Naa Clones Are Frequently Committed To Malignant Transformation

Accumulated evidence indicates that this autoreactive B-cell repertoire frequently undergoes a malignant transformation. This evidence arose from the study of monoclonal immunoglobulins (MIg), chronic lymphocytic leukemia (CLL) and follicular non-Hodgkin lymphomas (FNHL).

3.1. Antibody Activity of MIg

MIg correspond to normal synthetic products whose counterpart can be found in the heterogenous normal Ig compartment. The antibody-like activity of MIg has been described against a large number of antigens, e.g., bacterial antigens, plasma proteins, tissue antigens and nonbiological haptens [33]. However, an impressive and unexpected frequency of activities has been reported against: (1) I blood group antigen (cold agglutinins, [CA]); (2) the Fc fraction of IgG (rheumatoid factor [RF]); (3) cytoskeleton proteins and DNA (polyreactive autoantibodies); and (4) antimyelin associated glycoproteins (MAG).

3.1.1. MIg with CA activity

An interesting exception to the usage of multiple VH segments in the case of pathogenic human autoantibodies, is given by anti-Ii cold agglutinins. Pioneer work by Williams et al. [34] demonstrated that these cold agglutinins were sharing recurrent idiotopes. This result suggested the presence of common V-region structures shared by these pathogenic autoantibodies leading to hemolytic anemia. Recent structural studies substantiated this theory by demonstrating that anti-Ii autoantibodies are constantly encoded by the VH4-21 gene segment, which is frequently associated to a VkTU gene [35-37]. The structural basis of the recurrent idiotope detected by the rat anti-idiotypic antibody 9G4, which binds to these autoantibodies and inhibits the binding of these to red blood cells, was recently elucidated by Potter et al. [38], who showed that the presence of an AVY motif in positions 2325 of the FR1 region of the antibody, constituted the reactive site of the idiotope. The structural relationship between this binding site and that of the antigen is unclear as yet. Cold agglutinins share a gross antigenic specificity but differ in their fine antigenic specificity. According to Silberstein et al. [36], the gross anti-Ii specificity could be regulated by the VH4-21 region, whereas, the fine specificity could be determined by the CDR3 regions, which differ among the different cold agglutinins. CA paraproteins are almost constantly IgMr paraproteins, which usually react with a set of antigenic determinants directed against the Ii system, or compound antigens including Ii (Al, HI, etc.). Their activity is increased by cold, but thermal amplitude is variable [39],

3.1.2. MIg with RF activity

Since the first report by Kritzman et al. [40] of a monoclonal IgM>c paraprotein with anti-IgG activity, an increasing number of cases have been reported, and the frequency of this specificity has been estimated at more than 10% of total IgM, paraproteins [41]. Most monoclonal components with reported RF activity, were found to form a cryoprecipitate. Almost all cases corresponded to IgM/c MIg, but rare cases of human monoclonal IgG and IgA with RF activity and cryo-precipitables have been described. Agnello et al. [42] first reported the presence of cross-reactive idiotypic (CRI) specificities among these MIg. Sixty percent of these MIg displaying RF activity were found to share a major CRI called Wa; 20% belonged to a less common CRI designated Po, and a few expressed a more rare CRI, named Bla.

During recent years, considerable work, largely emanating from the group of Dennis Carson, has contributed important information concerning this type of MIg, by precisely defining genetic origins in serological and structural terms [43]. These studies were mainly focused on MIg sharing the Wa CRI. It was found that: (a) almost all Wa+ RF share the 17109 CRI related to light chains and the G6 idiotype related to heavy chains; (b) they constantly express the minor subgroup V/cIIIb light chain; (c) the Vk light chain is derived from a single germinal gene (Hum/c v325), since most Wa+ paraproteins display an identical or nearly identical light chain sequence, as stated by 13 complete light chain sequences; and (d) there is strong sequence homology among /i chains expressing the Wa idiotype. Most of them use the VH1 family (80%) and the minority use VH2 and VH3 families. Although information derived from Po+ RF MIg is less extensive, they appear to constantly use a V/c germinal gene (Hunwv328) and to use a conserved VH3 sequence [43, 44]. More recently, it has been demonstrates that the idiotype Bla was encoded by a gene of the VH4 gene family [45].

