Neonatal murine B lymphocytes respond to polysaccharide antigens in the presence of IL-1 and IL-6

Unlike adults, neonates are unable to respond to polysaccharide Ags, making them especially vulnerable to pathogenic encapsulated bacteria. Since the Ab response to polysaccharides in adult mice requires certain cytokines, it was hypothesized that neonatal murine B cells may be competent to respond to such Ags, but may fail to do so due to a deficiency of cytokines. Neonatal splenocyte cultures, which were otherwise unresponsive to trinitrophenyl (TNP)-Ficoll, a haptenated polysaccharide Ag, mounted an adult-like Ab response when supplemented with IL-1. However, IL-1 failed to induce such a response to TNP-Ficoll when purified B cells were used instead. Although IL-6 alone did not induce a response in whole spleen cells or purified B cells from neonates, it synergized with IL-1 in inducing purified neonatal B cells to respond to TNP-Ficoll. The avidity of the cytokine-induced neonatal anti-TNP Abs was comparable to that of Abs made by adult splenocyte cultures. One effect of IL-1 may be at the level of clonal expansion, since it induced neonatal B cells to proliferate in response to anti-IgM, which was further enhanced by IL-6. The spontaneous secretion of IL-1 by neonatal splenocytes was below the detection limit, while adult splenocytes secreted 30.8 +/- 5.2 U/ml, which is of the same order of magnitude as what was required to stimulate neonatal B cells to respond to TNP-Ficoll. Thus, the neonatal unresponsiveness to polysaccharide Ags could be due to the inability of a non-B cell population resident in the neonatal spleen to secrete sufficient quantities of IL-1.     

IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.     

Deficient interleukin-10 production by neonatal T cells does not explain their ineffectiveness at promoting neonatal B cell differentiation

Neonatal T cells are poor promoters of Ig secretion by neonatal B cells. Since IL-10 has been shown to play a role in B cell differentiation, we investigated the relationship of IL-10 production by neonatal T cells and their ability to provide B cell help. Neonatal CD4+(CD8-) T cells and adult naive CD4+ (CD8-/CD45RO-) T cells activated with immobilized anti-CD3 produced consistently less IL-10 than adult memory CD4+(CD8-/CD45RA-) T cells. Production of IL-10 by adult and neonatal T cells was dependent on IL-2, but was unaffected by supplemental IL-4. Despite diminished IL-10 production, supplemental IL-10 increased neonatal T cell-dependent Ig secretion only modestly, but did not increase Ig heavy chain isotype switching. This contrasted with the ability of IL-10 to enhance the secretion of all Ig isotypes by adult B cells stimulated in the presence of either IL-2 or IL-4. These results suggest that IL-10 can promote T cell-dependent Ig secretion but not Ig heavy chain isotype switching by neonatal B cells. However, deficient IL-10 production alone does not account for the poor ability of neonatal T cells to support neonatal B cell Ig production.     

Lack of type 2 T cell-independent B cell responses and defect in isotype switching in TNF-lymphotoxin alpha-deficient mice

TNF is implicated in in vitro Ig production, but its role in vivo is not clearly defined. Our previous studies had shown that TNF-LT alpha double-deficient mice have defective IgM and IgG primary Ab responses to the T cell-dependent (TD) Ag SRBC. We now extend these studies to secondary responses and to T cell-independent (TI) B cell responses. Injections of the TD Ag SRBC did not induce germinal center formation in the spleen of TNF-LT alpha-deficient mice. Associated with the morphologic defect, there was a defective IgG Ab response and a secondary hyper-IgM response to the TD Ag in TNF-LT alpha-deficient mice. The response to the TI Ag type 2 DNP-alanyl-glycyl-glycyl-Ficoll was essentially absent in TNF-LT alpha-deficient mice, while that to the TI Ag type 1 TNP-LPS was significantly reduced only for IgG2b isotype. Transplantation of bone marrow cells from wild-type mice into irradiated TNF-LT alpha-deficient mice restored the formation of splenic germinal centers and corrected the IgM and IgG responses to both TD and TI Ags. These data suggest that TNF and/or LT alpha signaling are critically required for germinal center formation and for the IgM and IgG responses to both TD and TI type 2 Ags.     

