Since spontaneous PCs occur rarely in mice, studies in plasmacytomagenesis rely on induction models. All PCs arise in the peritoneum, where the presence of nonmetabolizable paraffin oils (pristane) or plastic implants induces chronic inflammation, granuloma formation, and finally development of the PC
1. The SIPC may differ by having fewer numbers of atypical foci
8. Binding studies of pristane-induced PCs showed that PCs can display binding activities against phosphorylcholine, various dextrans, and fructofuranans
2. The SIPCs, as we show in this study, possess a set of binding specificities against cytoskeletal proteins and DNA. In fact, in the initial panel of ascites from 33 SIPC tumors, binding activity against at least 1 of the Ags of the panel, and most often polyreactive binding activity, is commonly observed. To ascertain whether the SIPC tumors possess a characteristic set of binding properties and whether these binding specificities could assist in identifying a precursor cell population, we focused on the binding activity of purified proteins from five representative SIPC tumors.
We have found that three independent SIPC tumors, each derived from different generations (SIPC3282, SIPC3308, and SIPC3336), share identical V
H domains including D
H and J
H regions (). Several facts argue strongly in favor of the independent derivation of these tumors: (a) each tumor was harvested at different times as a result of variable latency periods (see Materials and Methods); (b) reactivity patterns differ between each tumor (); (c) SIPC3282 shows an additional rearrangement (J
H) not found in other tumors, as well as additional somatic mutations not found in other tumors; (d) SIPC3385 and SIPC3308 both lack V
λ1 rearrangements; and (e) no RAG-1/2 expression is found in SIPC3282 or SIPC3336. These results also support the existence of a strong restriction in V gene usage for the SIPCs. These results are in contrast with reports for human myeloma, where no particular selection for V genes has been observed
24. In contrast, Waldenström macroglobulinemia patients exhibiting rheumatoid or cold agglutinin anti-I specificities exhibit a strong restriction for V genes, as the V1-69 V
H1 gene member is almost constantly expressed in the case of cryoglobulins expressing the WA recurrent idiotype (60% of cases), and the V4-34 (V
H4-21) gene is constantly expressed by cold agglutinins with anti-I specificity (95% of cases;
25). Although the D
H region is not identical among these Abs, a report by Fais et al.
26 shows that among 50% of CLL patients expressing the 1-69 gene, the D
H3-3 region is used. In addition, a report from the same group indicated that five different cases of CD5
+IgG
+ CLLs expressed virtually identical Ag receptors, by recombining (unmutated) the V
H-4-39 gene to D6-13-J
H5b and the V
κ012 gene to J
κ1
27.
| Table 4Heavy and Light Chain Gene Usage in SIPC Tumors |
Ig gene assembly is an ordered process that begins with heavy chain gene rearrangement
28 under the regulation of recombinase genes (RAG-1/RAG-2). As the Ig rearrangement process is error prone, only a successful V(D)J assembly leads to pre-B cell receptor assembly
2930 and eventual downregulation of RAG-1/RAG-2 activity
31. Further differentiated and proliferating B cells reactivate the recombinases to rearrange the light chain genes, which once again are error prone, i.e., wherein only successful rearrangement leads to maturation rather than cell death
2930. Therefore, allelic exclusion, typically the hallmark of Ig gene expression and of an active process, may also stem more from the low frequency of productive or successful rearrangement. With the elucidation of V gene receptor editing
32333435363738, we now know that lymphocytes expressing self-reactivity in the bone marrow, germinal center (GC), or even in the periphery can be rescued from cell death by exchanging already rearranged V regions with other available V genes through reactivation of the RAG-1/RAG-2 recombination pathway
23383940. It has been shown that recombinase activity is stimulated by low-affinity Abs and is inhibited by high-affinity Abs
41, and accordingly, secondary Ig rearrangements actually occur quite frequently
42434445. RAG-1/RAG-2 activity is also found in GC B cells bearing low-affinity receptors, or in peripheral B-1 cells
23. Indeed, there is increasing evidence that productive and successful light chain rearrangements do not effectively arrest further rearrangement
46474849505152. Thus, one would then expect frequent violations of allelic exclusion in that κ:λ dual expressors
53, as well as dual expressors of Ig
κ 5455 subtype, should be quite common.