3.1.3. MIg with polyreactive activity

Prompted by our results in normal human serum in the early 80s, we screened 612 MIg for the presence of antibody activity directed against cytoskeleton proteins and DNA. Our results indicated that about 6% of all MIg and 10% of IgM paraproteins bound to these antigens, and that most of them displayed a polyreactive pattern of binding comparable to that found in normal human serum, indicating that MIg frequently correspond to expansion of a clone normally producing a NAA [18, 19]. Dellagi et al. [46] also reported the presence of IgM paraproteins binding to intermediate filaments, and Shoenfeld et al. [47] found that more than 10% MIg shared the 16-6 CRI derived from a monoclonal Ig with anti-DNA activity. However, only 25% of these MIg were demonstrated to possess anti-DNA activity. Another anti-idiotypic reagent (F4) was found to be present in 12% of MIg and was found to be strongly associated with IgG isotype and anti-DNA activity [48]. The sequences of myeloma immunoglobulin genes do reveal a lot of information about the stage in the B-cell differentiation pathway in which the oncogenic event might have taken place. The presence of somatic mutations in a nonrandom fashion without intraclonal variation leads to the conclusion that the precursor myeloma cell could not possibly be a pre-B cell or stem cell but has to be a mature B cell that has been in contact with antigen and has past through the phase of somatic mutation, like a memory B cell or plasmablast [49],

3.1.4. MIg with anti-MAG activity

A peripheral neuropathy is observed in about 5% of Waldenstrom's macroglobulinemia patients [50, 51]. In a majority of these cases MIg display an antibody activity against a myelin associated glycoprotein (MAG). The epitope recognized corresponds to a gly-curonyl sulfate group. However, the pathogenic role of the MIg is not definitively established. Brouet et al. [51], reported a recurrent idiotype among 9 MIg with anti-MAG activity. Six out of these 7 MIgs for which studies could be performed were expressing VH3 and the remaining VH2. Interestingly the rare V/cIV family was found in 3 cases, VkI in 2 and V/cII in 1 and the remaining patient expressed X light chain.

3.1.5. Antibody activity of the CD5+ CLL-B lymphocytes

One of the main difficulties in working with CLL-B lymphocytes arises from the fact that these cells are highly resistant to transformation by Epstein-Barr virus (EBV) and only a few EBV cell lines have been obtained from CLL-B lymphocytes [52], Given this difficulty, recent work was performed by using mitogenic stimulation of CLL-B lymphocytes and succeeded to demonstrate autoantibody production by these cells [53, 54], With the aim to obtain long-term cell lines that would enable production of high level of Ig and permit studies at the molecular level, we have fused leukemic lymphocytes from 27 different CLL patients with the nonsecreting X-63 mouse myeloma.

We have found that 11 out of the 19 patients for which we could study antibody activity were expressing autoantibody activity [55]. These results were indicating that CD5+ B-CLL lymphocytes are frequently committed to the production of natural autoantibodies. In addition, the surprising high frequency of this autoantibody activity among CLL-B lymphocytes favors the idea that CLL-B cells are expressing a restricted set of genes. In a recent work, Kipps et al. [56, 57] found that a high proportion of B-CLL cells expressing k at the membrane reacted with a murine anti-idiotypic antibody raised against a monoclonal IgM rheumatoid factor expressing the idiotype Wa. Analysis of V/c genes expressed by leukemic cells sharing idiotype Wa, enabled these authors to demonstrate that they were employing the germinal ummutated Hurrwv 325 germinal gene. Humphries et al. [58] reported that 30% of CLL patients were expressing the 5-51, which is one of the two germinal members of the VH5 family. Logtenberg et al. [59] found that CLL-B lymphocytes express VH4 in 50% of cases, VH5 in 20% and VH6 in 15%. Similar restriction was found for VH genes, since the germinal VH1 1-69 gene, was found to be expressed in 20% of CLL cases [57]. We have studied VH expression in 40 CD5+ B-CLL and found that VH1 was employed in 17%, VH2 in 8%, VH3 in 36%, VH4 in 17%, VH5 in 8% and VH6 in 14% [60], Although, our study failed to find the same incidence of the small VH4, VH5 and VH6 families; it shows a clear overrepresentation of these.

