RESUMO
Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.
Assuntos
Citidina Desaminase , Hipermutação Somática de Imunoglobulina , Animais , Camundongos , Anticorpos/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , DNA de Cadeia Simples , Mutação , Evolução Molecular , Regiões Determinantes de Complementaridade/genética , Motivos de NucleotídeosRESUMO
In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss the implications of these findings for harnessing antibody diversification mechanisms.
Assuntos
Linfócitos B/metabolismo , Citidina Desaminase/genética , Switching de Imunoglobulina , Hipermutação Somática de Imunoglobulina , Recombinação V(D)J , Animais , Humanos , Camundongos , Mutação , Globinas beta/genéticaRESUMO
The antigen-binding variable regions of the B cell receptor (BCR) and of antibodies are encoded by exons that are assembled in developing B cells by V(D)J recombination1. The BCR repertoires of primary B cells are vast owing to mechanisms that create diversity at the junctions of V(D)J gene segments that contribute to complementarity-determining region 3 (CDR3), the region that binds antigen1. Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs)2,3. Although most GCs are transient3, those in intestinal Peyer's patches (PPs)-which depend on the gut microbiota-are chronic4, and little is known about their BCR repertoires or patterns of somatic hypermutation. Here, using a high-throughput assay that analyses both V(D)J segment usage and somatic hypermutation profiles, we elucidate physiological BCR repertoires in mouse PP GCs. PP GCs from different mice expand public BCR clonotypes (clonotypes that are shared between many mice) that often have canonical CDR3s in the immunoglobulin heavy chain that, owing to junctional biases during V(D)J recombination, appear much more frequently than predicted in naive B cell repertoires. Some public clonotypes are dependent on the gut microbiota and encode antibodies that are reactive to bacterial glycans, whereas others are independent of gut bacteria. Transfer of faeces from specific-pathogen-free mice to germ-free mice restored germ-dependent clonotypes, directly implicating BCR selection. We identified somatic hypermutations that were recurrently selected in such public clonotypes, indicating that affinity maturation occurs in mouse PP GCs under homeostatic conditions. Thus, persistent gut antigens select recurrent BCR clonotypes to seed chronic PP GC responses.
Assuntos
Afinidade de Anticorpos/genética , Centro Germinativo/citologia , Centro Germinativo/imunologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Fezes/microbiologia , Microbioma Gastrointestinal/imunologia , Genes de Cadeia Pesada de Imunoglobulina/genética , Vida Livre de Germes , Homeostase , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Seleção Genética , Hipermutação Somática de Imunoglobulina/genética , Recombinação V(D)J/genéticaRESUMO
Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.
Assuntos
Citidina Desaminase/metabolismo , DNA/metabolismo , Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Recombinação Genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Diversidade de Anticorpos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Citidina Desaminase/química , Citidina Desaminase/genética , DNA/química , DNA/genética , Humanos , Camundongos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Baço/enzimologia , Baço/imunologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1-infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Linfócitos B/metabolismo , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Mutação , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais Murinos/genética , Anticorpos Neutralizantes/genética , Anticorpos Anti-HIV/genética , CamundongosRESUMO
For decades, intravenous immunoglobulin (IVIg) has provided safe and effective therapy for immunodeficient patients. This proof-of-principle study describes a novel approach to generate personalized IVIg for chronic, antibiotic-resistant infection in real time.
Assuntos
Antibacterianos/uso terapêutico , Terapia Biológica , Imunoglobulinas Intravenosas/uso terapêutico , Infecções por Mycoplasma/terapia , Medicina de Precisão , Infecção da Ferida Cirúrgica/terapia , Idoso , Animais , Bovinos , Humanos , Imunoglobulinas Intravenosas/efeitos adversos , Síndromes de Imunodeficiência/terapia , Masculino , Infecções por Mycoplasma/diagnóstico , Mycoplasma hominis/efeitos dos fármacos , Estudo de Prova de Conceito , Infecção da Ferida Cirúrgica/microbiologiaRESUMO
Natural killer (NK) cells kill target cells following triggering via germline-encoded receptors interacting with target cell-expressed ligands (direct killing), or via antibody-dependent cellular cytotoxicity (ADCC) mediated by FcγRIIIa. NK cytotoxicity is modulated by signaling through activating or inhibitory receptors. A major checkpoint is mediated by the NK inhibitory receptor NKG2A/CD94 and its target cell ligand, HLA-E, which is complexed with HLA signal sequence-derived peptides termed VL9 (HLA-E-VL9). We have previously reported the isolation of a murine HLA-E-VL9-specific IgM antibody 3H4 and the generation of a higher affinity IgG version (3H4v3). Here we have used phage display library selection to generate a high affinity version of 3H4v3, called 3H4v31, with an â¼700 fold increase in binding affinity. We show using an HLA-E-VL9+ K562 tumor model that, in vitro, the addition of 3H4v31 to target cells increased direct killing of targets by CD16-negative NK cell line NK-92 and also mediated ADCC by NK-92 cells transfected with CD16. Moreover, ADCC by primary NK cells was also enhanced in vitro by 3H4v31. 3H4v31 was also able to bind and enhance target cell lysis of endogenously expressed HLA-E-VL9 on human cervical cancer and human pancreatic cancer cell lines. In vivo, 3H4v31 slowed the growth rate of HLA-E-VL9+ K562 tumors implanted into NOD/SCID/IL2rγ null mice compared to isotype control when injected with NK-92 cells intratumorally. Together, these data demonstrate that mAb 3H4v31 can enhance NK cell killing of HLA-E-VL9-expressing tumor cells in vitro by both direct killing activity and by ADCC. Moreover, mAb 3H4v31 can enhance NK cell control of tumor growth in vivo. We thus identify HLA-E-VL9 monoclonal antibodies as a promising novel anti-tumor immunotherapy. One Sentence Summary: A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing by checkpoint inhibition and antibody dependent cellular cytotoxicity.
