RESUMEN
Activation-induced cytidine deaminase (AID) initiates class-switch recombination and somatic hypermutation (SHM) in antibody genes. Protein expression and activity are tightly controlled by various mechanisms. However, it remains unknown whether a signal from the extracellular environment directly affects the AID activity in the nucleus where it works. Here, we demonstrated that a deubiquitinase USP10, which specifically stabilizes nuclear AID protein, can translocate into the nucleus after AKT-mediated phosphorylation at its T674 within the NLS domain. Interestingly, the signals from BCR and TLR1/2 synergistically promoted this phosphorylation. The deficiency of USP10 in B cells significantly decreased AID protein levels, subsequently reducing neutralizing antibody production after immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or human immunodeficiency virus type 1 (HIV-1) nanoparticle vaccines. Collectively, we demonstrated that USP10 functions as an integrator for both BCR and TLR signals and directly regulates nuclear AID activity. Its manipulation could be used for the development of vaccines and adjuvants.
Asunto(s)
Vacunas contra el SIDA/inmunología , Factor Activador de Células B/inmunología , Vacunas contra la COVID-19/inmunología , Citidina Desaminasa/inmunología , VIH-1/inmunología , Nanopartículas , SARS-CoV-2/inmunología , Transducción de Señal/inmunología , Ubiquitina Tiolesterasa/inmunología , Ubiquitinación/inmunología , Vacunas contra el SIDA/genética , Animales , Factor Activador de Células B/genética , Vacunas contra la COVID-19/genética , Citidina Desaminasa/genética , Células HEK293 , VIH-1/genética , Humanos , Ratones , Ratones Noqueados , SARS-CoV-2/genética , Transducción de Señal/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
Ovarian cancer (OC) is one of the most malignant tumors whose mortality rate ranks first in gynecological tumors. Although immunotherapy sheds new light on clinical treatments, the low response still restricts its clinical use because of the unique characteristics of OC such as immunosuppressive microenvironment and unstable genomes. Further exploration on determining an efficient biomarker to predict the immunotherapy response of OC patients is of vital importance. In this study, integrative analyses were performed systematically using transcriptome profiles and somatic mutation data from The Cancer Genome Atlas (TCGA) based on the immune microenvironment and genomic instability of OC patients. Firstly, intersection analysis was conducted to identify immune-related differentially expressed genes (DEGs) and genomic instability-related DEGs. Secondly, Apolipoprotein B MRNA Editing Enzyme Catalytic Subunit 3A (APOBEC3A) was recognized as a protective factor for OC, which was also verified through basic experiments such as quantitative reverse transcription PCR (RT-qPCR), immunohistochemistry (IHC), Cell Counting Kit-8 (CCK-8), and transwell assays. Thirdly, the correlation analyses of APOBEC3A expression with tumor-infiltrating immune cells (TICs), inhibitory checkpoint molecules (ICPs), Immunophenoscores (IPS), and response to anti-PD-L1 immunotherapy were further applied along with single-sample GSEA (ssGSEA), demonstrating APOBEC3A as a promising biomarker to forecast the immunotherapy response of OC patients. Last, the relationship between APOBEC3A expression with tumor mutation burden (TMB), DNA damage response (DDR) genes, and m6A-related regulators was also analyzed along with the experimental verification of immunofluorescence (IF) and RT-qPCR, comprehensively confirming the intimate association of APOBEC3A with genomic instability in OC. In conclusion, APOBEC3A was identified as a protective signature and a promising prognostic biomarker for forecasting the survival and immunotherapy effect of OC patients, which might accelerate the clinical application and improve immunotherapy effect.
Asunto(s)
Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Proteínas/genética , Proteínas/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Antígeno B7-H1/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Daño del ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Humanos , Inmunoterapia , Persona de Mediana Edad , Mutación , Neoplasias Ováricas/tratamiento farmacológico , PronósticoRESUMEN
As the world is racing to develop perpetual immunity to the SARS-CoV-2 virus. The emergence of new viral strains, together with vaccination and reinfections, are all contributing to a long-term immunity against the deadly virus that has taken over the world since its introduction to humans in late December 2019. The discovery that more than 95 percent of people who recovered from COVID-19 had long-lasting immunity and that asymptomatic people have a different immune response to SARS-CoV-2 than symptomatic people has shifted attention to how our immune system initiates such diverse responses. These findings have provided reason to believe that SARS-CoV-2 days are numbered. Hundreds of research papers have been published on the causes of long-lasting immune responses and variations in the numbers of different immune cell types in COVID 19 survivors, but the main reason of these differences has still not been adequately identified. In this article, we focus on the activation-induced cytidine deaminase (AID), which initiates molecular processes that allow our immune system to generate antibodies against SARS-CoV-2. To establish lasting immunity to SARS-CoV-2, we suggest that AID could be the key to unlocking it.
