RESUMO
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 BCR recognizes foreign Ags to initiate humoral immunity that needs isotype-switched Abs generated via class switch recombination (CSR); however, stimulating the BCR in the absence of costimulation (e.g., CD40) does not induce CSR; thus, it remains elusive whether and how the BCR induces CSR mechanistically. Autoreactive B cells can maintain anergy via unresponsiveness of their BCRs to self-antigens. However, it remains unknown what molecule(s) restrict BCR signaling strength for licensing BCR-induced CSR and whether deficiency of such molecule(s) disrupts autoreactive B cell anergy and causes B cell-mediated diseases by modulating BCR signaling. In this study, we employ mouse models to show that the BCR's capacity to induce CSR is restrained by B cell-intrinsic checkpoints TRAF3 and TRAF2, whose deletion in B cells enables the BCR to induce CSR in the absence of costimulation. TRAF3 deficiency permits BCR-induced CSR by elevating BCR-proximal signaling intensity. Furthermore, NF-κB2 is required for BCR-induced CSR in TRAF3-deficient B cells but not for CD40-induced or LPS-induced CSR, suggesting that TRAF3 restricts NF-κB2 activation to specifically limit the BCR's ability to induce CSR. TRAF3 deficiency also disrupts autoreactive B cell anergy by elevating calcium influx in response to BCR stimulation, leading to lymphoid organ disorders and autoimmune manifestations. We showed that TRAF3 deficiency-associated autoimmune phenotypes can be rectified by limiting BCR repertoires or attenuating BCR signaling strength. Thus, our studies highlight the importance of TRAF3-mediated restraint on BCR signaling strength for controlling CSR, B cell homeostasis, and B cell-mediated disorders.
Assuntos
Linfócitos B/imunologia , Anergia Clonal , Switching de Imunoglobulina/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Animais , Linfócitos B/citologia , Camundongos , Camundongos Transgênicos , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/imunologia , Transdução de Sinais/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/genéticaRESUMO
The question as to why some hosts can eradicate their tumors while others succumb to tumor-progression remains unanswered. Here, a provocative concept is proposed that intrinsic differences in the T cell receptor (TCR) repertoire of individuals may influence the outcome of anti-tumor immunity by affecting the frequency and/or variety of tumor-reactive CD8 and/or CD4 tumor-infiltrating lymphocytes. This idea implicates that the TCR repertoire in a given patient might not provide sufficiently different TCR clones that can recognize tumor antigens, namely, "a hole in the TCR repertoire" might exist. This idea may provide a novel perspective to further dissect the mechanisms underlying heterogeneous anti-tumor immune responses in different hosts. Besides tumor-intrinsic heterogeneity and host microbiome, the various factors that may constantly shape the dynamic TCR repertoire are also discussed. Elucidating mechanistic differences in different individuals' immune systems will allow to better harness immune system to design new personalized cancer immunotherapy.
