Biomedical subjects

Michel C Nussenzweig

Publications and source records attributed to Michel C Nussenzweig.

At least 19 recordsLinked to original sources

PubMed2007

DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes.

Optimal HIV vaccines should elicit CD8+ T cells specific for HIV proteins presented on MHC class I products, because these T cells contribute to host resistance to viruses. We had previously found that the targeting of antigen to dendritic cells (DCs) in mice efficiently induces CD8+ T cell responses. To extend this finding to humans, we introduced the HIV p24 gag protein into a mAb that targets DEC-205/CD205, an endocytic receptor of DCs. We then assessed cross-presentation, which is the processing of nonreplicating internalized antigen onto MHC class I for recognition by CD8+ T cells. Low doses of alphaDEC-gag, but not control Ig-gag, stimulated proliferation and IFN-gamma production by CD8+ T cells isolated from the blood of HIV-infected donors. alphaCD205 fusion mAb was more effective for cross-presentation than alphaCD209/DC-SIGN, another abundant DC uptake receptor. Presentation was diverse, because we identified eight different gag peptides that were recognized via DEC-205 in 11 individuals studied consecutively. Our results, based on humans with highly polymorphic MHC products, reveal that DCs and DEC-205 can cross-present several different peptides from a single protein. Because of the consistency in eliciting CD8+ T cell responses, these data support the testing of alphaDEC-205 fusion mAb as a protein-based vaccine.

Amino Acid Sequence↗

PubMed2007

Differential antigen processing by dendritic cell subsets in vivo.

Dendritic cells (DCs) process and present self and foreign antigens to induce tolerance or immunity. In vitro models suggest that induction of immunity is controlled by regulating the presentation of antigen, but little is known about how DCs control antigen presentation in vivo. To examine antigen processing and presentation in vivo, we specifically targeted antigens to two major subsets of DCs by using chimeric monoclonal antibodies. Unlike CD8+ DCs that express the cell surface protein CD205, CD8- DCs, which are positive for the 33D1 antigen, are specialized for presentation on major histocompatibility complex (MHC) class II. This difference in antigen processing is intrinsic to the DC subsets and is associated with increased expression of proteins involved in MHC processing.

Amino Acid Sequence↗

PubMed2007

Enhanced intra-switch region recombination during immunoglobulin class switch recombination in 53BP1-/- B cells.

Immunoglobulin class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. U:G mismatches created by AID are processed to produce lesions that recruit and activate DNA damage response proteins including Ataxia-telangiectasia mutated (ATM), histone H2AX, Nijmegen breakage syndrome 1 (Nbs1), and p53 binding protein 1 (53BP1). Among these proteins, absence of 53BP1 produces the most severe impairment of class switching. Here, we demonstrate that AID is targeted normally to switch region DNA and that intra-switch region recombination is enhanced in 53BP1-/- B cells. In addition, Smicro-Sgamma1 switch region junctions cloned from 53BP1-/- B cells show unusual insertions suggestive of failed class switching. Our data are consistent with a role for 53BP1 in stabilizing the synapsis of switch regions during CSR.

Animals↗

PubMed2006

The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites.

Malaria infection starts when mosquitoes inject sporozoites into the skin. The parasites enter the blood stream and make their way to the liver where they develop into the exo-erythrocytic forms (EEFs). Immunization with irradiated sporozoites (IrSp) leads to robust protection against malaria infection in rodents, monkeys and humans by eliciting antibodies to circumsporozoite protein (CS) that inhibit sporozoite infectivity, and T cells that destroy the EEFs. To study the role of non-CS antigens in protection, we produced CS transgenic mice that were tolerant to CS T-cell epitopes. Here we show that in the absence of T-cell-dependent immune responses to CS, protection induced by immunization with two doses of IrSp was greatly reduced. Thus, although hundreds of other Plasmodium genes are expressed in sporozoites and EEFs, CS is a dominant protective antigen. Nevertheless, sterile immunity could be obtained by immunization of CS transgenics with three doses of IrSp.

Animals↗

PubMed2006

Persistent expression of autoantibodies in SLE patients in remission.

A majority of the antibodies expressed by nascent B cells in healthy humans are self-reactive, but most of these antibodies are removed from the repertoire during B cell development. In contrast, untreated systemic lupus erythematosus (SLE) patients fail to remove many of the self-reactive and polyreactive antibodies from the naive repertoire. Here, we report that SLE patients in clinical remission continue to produce elevated numbers of self-reactive and polyreactive antibodies in the mature naive B cell compartment, but the number of B cells expressing these antibodies is lower than in patients with active disease. Our finding that abnormal levels of self-reactive mature naive B cells persist in the majority of patients in clinical remission suggests that early checkpoint abnormalities are an integral feature of SLE.