Asunto(s)
COVID-19/inmunología , Citidina Desaminasa/genética , Inmunidad/genética , SARS-CoV-2/inmunología , COVID-19/virología , Citidina/genética , Citidina/inmunología , Citidina Desaminasa/inmunología , Desaminación/inmunología , Humanos , SARS-CoV-2/patogenicidad , VacunaciónRESUMEN
APOBEC3B (A3B) is one of seven human APOBEC3 DNA cytosine deaminases that restrict viral infections as part of the overall innate immune response, but it also plays a major role in tumor evolution by mutating genomic DNA. Given the importance of A3B as a restriction factor of viral infections and as a driver of multiple human cancers, selective antibodies against A3B are highly desirable for its specific detection in various research and possibly diagnostic applications. Here, we describe a high-affinity minimal antibody, designated 5G7, obtained via a phage display screening against the C-terminal catalytic domain (ctd) of A3B. 5G7 also binds APOBEC3A that is highly homologous to A3Bctd but does not bind the catalytic domain of APOBEC3G, another Z1-type deaminase domain. The crystal structure of 5G7 shows a canonical arrangement of the heavy and light chain variable domains, with their complementarity-determining region (CDR) loops lining an antigen-binding cleft that accommodates a pair of α-helices. To understand the mechanism of A3Bctd recognition by 5G7, we used the crystal structures of A3Bctd and 5G7 as templates and computationally predicted the A3B-5G7 complex structure. Stable binding poses obtained by the simulation were further tested by site-directed mutagenesis and in vitro binding analyses. These studies mapped the epitope for 5G7 to a portion of C-terminal α6 helix of A3Bctd, with Arg374 playing an essential role. The same region of A3Bctd was used previously as a peptide antigen for generating a rabbit monoclonal antibody (mAb 5210-87-13), suggesting that this region is particularly immunogenic and that these antibodies from very different origins may share similar binding modes. Our studies provide a platform for the development of selective antibodies against A3B and other APOBEC3 family enzymes.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Cristalización , Células HEK293 , Humanos , Inmunidad Innata , Simulación de Dinámica Molecular , Unión Proteica , Anticuerpos de Cadena Única/metabolismoRESUMEN
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy. The two-step BCP-ALL pathogenesis requires in utero-induced chromosomal aberrations and additional mutagenic events for overt leukemia. In mouse models, activation-induced cytidine deaminase (AID/AICDA) was suggested to contribute to BCP-ALL pathogenesis by off-target mutagenic activity. The role of AID in patients, however, remains unclear. Moreover, AID is usually not expressed in precursor B-cells but in germinal center B-cells, where it is induced upon T-helper (Th) cell stimulation. We have previously demonstrated that autologous Th-cells supportively interacted with BCP-ALL-cells. Here, we hypothesize that this interaction additionally induces AID expression in BCP-ALL-cells, leading to off-target mutagenic activity. We show that co-culture with autologous bone marrow Th-cells induced high AICDA expression in primary BCP-ALL-cells. This induction was mediated by a mechanism similar to the induction in mature B-cells involving IL-13/Stat6, CD40L/NF-κB and TGFß/Smad2/3 signaling. Even though Th-cell-induced AID seemed to be active in vitro in a BCP-ALL reporter cell line, extensive mutational signature analysis revealed no major contribution of AID activity to the mutational landscape in BCP-ALL patients. AID activity was neither detected in mutation clusters nor in known AID targets. Moreover, no recurrently mutated gene showed a relevant enrichment of mutations in the AID motif. Together, the lack of AID-induced mutational consequences argues towards a Th-cell-promoted yet AID-independent BCP-ALL pathogenesis and favors therapeutic research focusing on Th-cell-derived support of BCP-ALL-cells rather than AID-induced effects.
Asunto(s)
Médula Ósea/inmunología , Citidina Desaminasa/inmunología , Linfoma de Células B/inmunología , Mutagénesis/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adolescente , Adulto , Linfocitos B/inmunología , Línea Celular Tumoral , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Mutación/inmunología , Transducción de Señal/inmunología , Adulto JovenRESUMEN
Immunoglobulins and T cell receptors (TCR) have obvious structural similarities as well as similar immunogenetic diversification and selection mechanisms. Nevertheless, the two receptor systems and the loci that encode them are distinct in humans and classical murine models, and the gene segments comprising each repertoire are mutually exclusive. Additionally, while both B and T cells employ recombination-activating genes (RAG) for primary diversification, immunoglobulins are afforded a supplementary set of activation-induced cytidine deaminase (AID)-mediated diversification tools. As the oldest-emerging vertebrates sharing the same adaptive B and T cell receptor systems as humans, extant cartilaginous fishes allow a potential view of the ancestral immune system. In this review, we discuss breakthroughs we have made in studies of nurse shark (Ginglymostoma cirratum) T cell receptors demonstrating substantial integration of loci and diversification mechanisms in primordial B and T cell repertoires. We survey these findings in this shark model where they were first described, while noting corroborating examples in other vertebrate groups. We also consider other examples where the gnathostome common ancestry of the B and T cell receptor systems have allowed dovetailing of genomic elements and AID-based diversification approaches for the TCR. The cartilaginous fish seem to have retained this T/B cell plasticity to a greater extent than more derived vertebrate groups, but representatives in all vertebrate taxa except bony fish and placental mammals show such plasticity.
Asunto(s)
Inmunoglobulinas/genética , Mamíferos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos/genética , Tiburones/inmunología , Inmunidad Adaptativa , Animales , Citidina Desaminasa/inmunología , Evolución Molecular , Humanos , Mamíferos/genética , Tiburones/genéticaRESUMEN
There has been a longstanding question of whether affinity maturation occurs in ectotherms, and if it does, where in tissues this happens. Although cold-blooded vertebrates (ectotherms) lack histologically discernible germinal centers, they have a fully functional Ig gene mutator enzyme (activation-induced cytidine deaminase: AID or Aicda). Protein and Ig cDNA transcript analyses provide evidence that ectotherms can, under certain conditions, demonstrate antibody affinity maturation, and somatic hypermutation of their Ig genes during secondary immune responses. Here, we review the evidence for antibody affinity maturation and somatic hypermutation of Ig V(D)J exons. We argue that past evidence of long-term intact antigen retention, and recent studies of in situ expression of AID transcripts, point to fish melanomacrophage clusters as sites functionally analogous to a germinal center. Recent work in zebrafish provides a way forward to test these predictions through V(D)J repertoire analyses on isolated, intact melanomacrophage clusters. This work has implications not only for vaccine use in aquaculture, but also for antibody affinity maturation processes in all ectothermic vertebrates.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Centro Germinal/inmunología , Inmunoglobulinas/inmunología , Xenopus/inmunología , Pez Cebra/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Hipermutación Somática de Inmunoglobulina/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
Jawless vertebrates diverged from an ancestor of jawed vertebrates approximately 550 million years ago. They mount adaptive immune responses to repetitive antigenic challenges, despite lacking major histocompatibility complex molecules, immunoglobulins, T cell receptors, and recombination-activating genes. Instead of B cell and T cell receptors, agnathan lymphocytes express unique antigen receptors named variable lymphocyte receptors (VLRs), which generate diversity through a gene conversion-like mechanism. Although gnathostome antigen receptors and VLRs are structurally unrelated, jawed and jawless vertebrates share essential features of lymphocyte-based adaptive immunity, including the expression of a single type of receptor on each lymphocyte, clonal expansion of antigen-stimulated lymphocytes, and the dichotomy of cellular and humoral immunity, indicating that the backbone of the adaptive immune system was established in a common ancestor of all vertebrates. Furthermore, recent evidence indicates that, unlike previously thought, agnathans have a unique classical pathway of complement activation where VLRB molecules act as antibodies instead of immunoglobulins. It seems likely that the last common ancestor of all vertebrates had an adaptive immune system resembling that of jawless vertebrates, suggesting that, as opposed to jawed vertebrates, agnathans have retained the prototype of vertebrate adaptive immunity.
