Bourke R. Maddox and David W. Scott

Immunology Department, American Red Cross Holland Laboratory, Rockville, MD 20855 USA

Received 03/29/96; Accepted 06/10/96; On-line 06/25/96

Transgenic mice, expressing IgM receptors typical of either B1 or conventional B cells, were used to compare susceptibility of these B cell types to the induction of PCD by anti-IgM. We used three lines of transgenics, one expressing IgM receptors typically of the B1 repertoire, and two lines expressing receptors typical of conventional B cells. In each case, we used non-transgenic littermates for each line as controls, as well as normal C57Bl/6 x DBA/2J F1 (B6D2) mice. In particular, line 6.1 spleen cells expressed >96% of the CH27 V_H12 idiotype seen in peritoneal B1 cells, whereas the 3.83 and Sp6 lines contained primarily conventional (B2) cells in the spleen. Importantly, more than 66-75% of splenic B cells in line 6.1 mice are CD5⁺, the remainder having the characteristics of B1 "sister cells" (4). Therefore, this allows us to compare splenic populations from different IgM transgenic lines in terms of their susceptibility to anti-IgM-induced apoptosis and eliminates problems associated with purification of peritoneal B cells. The standard protocol of hyper-crosslinking sIgM with anti-µ specific goat antibodies, followed by a 24 hour incubation period (9, 10) was employed. Cultures were then examined by FACScan^® for apoptotic B-cell populations after gating on B220⁺ cells. The results in Figure 1-2 summarize the observed differences.

Susceptibility of Sp6 anti-TNP transgenic B cells to programmed cell death with anti-æ.

It has been reported that the majority of splenic B cells in the Sp6 anti-TNP line are transgenic, CD5-, and express IgM and B220 at levels typical of normal splenic B cells. This phenotype suggests that these transgenic B cells are of the conventional B2 cell type (4, 8, 12), except that they do not express sIgD due to lack of endogenous rearrangements. We have shown in prior studies that splenic B cells from these animals can be rendered unresponsive to subsequent challenge through sIgM crosslinking in a manner similar to non-transgenic conventional B cells (4). Moreover, Carsetti and colleagues demonstrated that Sp6 B cells would undergo programmed cell death, in vivo and in vitro, with a specific antigen like TNP-dextran (12). This suggests an intact and functioning membrane IgM signaling pathway. We report here that Sp6 splenic B cells also undergo induced PCD in a normal, dose-dependent manner (Figure 1 middle). In repeat experiments, the dose response curves for Sp6 transgenic B cells were similar to both non-transgenic littermates and B6D2 controls (Figure 1 left and data not shown).

Figure 1. Induction of apoptosis by anti-IgM in non-transgenic and transgenic B cells. Assay for apoptotic populations was performed with propidium iodide staining and FACScan analysis as described in Materials and Methods. Splenic B cells from B6D2/F1, Sp6 (anti-TNP), and 3.83 (anti-MHC) mice were analyzed for spontaneous and inducible apoptosis. Top panels contain background apoptosis controls (medium alone); the bottom panels show apoptosis induced with 30µg/ml anti-IgM. Histograms are from a single experiment, but are representative of at least three similar experiments. These data show that Sp6 and 3.83 transgenic B cells exhibit similar levels of apoptosis when compared to normal B6D2/F1 control mice, and non-transgenic littermates (data not shown).

Susceptibility of 3.83 anti-MHC transgenic B cells to programmed cell death with anti-µ.

The results above suggest that transgenic anti-TNP specific B cells respond normally to hyper-crosslinking signals with anti-µ. It was important to repeat these experiments in another transgenic strain expressing Ig receptors of a different specificity. We chose to use the 3.83 anti-MHC transgenic line both because it had been used previously to demonstrate deletion of the developing B-cell repertoire (7) and to be susceptible to tolerance in vitro in our anti-µ protocol (4). Spleen cells from 3.83 transgenic and non-transgenic control littermates were treated with anti-µ as above and analyzed for apoptosis. The results in Figure 1 (right) indicate that the anti-MHC transgenics also responded normally to hyper-crosslinking with anti-µ by undergoing PCD, as was seen in B6D2 and Sp6 conventional splenocytes.

V_H12 anti-phosphatidyl choline transgenic B cells are resistant to programmed cell death with anti-µ.

We have previously reported that V_H12 transgenic mice, as well as CH12 transgenics, are resistant to tolerance induction with anti-µ (4). Both strains contain large numbers of B1 cells, either CD5⁺ or CD5^- "sister" cells in their spleens. To establish the susceptibility of V_H12 transgenic B cells to apoptosis, we repeated the anti-µ protocol above. The results in Figure 2 show that V_H12 transgenic B1 B cells are relatively resistant to anti-µ-driven PCD. Note that non-transgenic control spleen cells, which possess similar numbers of B cells, examined simultaneously were susceptible to PCD just like the previous conventional strains tested (see Figure 1).

Figure 2. V_H12 line 6.1 transgenic exhibit an impaired ability to undergo induced apoptosis via hypercrosslinking of sIgM. Dose responses to anti-IgM treatment of non-transgenic littermate (left) versus 6.1 transgenic B cells are shown. Methods were identical to those used Figure 1.

Moreover, this is not simply a difference in dose response curves because a full range of anti-µ concentrations was done in each experiment (Figure 2). This suggests that one explanation of the resistance to tolerance of B1 cells is their failure to process the hyper-crosslinking signals that lead to apoptotic cell death.

Strain differences in the proliferative response to anti-µ.

We previously suggested that initial signaling differences between V_H12 (B1) and the conventional transgenic B cells tested could account for differential susceptibility to tolerance induction (4).

Figure 3. Proliferation of splenic B cells in response to anti-IgM treatment. Lines 6.1 and Sp6 both exhibit inhibited proliferation when compared to non-transgenic littermates and B6D2/F1 controls. Line 3.83 shows a strain related reduction in proliferation, though proliferation appears normal when compared to non-transgenic littermates.

Moreover, if one bypassed the Ig receptors using phorbol esters or ionomycin (or both), then unresponsiveness ensued. In these experiments, it was not known whether V_H12 transgenic B cells (or the other strains tested) were able to proliferate normally to anti-µ. Therefore, we examined splenocytes from all three strains and normal B6D2 F1 mice for their ability to proliferate with polyclonal anti-µ. In these studies, spleen cells are cultured with 3, 10 and 30 µg/ml anti-µ for 44-50 hours in order to drive significant numbers of B cells into cycle. The results in Figure 3 show that V_H12 transgenic B cells failed to proliferate with anti-m, whereas the other transgenic lines were more responsive with peak proliferation at 10µg/ml. However, it should be noted that Sp6 transgenic B cells (which represent conventional B2 cells) responded less well than their non-transgenic counterparts and below the levels of B6D2 control B cells. Importantly, V_H12 B cells failed to proliferate at any dose of anti-µ.