Polyclonal immunoglobulin response of thymic, hepatic and splenic lymphocytes from fetal, germ-free and conventionally reared pigs to different B-cell activators

Immunoglobulin (Ig) response to different polyclonal B-cell activators was measured by ELISA in cell culture media of thymocytes, splenocytes and liver cells isolated from pig fetuses, 8-d-old germ-free piglets and conventionally reared pigs. Both in fetal and in postnatal life polyclonally stimulated lymphocytes were found to produce predominantly the IgM isotype; the first IgM formation was detected in 50-d-old fetal liver (gestation in pigs lasts 114 d). Surprisingly, 73-d-old fetal thymic cells were shown to be induced to Ig synthesis and secretion. In contrast to splenocytes of the same age, which secreted exclusively IgM, fetal thymocytes produced IgM, IgG and IgA. Polyclonally stimulated splenic cells as compared with thymic cells started to produce IgA later in fetal ontogeny, whereas the IgG response was not detectable in splenic cell culture media during the whole embryonal development and appeared only after birth. The earliest and the highest Ig stimulation was found after cultivation of lymphocytes with Nocardia delipidated cell mitogen. Interestingly, the moderate stimulatory effect of 65-kDa heat shock protein (Hsp-65) in polyclonal IgM response of fetal splenocytes was observed. We showed that thymic B lymphocytes represent probably the first maturing B cell population detectable in fetal life, which is able to differentiate after polyclonal stimulation into IgM as well as IgA and IgG producing cells.     

Identification and characterization of B cell precursors in rat lymphoid tissues. I. Adoptive transfer assays for precursors of TI-1, TI-2, and TD antigen-reactive B cells

Quantitative adoptive transfer assays were developed to detect the precursors of TI-1, TI-2, and TD antigen-reactive B cells in rat lymphoid tissues. Studies on the immune responses in normal and athymic nude rats validate the use of TNP-lipopolysaccharide as a TI-1 antigen, TNP-Ficoll as a TI-2 antigen, and SRBC as a TD antigen in rats. The precursors to these immunologically competent B cells are detected, following transfer into irradiated histocompatible recipients, by their ability to generate expanded populations of antigen-reactive B cells capable of mounting antibody responses (splenic IgM plaque-forming cells) to these antigens. Maximal numbers of antigen-reactive B cells emerge in antigenically naive rats after an interval of 7-12 days following transfer of donor lymphoid cells and decline rapidly thereafter. The delayed responses in adoptive recipients reconstituted with spleen cells are proportional to the numbers of spleen cells transferred and are shown to be primarily donor derived using histocompatible Ig kappa chain alloantigen disparate rat strain combinations. The precursors of TI-1, TI-2, and TD antigen-reactive B cells are present in both donor spleen and bone marrow. However, precursor cells to TI-1 and TD antigens are largely absent from donor lymph node cells, whereas precursors to the TI-2 antigen are as prevalent in donor lymph node as in donor spleen. These results support the hypothesis that newly formed virginal B cells represent transient populations of precursor cells that undergo further proliferation and differentiation in the spleen before acquiring immunological competence. The results also suggest that the precursors of TI-2 antigen-reactive B cells differ developmentally from those of TI-1 and TD antigen-reactive B cells, and that the antigen-reactive progeny of these precursors require additional stimulation in order to join the pool of long-lived peripheral B cells.     

Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3

During antigen-induced immune responses, human B cells switch isotype from immunoglobulin M (IgM)-IgD to IgG1-4, IgA1-2, or IgE. In the human, no cytokines have yet been demonstrated to act as switch factors for IgG1, IgG2, and IgG3. In this paper, we report that in response to interleukin 10 (IL-10), anti-CD40 activated tonsillar surface IgD+ (sIgD+) B cells are induced to secrete large amounts of IgM, IgG1, and IgG3 but neither IgG2 nor IgG4. Cord blood purified B cells and lymphocytes from Hyper-IgM patients also produced IgG1 and IgG3 after culture with anti-CD40 and IL-10. In contrast, sIgD- isotype-committed B cells produce IgG1, IgG2, and IgG3 when activated through CD40 in the presence of IL-10. Thus, in addition to its growth-promoting and differentiating activities on human B cells, IL-10 may represent a switch factor for IgG1 and IgG3.     

Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM

The role of endogenous natural IgM in promoting the adaptive Ab response was investigated in newly constructed mutant mice in which B cells do not secrete IgM but still express surface IgM and IgD and undergo class switching to express other Ig isotypes. While the mutant mice had relatively normal numbers of conventional B (B-2) cells in all tissues examined, unexpectedly, B-1 cells in the peritoneum and spleen were approximately threefold more abundant. The elevated levels of B-1 cells were already detectable at 4 wk of age and were stably maintained throughout life. The levels of serum IgG2a, IgG3, and IgA were also elevated in the mutant mice at an early age. IgG2a response to a T cell-independent Ag was augmented, whereas IgG Ab responses to suboptimal doses of a T cell-dependent Ag were impaired. The latter defect was associated with fewer splenic germinal centers, impaired Ab affinity maturation, and less Ag trapping on follicular dendritic cells. Together, these findings demonstrate a physiologic role of natural IgM in the feedback regulation of B-1 cell development, the regulation of IgG2a production, and the promotion of efficient B-2 cell Ab responses.     

