Immunology Graduate Program | University of California, San Francisco

We study B lymphocyte differentiation and the events when mutations interfere with this differentiation and result in cancer. The raison d’etre of B lymphocytes is to produce antibodies, but only when called for. Thus, the first part of a B lymphocyte’s life is to rearrange its immunoglobulin genes in order to display their products on the cell surface as antigen receptor. Once this is accomplished, the second part of its life is spent to police the organism for microbes and their products. When a B lymphocyte meets its cognate antigen, it becomes activated to proliferate into a clone and differentiate into plasma cells, which secrete large amounts of antibodies with specificity similar to the one displayed by the precursor cell. During the clonal expansion phase, lymphocytes may hypermutate the variable (V) regions of their antigen receptors and switch the constant (C) regions of the receptors.

In the following, the lab activities are broken down in three themes.

Gene-targeted mice with a simplified immune system
Clonal selection demands that individual B cells be monospecific. Allelic and isotypic exclusion ensure that there is but one functional heavy (H) chain and one functional light (L) chain gene. While there is general agreement that allelic and isotypic exclusion of L chain is accomplished by turning off the enzymes that assemble the genes from gene segments, various competing theories have been proposed to explain allelic exclusion at the H-chain locus. The goal of our experiments is to assess the contributions of the stochastic, genetic regulation, and cellular regulation models to the understanding of allelic exclusion at the immunoglobulin H-chain locus.

We test the various hypotheses in the monoclonal B-cell mouse generated by nuclear transfer from a B lymphocyte. In these experiments, the H and L alleles from the original B lymphocyte is shuffled and combined with germ line or nonfunctional alleles. The aim is to create mice that represent various pre-B- and B-cell genotypes, and to investigate the effect of the various preformed alleles on the germ line or rearranged allele(s) and on B-cell development as it relates to allelic exclusion.

B-lymphocytes as mutator mutants
Whether they lead to cancer, aging, or both, somatic mutations are usually detrimental to the individual. Mutations at the immunoglobulin (Ig) loci are an exception, because they generate high-affinity antibodies, which are important in memory responses to pathogens. Thus, in the segments of immunoglobulin genes that encode the V regions of antibodies, mutations are beneficial. Since such mutations arise at a rate a million times higher than the normal, spontane-ous mutation rate at other loci, the process is called hypermutation. With our experiments we define cis and trans-acting elements for hypermutation at the immunoglobulin loci.

But hypermutation is not the only change mature B lymphocytes undergo. Small, resting B lymphocytes all start out producing IgM antibodies. Upon encountering antigen, the cells become activated and make a switch from IgM to other immunoglobulin classes. This class switch serves to distribute a particular variable region to different Ig constant regions. Each constant region mediates a specialized effector function and so through switching an organism can guide its antibodies to various sites. Creating the new heavy chain requires loop out and deletion of DNA between switch regions. These DNA acrobatics require transcription of the switch regions, presumably so that necessary factors can gain access to the DNA. These requisite switching factors include the cytidine deaminase AID and components of general DNA repair, including mismatch repair, and double strand break repair. Yet many important factors remain to be discovered, especially those that may guide recombination to a particular subclass, and these are the factors we concentrate on.

Tumor suppressors in lymphocytes
The genes that are necessary for normal, controlled cell growth are called tumor suppressor genes, and inactivation of these genes can lead to tumor formation. The majority of known tumor suppressors were located by the genetic mapping of organisms with an inherited predisposition for cancer. However, familial predisposition to cancer is responsible for only approximately 10% of human cancers and identification of tumor suppressors involved in non-familial cancers has been slower. The goal in the lab is to define the set of tumor suppressors that when deleted contribute to the formation of lymphocyte tumors in mice; and their interactions with protooncogenes, as completely as possible.
We define cancer genes by retroviral insertional mutagenesis, combined with chemical mutagenesis, to inactivate both alleles in cells of a given mouse. The offspring of chemically mutagenized male mice are subjected to insertional mutagenesis by retrovirus that induces tumors of lymphocyte origin. The viral genome disrupts potential suppressor genes, leading to their inactivation, and at the same time creates a marker for identifying the insertion loci. The tagged cancer genes are cloned and sequenced. In collaboration with several other labs, we characterize a subset of the cancer genes and determine the nature of their cooperation with other oncogenes (co-mutations).