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Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

Monoclonal antibodies are used in the treatment and diagnosis of diseases and to study the protective and adverse functions of antibodies in vitro and in vivo. Since the isotype determines the effector function, half-life in the serum and distribution throughout the body, it would be useful to have a battery of antibodies with the same binding site associated with different isotypes. However, since hybridomas switch isotypes at very low frequencies in tissue culture, it has been difficult and very labor intensive to isolate panels of class switch variants. We show here that stable transfection of activation-induced cytidine deaminase (AID) in hybridomas increased their frequency of switching to a level that greatly facilitated the isolation of subclones expressing monoclonal antibodies of different isotypes. Although forced expression of AID also increased the frequency of somatic hypermutation in the immunoglobulin variable regions that encode the antigen-binding site, antigen recognition was retained in the isotype switched antibodies.

A pharmacogenetic mouse model was utilized to determine the role of Cyplal expression on the formation of Ki-ras mutations in lung tumors following transplacental exposure to polycyclic aromatic hydrocarbons (PAHs). A backcross between Ah responsive male B6D2F1 mice and nonresponsive female DBA mice resulted in a litter in which both responsive and nonresponsive fetuses resided in the same nonresponsive maternal environment. Pregnant mothers received a single i.p. injection of either 10 or 30 mg/kg of 3-methylcholanthrene (MC) or olive oil vehicle on day 17 of gestation. At the higher dose of MC, the responsive offspring of both sexes had significantly (P < 0.05) higher incidences of lung tumors than their nonresponsive littermates. The male responsive mice also exhibited a significantly increased liver tumor incidence over the nonresponsive mice at the P < 0.05 level. Administration of 10 mg/kg of MC caused a very low incidence of lung tumors and did not result in the appearance of macroscopically visible liver tumors. Exons 1 and 2 of the Ki-ras gene were amplified from paraffin-embedded tissue samples. The PCR products were screened by allele-specific oligonucleotide hybridization (ASO). Thirteen of 16 lung tumors (81%) screened exhibited point mutations in the 12th or 13th codon, including seven tumors that contained GGT→GTT (GLY 12 →VAL 12 ) transversions, four which exhibited GGT→TGT (GLY 12 →CYS 12 ) transversions, and two which contained GGC→CGC (GLY 13 → ARG 13 ) transversions. None of the tumors had mutations at codon 61. The results obtained by ASO were confirmed by cloning and sequence analysis of the PCR products from four of these tumors. Within the subset of 16 tumors examined in this study, the same types of mutations in the Ki-ras gene were generally present in both responsive and nonresponsive mice, although G

Affinity for DNA and cross-reactivity with renal antigens are associated with enhanced renal pathogenicity of lupus autoantibodies. In addition, certain IgG subclasses are enriched in nephritic kidneys, suggesting that isotype may determine the outcome of antibody binding to renal antigens. To investigate if the isotype of DNA antibodies affects renal pathogenicity by influencing antigen binding, we derived IgM, IgG1, IgG2b and IgG2a forms of the PL9–11 antibody (IgG3 anti-DNA) by in vitro class switching or PCR cloning. The affinity and specificity of PL9–11 antibodies for nuclear and renal antigens were analyzed using ELISA, Western blotting, surface plasmon resonance (SPR), binding to mesangial cells, and glomerular proteome arrays. Renal deposition and pathogenicity were assayed in mice injected with PL9–11 hybridomas. We found that PL9–11 and its isotype-switched variants had differential binding to DNA and chromatin (IgG3 > IgG2a > IgG1 > IgG2b > IgM) by direct and competition ELISA, and SPR. In contrast, in binding to laminin and collagen IV the IgG2a isotype actually had the highest affinity. Differences in affinity of PL9–11 antibodies for renal antigens were mirrored in analysis of specificity for glomeruli, and were associated with significant differences in renal pathogenicity in vivo and survival. Our novel findings indicate that the constant region plays an important role in the nephritogenicity of antibodies to DNA by affecting immunoglobulin affinity and specificity. Increased binding to multiple glomerular and/or nuclear antigens may contribute to the renal pathogenicity of anti-DNA antibodies of the IgG2a and IgG3 isotype. Finally, class switch recombination may be another mechanism by which B cell autoreactivity is generated.