Adolescent↗

PubMed2006

Switching on chromosomal translocations.

Activation-induced deaminase initiates three different antibody diversification reactions: class switch recombination, somatic hypermutation (SHM), and gene conversion. We have shown that, in addition to antibody diversification, activation-induced deaminase can also initiate Burkitt's lymphoma-like c-myc/IgH translocations. However, distinct DNA damage- and oncogene-induced checkpoints operate in B cells to produce a normal intrachromosomal class switch recombination event or an aberrant interchromosomal fusion. These findings open the way to study the molecular pathways taking place at the early stages of malignant transformation.

Cell Transformation, Neoplastic↗

PubMed2006

Ig beta tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization.

Immunoglobulin (Ig)alpha and Igbeta initiate B cell receptor (BCR) signaling through immune receptor tyrosine activation motifs (ITAMs) that are targets of SH2 domain-containing kinases. To examine the function of Igbeta ITAM tyrosine resides in mature B cells in vivo, we exchanged these residues for alanine by gene targeting (Igbeta(AA)). Mutant mice showed normal development of all B cell subtypes with the exception of B1 cells that were reduced by fivefold. However, primary B cells purified from Igbeta(AA) mice showed significantly decreased steady-state and ligand-mediated BCR internalization and higher levels of cell surface IgM and IgD. BCR cross-linking resulted in decreased Src and Syk activation but paradoxically enhanced and prolonged BCR signaling, as measured by cellular tyrosine phosphorylation, Ca(++) flux, AKT, and ERK activation. In addition, B cells with the ITAM mutant receptor showed an enhanced response to a T-independent antigen. Thus, Igbeta ITAM tyrosines help set BCR signaling threshold by regulating receptor internalization.

Alanine↗

PubMed2006

Regulation of hypermutation by activation-induced cytidine deaminase phosphorylation.

Activation-induced cytidine deaminase (AID) initiates Ig class switch recombination and somatic hypermutation by producing U:G mismatches in DNA. These mismatches also have the potential to induce DNA damage including double-stranded breaks and chromosome translocations; therefore, strict regulation of AID is important for maintaining genomic stability. In addition to transcriptional regulation, it has been proposed that phosphorylation can also modulate AID activity. Using a combination of MS and immunochemical approaches we found that 5-15% of the AID expressed in activated B cells was phosphorylated at serine-38 (p38AID). This form of AID was enriched in the chromatin fraction in activated B cells, suggesting a role for phosphorylation in targeting AID to DNA. Consistent with this idea, serine-38 to alanine mutant AID (AID(S38A)) showed diminished somatic hypermutation activity on artificial and physiological DNA targets. We conclude that a small fraction of AID is phosphorylated in activated B cells and that the modified form contributes disproportionately to hypermutation.

Amino Acid Sequence↗

PubMed2006

Antigen targeting to dendritic cells elicits long-lived T cell help for antibody responses.

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses.

Adjuvants, Immunologic↗

PubMed2006

Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine.

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.

AIDS Vaccines↗

PubMed2006

T-independent type II immune responses generate memory B cells.

Unlike T-dependent immune responses against protein antigens, T-independent responses against polysaccharides confer long-lasting humoral immunity in the absence of recall responses and are not known to generate memory B cells. Here we report that polysaccharide antigens elicit memory B cells that are phenotypically distinct from those elicited by protein antigens. Furthermore, memory B cell responses against polysaccharides are regulated by antigen-specific immunoglobulin G antibodies. As the generation and regulation of immunologic memory is central to vaccination, our findings help explain the mode of action of the few existing polysaccharide vaccines and provide a rationale for a wider application of polysaccharide-based strategies in vaccination.

Animals↗

PubMed2006

A checkpoint for autoreactivity in human IgM+ memory B cell development.

