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To examine the in vivo function of IgD we generated mice deficient for IgD by gene targeting. The IgDmice show a reduced B-cell compartment with 30-50% less B cells in the spleen and lymph nodes but show a normal pre-B-cell compartment. The surface-IgD-B cells express two to three times more surface IgM than B cells ofcontrol animals. Serum concentrations of the immunoglobuln isotpes of IgDmice are almost normal, indicating that surface-IgD expression is not necessary for class switching of B cells. Immunization experiments showed that IgD-mice could respond well to thymus-dependent and -independent antigens. After immunization normal germinal centers developed in the IgD-mice. cells (7, 8). In vivo the functions attributed to the engagement of sIgD vary from the acquisition of resistance to tolerance induction (10-12) and the initiation ofthe B-cell response (13) to a role in B-cell memory (6,14,15). Some ofthese functions were studied by using chronic anti-IgD treatment from birth (16,17), by in vitro sorting of sIgD+ and sIgD-B cells (10,18,19), by enzymatic removal of sIgD in vitro (11), or by transgenic mouse models (20). Discrepancies of results were found in these studies that could be generated by the nature of the indirect experimental systems used. Roes and Rajewsky (21) PCR and Southern Blots. ES cell clones were picked and prepared for a nested primer PCR analysis, as described (35). Genomic DNA for Southern blotting was prepared from ES cells or from the tail ends of mice.Flow Cytometric Analysis (FCM). FCM of single-cell suspensions of lymphatic organs was done on a FACScan flow cytometer (Becton Dickinson). The antibodies used for staining were either (i) biotinylated and counterstained with streptavidin-phycoerythrin 11-26C (anti-IgD, from J. Kearney, University of Alabama, Birmingham) ( Fig. 2 a and b), M41 (anti-IgM) (27) (Fig. 2 c and d), B7/6 (anti-IgM) (27) (Fig. 2 g and h) or (ii) conjugated with fluorescein isothiocyanate M41 (anti-IgM) (Fig. 2 a and

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Pre-B cell lines proliferating for several months on stromal cells in the presence of interleukin 7 (IL-7) were established from fetal liver of (NZB x NZW)F1 mice. They express the B lineage-specific markers PB76, B220, and VpreB, but do not express surface immunoglobulin (sIg). Upon removal of IL-7 from the culture, they differentiate to sIg+ B cells that can then be stimulated by lipopolysaccharide to become IgM-secreting cells. Transfer of these pre-B cell lines into SCID mice leads to hypergammaglobulinemia of IgM (600-900 micrograms/ml), IgG2a (1-3 mg/ml), and IgG3 (300-500 micrograms/ml) for the next 3-5 mo. The spleen appears populated with (NZB x NZW)F1-derived pre-B cells, few B cells, and many IgM and/or IgG-producing plasma cells. In contrast, SCID mice populated with pre-B cell lines of normal (C57BL/6 x DBA/2)F1 mouse fetal liver develop normal levels of serum IgM (approximately 100-300 micrograms/ml), almost no detectable levels of IgG, and no plasma cell hyperplasia. The (NZB x NZW)F1 pre-B cell-populated SCID mice contain elevated serum titers of IgG antinuclear autoantibodies, but no retroviral gp70-specific nor erythrocyte-specific autoantibodies. Up to 20% of the SCID mice develop proteinuria as a consequence of IgG deposits in the kidney glomeruli during a 7-mo period of observation. All signs of autoimmune disease seen in these mice are independent of the sex of the SCID host. This experimental system provides a distinction between the disease-determining (NZB x NZW)F1 genes, which are expressed in the B lymphocyte lineage and cause the development of the disease, from those expressed in other cell lineages which only modulate its progression.

Recent findings imply that germinal center paucity in old mice, at least in part, results from a defect in the mechanisms responsible for the transport of antigens to lymphoid nodules (follicles) and the consequent impairment of the antigen retaining reticulum (ARR) of follicular dendritic cells (FDCs). The present objective was to observe the kinetics of lymph node germinal center development in old mice having antigen transport and ARR deficits. Germinal center development was monitored in popliteal (PLN) and axillary (AXLN) lymph nodes of 6-8 wk and 23-mo-old horseradish peroxidase (HRP) immune C57BL/6 mice. Using the selective binding of germinal center B cells for peanut agglutinin (PNA), germinal centers were identified in serial vibratome sections following histochemical labeling with PNA-peroxidase conjugates at times 0, 15 min, 1, 3, 5, and 10 days after footpad challenge with 8 micrograms HRP. To follow the fate of preexisting (environmental antigen-induced) germinal centers and the development of de novo (HRP-induced) germinal centers, it was essential to distinguish between these germinal centers. Accordingly, PNA positive germinal centers associated with HRP-retaining (peroxidase positive) ARR were identified as de novo germinal centers and germinal centers not associated with a peroxidase positive ARR were classified as preexisting germinal centers. Kinetic analysis of PNA positive germinal centers showed the following: 1) Preexisting, environmentally-induced germinal centers dissociated and disappeared by day 3 as indicated by a decline in their numbers after antigen injection: the process of germinal center dissociation remained unaffected by aging. 2) The latency of de novo germinal center appearance was approximately equal in duration (approximately 3 days) to the disappearance of pre-existing germinal centers. 3) The number and size of de novo HRP-induced germinal centers increased through the experimental period in young lymph nodes, but in old mice these parameters were depressed, resulting in a significant germinal center deficit. 4) The ratio of HRP-retaining ARR to de novo induced germinal centers was 1:1 in young and responder old mice. This ratio was not affected by aging. This finding favored the concept that antigen retention in ARR is a requirement of germinal center development. The observations supported our hypothesis that germinal center development, at least in part, depends on a normal antigen transport by showing that in aged mice with defective antigen transport-related ARR and iccosome deficits there is an impaired development of germinal centers.

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