nonfluoresceinated CD32 mAb
inhibited B-cell proliferation,
indicating that
specific
2B6 U-DART and
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