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Porcine organs are rapidly rejected after transplantation into primate recipients due to the presence of preexisting immunoglobulins that bind to terminal galactose alpha1,3 galactose residues (alpha-galactosyl) present on porcine glycoproteins and glycolipids. Currently available immunosuppressive reagents have been largely ineffective at controlling the synthesis of these anti-Gal antibodies. Nonantigenic hapten polymers have been shown to be effective materials for blocking humoral immune responses in various model systems. We have developed a series of alpha-galactosyl glycoconjugate polymers and tested their ability to block anti-Gal antibody binding in vitro and in vivo. A galactose alpha1,3 galactose beta 1,4 GlcNAc trisaccharide free acid (TRFA) with a hexanoic acid spacer, containing five methylene groups and a carboxylic acid, was produced and coupled to a variety of polymeric backbones including dextran, branched poly(ethylene glycol) (PEG), and poly-L-lysine. The ability of monomeric TRFA and the alpha-galactosyl conjugates to block anti-Gal IgG and IgM binding was determined using a competition ELISA assay on defined HSA-Gal glycoconjugates and porcine microvascular endothelial cell substrates. We show that branched PEG carriers, with a TRFA sugar attached to each branch, exhibit enhanced antibody blocking ability compared to TRFA, but at higher target antigen densities these simple PEG conjugates are no more effective then an equivalent amount of TRFA in blocking anti-Gal IgM antibody interactions. In contrast, polymers of the branched PEG conjugates and linear conjugates made using dextran and poly-L-lysine were 2000 to 70000-fold more effective inhibitors of anti-Gal antibodies. In a study using nonhuman primates, a single dose infusion of polymeric PEG or dextran glycoconjugates dramatically reduced the level of circulating anti-Gal antibodies in cynomologus monkeys for at least 72 h. Glycoconjugates similar to these might be useful both to block anti-Gal interactions in vivo and to specifically control the induced anti-Gal immune response.

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.