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Long-term survivors of human immunodeficiency virus (HIV) infection have been shown to have a greatly increased incidence of B cell lymphomas. This increased lymphomagenesis suggests some link between HIV infection and the destabilization of the host B cell genome, a phenomenon also suggested by the extraordinary high frequency of mutation, insertion, and deletion in the broadly neutralizing HIV antibodies. Since HIV does not infect B cells, the molecular mechanisms of this genomic instability remain to be fully defined. Here, we demonstrate that the cell membrane-permeable HIV Tat proteins enhance activation-induced deaminase (AID)-mediated somatic hypermutation (SHM) of antibody V regions through their modulation of the endogenous polymerase II (Pol II) transcriptional process. Extremely small amounts of Tat that could come from bystander HIV-infected cells were sufficient to promote SHM. Our data suggest HIV Tat is one missing link between HIV infection and the overall B cell genomic instability in AIDS patients., IMPORTANCE Although the introduction of antiretroviral therapy (ART) has successfully controlled primary effects of human immunodeficiency virus (HIV) infection, such as HIV proliferation and HIV-induced immune deficiency, it did not eliminate the increased susceptibility of HIV-infected patients to B cell lymphomas. We find that a secreted HIV protein, Tat, enhances the intrinsic antibody diversification mechanism by increasing the AID-induced somatic mutations at the heavy-chain variable (VH) regions in human B cells. This could contribute to the high rate of mutation in the variable regions of broadly neutralizing anti-HIV antibodies and the genomewide mutations leading to B cell malignancies in HIV carriers.

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.

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.

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.

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.