Assuntos
Neoplasias , Receptores de Antígenos de Linfócitos T , Linfócitos T CD8-Positivos , Humanos , Imunidade , Linfócitos do Interstício Tumoral , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
Head and neck cancers are a heterogeneous group of tumors that are highly aggressive and collectively represent the sixth most common cancer worldwide. Ninety percent of head and neck cancers are squamous cell carcinomas (HNSCCs). The tumor microenvironment (TME) of HNSCCs consists of many different subsets of cells that infiltrate the tumors and interact with the tumor cells or with each other through various networks. Both innate and adaptive immune cells play a crucial role in mediating immune surveillance and controlling tumor growth. Here, we discuss the different subsets of immune cells and how they contribute to an immunosuppressive TME of HNSCCs. We also briefly summarize recent advances in immunotherapeutic approaches for HNSCC treatment. A better understanding of the multiple factors that play pivotal roles in HNSCC tumorigenesis and tumor progression may help define novel targets to develop more effective immunotherapies for patients with HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço/imunologia , Microambiente Tumoral/imunologia , Animais , Transformação Celular Neoplásica/imunologia , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Imunoterapia/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapiaRESUMO
Effective humoral immunity requires class switch recombination (CSR) catalyzed by activation-induced cytidine deaminase (AID). In response to T cell-dependent (TD) Ags, CSR can be induced by CD40 signaling in B cells. TNFR-associated factors 2 and 3 (TRAF2/TRAF3) function as adaptors of the CD40 signaling pathway. B cell-intrinsic TRAF2 or TRAF3 (B-TRAF2 or B-TRAF3) knockout mice were previously reported to have indistinguishable phenotypes in gene expression, B cell survival and development, and enlarged peripheral lymphoid organs. However, it remains unknown whether deficiency of B-TRAF2 or B-TRAF3 differentially affects TD humoral immune responses and CD40-induced CSR. In this article, we show that B-TRAF2 is essential for optimal isotype switching induced by in vivo TD Ag immunization or by engaging CD40 in vitro. Our data clarify the controversial role of B-TRAF3 and confirm its dispensability in CD40-induced CSR. Mechanistically, CD40-induced AID expression was markedly impaired by B-TRAF2, but not B-TRAF3, deficiency. Moreover, B-TRAF2 deficiency causes defective activation of the NF-κB1 complex in a CD40-autonomous manner, and restoring CD40-induced NF-κB1 activation in TRAF2-deficient B cells rescues AID expression and CSR. We conclude that TRAF2 is essential but TRAF3 is dispensable for TD humoral immunity and CD40-induced CSR. Our studies provide significant biological bases for optimizing treatment of B cell-associated immune disorders by targeting CD40 signaling.
Assuntos
Linfócitos B/imunologia , NF-kappa B/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Fator 2 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/genética , Animais , Antígenos CD40/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Imunidade Humoral , Switching de Imunoglobulina , Camundongos , Camundongos Knockout , Transdução de Sinais , Ativação TranscricionalRESUMO
Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.
Assuntos
Carcinoma de Células Escamosas/etiologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Genes p53 , Neoplasias de Cabeça e Pescoço/etiologia , Queratina-15/metabolismo , Animais , Carcinoma de Células Escamosas/mortalidade , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Linfócitos do Interstício Tumoral , Camundongos Transgênicos , Neoplasias Experimentais , Microambiente TumoralRESUMO
SMAD4 is a potent tumor suppressor and a central mediator of the TGFß signaling pathway. SMAD4 genetic loss is frequent in squamous cell carcinomas (SCCs). Reports of SMAD4 expression in SCCs vary significantly possibly due to inter-tumor heterogeneity or technical reasons. SMAD4 loss is an initiation event for SCCs. In tumor epithelial cells, SMAD4 loss causes increased proliferation, decreased apoptosis, and "Brca-like" genomic instability associated with DNA repair defects. SMAD4 loss also plays a role in the expansion of cancer stem cells. Epithelial SMAD4 loss causes overexpression of TGFß that is released into the tumor microenvironment and contributes to SCC progression through proinflammatory and immune evasive mechanisms. SMAD4 loss, while not a direct therapeutic target, is associated with multiple targetable pathways that require further therapeutic studies. Altogether, SMAD4 loss is a potential biomarker in SCCs that should be further studied for its values in prognostic and therapeutic predictions. Such information will potentially guide future biomarker-driven clinical trial designs and improve SCC patient outcomes.