The analysis of individual expression of VH genes reveals a skewed expression. According to the complexity of the system, if gene expression follows a stochastic process, each VH gene should be expressed in about 2% of cases. However, by pooling the results from 156 sequences derived from different published series [61-69] it appears that some genes like 4-34 and 1-69 (10% each), 4-39, 3-07 and 3-23 (6% each), and 1-02 and 1-18 (5% each), are overexpressed in B CLL and that these seven different genes account for 48% expression in CLL, as compared to the 14% expression that would be expected in the case of a stochastic process. In contrast, 15 different VH genes among the 51 functionally existing VH genes, i.e., the 1-24,1-45, 1-e, 1-f, 2-70,3-20,3-43,3-d, 3-64,3-72,4-28,4-b, 461,4-30.1 and 4-30.4 genes, have never been reported to be expressed in B-CLL [61-69] and Pritsch et al. (unpublished).

Although, skewed expression of VH genes could result as a consequence of a selective process driven by the antigen, overexpression for most of these genes (1-18, 3-07,3-23, 4-34, 4-39) has also been found in the fetal and adult normal repertoires [34-37], Thus, the reasons for the overexpression of these genes in CLL and the normal repertoire remain unclear. Several explanations have been proposed, including the occurrence of several genomic copies, particular regulatory regions and selection through certain antigen binding or idiotypic determinants [36, 37]. In the case of the 3-23 gene, the fact that the same degree of overexpression has been observed in both productive and nonproductive rearrangements of normal B cells favors an intrinsic genetic mechanism [38]. Based on JH family usage and the CDR3 length it was found that CLL-B cells express H chain variable domains typical of postnatal rather than fetal tissues [61], This study also showed that some genes like 1-69 and 4-39 were in most cases expressed in a germ line configuration, whereas, others like 4-34 and 5-51 contained in most cases somatic mutations. Whether, or not, CLLs expressing genes in germinal configuration represent a more immature set of cases, and CLLs expressing genes containing somatic mutations represent a more mature population selected through an antigen-driven process, remains an open question. Recent work from Chiorazzi's laboratory, reported studies carried out on 7 IgG expressing CLLs. This work indicates that the switch is biased in favor of y I, and that there is evidence favoring an antigen-driven process in at least some of these cases [62],

3.1.6. Antibody activity of the CD5~ B lymphocyte from follicular non-Hodgkin lymphomas (FNHL)

Our results with CLL were in aid to the hypothesis that CD5+ B mostly secrete autoantibodies. However, in a recent work, based on the detection of mRNA transcript of Lyl gene among 40 murine hybridomas displaying natural autoantibody activity, we could demonstrate that both Lyl- and Lyl+ B lymphocyte subsets were involved in the production of natural autoantibodies [70]. To gain better insight into this problem, we have recently studied 31 hybridomas obtained in the laboratories of Miller and Levy, from CD5" B-cell NHL. Our results indicated that 8 out of the 31 hybridomas displayed rheumatoid factor activity, and 2 out of these 11 displayed a multispecific activity [71]. These results obtained with Igs derived from CD5- B-cell tumors strongly support the idea that CD5" B cells are also involved in the production of natural autoantibodies. Cleary et al. [72] reported that at the difference of CLL and acute lymphoblastic leukemia, where a bias in expression of VH4, VH5 and VH6 families has been demonstrated, CD5- B cells proliferating in NHL appear to employ VH gene families in a more stochastic way, by privilegiating the multigenic VH3 family. In addition, there is an active somatic mutational process in B-cell follicular NHL, that is rarely observed in B-CLL.

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