Rules for the interpretation of 1B11 reactivity

Rules for the interpretation of 1B11 reactivity*

1B11 recognition of CD43 depends on core 2 O-glycan branches (catalyzed by C2GlcNAcT enzymes) and sialic acid residues.

1B11 recognition of CD45RB occurs only in absence of core 2 O-glycan branches and absence (or reduced levels) of sialic acid residues.

FACS analysis:

CD8 T cells:

1B11 binding on CD8 T cells is relatively complex.

Naïve CD8 T cells and CD8 memory cells: These cells have no C2GlcNAcT activity and 1B11 will therefore recognize CD45RB (but not CD43) with a medium signal. Endogenous neuraminidase in CD8 T cells likely contributes to formation of hyposialylated CD45RB recognized by 1B11.

Activated CD8 T cells: Depending on culture conditions, these cells have significant core 2 activity and 1B11 will recognize CD43 with a strong signal while there is no recognition of CD45RB, as 1B11-CD45RB recognition is inhibited by C2GlcNAcT activity.

Note: We have found that 1B11 reactivity in C2GlcNAcT-I null mice has a similar pattern as observed in CD43null mice, thus indicating that 1B11-CD43 reactivity is dependent on C2GlcNAcT-I activity.

CD4 T cells and myeloid cells: 1B11 binding on myeloid cells and on CD4 T cells reflects the presence of core 2 O-glycans whereas lack of 1B11 reactivity indicates absence of core 2 O-glycan branches.

Note: Neuraminidase treatment will unmask the 1B11-CD45RB epitope on CD4 T cells (but not on myeloid cells). While we have failed to find an endogenous neuraminidase that acts on CD4 T cells we cannot exclude such a mechanism.

Western blotting:

The 1B11-CD45RB epitope is sensitive to denaturation while the 1B11-CD43 epitope is not affected.

If samples are boiled and reduced and SDS-gels are run under reducing conditions, only CD43 (MW 130kDa) is recognized in 1B11 immunoblots.

For SDS-Gels run under non-reducing conditions both CD45RB (MW 200kDa) and CD43 (MW 130kDa) can be detected with 1B11.

*Carlow, D. A., B. Ardman and H. J. Ziltener, 1999 “A novel CD8 T cell-restricted CD45RB epitope shared by CD43 is differentially affected by glycosylation.” J Immunol 163: 1441-8.