Assuntos
Carcinoma de Células Escamosas/genética , Proteína Smad4/genética , Animais , Biomarcadores Tumorais/genética , Reparo do DNA/genética , Células Epiteliais/patologia , Genes Supressores de Tumor/fisiologia , Humanos , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Chemotherapeutic agents, e.g., cytarabine and doxorubicin, cause DNA damage. However, it remains unknown whether such agents differentially regulate cell cycle arrest in distinct types of B-cell lymphomas, and whether this phenotype can be exploited for developing new therapies. We treated various types of B cells, including primary and B lymphoma cells, with cytarabine or doxorubicin, and determined DNA damage responses, cell cycle regulation and sensitivity to a Wee1 inhibitor. We found that cyclin A2/B1 upregulation appears to be an intrinsic programmed response to DNA damage; however, different types of B cells arrest in distinct phases of the cell cycle. The Wee1 inhibitor significantly enhanced the apoptosis of G2 phase-arrested B-cell lymphomas by inducing premature entry into mitosis and mitotic catastrophe, whereas it did not affect G1/S-phase-arrested lymphomas. Cytarabine-induced G1-arrest can be converted to G2-arrest by doxorubicin treatment in certain B-cell lymphomas, which correlates with newly acquired sensitivity to the Wee1 inhibitor. Consequently, the Wee1 inhibitor together with cytarabine or doxorubicin inhibited tumor growth in vitro and in vivo more effectively, providing a potential new therapy for treating B-cell lymphomas. We propose that the differential cell cycle arrest can be exploited to enhance the chemosensitivity of B-cell lymphomas.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linfoma de Células B/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Citarabina/farmacologia , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Humanos , Linfoma de Células B/tratamento farmacológico , CamundongosRESUMO
Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sµ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity.
Assuntos
Sítios de Ligação , Citidina Desaminase/metabolismo , Reparo do DNA , Motivos de Nucleotídeos , Alelos , Animais , Sequência de Bases , Reparo de Erro de Pareamento de DNA , Técnicas de Introdução de Genes , Ordem dos Genes , Marcação de Genes , Loci Gênicos , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS/metabolismo , Mutação , Taxa de Mutação , Especificidade por Substrato , Uracila-DNA Glicosidase/metabolismo , Éxons VDJ/genéticaRESUMO
Class switch recombination (CSR) generates isotype-switched Abs with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA PI3K catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. In this article, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role for p110α in CSR. Furthermore, we identify a cooperative role for p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN inversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.
Assuntos
Linfócitos B/imunologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Switching de Imunoglobulina/imunologia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Células Cultivadas , Citidina Desaminase/biossíntese , Citidina Desaminase/metabolismo , Switching de Imunoglobulina/genética , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). RESULTS: Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. CONCLUSIONS: We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.
Assuntos
Rearranjo Gênico , Patrimônio Genético , Genoma , Genômica , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biologia Computacional/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Linfoma/genética , Camundongos , Camundongos Transgênicos , Reprodutibilidade dos Testes , Translocação GenéticaRESUMO
Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at Ig loci. How AID-initiated lesions are prevented from inducing genome-wide damage remains elusive. A differential DNA repair mechanism might protect certain non-Ig loci such as c-myc from AID attack. However, determinants regulating such protective mechanisms are largely unknown. To test whether target DNA sequences modulate protective mechanisms via altering the processing manner of AID-initiated lesions, we established a knock-in model by inserting an Sγ2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance c-myc genomic instability, due to error-free repair of AID-initiated lesions, in Ag-stimulated germinal center B cells. In contrast, in vitro cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Furthermore, we observe a comparable frequency of AID deamination events between the c-myc intronic sequence and inserted S region in different B cell populations, demonstrating a similar frequency of AID targeting. Thus, our study reveals a clear difference between germinal center and cytokine-activated B cells in their ability to develop genomic instability, attributable to a differential processing of AID-initiated lesions in distinct B cell populations. We propose that locus-specific regulatory mechanisms (e.g., transcription) appear to not only override the effects of S region sequence on AID targeting frequency but also influence the repair manner of AID-initiated lesions.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/fisiologia , Citidina Desaminase/metabolismo , Centro Germinativo/imunologia , Animais , Células Cultivadas , Citidina Desaminase/genética , Citocinas/metabolismo , Reparo do DNA/imunologia , Técnicas de Introdução de Genes , Loci Gênicos/genética , Instabilidade Genômica , Humanos , Íntrons/genética , Camundongos da Linhagem 129 , Mutação/genética , Proteínas Proto-Oncogênicas c-myc/genética , Hipermutação Somática de Imunoglobulina , Especificidade por SubstratoRESUMO
Variable, diversity and joining gene segment (V(D)J) recombination assembles immunoglobulin heavy or light chain (IgH or IgL) variable region exons in developing bone marrow B cells, whereas class switch recombination (CSR) exchanges IgH constant region exons in peripheral B cells. Both processes use directed DNA double-strand breaks (DSBs) repaired by non-homologous end-joining (NHEJ). Errors in either V(D)J recombination or CSR can initiate chromosomal translocations, including oncogenic IgH locus (Igh) to c-myc (also known as Myc) translocations of peripheral B cell lymphomas. Collaboration between these processes has also been proposed to initiate translocations. However, the occurrence of V(D)J recombination in peripheral B cells is controversial. Here we show that activated NHEJ-deficient splenic B cells accumulate V(D)J-recombination-associated breaks at the lambda IgL locus (Igl), as well as CSR-associated Igh breaks, often in the same cell. Moreover, Igl and Igh breaks are frequently joined to form translocations, a phenomenon associated with specific Igh-Igl co-localization. Igh and c-myc also co-localize in these cells; correspondingly, the introduction of frequent c-myc DSBs robustly promotes Igh-c-myc translocations. Our studies show peripheral B cells that attempt secondary V(D)J recombination, and determine a role for mechanistic factors in promoting recurrent translocations in tumours.