RESUMO
The discrimination of benign from malignant lymphoproliferative disorders is sometimes difficult because there can be overlap in their histological and immunophenotypic features. In such situations, molecularly based clonality testing is often used to discriminate benign (polyclonal) from malignant (monoclonal) processes. Clonality testing by polymerase chain reaction (PCR) has a number of pitfalls that may result in spurious results. Here we report the case of a woman diagnosed by 2 major academic institutions with hepatosplenic T-cell lymphoma based on a dense infiltration of the spleen by a γδ T-cell population with mild cytologic atypia, resulting in expansion of the splenic red pulp, and a positive T-cell receptor clonality test by PCR. There was likewise mild involvement of the liver and bone marrow by the "atypical" T-cell population. Close attention to her uncharacteristically well clinical appearance led to repeat T-cell receptor clonality testing using next-generation sequencing. Definitive demonstration of polyclonality by this test showed that she in fact did not have hepatosplenic T-cell lymphoma but rather a reactive condition, and allogeneic stem cell transplantation could be safely avoided. As molecular clonality testing is widely used in the practice of hematology, this case brings attention to the pitfalls of clonality testing by PCR that practitioners may encounter. It is therefore a cautionary tale highlighting the need for critical interpretation of test results in full clinical context.
Assuntos
Erros de Diagnóstico , Reação em Cadeia da Polimerase/métodos , Neoplasias Esplênicas/patologia , Linfócitos T/metabolismo , Proliferação de Células , Feminino , HumanosRESUMO
Less than a year since the start of the COVID-19 pandemic, ten vaccines against SARS-CoV-2 have been approved for at least limited use, with over sixty others in clinical trials. This swift achievement has generated excitement and arrives at a time of great need, as the number of COVID-19 cases worldwide continues to rapidly increase. Two vaccines are currently approved for full use, both built on mRNA and lipid nanotechnology platforms, a success story of mRNA technology 20 years in the making. For patients with cancer, questions arise around the safety and efficacy of these vaccines in the setting of immune alterations engendered by their malignancy and/or therapies. We summarize the current data on leading COVID-19 vaccine candidates and vaccination of patients undergoing immunomodulatory cancer treatments. Most current cancer therapeutics should not prevent the generation of protective immunity. We call for more research in this area and recommend that the majority of patients with cancer receive COVID vaccinations when possible.
Assuntos
Vacinas contra COVID-19/uso terapêutico , COVID-19/complicações , COVID-19/prevenção & controle , Neoplasias/complicações , Animais , Antineoplásicos/uso terapêutico , COVID-19/imunologia , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/imunologia , Humanos , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Pandemias/prevenção & controleRESUMO
Subset analysis of patients with sarcomatoid renal cell carcinoma (sRCC) included in the CheckMate 214 trial of ipilimumab-nivolumab versus sunitinib showed improved outcomes in sRCC with ipilimumab-nivolumab. The use of checkpoint inhibitor-based regimens in sRCC, for which therapeutic options were once limited, is further supported by additional clinical trials.See related article by Tannir et al., p. 78.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/tratamento farmacológico , Via de Sinalização Hippo , Humanos , Imunoterapia/efeitos adversos , Ipilimumab , Neoplasias Renais/tratamento farmacológico , Nivolumabe/efeitos adversos , Proteínas Serina-Treonina Quinases/genética , SunitinibeRESUMO
Since the approval of immunotherapy checkpoint inhibitors for first-line treatment of metastatic renal cell carcinoma, new and clinically relevant questions have emerged that ongoing clinical trials and trials in development will address. These questions include how to integrate combination immunotherapy approaches like ipilimumab/nivolumab with targeted therapies against vascular endothelial growth factor (VEGF) receptors, which patients can discontinue treatment, and who needs ipilimumab to maximize clinical responses. Furthermore, with new approvals of treatment regimens combining checkpoint inhibitors with targeted therapies, new questions arise in the clinic regarding optimal treatment selection for first-line clear cell renal cell carcinoma. This review will highlight the contemporary clinical trials in metastatic clear cell renal cell carcinoma that try to address some of these knowledge gaps.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/terapia , Neoplasias Renais/terapia , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Quimioterapia Adjuvante/métodos , Tomada de Decisão Clínica , Procedimentos Cirúrgicos de Citorredução/métodos , Procedimentos Cirúrgicos de Citorredução/estatística & dados numéricos , Progressão da Doença , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Rim/patologia , Rim/cirurgia , Neoplasias Renais/imunologia , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Terapia Neoadjuvante/métodos , Nefrectomia/métodos , Nefrectomia/estatística & dados numéricos , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fatores de Tempo , Tempo para o TratamentoRESUMO
The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination.