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Summary: Max Cooper's laboratory pursues ontogenetic and phylogenetic studies of the adaptive immune system in parallel with the analysis of immunological diseases in humans.

Originally defined in comparative analysis of immune system development in birds and mammals, T and B lymphocytes serve as the primary antigen recognition and effector cells of specific adaptive immunity. T cells provide specific immunity against viruses, fungi, and other intracellular pathogens, and they help B cells produce antibodies against extracellular pathogens and toxins. The T cells are generated from precursor cells within the thymus of all jawed vertebrates. Immunoglobulin (Ig)-bearing B cells are generated from precursor cells within the bursa of Fabricius in birds and in the blood-forming tissues of other jawed vertebrates, primarily the fetal liver and adult bone marrow in mammals. In these central lymphoid tissues, the progeny of hematopoietic stem cells are influenced by neighboring stromal cells to initiate elaborate gene programs and undergo proliferation to generate millions of T and B cell clones, each of which expresses a T cell receptor (TCR) or a B cell receptor (BCR) of different antigen specificity. Before migrating from the thymus or bone marrow through the bloodstream to the periphery, newly formed T and B cells are positively or negatively selected on the basis of receptor-binding affinity for self-antigens. In peripheral lymphoid tissues, such as the spleen, intestine, and lymph nodes, they collaborate with antigen-presenting cells and each other to undergo antigen-driven clonal proliferation and differentiation to provide protective immediate immunity and subsequent memory of the antigen encounter. Inherited or acquired gene defects can alter T and B cell development to cause life-threatening immunodeficiencies, autoimmunity, or lymphoid malignancies.

B Cell Development
In parallel with development of the diverse T cell repertoire, the primary B cell repertoire is generated by combinatorial Ig V(D)J gene segment rearrangements. These preferentially occur in the sequence of D—>J_H, V—>DJ_H, and V—>J_L during the pro–B and pre–B cell stages. Because cellular proliferation occurs during these rearrangement events, we hypothesized that intraclonal diversification may occur during B cell development through the use of different V gene segments by sister cells. The identification of an unusual acute lymphoblastic leukemia cell line, EU12, provided an opportunity to test this idea. This cell line spontaneously undergoes differentiation from pro–B cell to the pre–B cell and B cell stages, along with changes in its gene expression profile that recapitulate the normal B cell differentiation pattern. After confirming the monoclonality of EU12 cells, we analyzed their Ig gene usage. This analysis indicated that pro–B cell members of the clone give rise to B cell progeny with different V_H and V_L gene segment rearrangements. These findings, in a dynamic model of human B cell differentiation, validate the principle of intraclonal V(D)J diversification during B cell generation.

	FC receptors and Fc receptor homologs...

Surprisingly, the mechanism employed for repertoire diversification in the EU12 cell line involved serial V_H gene replacement that is accomplished through the use of cryptic recombination signal sequences (cRSS) located within the 3' end of almost all V_H genes. Recombination-activating gene products RAG-1 and RAG-2 bind and cleave the cRSS to allow an upstream V_H gene to be brought in as a replacement for the originally rearranged V_H gene segment. Evidence for V_H replacement during normal B cell development was obtained through the detection of double-stranded DNA breaks at the V_H cRSS sites in immature B cells and identification of the V_H replacement products in normal IgH sequences. These V_H replacement “footprints, in the form of residual 3' V_H nucleotide sequences, contribute additional length and charged amino acids to the complementarity-determining region 3 (CDR3) of human Ig heavy-chain sequences. Since both of these CDR3 features are seen in autoreactive antibodies, current studies address the V_H replacement regulatory mechanism and its contribution to the antibody repertoire in healthy individuals and in individuals with autoimmune diseases.

Fc Receptor Homologs
Using a consensus sequence derived from the Ig Fc-binding sites of classical Fc receptors, we identified a family of genes that encode Fc receptor homologs (FcRHs). The FcRH genes are located near the classical FcR genes on human chromosome 1q21. They encode 3–9 Ig-like extracellular domains, a transmembrane region, and cytoplasmic domains containing consensus immunoreceptor tyrosine-based activating and/or inhibitory motifs. The first five FcRHs are expressed at different times during B cell differentiation. One of them, FcRH1, is expressed at highest levels on naïve B cells, where it may serve as an activating coreceptor. Conversely, FcRH4 has potent inhibitory potential and is preferentially expressed on a subpopulation of memory B cells, where it may inhibit BCR-mediated proliferation to favor plasma cell differentiation and antibody production. Two other Fc receptor relatives, FcRX and FcRY, are found within mature B-lineage cells, where their function is presently unknown. The intrigue of the FcRH gene family is heightened by their highly conserved nature and the immunomodulatory potential of their protein products. Ongoing studies seek to define FcRH ligands, signaling pathways, and function in antibody responses.

Phylogenetic Origin of Specific Adaptive Immunity
Since the recognition of separate T and B cell lineages, a long-standing question has been which came first. Although all multicellular organisms possess innate immune defense mechanisms, only the jawed vertebrates have been found to have T, B, and antigen-presenting cells. The requisite genes for BCR, TCR, and the major histocompatability complex (MHC) have not been identified in jawless vertebrates or invertebrates. In a search for insight into the origin of adaptive immunity in jawless vertebrates, we analyzed transcripts expressed by lymphocyte-like cells in the lamprey, one of two surviving jawless vertebrates. This analysis identified genes of the innate immune response and others that may control lamprey lymphocyte development, intracellular signaling, proliferation, and migration. Lamprey relatives of mammalian genes involved in antigen processing and transport of antigenic peptides were also found, but MHC-like genes were not. A single lamprey TCR-like gene was identified, but its V-region exon encodes both V-like and J-like sequences. This TCR-like gene therefore lacks the capacity to generate receptor variability through V(D)J recombinatorial diversification. Our analysis suggested that another type of immune recognition must underlie the lamprey's putative immune responsiveness.

Reasoning that immunocompetent cells in the circulation would be more likely to express genes involved in adaptive responses, we surveyed the transcriptome of activated lymphocytes from immunostimulated lamprey. This search identified a new type of variable lymphocyte receptors (VLRs) composed of highly diverse leucine-rich repeats (LRRs) sandwiched between amino- and carboxyl-terminal LRRs. An invariant stalk region tethers the VLRs to the cell surface via a glycosylphosphatidylinositol anchor. To generate rearranged VLR genes of the diversity needed for an anticipatory immune system, the single lamprey VLR locus contains a large bank of diverse LRR cassettes, available for insertion into an incomplete germline VLR gene. Individual lamprey lymphocytes express a uniquely rearranged VLR gene in monoallelic fashion. Hagfish, the other contemporary jawless vertebrate, are found in recent studies to have two VLR genes. As in the lamprey lymphocytes, the incomplete germline VLR-A and VLR-B genes are modified in hagfish lymphocyte-like cells to generate highly diverse repertoires of VLR-A and VLR-B proteins. Different evolutionary strategies were thus used to generate highly diverse lymphocyte receptors, through rearrangement of LRR modules in agnathans (jawless fish) and of Ig gene segments in gnathostomes (jawed vertebrates). We seek now to understand the recombinatorial mechanism for VLR diversification and how the VLR-bearing lymphocytes mediate adaptive immunity.

Last updated July 15, 2005