Latent intact HIV-1 proviruses persist in a small subset of long-lived CD4+ T cells that can undergo clonal expansion in vivo. Expanded clones of CD4+ T cells dominate latent reservoirs in ...individuals on long-term antiretroviral therapy (ART) and represent a major barrier to HIV-1 cure. To determine how integration landscape might contribute to latency, we analyzed integration sites of near full length HIV-1 genomes from individuals on long-term ART, focusing on individuals whose reservoirs are highly clonal. We find that intact proviruses in expanded CD4+ T cell clones are preferentially integrated within Krüppel-associated box (KRAB) domain-containing zinc finger (ZNF) genes. ZNF genes are associated with heterochromatin in memory CD4+ T cells; nevertheless, they are expressed in these cells under steady-state conditions. In contrast to genes carrying unique integrations, ZNF genes carrying clonal intact integrations are down-regulated upon cellular activation. Together, the data suggest selected genomic sites, including ZNF genes, can be especially permissive for maintaining HIV-1 latency during memory CD4+ T cell expansion.
Despite suppressive combination antiretroviral therapy (ART), latent HIV-1 proviruses persist in patients. This latent reservoir is established within 48-72 h after infection, has a long half-life
, ...enables viral rebound when ART is interrupted, and is the major barrier to a cure for HIV-1
. Latent cells are exceedingly rare in blood (∼1 per 1 × 10
CD4
T cells) and are typically enumerated by indirect means, such as viral outgrowth assays
. We report a new strategy to purify and characterize single reactivated latent cells from HIV-1-infected individuals on suppressive ART. Surface expression of viral envelope protein was used to enrich reactivated latent T cells producing HIV RNA, and single-cell analysis was performed to identify intact virus. Reactivated latent cells produce full-length viruses that are identical to those found in viral outgrowth cultures and represent clones of in vivo expanded T cells, as determined by their T cell receptor sequence. Gene-expression analysis revealed that these cells share a transcriptional profile that includes expression of genes implicated in silencing the virus. We conclude that reactivated latent T cells isolated from blood can share a gene-expression program that allows for cell division without activation of the cell death pathways that are normally triggered by HIV-1 replication.
Chronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt’s lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc ...oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells.
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•Capture of chromosome translocations occurring in germinal center B cells in vivo•AID promotes Plasmodium-associated B cell lymphoma•Chronic infection causes a shift toward a more mature lymphoma phenotype•Plasmodium-induced lymphomas bear chromosome translocations, including c-myc/Igh
Chronic infection with Plasmodium parasites induces widespread genomic instability and favors mature B cell cancers by eliciting protracted AID expression in GC B cells, explaining the association between malaria and development of Burkitt’s lymphoma.
HIV-1–infected individuals harbor a latent reservoir of infected CD4⁺ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and ...has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.
The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are ...susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA+ enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.
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•AID targets are interconnected in 3D nuclear space•Targeted networks overlap predominantly with super-enhancer domains•Both promoters and transcriptionally active cognate enhancers are damaged by AID•Tethered regulatory elements cooperate to recruit AID activity
AID targets in the genome are interconnected in 3D networks that overlap with super-enhancer domains, revealing the role of the nuclear architecture and the B cell regulome in recruiting AID activity.
Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate ...oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.
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► The Pol II stalling factor, Spt5, is required for CSR ► Spt5 interaction with AID is important for AID-Pol II association ► Spt5 colocalizes with AID at sites of Pol II stalling genome-wide in B cells ► Sites of high-density Spt5 and Pol II stalling are predictive of AID-mediated mutation
Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The ...circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4⁺ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti–HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.