Fetal immune response following prematurely ruptured membranes

Concentrations of immunoglobulins (Ig)A1, and IgA2, IgD, IgE, IgG, and IgM have been determined in cord blood, amniotic fluid, and maternal serum in a group of patients with a history of prematurely ruptured membranes (PRM) prior to the onset of labor and in a control group of patients undergoing normal delivery and without a history of infection during pregnancy. IgA and IgD were determined by sensitive hemagglutination-inhibition tests; IgG and IgM, by radial immunodiffusion; IgE, by a radioimmunoassay. There was evidence for an immune response in 10 of 16 cases of PRM: five of 16 had increased IgA but normal IgM; three of 16 had increased IgA and IgM; two of 16 had high IgM and normal IgA in cord blood. In patients with significantly increased levels of either IgA or IgM or both, there was a decreased level of IgD. These changes are most likely the result of the immune response to ascending infection from the maternal genitals. The sensitive testing method employed could demonstrate the presence of IgD in 53 per cent of normal cord blood samples and 72 per cent of amniotic fluid samples obtained at term. IgE was found in all normal cord blood and amniotic fluid samples tested. By concentrating the amniotic fluid up to 180-fold, IgM was demonstrated in all normal samples tested. The potential importance of IgA determinations in cord blood in addition to IgM determination for detection of intrauterine infections is stressed.     

A tyrosine-based signal present in Ig alpha mediates B cell receptor constitutive internalization

B lymphocytes express Ag receptors (BCR) that are composed of ligand binding subunits, the membrane Igs, associated with Ig alpha/Ig beta heterodimers. One main BCR function is to bind and to internalize Ags. Peptides generated from these internalized Ags may be presented to T lymphocytes. Here, we have analyzed the involvement of BCR Ig alpha/Ig beta components in BCR constitutive endocytosis. The role of Ig alpha subunit in BCR constitutive endocytosis was first determined in the context of an IgM-based BCR. In contrast with BCR that contain wild-type Ig alpha, surface BCR lacking Ig alpha cytoplasmic domain were not constitutively internalized. The respective roles of Ig alpha and Ig beta subunits were then analyzed by expressing chimeric molecules containing the cytoplasmic domains of either subunits in a B cell line. Only the Ig alpha cytoplasmic domain contained an internalization signal that allowed constitutive endocytosis of Ig alpha chimeras via coated pits and accumulation in sorting-recycling endosomes. This internalization signal is contained in its immunoreceptor tyrosine-based activation motif. These results indicate that Ig alpha, through its immunoreceptor tyrosine-based activation motif, may account for the ability of IgM/IgD BCR to constitutively internalize monovalent Ags.     

B cells are anergic in transgenic mice that express IgM anti-DNA antibodies

B lymphocytes in individuals with systemic lupus erythematosus (SLE) secrete pathogenic autoantibodies to DNA which cause clinical nephritis. (NZB X NZW) F1 (BW) female mice also secrete pathogenic anti-DNA autoantibodies, and therefore are considered to be an animal model of SLE. The rearranged immunoglobulin (Ig) genes that encode an anti-DNA antibody from a diseased BW mouse have been cloned, and transgenic (Tg) mice have been created by microinjection of these constructs into fertilized eggs from normal mice. As we reported previously, when the construct contains the C gamma 2a heavy chain constant (CH) region, the mice spontaneously secrete anti-DNA IgG and they develop mild nephritis. This demonstrated that the Ig encoded by the transgene is pathogenic. In contrast, here we report that when the construct contains the same anti-DNA Ig variable (V) regions used previously, along with the C mu region, the autoreactive B cells are rendered tolerant. Most B cells in the Tg mice express the mu transgene product on their surface, and rearrangement of endogenous light chain genes is partially suppressed. Furthermore, most hybridomas made from Tg B cells secrete IgM anti-DNA. Despite this, the Tg mice have reduced levels of total serum Ig and they do not secrete anti-DNA IgM either spontaneously or following immunization with DNA. We conclude that most B cells in the Tg mice have been rendered anergic. Anergy is however reversible in vitro; lipopolysaccharide stimulation of Tg B cells leads to the production of a significant amount of IgM anti-DNA antibody. The studies demonstrate that in this line of Tg mice on a normal mouse genetic background potentially pathogenic B cells that express a high-affinity Ig specific for a natural autoantigen are subject to tolerance by induction of anergy.     