Asunto(s)
Inmunidad Adaptativa/genética , Inmunidad Adaptativa/inmunología , Vertebrados/inmunología , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Evolución Biológica , Vía Clásica del Complemento , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Citocinas/genética , Citocinas/inmunología , Inmunidad Innata , Linfocitos/citología , Linfocitos/inmunología , Receptores de Antígenos/genética , Receptores de Antígenos/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Vertebrados/genéticaRESUMEN
Allogeneic hematopoietic stem cell transplants can lead to dramatic reductions in human immunodeficiency virus (HIV) reservoirs. This effect is partially mediated by donor T cells recognizing lymphocyte-expressed minor histocompatibility antigens (mHAgs). The potential to mark malignant and latently infected cells for destruction makes mHAgs attractive targets for cellular immunotherapies. However, testing such HIV reservoir reduction strategies will likely require preclinical studies in non-human primates (NHPs). In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify an mHAg in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1∗063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8+ T cells recognized immortalized B cells but not fibroblasts from an mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs.
Asunto(s)
Citidina Desaminasa/inmunología , Macaca fascicularis/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunologíaRESUMEN
Gammaherpesviruses (GHVs) are DNA tumor viruses that establish lifelong, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immunocompromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, colonization of neither draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCE Gammaherpesviruses establish lifelong chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/inmunología , Infecciones por Herpesviridae/virología , Rhadinovirus/fisiología , Proteínas Virales/inmunología , Activación Viral , Animales , Antígenos CD19/metabolismo , Linfocitos B/virología , Diferenciación Celular , Proliferación Celular , Centro Germinal/inmunología , Centro Germinal/virología , Infecciones por Herpesviridae/inmunología , Tejido Linfoide/inmunología , Tejido Linfoide/virología , Ratones , Rhadinovirus/genética , Rhadinovirus/metabolismo , Proteínas Virales/genética , Latencia del VirusRESUMEN
Patients with common variable immunodeficiency associated with autoimmune cytopenia (CVID+AIC) generate few isotype-switched B cells with severely decreased frequencies of somatic hypermutations (SHMs), but their underlying molecular defects remain poorly characterized. We identified a CVID+AIC patient who displays a rare homozygous missense M466V mutation in ß-catenin-like protein 1 (CTNNBL1). Because CTNNBL1 binds activation-induced cytidine deaminase (AID) that catalyzes SHM, we tested AID interactions with the CTNNBL1 M466V variant. We found that the M466V mutation interfered with the association of CTNNBL1 with AID, resulting in decreased AID in the nuclei of patient EBV-transformed B cell lines and of CTNNBL1 466V/V Ramos B cells engineered to express only CTNNBL1 M466V using CRISPR/Cas9 technology. As a consequence, the scarce IgG+ memory B cells from the CTNNBL1 466V/V patient showed a low SHM frequency that averaged 6.7 mutations compared with about 18 mutations per clone in healthy-donor counterparts. In addition, CTNNBL1 466V/V Ramos B cells displayed a decreased incidence of SHM that was reduced by half compared with parental WT Ramos B cells, demonstrating that the CTNNBL1 M466V mutation is responsible for defective SHM induction. We conclude that CTNNBL1 plays an important role in regulating AID-dependent antibody diversification in humans.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Linfocitos B , Inmunodeficiencia Variable Común , Homocigoto , Memoria Inmunológica/genética , Mutación Missense , Proteínas Nucleares , Hipermutación Somática de Inmunoglobulina , Sustitución de Aminoácidos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular , Preescolar , Inmunodeficiencia Variable Común/genética , Inmunodeficiencia Variable Común/inmunología , Inmunodeficiencia Variable Común/patología , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Femenino , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/inmunologíaRESUMEN
The adaptive immune systems of all vertebrates rely on self-DNA mutating enzymes to assemble their antigen receptors in lymphocytes of their two principal lineages. In jawed vertebrates, the RAG1/2 recombinase directs V(D)J recombination of B cell and T cell receptor genes, whereas the activation-induced cytidine deaminase AID engages in their secondary modification. The recombination activating genes (RAG) 1 and 2 evolved from an ancient transposon-encoded genome modifier into a self-DNA mutator serving adaptive immunity; this was possible as a result of domestication, involving several changes in RAG1 and RAG2 proteins suppressing transposition and instead facilitating-coupled cleavage and recombination. By contrast, recent evidence supports the notion that the antigen receptors of T-like and B-like cells of jawless vertebrates, designated variable lymphocyte receptors (VLRs), are somatically assembled through a process akin to gene conversion that is believed to be dependent on the activities of distant relatives of AID, the cytidine deaminases CDA1 and CDA2, respectively. It appears, therefore, that the precursors of AID and CDAs underwent a domestication process that changed their target range from foreign nucleic acids to self-DNA; this multi-step evolutionary process ensured that the threat to host genome integrity was minimized. Here, we review recent findings illuminating the evolutionary steps associated with the domestication of the two groups of genome editors, RAG1/2 and cytidine deaminases, indicating how they became the driving forces underlying the emergence of vertebrate adaptive immune systems.
