Chemokine-mediated B cell trafficking during early rabbit GALT development.

Microbial and host cell interactions stimulate rabbit B cells to diversify the primary Ab repertoire in GALT. B cells at the base of appendix follicles begin proliferating and diversifying their V-(D)-J genes around 1 wk of age, ∼5 d after B cells first begin entering appendix follicles. To gain insight into the microbial and host cell interactions that stimulate B cells to diversify the primary Ab repertoire, we analyzed B cell trafficking within follicles during the first week of life. We visualized B cells, as well as chemokines that mediate B cell homing in lymphoid tissues, by in situ hybridization, and we examined B cell chemokine receptor expression by flow cytometry. We found that B cells were activated and began downregulating their BCRs well before a detectable B cell proliferative region appeared at the follicle base. The proliferative region was similar to germinal center dark zones, in that it exhibited elevated CXCL12 mRNA expression, and B cells that upregulated CXCR4 mRNA in response to signals acquired from selected intestinal commensals localized in this region. Our results suggest that after entering appendix follicles, B cells home sequentially to the follicle-associated epithelium, the follicular dendritic cell network, the B cell/T cell boundary, and, ultimately, the base of the follicle, where they enter a proliferative program and diversify the primary Ab repertoire.

Generation of recombinant guinea pig antibody fragments to the human GABAC receptor.

To generate monoclonal antibodies to the human ρ1 GABA(C) receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (V(H)) and light chain (V(L)) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the V(H) and V(L) regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned V(H) and V(L) regions were transferred together into a phagemid vector, yielding a library of 5×10(6) members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABA(C), we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABA(C) receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABA(C) receptor.

High-throughput biotinylation of proteins.

One of the more useful tags for a protein in biochemical experiments is biotin, because of its femtomolar dissociation constant with streptavidin or avidin. Robust methodologies have been developed for other the in vivo addition of a single biotin to recombinant protein or the in vitro enzymatic or chemical addition of biotin to a protein. Such modified proteins can be used in a variety of experiments, such as affinity selection of phage-displayed peptides or antibodies, pull-down of interacting proteins from cell lysates, or displaying proteins on arrays. We present three complementary approaches for biotinylating proteins in vivo in Escherichia coli or in vitro using chemical or enzymatical reactions all of which can be scaled up to tag large numbers of proteins in parallel.

Scarcity of lambda 1 B cells in mice with a single point mutation in C lambda 1 is due to a low BCR signal caused by misfolded lambda 1 light chain.

The presence of valine-154 instead of glycine in the constant region of lambda1 causes a severe lambda1 B cell defect in SJL and lambda1-valine knock-in mice with a compensatory increase in lambda2,3 B cells. The defect is due to low signaling by the lambda1-valine BCR. lambda1-Valine B cells deficient in the SHP-1 phosphatase survive better than lambda2,3 B cells in these mice, or lambda1 B cells in lambda1 wildtype mice. Low signaling is apparently due to misfolding of the lambda1-valine light chain as demonstrated by the absence of a regular beta-sheet structure determined by circular dichroism, the sedimentation of the light chain in solution, and the association of valine-valine constant regions in a yeast two-hybrid assay. lambda1-Valine B cells that survive apparently have a higher BCR signal, presumably because of their specific lambda1-heavy chain combination or having encountered a high-affiniy antigen. lambda1-Valine mice have increased B1 cells which were shown by others to have a higher signaling potential. Valine mice crossed with non-conventional gamma2b transgenic mice, in which B cell development is accelerated and in which B1 cells and high signaling cells are greatly reduced, have essentially no, lambda2,3 B cells, but increased numbers of lambda1-valine B cells. This supports the conclusion that the major defect in lambda1-valine mice is the inability of valine-preB cells to produce a threshold signal for B cell development. The reduction of lambda2,3 B cells in valine mice with a gamma2b transgene shows that the majority of their compensatory increase is almost entirely of the B1 cell type.