Activation-induced cytidine deaminase (AID) mediates the somatic hypermutation (SHM) of immunoglobulin (Ig) variable (V) regions that is required for the generation of antibody diversity and for the affinity maturation of the antibody response against infectious agents and toxic substances. AID preferentially targets WRC (W = A/T, R = A/G) hot spot motifs, particularly WGCW motifs that create overlapping hot spots on both strands. In order to gain a better understanding of the generation of antibody diversity and to create a platform for the in vitro generation of affinity-matured antibodies, we have established a system involving recombinase-mediated cassette exchange (RMCE) to replace the V region and its flanking sequences. This makes it possible to easily manipulate the sequence of the Ig gene within the endogenous heavy chain of the Ramos human Burkitt’s lymphoma cell line. Here we show that the newly integrated wild-type (WT) VH regions introduced by RMCE undergo SHM similarly to non-RMCE-modified Ramos cells. Most importantly, we have shown that introducing a cluster of WGCW motifs into the complementary determining region 2 (CDR2) of the human heavy chain V region significantly raised the mutation frequency and number of mutations per sequence compared to WT controls. Thus, we have demonstrated a novel platform in Ramos cells whereby we can easily and quickly manipulate the endogenous human VH region to further explore the regulation and targeting of SHM. This platform will be useful for generating human antibodies with changes in affinity and specificity in vitro., IMPORTANCE An effective immune response requires a highly diverse repertoire of affinity-matured antibodies. Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Although a great deal has been learned about the regulation of AID, it remains unclear how it is preferentially targeted to particular motifs, to certain locations within the Ig gene and not to other highly expressed genes in the germinal center B cell. This is an important question because AID is highly mutagenic and is sometimes mistargeted to other highly expressed genes, including proto-oncogenes, leading to B cell lymphomas. Here we describe how we utilize recombinase-mediated cassette exchange (RMCE) to modify the sequence of the endogenous heavy chain locus in the Ramos Burkitt’s lymphoma cell line. This platform can be used to explore the regulation and targeting of SHM and to generate human antibodies with changes in affinity and specificity in vitro.

To create high-affinity antibodies, B cells target a high rate of somatic hypermutation (SHM) to the Ig variable-region genes that encode the antigen-binding site. This mutational process requires transcription and is triggered by activation-induced cytidine deaminase (AID), which converts deoxycytidine to deoxyuridine. Mistargeting of AID to non-Ig genes is thought to result in the malignant transformation of B cells, but the mechanism responsible for targeting SHM to certain DNA regions and not to others is largely unknown. Cis-acting elements have been proposed to play a role in directing the hypermutation machinery, but the motifs required for targeting SHM have been difficult to identify because many of the candidate elements, such as promoters or enhancers, are also required for transcription of Ig genes. Here we describe a system in cultured hybridoma cells in which transcription of the endogenous heavy-chain Ig gene continues in the absence of the core intronic enhancer (Eμ) and its flanking matrix attachment regions (MARs). When AID is expressed in these cells, SHM occurred at the WT frequency even when Eμ and the MARs were absent together. Interestingly, SHM occurred at less than the WT frequency when Eμ or the MARs were individually absent. Our results suggest that these intronic regulatory elements can exert a complex influence on SHM that is separable from their role in regulating transcription.

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts1, is required for this process, but its exact role is unknown2. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G·C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.

Cryptococcus neoformans causes life-threatening infections in immunocompromised patients, including those with AIDS and hematological malignancies (28). Cryptococcal infection in individuals with impaired immunity has high mortality, and those who survive the acute infection often have chronic infections. Given that the therapy of cryptococcosis is unsatisfactory, there is interest in vaccine development (14). Control of C. neoformans infection is associated with cell-mediated immunity (28) and granuloma formation (23). However, there is strong evidence that humoral immunity can also be important. Antibody to the capsular glucuronoxylomannan (GXM) can prolong survival, reduce organ fungal burden, enhance granulomatous inflammation, and clear capsular polysaccharide antigen in infected mice (reviewed in references 3, 21, and 36). This suggests that vaccines which elicit high titers of protective antibodies may be useful for prevention of C. neoformans infection. GXM alone is unlikely to be an effective vaccine because it is poorly immunogenic and can be immunosuppressive (10, 26, 34). Conjugation of GXM to a protein carrier produces a highly immunogenic vaccine (6, 12, 13, 42). Mice immunized with a GXM-tetanus toxoid (GXM-TT) vaccine live longer and have lower CFU counts than controls when challenged with C. neoformans infection (12). Molecular and idiotypic analyses of antibodies to GXM produced in response to infection or GXM-TT immunization revealed restriction in variable gene usage (5, 8, 29). Murine monoclonal antibodies (MAbs) to GXM can be classified into five groups based on molecular and idiotypic characteristics (5). Group II MAbs include several protective antibodies and are defined by a heavy-chain V (variable)-region using a VH from the 7183 gene family, a seven-amino-acid D/N segment which results in a fixed-length third complementarity-determining (CDR3) region, a light-chain V region using Vκ5.1, and reactivity with the 2H1 idiotype (Id) (5). The 2H1 Id is expressed by MAb 2H1, which is the prototypical group II antibody to GXM (5). MAb 2H1 has been demonstrated to protect against C. neoformans in murine intravenous (33), intracerebral (31), intraperitoneal (i.p.) (32), and intratracheal (20) infection. The crystal structure of MAb 2H1 has been solved with and without peptide mimetics (43). A MAb with a structure similar to that of 2H1 is in advanced preclinical development for use as adjunctive therapy in cryptococcal infections (4). However, expression of 2H1 Id is not sufficient for protection since antibodies with the same V-region usage manifest large differences in protective efficacy based on isotype (44) and fine specificity (30). The molecular and cellular mechanisms which produce V-region-restricted responses are poorly understood. Here we studied the antibody response to a GXM conjugate vaccine in genetically different strains of mice, including three autoimmune strains. Our initial goal was to study the extent of V-region restriction in GXM-binding antibodies and generate MAbs different in specificities and molecular structure from those already available. We hypothesized that if restriction was the result of clonal deletion of self-reactive antibodies, then autoimmune mice would have a less restricted antibody response. Furthermore, we hypothesized that antibody responses in genetically different mouse strains might be quite different. This appeared to be confirmed by preliminary experiments with anti-idiotypic reagents to the 2H1 Id. However, molecular analysis of hybridomas from mice with 2H1 Id-negative (Id−) responses does not support either hypothesis since all mice studied made antibodies to GXM which are structurally similar to MAb 2H1. Instead, our findings suggest that V-region restriction in GXM-binding antibodies is a result of constrained requirements for antigen binding.

