High-Dose IV Administration of Rasburicase Suppresses Anti-rasburicase Antibodies, Depletes Rasburicase-Specific Lymphocytes, and Upregulates Treg Cells.

Therapeutic proteins can be potent agents for treating serious diseases, but in many patients these proteins provoke antibody responses that blunt therapeutic efficacy. Intravenous administration of high doses of some proteins induces immune tolerance, but the mechanisms underlying this effect are poorly understood. As a model to study tolerance induction in mice, we used rasburicase, a commercial recombinant uricase used for the treatment of hyperuricemia. Intraperitoneal (i.p.) injection of rasburicase without or with alum adjuvants induced a clear anti-rasburicase antibody response, but intravenous (i.v.) injection did not. The lack of response to i.v. rasburicase was apparently due to active immune suppression since i.v.-treated mice showed blunted antibody and reduced T cell responses to subsequent i.p. injections of rasburicase. This blunted response was associated with a decrease in rasburicase-specific B cell and T cell responses and an increase in proportion of CD4+ FoxP3+ regulatory T cells (Treg) in the spleen. We examined the number of lymphocytes in peripheral blood after rasburicase i.v. injection. Rasburicase caused a transient reduction in B and T cells, but a robust and sustained depletion of rasburicase-specific B cells. Further experiments showed that rasburicase i.v. injection decreased the number of lymphocytes and was associated with apoptosis of both B cells and activated T cells and that the enhanced percentage of Treg cells was likely mediated by a macrophage-dependent pathway. Thus, our data suggest that apoptosis and depletion of antigen-specific B lymphocytes and upregulation of Treg cells may play important roles in the immune suppression induced by intravenous administration of a therapeutic protein.

A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus.

UNLABELLED: Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design. IMPORTANCE: Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity. This question is particularly relevant to developing more effective vaccines for influenza A virus (IAV) and other viruses that are difficult vaccine targets. Limitations in methods for characterizing antigen-specific B cells have impeded progress in characterizing the quality of immune responses to vaccine and natural immunogens. In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies. Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses. This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.

Single-strand DNA breaks in Ig class switch recombination that depend on UNG but not AID.

B lymphocytes switch from secreting IgM to secreting IgG, IgA or IgE through a DNA recombination, class switch recombination (CSR), whose mechanism is incompletely understood. CSR is thought to be triggered by activation-induced deaminase (AID), which is believed to deaminate cytosines to uracil in single-strand regions of switch region DNA. Subsequent excision of uracils by uracil DNA glycosylase (UNG) (product of the UNG gene) generates abasic sites, which are targeted for DNA cleavage, producing DNA breaks that are critical intermediates in CSR. Consistent with this model, CSR-related double-strand breaks (DSBs)--detected by ligation-mediated PCR (LMPCR)--have been reported to be dramatically reduced in B cells from either AID(-/-) or UNG(-/-) mice. Here we examine single-strand breaks (SSBs) using LMPCR and report, surprisingly, that CSR-related anti-sense strand breaks in Sgamma regions are dependent only on UNG, and not AID, suggesting participation of a cytosine deaminase other than AID. This conclusion is supported by the sequences at these DNA breaks, which show a bias for a consensus sequence different from that reported for AID. The SSBs appear to be part of the normal CSR pathway since in B cells in which CSR is blocked by deletion of Smu, the content of Sgamma SSBs is elevated as though the breaks resolve inefficiently owing to the lack of a recombination partner for completing mu-to-gamma CSR. These results suggest a narrower role for AID in CSR than previously recognized and prompt a search for a putative alternative cytosine deaminase participating in CSR.

Dynamic changes in accessibility, nuclear positioning, recombination, and transcription at the Ig kappa locus.

The 3-megabase Igkappa locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Igkappa locus was determined in a murine pre-B cell line that can be induced to recombine at the Igkappa locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Igkappa locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific Vkappa genes were observed. Histone modifications of the Vkappa genes did not vary upon induction but the levels of modifications correlated with the levels of germline Vkappa gene transcripts and recombination. Upon induction, but before VkappaJkappa recombination, centromeric recruitment of single Igkappa alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the Vkappa genes was increased in the silencer regions upstream of Jkappa1, within the Igkappa sterile transcript, the kappa constant region, the Ekappai and Ekappa3' enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the Vkappa genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.

Immunoglobulin class switch recombination is impaired in Atm-deficient mice.

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell-dependent antigen (Ag), Atm-/- mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm-/- B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm-/- cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm-/- B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm-/- B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm-/- B cells occurs at the level of genomic DNA recombination as measured by digestion-circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the mu-gamma1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

Single-stranded DNA breaks adjacent to cytosines occur during Ig gene class switch recombination.

Class switch recombination (CSR) at the DNA level underlies ability of B lymphocytes to switch from expressing IgM to expressing IgG, IgA, or IgE. The mechanism of CSR is largely unknown, but it is clear that CSR is stimulated by T cell signals and is mediated in part by activation-induced deaminase (AID), an enzyme that is also required for somatic hypermutation of Ig genes. In one current model, AID is proposed to initiate CSR by deaminating cytosines in the unpaired nontemplate strand of DNA displaced from its complementary strand by the "sterile" RNA transcript across the switch region. We have used LM-PCR to analyze single-strand breaks in CH12F3-2, a murine cell line that switches in vitro to IgA expression. In contrast to the above model, we have detected CSR-associated ssDNA breaks in the template strand of the H chain alpha switch region, the strand thought to be complexed with RNA. Most breaks are adjacent to cytosines, consistent with mediation by AID, and occur within the novel consensus sequence C*AG, which occurs much more frequently on the template strand than on the putatively displaced nontemplate strand. These results suggest that AID may target the DNA strand bound to RNA, perhaps resembling APOBEC-3G, a cytosine deaminase related to AID that inhibits HIV replication by mutating viral DNA. Furthermore, the absence of detectable breaks in the nontemplate strand within the DNA segment under study suggests that the two DNA strands are handled differently in the generation or processing of strand breaks.

Complex mechanisms for inhibition of immunoglobulin gene expression in a germinal center B cell line.

CD40 ligation and IL-4 stimulation are critical Th2 cell-derived signals that act on germinal center B cells to stimulate immunoglobulin isotype switching. In addition to this well-known effect, these same Th2 signals have also been reported to inhibit ongoing immunoglobulin synthesis in germinal center B cells. To study the mechanism of this inhibition, we have investigated which immunoglobulin gene regulatory regions might be affected by IL-4 and CD40 Ligand (CD40L). CL-01 cells, a human B cell line of germinal center phenotype, were transiently transfected with luciferase reporter constructs containing various light and heavy chain enhancers and promoters; the cells were then incubated with or without CD40L and IL-4 and then assayed for luciferase expression. We find that the intronic enhancer of the kappa light chain (but not the heavy chain) is upregulated by CD40 ligation, but that VH and Vkappa promoters and the 3' enhancers of both the kappa and heavy chain loci are inhibited by CD40 ligation and the Th2 cytokines IL-4 and IL-10. The inhibitory response of the 3'alpha enhancer can be observed with a 130 bp core fragment of the enhancer, and remains unaffected by mutations in several motifs known or suspected to contribute to enhancer function. The ultimate effects of cytokines and CD40 ligation on immunoglobulin gene transcription therefore represent a complex integration of positive and negative stimuli acting on enhancers and promoters.

Detection of isotype switch rearrangement in bulk culture by PCR.

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)-based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion-circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.