Alan M. Stall

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.

The BCL-6 proto-oncogene controls germinal-centre formation and Th2- type inflammation

Structural alterations of the promoter region of the BCL-6 proto-oncogene represent the most frequent genetic alteration associated with non-Hodgkin lymphoma, a malignancy often deriving from germinal-centre B cells. The BCL-6 gene encodes a zinc-finger transcriptional represser normally expressed in both B cells and CD4+ T cells within germinal centres, but its precise function is unknown. We show that mice deficient in BCL-6 displayed normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell-dependent antibody responses. This defect included a complete lack of affinity maturation and was due to the inability of follicular B cells to proliferate and form germinal centres. In addition, BCL-6-deficient mice developed an inflammatory response in multiple organs characterized by infiltrations of eosinophils and IgE-bearing B lymphocytes typical of a Th2-mediated hyperimmune response. Thus, BCL-6 functions as a transcriptional switch that controls germinal centre formation and may also modulate specific T-cell-mediated responses. Altered expression of BCL-6 in lymphoma represents a deregulation of the pathway normally leading to B cell proliferation and germinal centre formation.

The LY-1B Cell Lineage

The murine Ly-I lymphocyte surface glycoprotein was defined initially with conventional antisera in cytotoxic assays (Cantor & Boyse 1977). As such, it appeared to be expressed exclusively on helper T cells (Cantor & Boyse 1975). Later, however. Fluorescence Activated Cell Sorter (FACS) analyses and sorting studies with monoclonal antibody reagents showed that all T cells express Ly-1, regardless of functional subclass (Ledbetter et al. 1980). Furthermore, these studies (Lanier et al. 1981a, 1981b) showed that Ly-1 is expressed on several murine B cell tumors and introduced evidence suggesting that this glycoprotein may also expressed on a small proportion of normal murine splenic B cells (Manohar et al. 1982, Hayakawa et al. 1983). Similar studies with human lymphocytes demonstrated the homologous {LeuI) cell surface antigen on all normal T cells (Ledbetter et al. 1981), on some B cell tumors (particularly chronic lymphocytic leukemias) (Martin et al. 1981) and, as in the mouse, on a small proportion of apparently normal B cells (CalligarisCappio et al. 1982). Thus, a series of earlier findings foreshadowed contemporary evidence demonstrating Ly-I and Leu-1, respectively, on subsets of murine and human B cells and showing further that Ly-1 marks functionally distinct B cells that play a major role in autoimmunity in the mouse. In this paper, we summarize the physical and functional characteristics that distinguish Ly-1 B cells from the majority of splenic and lymph node (conventional) B cells. We focus on data from cell transfer and antibody treatment studies, which locate Ly-I B cells in a separate developmental lineage that branches off from the conventional lymphocyte developmental lineage during prenatal or early neonatal life. We then consider various genetic defects that influence autoantibody production and Ly-I B representation and, finally, we discuss potential homolog-

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations.

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.

Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing Vβ8 TCR: a role of the Qa-1 molecule

Interactions mediated by TCRs expressed on different T cell subsets may play a role in immunoregulation. To investigate this idea, we studied the regulation of superantigen-induced TCR V beta-restricted responses. We asked whether the in vivo regulation of CD4+ V beta 8+ T cells following SEB injection is controlled by CD8+ T cells. We found that in mice deficient in CD8+ T cells, the down-regulation of CD4+ V beta 8+ T cells below baseline is not observed. Moreover, following SEB administration, CD8+ T cells emerge that preferentially kill subpopulations of activated CD4+ V beta 8+ but not CD4+ V beta 8- T cells in vitro. This TCR V beta-specific cytotoxicity is dependent on beta 2-microglobulin and is inhibited by antisera specific for Qa-1 but not by antibody to MHC class Ia. These data suggest the idea that the specificity of immune regulation may involve CD8+ T cell recognition of TCR V beta determinants and Qa-1 molecules expressed on CD4+ T cells.

Characteristics and Development of the Murine B‐lb (Ly‐1 B Sister) Cell Population

In this paper we have outlined the evidence for two distinct branches of the B-1 cell lineage. The data show that phenotypically B-1a and B-1b cells are essentially identical, distinguished only by the presence or absence of the CD5 antigen. Functionally no differences between the two populations have yet been identified. Both produce anti-PtC antibodies, a specificity not observed in conventional B cells. Both produced high levels of IgM as measured in adoptive transfer experiments. Developmentally, B-1a and B-1b cells are indistinguishable with respect to generation from progenitors present in fetal liver and omentum, feedback regulation of new B-1a and B-1b cells from bone marrow, self-replenishment from Ig+ cells following adoptive transfer, and the generation of clonal populations. The major difference in the two populations is seen in the development of B-1a and B-1b cells from B220- progenitors in the adult bone marrow. Although B220- B-1a progenitors are rare in adult (greater than 6 weeks) bone marrow, the progenitors for B-1b cells persist well into adulthood. Our understanding of B-1b cell ontogeny is at a stage similar to that of B-1a cells five years ago. We have evidence from transfer experiments that strongly suggests the existence of two distinct progenitors for B-1a and B-1b, but we have yet to physically separate these progenitors as Solvansen et al. have done for B-1 and conventional B cells. Furthermore we must determine whether the B-1b cells that develop from fetal liver and bone marrow are functionally and developmentally equivalent to those that develop from adult bone marrow. As with B-1a cells, the role of B-1b cells in the immune system is unclear. Although we have not yet discerned functional differences between B-1a and B-1b, given the recent identification of CD72 (Lyb-2) as the ligand for CD5, it is tempting to speculate that B-1a cells are more involved in B-B cell interactions such as idiotype-anti-idiotype regulation of the early B-cell repertoire and that B-1b cells are more involved in B-T cell interactions. Whatever their function, it is clear that in trying to understand the role of the B-1 lineage it is important to consider both the B-1a and B-1b lineages.

Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines.

Transgenic mice carrying either the c‐myc or N‐myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre‐B and B cell lymphomas (E mu‐c‐myc and E mu‐N‐myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v‐myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre‐B stage of development‐including the recombinase activating genes RAG‐1 and RAG‐2. Continued RAG‐1 and RAG‐2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross‐linking of the surface immunoglobulin on these lines with an anti‐mu antibody leads to rapid, specific and reversible down‐regulation of RAG‐1 and RAG‐2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG‐1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre‐B to B cell transition.

Differences in glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially enable transduction of different signals.

Studies presented here demonstrate that IgM and IgD molecules on normal murine B lymphocytes exist in different, noncovalently associated molecular complexes containing distinct but potentially related glycoproteins. The glycoproteins in these complexes, particularly those associated with IgD, show striking differences in various lymphoid organs and in X‐linked immunodeficient (Xid) mice. These differences are due in part to post‐translational processing. They apparently reflect the differential expression of the Ig‐associated glycoproteins in the various B cell subpopulations and lineages and the differential distribution of the subpopulations and lineages in the various lymphoid organs. In addition, they reflect structural differences in the IgM and IgD complexes which, we suggest, permit differential signal transduction by IgM and IgD on the same B cell.

A Major Peritoneal Reservoir of Precursors for Intestinal IgA Plasma Cells

Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyers patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.

Repetitive usage of immunoglobulin VH and D gene segments in CD5+ Ly-1 B clones of (NZB x NZW)F1 mice.

The usually small Ly‐1 B cell population is markedly increased in older mice by expansion of certain clones. This results in a cellular picture very similar to human B chronic lymphocytic leukemia. Here we report a molecular analysis of the immunoglobulin gene rearrangements of the Ly‐1 B cell populations in (NZB x NZW)F1 females. We find that (i) the number of clones found in the peritoneum (a major tissue source of Ly‐1 B cells) decreases with age till mono‐ or biclonality is common by approximately 6 months, (ii) many clones from different mice show the same size rearrangements at both the Ig heavy and light chain loci and (iii) the IgH rearrangements found in a clone isolated from the spleen of one mouse are a subset of those found in the peritoneum of the same mouse, implying migration occurs from the peritoneum to the spleen. Molecular cloning and sequencing of the IgH rearrangements from the peritoneal clones of one B/W mouse revealed that all productive rearrangements used the identical unmutated VH and D elements joined to different JHS. Indeed, two VDJH4 rearrangements were recovered which were identical but for six junctional (N region) nucleotides. The conservation of VH and D segment usage in the rearrangements of these Ly‐1 B cell clones could indicate some strong selective pressure for clonal expansion (for example antigen selection) operates via the immunoglobulin molecules of these cells. Southern analyses of other (NZB x NZW)F1 mice with this cloned VH and the usage of the same or similar VH genes among a number of Ly‐1 B origin tumors in other mouse strains indicate the generality of this repetitive VH gene usage in individual mice.