Previous studies from this and other laboratories have shown that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis of paraffin-embedded lung tissue for Ki-ras-2 mutations indicated that 79% of the lesions examined contained point mutations in codons 12 and 13 of the Ki-ras-2 gene locus, the majority of which (84%) were G--T transversions. The mutational spectrum was dependent on the tumor stage, as both the incidence of mutation and type of mutation produced correlated with malignant progression of the tumor. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas, and 100% of the adenocarcinomas. In the tumors with mutations, GLY12--CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas, and 14% of the adenocarcinomas. GLY12--VAL12 transversions were not observed in hyperplasias and occurred in 42% of the adenomas and 57% of the adenocarcinomas. The remaining ASP12 and ARG13 mutations occurred only in adenomas (17%) and adenocarcinomas (29%). The tumors were also analyzed for alterations in the structure or function of the tumor suppressor genes Rb, p53, and Cdkn2a. No mutations were observed in exons 5-8 of the p53 gene. SSCP analysis demonstrated that 2 of 15 lung tumors contained shifted bands at the Cdkn2a gene locus. Sequence analysis had identified these as mutations in exon 2, with a CAC--TAC transition at base 301 (HIS74--TYR74) in tumor 23-1 and GGG--GAG transition at base 350 (GLY90--GLU90) in tumor 36-1. Northern blot analysis of the larger tumors revealed that 14 of 14 of these large lung tumors exhibited markedly decreased expression of Rb gene transcripts. These results were confirmed by immunohistochemistry. The larger tumors, which exhibited features of adenocarcinomas, showed a marked reduction or almost complete absence of nuclear pRb staining compared with smaller adenomas and normal lung tissue. The results suggest that Ki-ras-2 mutations are an early and frequent event in lung tumorigenesis, and that the type of mutation produced by environmental chemicals can influence the carcinogenic potential of the tumor. The results obtained with the Cdkn2a and Rb genes suggest that alterations in the Rb regulatory axis may play a key role in the pathogenesis of the pulmonary tumors and appear to occur later in the neoplastic process. It appears from these experiments that the combination of mutated Ki-ras-2 and alterations in the Rb regulatory gene locus, which are frequent alterations in human lung tumors, may be the preferred pathway for lung tumor pathogenesis in mice exposed transplacentally to environmental carcinogens.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.

After encounter with antigen, the antibody repertoire is shaped by somatic hypermutation (SHM), which leads to an increase in the affinity of antibodies for the antigen, and class-switch recombination (CSR), which results in a change in the effector function of antibodies. Both SHM and CSR are initiated by activation-induced cytidine deaminase (AID), which deaminates deoxycytidine to deoxyuridine in single-stranded DNA (ssDNA). The precise mechanism responsible for the formation of ssDNA in V regions undergoing SHM has yet to be experimentally established. In this study, we searched for ssDNA in mutating V regions in which DNA–protein complexes were preserved in the context of chromatin in human B cell lines and in primary mouse B cells. We found that V regions that undergo SHM were enriched in short patches of ssDNA, rather than R loops, on both the coding and noncoding strands. Detection of these patches depended on the presence of DNA-associated proteins and required active transcription. Consistent with this, we found that both DNA strands in the V region were transcribed. We conclude that regions of DNA that are targets of SHM assemble protein–DNA complexes in which ssDNA is exposed, making it accessible to AID.