In this study of SIPC tumors, we have observed only a single productive rearrangement of the light chain genes at the Southern hybridization level for V
κ24C-J
κ4. For SIPC3385 (expressing V
κ21G-J
κ2) and SIPC3301 (expressing V
κ34C-J
κ2), we find rearrangements in both EcoR1 and BamH1 Southern blots that are, in fact, consistent with predicted sizes from germline restriction maps for both V
κ and J
κ gene segments
1617. We only detected secondarily expressed alleles (κ or λ) by RT-PCR at levels below the Southern detection levels, i.e., subthreshold. We can eliminate contaminating host tissue as contributing to this observation by several lines of argument. (a) A restricted subset (V
κ1 or V
λ1) of V genes are found, rather than simply random usage. Furthermore, since V
λ1 is rarely expressed in the mouse, finding V
λ1 in 3/5 tumors is highly unlikely. (b) We have recently colinked V
κ24 and V
λ1 expression at the single cell level by RT-PCR amplification (our unpublished results). A more likely explanation, therefore, is that we must be observing only a subset of B cells in these tumors expressing both alleles. Thus, the presence of dual expressing κ:λ or κ:κ alleles indicates that secondary rearrangements are occurring in the SIPC tumors, but whether this rearrangement occurs at significant levels to alter the tumor reactivity patterns must be considered further. Interestingly, it is conceivable that the V
κ24C-J
κ4 rearrangement that is shared by three of the tumors may already be an example of secondary rearrangements after a highly selectable process, as secondary rearrangements of the same allele are often characterized by upstream V
κ segments associated with downstream J
κ segments. An alternative explanation, that the observed biallelic expression arises from an outgrowth of a subset of tumor cells, cannot formally be discounted. Indeed, when we compare the reactivities () of two SIPC tumors (SIPC3308, SIPC3336) that exhibit identical V
κ24C-J
κ4 and V(D)J rearrangements, including amino acid substitutions, we find greater reactivity to ssDNA with SIPC3308. Thus, a perceived difference in reactivity between these tumors may stem from the “subthreshold” levels of V
κ1 or V
λ. These “subthreshold” rearrangements do not necessarily have to occur in the bone marrow or GC, but could occur in the periphery where RAG-1/2 can be reactivated
56. However, as recent studies suggest that RAG-1/2 activity may not actually be reinduced, but may reflect differing levels of B cell development
57, we may be observing a small self-renewing B cell population in the tumors presented here. Interestingly, we find RAG-1/2 still active in most of the SIPCs, with the exception of two tumors (SIPC3336 and SIPC3282), both of which also express IgV
λ.
The precursor to PCs has long been thought to be the B-1 cell through two lines of evidence: (a) B-1 cells are most abundant in the peritoneum, and are associated primarily with IgA in the lamina propria
58; and (b) in addition to dextran, phosphorylcholine is one of the more common Ags associated with the gut flora and is dominated by the T15 idiotype
1. It has been shown by X-irradiation and failure to regenerate the T15 idiotype by bone marrow reconstitution
5960 that the T15 idiotype can only be restored by peritoneal B cells (i.e., B-1 cells). While B-1 cells express Ly1, it is uncertain as to whether Ly1 is activated as a consequence of immortalization, or whether this represents a distinct B cell lineage. We have determined that several pristane-induced PCs (including M104E and ABPC18) express Ly1 by RT-PCR amplification. Conversely, we have found that the SIPC tumors do not express Ly1, but further studies will be needed to rigorously determine whether the SIPCs are truly B-1a, B-1b, or even B-2 cells.
We now show that the SIPC tumors exhibit secondary Ig light chain gene rearrangements (and accompanying RAG-1/2 activity), exhibit low levels of somatic mutation in V
κ or V
λ, and show some evidence of clonal heterogeneity. While B-2 cells are mutated and most frequently found in the GC, the B-1 population is most often found in the mantle zone with few somatic mutations
61. The fact that we find evidence of tumors bearing somatic mutations with intraclonal heterogeneity and with high R/S ratios suggests Ag selection is occurring. V region analysis in several tumor systems besides SIPCs, including Burkitt's lymphoma
6263, diffuse large cell lymphoma
64, mantle cell lymphoma
65, and follicular lymphomas
64, demonstrates clonal heterogeneity in that somatic mutations appear to be ongoing during the progression of the tumor. Temporally, many of these tumors arise at different stages of lymphoid maturation and in different lymphoid compartments. In contrast, more mature tumors such as multiple myeloma
6667 and pristane-induced mouse PCs
2 have traditionally been found to exhibit homogeneous Abs, suggesting that these transformed cells must have been immortalized post-GC. Based on these findings, we propose that the SIPC tumors may have become an immortalized B cell population in the periphery.
This article has been 