Deletion of bone marrow stromal cell antigen-1 (CD157) gene impaired systemic thymus independent-2 antigen-induced IgG3 and mucosal TD antigen-elicited IgA responses

Bone marrow stromal cell Ag-1 (BST-1; CD157)-deficient mice were generated to examine the immunologic roles of the molecule in vivo. In BST-1(-/-) mice, the development of peritoneal B-1 cells was delayed, and CD38(low/-) B-lineage cells were increased in the bone marrow and spleen. Partial impairment of thymus-independent (TI-2) and thymus-dependent (TD) Ag-specific immune responses was noted in the systemic and mucosal compartments of BST-1(-/-) mice, respectively. Although serum Ig levels as well as TD and TI-1 Ag-specific systemic immune responses were normal, the TI-2 Ag-induced IgG3 response was selectively impaired. Oral immunization of BST-1(-/-) mice with cholera toxin, a potent TD Ag for the induction of IgA response, resulted in the poor production of Ag-specific Abs at the intestinal mucosa accompanied by the reduced number of Ag-specific IgA-producing cells in the lamina propria. These results indicate that BST-1 has roles in B cell development and Ab production in vivo.     

Fc epsilonRI-deficient mice infected with Schistosoma mansoni mount normal Th2-type responses while displaying enhanced liver pathology

The IgE/Fc epsilonRI interaction is postulated to play an important role in resistance to helminths both at the level of anti-parasitic effector cell function and in the initiation of Th2 responses through IL-4 produced by Fc epsilonRI+ non-B, non-T (NBNT) cells. To formally evaluate the role of IgE/Fc epsilonRI signaling in the host response to helminths we studied Schistosoma mansoni infection in Fc epsilonRI knockout (KO) mice. Infected wild-type (wt) and KO animals showed comparable adult worm and tissue egg burdens, arguing against a role for Fc epsilonRI interactions in host resistance. Significantly, NBNT cells from infected KO, in contrast to wt animals, did not secrete IL-4 when stimulated with anti-IgE Ab or soluble parasite Ag. Nevertheless, serum IgE levels and Th2 cytokine production profiles were comparable in both strains of mice, demonstrating that the Ag-dependent stimulation of IL-4 secretion by NBNT cells is not essential for helminth-induced Th2 differentiation. However, when stimulated with low Ag doses, splenocytes from infected Fc epsilonRI-deficient mice produced less IL-4 in vitro than similar cultures from infected wt animals, an effect attributable to their defective NBNT cell function. Moreover, infected KO mice showed enhanced egg granuloma formation and hepatic fibrosis, revealing that the IgE/Fc epsilonRI interaction, while not essential for Th2 response development or resistance to primary infection, plays a significant role in down-regulating host pathology.     

Effect of cytokine-specific antisense oligonucleotides on the immunoglobulin production by rat spleen cells in vitro

We examined in this study the regulation of immunoglobulin (IgM, IgG, IgE) production by spleen cells from N brasiliensis infected rats following addition of antisense oligodeoxynucleotides (ODNs). The ODNs were selected near the AUG initiation codon of mRNA specific for interleukin 4 (IL-4) or interleukin 2 (IL-2). Results show that addition of antisense to IL-4 inhibited IgE production, while the production of IgG and IgM increased. The use of sense IL-4 sequence did not affect immunoglobulin production. In contrast, the use of antisense IL-2 ODN induced an enhancement of IgE as well as of IgM and IgG responses. Both the Ig secretion in culture supernatants and the number of Ig secreting cells, as detected by an Elispot assay, were influenced by the presence of antisense IL-4 ODNs. These results clearly show the involvement of IL-2 and IL-4 in the regulation of isotype selection during antibody synthesis and that IL-2 and IL-4 do operate differently on IgE production. They also argue that antisense strategy represents a useful tool for the antibody regulation.     