Asunto(s)
Inmunidad Adaptativa/genética , Citidina Desaminasa/genética , Proteínas de Unión al ADN/genética , Edición Génica , Inmunidad Adaptativa/inmunología , Animales , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN/inmunología , Humanos , Vertebrados/inmunologíaRESUMEN
Previously, we developed a CHO cell display-based antibody maturation procedure in which an antibody (or other protein) gene of interest was induced to mutate by activation-induced cytidine deaminase (AID) and then form a library by simply proliferating the CHO cells in culture. In this study, we further improved the efficiency of this maturation system by reengineering AID, and optimizing the nucleic acid sequence of the target antibody gene and AID gene as well as the protocol for AID gene transfection. These changes have increased both the mutation rate and the number of mutation type of antibody genes by more than 10 fold, and greatly improved the maturation efficiency of antibody/other proteins.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Biblioteca de Genes , Mutación , Anticuerpos de Cadena Única/biosíntesis , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Células CHO , Cricetinae , Cricetulus , Humanos , Tasa de Mutación , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
In addition to canonical TCR and BCR, cartilaginous fish assemble noncanonical TCR that employ various B-cell components. For example, shark T cells associate alpha (TCR-α) or delta (TCR-δ) constant (C) regions with Ig heavy chain (H) variable (V) segments or TCR-associated Ig-like V (TAILV) segments to form chimeric IgV-TCR, and combine TCRδC with both Ig-like and TCR-like V segments to form the doubly rearranging NAR-TCR. Activation-induced (cytidine) deaminase-catalyzed somatic hypermutation (SHM), typically used for B-cell affinity maturation, also is used by TCR-α during selection in the shark thymus presumably to salvage failing receptors. Here, we found that the use of SHM by nurse shark TCR varies depending on the particular V segment or C region used. First, SHM significantly alters alpha/delta V (TCRαδV) segments using TCR αC but not δC. Second, mutation to IgHV segments associated with TCR δC was reduced compared to mutation to TCR αδV associated with TCR αC. Mutation was present but limited in V segments of all other TCR chains including NAR-TCR. Unexpectedly, we found preferential rearrangement of the noncanonical IgHV-TCRδC over canonical TCR αδV-TCRδC receptors. The differential use of SHM may reveal how activation-induced (cytidine) deaminase targets V regions.
Asunto(s)
Citidina Desaminasa/inmunología , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T/genética , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T/genética , Cadenas Pesadas de Inmunoglobulina/genética , Tiburones/inmunología , Hipermutación Somática de Inmunoglobulina/genética , Animales , Citidina Desaminasa/genética , Tiburones/genéticaRESUMEN
The antibodies of jawless vertebrates consist of leucine-rich repeat arrays encoded by somatically assembled VLRB genes. It is unknown how the incomplete germline VLRB loci are converted into functional antibody genes during B lymphocyte development in lampreys. In Lampetra planeri larvae lacking the cytidine deaminase CDA2 gene, VLRB assembly fails, whereas the T lineage-associated VLRA and VLRC antigen receptor gene assemblies occur normally. Thus, CDA2 acts in a B cell lineage-specific fashion to support the somatic diversification of VLRB antibody genes. CDA2 is closely related to activation-induced cytidine deaminase (AID), which is essential for the elaboration of immunoglobulin gene repertoires in jawed vertebrates. Our results thus identify a convergent mechanism of antigen receptor gene assembly and diversification that independently evolved in the two sister branches of vertebrates.