Autoantibodies are removed from the repertoire at two checkpoints during B cell development in the bone marrow and the periphery. Despite these checkpoints, up to 20% of the antibodies expressed by mature naive B cells in healthy humans show low levels of self-reactivity. To determine whether self-reactive antibodies are also part of the antigen-experienced memory B cell compartment, we analyzed recombinant antibodies cloned from single circulating human IgM+ memory B cells. Cells expressing antibodies specific for individual bacterial polysaccharides were expanded in the IgM+ memory compartment. In contrast, B cells expressing self-reactive and broadly bacterially reactive antibodies were removed from the repertoire in the transition from naive to IgM+ memory B cell. Selection against self-reactive antibodies was implemented before the onset of somatic hypermutation. We conclude that a third checkpoint selects against self-reactivity during IgM+ memory B cell development in humans.

Antigens, Bacterial↗

PubMed2006

Role of genomic instability and p53 in AID-induced c-myc-Igh translocations.

Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc-Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations.

Animals↗

PubMed2006

Dynamic imaging of the immune system: progress, pitfalls and promise.

Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods.

Animals↗

PubMed2005

Inducing and expanding regulatory T cell populations by foreign antigen.

Evidence suggests that regulatory T cells expressing the transcription factor Foxp3 develop extrathymically and intrathymically. Mechanisms of extrathymic induction require further scrutiny, especially as proliferation and/or phenotypic changes of preexisting suppressor cells must be distinguished from true de novo generation. Here we report the conversion of truly naive CD4(+) T cells into suppressor cells expressing Foxp3 by targeting of peptide-agonist ligands to dendritic cells and by analysis of Foxp3 expression at the level of single cells. We show that conversion was achieved by minute antigen doses with suboptimal dendritic cell activation. The addition of transforming growth factor-beta or the absence of interleukin 2 production, which reduces proliferation, enhanced the conversion rate. In addition, regulatory T cell populations induced in subimmunogenic conditions could subsequently be expanded by delivery of antigen in immunogenic conditions. The extrathymic generation and proliferation of regulatory T cells may contribute to self-tolerance as well as the poor immunogenicity of tumors and may be exploited clinically to prevent or reverse unwanted immunity.

Animals↗

PubMed2005

AID from bony fish catalyzes class switch recombination.

Class switch recombination was the last of the lymphocyte-specific DNA modification reactions to appear in the evolution of the adaptive immune system. It is absent in cartilaginous and bony fish, and it is common to all tetrapods. Class switching is initiated by activation-induced cytidine deaminase (AID), an enzyme expressed in cartilaginous and bony fish that is also required for somatic hypermutation. Fish AID differs from orthologs found in tetrapods in several respects, including its catalytic domain and carboxy-terminal region, both of which are essential for the switching reaction. To determine whether evolution of class switch recombination required alterations in AID, we assayed AID from Japanese puffer and zebra fish for class-switching activity in mouse B cells. We find that fish AID catalyzes class switch recombination in mammalian B cells. Thus, AID had the potential to catalyze this reaction before the teleost and tetrapod lineages diverged, suggesting that the later appearance of a class-switching reaction was dependent on the evolution of switch regions and multiple constant regions in the IgH locus.

Animals↗

PubMed2005

Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo.

The maturation status of dendritic cells (DCs) determines whether they prime or tolerize T cells. We targeted ovalbumin peptide exclusively to DCs in situ using an antibody to DEC-205 and studied the interaction of DCs with naive CD4(+) T cells in tolerizing or priming conditions. We used two-photon microscopy to simultaneously track antigen-specific OT-II T cells, nonspecific T cells and DCs in lymph nodes of living mice. In both tolerance and immunity, OT-II cells arrested on DCs near high endothelial venules beginning shortly after extravasation and regained their baseline speed by 18 h. Thus, early antigen-dependent T cell arrest on DCs is a shared feature of tolerance and priming associated with activation and proliferation.

Animals↗

PubMed2005

Defective B cell tolerance checkpoints in systemic lupus erythematosus.

A cardinal feature of systemic lupus erythematosus (SLE) is the development of autoantibodies. The first autoantibodies described in patients with SLE were those specific for nuclei and DNA, but subsequent work has shown that individuals with this disease produce a panoply of different autoantibodies. Thus, one of the constant features of SLE is a profound breakdown in tolerance in the antibody system. The appearance of self-reactive antibodies in SLE precedes clinical disease, but where in the B cell pathway tolerance is first broken has not been defined. In healthy humans, autoantibodies are removed from the B cell repertoire in two discrete early checkpoints in B cell development. We found these checkpoints to be defective in three adolescent patients with SLE. 25-50% of the mature naive B cells in SLE patients produce self-reactive antibodies even before they participate in immune responses as compared with 5-20% in controls. We conclude that SLE is associated with abnormal early B cell tolerance.

Adolescent↗