Serum and intestinal fluid immunoglobulins and jejunal IgA secretion in Crohn's disease

Immunoglobulins in serum and proximal intestinal fluids and secretion of IgA by cultured jejunal mucosa were measured in 12 healthy subjects and 36 patients with Crohn's disease. Concentrations of IgA, IgG, IgM, and IgE in serum and intestinal fluids were similar in the two groups, except for increased serum IgA concentrations in the patients. Elevation of IgA and chronicity of disease were correlated, which suggests that the IgA alteration was a response to duration of disease rather than a primary pathogenetic factor. IgA secretion by cultured jejunum was similar in control and patient groups. Thus, no evidence was found that abnormalities of secretory immunoglobulins are pathogenetically involved in Crohn's disease.     

Energetics of vinca alkaloid interactions with tubulin isotypes: implications for drug efficacy and toxicity

Capture ELISAs, for canine IgG, IgM, IgA and albumin, were developed and used to analyse immunoglobulin (Ig) concentrations in both serum and secretions. Matched samples of serum, saliva and tears were taken from 31 dogs, assigned to two groups based on age, whilst bile samples were obtained from nine adult dogs at post-mortem. Serum and tear IgA concentrations were significantly lower in dogs < or = 12 months of age compared with dogs > 12 months of age (p = 0.006 and 0.045, respectively). There was no significant correlation between serum and secretory Ig levels, with the single exception of serum and tear IgM concentrations (rp = 0.553, p = 0.004). IgG and IgM concentrations were significantly correlated in matched tear and saliva samples (IgG: rp = 0.470, p = 0.023; IgM: rp = 0.651, p < 0.0001). Albumin concentrations were significantly correlated with IgG, but not IgM or IgA, in both saliva and tears (saliva, rp = 0.581, p = 0.004; tears, rp = 0.843, p < 0.0001) whilst IgA and IgM concentrations were significantly correlated with each other in both secretions (saliva, rp = 0.644, p = 0.001; tears, rp = 0.555, p = 0.009). Significantly, more Ig of all classes was secreted into saliva than tears as calculated by a secretory index. A large diurnal and day-to-day variation was observed in Ig concentrations in serial saliva and tear samples taken from a further four dogs. Serum Ig concentrations are therefore, poor indicators of mucosal secretion in this species and significant intra-individual variation exists in secretory Ig levels. Both findings should be taken into account in future studies of canine mucosal immunoglobulins.     

Resting B cells from autoimmune lupus-prone New Zealand Black and (New Zealand Black x New Zealand White)F1 mice are hyper-responsive to T cell-derived stimuli

To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.     

LPS-stimulated SJL macrophages produce IL-12 and IL-18 that inhibit IgE production in vitro by induction of IFN-gamma production from CD3intIL-2R beta+ T cells

SJL mice are known for their poor IgE production upon helminth infection. In this study, we have demonstrated that SJL standard B cells (85% IgM+ or B220+), prepared by complement-mediated T cell lysis, failed to proliferate and to produce IgE and IgG1 in response to LPS plus IL-4 in vitro. This diminished IgE production was restored by anti-IL-12 and enhanced by additional treatment with anti-IL-18, suggesting active suppression by the cells that produce IL-12 and IL-18. Indeed, SJL standard B cells were contaminated with Mac-1+ cells. Therefore, we removed macrophages by passing standard B cells through a Sephadex G-10 column (G10). Resultant cells (95% IgM+), designated as G10-B cells, responded to LPS and IL-4 by their proliferation and differentiation. G-10 treatment markedly diminished the proportion of B220- cells and Mac-1+ cells in SJL standard B cells. Furthermore, addition of SJL B220- cells dose dependently and MHC independently inhibited LPS plus IL-4-induced B cell growth and IgE production in SJL and BALB/c B cells. B220- cells in SJL standard B cells contained Mac-1+ cells (51%) and Fas ligand+ CD4-CD8- double-negative CD3intIL-2R beta+ T cells (26%). Thus, IL-12 and IL-18 produced by LPS-stimulated Mac-1+ cells stimulate this unique subpopulation of T cells to produce IFN-gamma, which in combination with Fas ligand, inhibits IgE production from the B cells. Our present results indicate that Mac-1+ cells and double-negative CD3intIL-2R beta+ T cells, uniquely abundant in the spleens of SJL mice, inhibit IgE production, indicating their new role in IgE response.     

Molecular characterization of IgA- and/or IgG-switched chronic lymphocytic leukemia B cells

The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a "subtype" of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/ IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH-D-JH gene sequence was joined with that of the C alpha gene in the B cells of the three IgA CLLs, and with that of the C gamma gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH-D-JH cDNA sequences were spliced to either C alpha or C gamma genes. In all five CLLs, the pattern of C mu DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the C mu exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.