Abstract
Attempts to create an effective, traditional vaccine against several pathogens such as HIV have so far failed and current research suggests that in the case of HIV protective serum responses ...mediated by broadly-neutralizing antibodies (bNAbs) will only be achieved by multiple, sequential immunizations. Identifying, producing and implementing a regimen consisting of a series of immunogens that lead to a relatively consistent, protective immune response in a heterogeneous population will be highly challenging. We are developing an alternative approach in which autologous primary B cells are edited to express an antibody of choice, in this case a bNAb against HIV. Following adoptive transfer the individual is immunized with a cognate antigen to activate the edited B cells and generate a protective, humoral immune response to HIV. This method can be adapted for any combination of high-affinity antibody and antigen. We have developed a CRISPR/Cas9-based method to target the endogenous IgH and IgK loci in mice. Both loci must be targeted in order to avoid producing B cells with chimeric and potentially self-reactive receptors. In addition, both of the bNAb encoding chains must be coordinately expressed. To do so we assemble an RNP consisting of Cas9 and single stranded DNA (ssDNA) encoding IgK followed by a P2A self-cleaving oligopeptide and the VDJ exon. A separate Cas9/guide complex is included to ablate the endogenous IgK locus. Since the heavy chain variable region is knocked into the endogenous locus in between the VDJ and constant regions normal isotype switching can occur and only on-target integrations can express the full BCR of choice. B cell survival is dependent on Ig expression and therefore all of the B cells that have lost Igk expression and do not properly integrate the bNAb will die. Using this approach, we have created cells carrying a synthetic intermediate of the anti-HIV bNAb 3BNC60. Transfer of 3BNC60 synthetic intermediate-edited cells into congenically marked wild-type mice followed by immunization with a cognate Env-derived antigen showed that the edited cells were able to start an immune response and create 3BNC60 synthetic intermediate serum antibody with the predicted epitope specificity of different isotypes. One of the potential benefits to this approach is that it would not be limited to a single monoclonal. B cells could be edited to express different antibodies and then be transferred together. Our initial experiments show that the approach is feasible but the process must be optimized to become practical; particularly, the number of correctly edited B cells needs to be increased for efficient transfer. We are also working on translating these experiments from mice to macaques in order to do protection experiments.We believe the approach presented here would have wide-ranging implications for both research and public health. It boasts several advantages compared to traditional vaccination, particularly in the context of HIV as it skips bNAb evolution, can potentially combine more than one type of bNAb and, as opposed to a sequential immunization, could potentially be achieved in a two-visit procedure (blood draw followed by injections). Since the approach uses primary B cells that are modified in their endogenous loci, which then mature into plasma cells in vivo, and not other cell types ectopically expressing an antibody, the immune response should in principle be boostable, long-lived and allow for bNAb diversification by somatic hypermutation. As opposed to an immunogen series in a sequential immunization, our approach is not limited to a particular bNAb and relies only on a functional bNAb and antigen combination as opposed to an entire series of antigens. Lastly, this approach is not limited to HIV and could be applicable to other diseases in which constant antibody intervention is indicated.
Citation Format: Harald Hartweger, Mila Jankovic, Michel C. Nussenzweig. Protective serum responses by CRISPR/Cas9-edited primary B cells expressing antibodies of choice abstract. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B113.
A clinical trial was performed to evaluate 3BNC117, a potent anti-HIV-1 antibody, in infected individuals during suppressive antiretroviral therapy and subsequent analytical treatment interruption ...(ATI). The circulating reservoir was evaluated by quantitative and qualitative viral outgrowth assay (Q
VOA) at entry and after 6 mo. There were no significant quantitative changes in the size of the reservoir before ATI, and the composition of circulating reservoir clones varied in a manner that did not correlate with 3BNC117 sensitivity. 3BNC117 binding site amino acid variants found in rebound viruses preexisted in the latent reservoir. However, only 3 of 217 rebound viruses were identical to 868 latent viruses isolated by Q
VOA and near full-length sequencing. Instead, 63% of the rebound viruses appeared to be recombinants, even in individuals with 3BNC117-resistant reservoir viruses. In conclusion, viruses emerging during ATI in individuals treated with 3BNC117 are not the dominant species found in the circulating latent reservoir, but frequently appear to represent recombinants of latent viruses.
Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear ...interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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