Asunto(s)
Anticuerpos Monoclonales/genética , Citidina Desaminasa/genética , Lampreas/genética , Receptores de Antígenos/genética , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Lampreas/inmunología , Lampreas/metabolismo , Receptores de Antígenos/inmunología , Receptores de Antígenos/metabolismoRESUMEN
Plasma cells (PCs) in human palatine tonsils are predominantly located in the germinal centres (GCs), in the subepithelial space and near the deep connective tissue septa surrounding each crypt. We analysed the location, phenotype, and proliferation of GC PCs by immunohistology comparing them to PCs in the other two locations. Most PCs in GCs were strongly positive for CD38, CD138, CD27, IRF4, and intracellular (ic) IgG. They often accumulated in the basal light zone, but could also be found scattered in the entire light zone. In addition, rows of PCs occurred at the surface of the GC bordering the mantle zone, i.e., in the outer zone, and at the surface of the dark zone. The latter cells were often continuous with PCs in the extrafollicular area. The vast majority of GC PCs were negative for Ki-67. Only a few Ki-67+ plasmablasts, predominantly icIgG+ or icIgM+, were found inside GCs. In certain GCs PCs accumulated around capillaries and the adjacent perikarya of follicular dendritic cells (FDCs). Newly formed PCs might migrate from the basal to the superficial part of the light zone and then back to the dark zone surface to leave the GC. This guarantees an even distribution of secreted Ig for exchange with immune complexes on FDCs. The surface of the dark zone may also be an exit site for Ki-67+CD30+ B lymphoblasts, which seed perifollicular and extrafollicular sites. We speculate that these cells tend to downmodulate CD20 and activation-induced deaminase and further up-regulate CD30 when developing into pre-plasmablasts.
Asunto(s)
Linfocitos B/citología , Citidina Desaminasa/inmunología , Centro Germinal/citología , Antígeno Ki-1/inmunología , Tonsila Palatina/citología , Células Plasmáticas/citología , Linfocitos B/inmunología , Proliferación Celular , Citidina Desaminasa/metabolismo , Centro Germinal/inmunología , Humanos , Tonsila Palatina/inmunología , Fenotipo , Células Plasmáticas/inmunologíaRESUMEN
While cancer cells gain aggressiveness by mutations, abundant mutations release neoantigens, attracting anti-cancer immune cells. We hypothesized that in breast cancer (BC), where mutation is less common, tumors with high mutation rates demonstrate aggressive phenotypes and attract immune cells simultaneously. High mutation rates were defined as the top 10% of the mutation rate, utilizing TCGA and METABRIC transcriptomic data. Mutation rate did not impact survival although high mutation BCs were associated with aggressive clinical features, such as more frequent in ER-negative tumors (p < 0.01), in triple-negative subtype (p = 0.03), and increased MKI-67 mRNA expression (p < 0.01) in both cohorts. Tumors with high mutation rates were associated with APOBEC3B and homologous recombination deficiency, increasing neoantigen loads (all p < 0.01). Cell proliferation and immune activity pathways were enriched in BCs with high mutation rates. Furthermore, there were higher lymphocytes and M1 macrophage infiltration in high mutation BCs. Additionally, T-cell receptor diversity, cytolytic activity score (CYT), and T-cell exhaustion marker expression were significantly elevated in BCs with high mutation rates (all p < 0.01), indicating strong immunogenicity. In conclusion, enhanced immunity due to neoantigens can be one of possible forces to counterbalance aggressiveness of a high mutation rate, resulting in similar survival rates to low mutation BCs.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Inmunidad/inmunología , Mutación/genética , Proliferación Celular/genética , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Inmunidad/genética , Linfocitos/inmunología , Macrófagos/inmunología , Persona de Mediana Edad , Mutación/inmunología , Tasa de Mutación , Fenotipo , ARN Mensajero/genética , ARN Mensajero/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Tasa de SupervivenciaRESUMEN
APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Citidina Desaminasa/inmunología , Inmunoterapia/métodos , Antígenos de Histocompatibilidad Menor/inmunología , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Resistencia a Antineoplásicos , Epítopos/inmunología , Femenino , Humanos , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Melanoma/terapia , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Mutación , Escape del Tumor/efectos de los fármacosRESUMEN
Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells, macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR), while decreasing at higher doses over a broad physiological range, AID and Blimp1 expression, CSR, somatic hypermutation and plasma cell differentiation. In human and mouse B cells, butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3'UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors, not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes, these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models.
Asunto(s)
Anticuerpos/genética , Epigénesis Genética/efectos de los fármacos , Ácidos Grasos Volátiles/farmacología , Microbioma Gastrointestinal/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Butiratos/farmacología , Citidina Desaminasa/antagonistas & inhibidores , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Citidina Desaminasa/metabolismo , Fibras de la Dieta , Ácidos Grasos Volátiles/aislamiento & purificación , Ácidos Grasos Volátiles/farmacocinética , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Inhibidores de Histona Desacetilasas/inmunología , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos C57BL , Ratones Mutantes , Factor 1 de Unión al Dominio 1 de Regulación Positiva/antagonistas & inhibidores , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Propionatos/farmacología , Distribución TisularRESUMEN
Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex-mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73+ B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.