Assuntos
Linfócitos B/metabolismo , Rearranjo Gênico do Linfócito B/genética , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , Translocação Genética/genética , Animais , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Feminino , Genes myc/genética , Proteínas de Homeodomínio/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Integrases/genética , Integrases/metabolismo , Interfase , Ativação Linfocitária , Masculino , Camundongos , Receptores de Complemento 3d/genética , Recombinação Genética/genética , Baço/citologia , Baço/imunologiaRESUMO
Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Animais , Linfócitos B/citologia , Linhagem da Célula , Camundongos , Translocação Genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on activated natural killer (NK) cells wherein it inhibits lysis of CEACAM1-bearing tumor cell lines. The mechanism for this is unknown. Here, we show that interleukin-2-induced expression of CEACAM1 on both mouse and primary human NK cells impairs the ability of NK gene complex group 2 member D (NKG2D) to stimulate cytolysis of CEACAM1-bearing cells. This process requires the expression of CEACAM1 on the NK cells and on the tumor cells, which is consistent with the involvement of trans-homophilic interactions between CEACAM1. Mechanistically, co-engagement of NKG2D and CEACAM1 results in a biochemical association between these two surface receptors and the recruitment of Src homology phosphatase 1 by CEACAM1 that leads to dephosphorylation of the guanine nucleotide exchange factor Vav1 and blockade of downstream signaling that is associated with the initiation of cytolysis. Thus, CEACAM1 on activated NK cells functions as an inhibitory receptor for NKG2D-mediated cytolysis, which has important implications for understanding the means by which CEACAM1 expression adversely affects tumor immunity.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Apoptose/imunologia , Antígeno Carcinoembrionário/genética , Linhagem Celular Tumoral , Humanos , Interleucina-2/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de SinaisRESUMO
Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.
Assuntos
Citidina Desaminase/genética , Técnicas de Introdução de Genes , Hipermutação Somática de Imunoglobulina/genética , Animais , Citidina Desaminase/metabolismo , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Técnicas de Introdução de Genes/métodos , Humanos , Região de Troca de Imunoglobulinas/genética , Camundongos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Polimerase II/genética , Recombinação Genética/imunologiaRESUMO
BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a 'just-right' level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAFLSL-V600E/+;Ptk2fl/fl mice, Fak deletion maximized BRAFV600E's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a 'just-right' ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.
Assuntos
Neoplasias do Ceco , Quinase 1 de Adesão Focal , Proteínas Proto-Oncogênicas B-raf , Animais , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Fosforilação , Camundongos , Humanos , Neoplasias do Ceco/metabolismo , Neoplasias do Ceco/genética , Neoplasias do Ceco/patologia , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Sistema de Sinalização das MAP Quinases , Receptores ErbB/metabolismo , Receptores ErbB/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , MasculinoRESUMO
Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.
Assuntos
Modelos Animais de Doenças , Linfócitos do Interstício Tumoral , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus , Microambiente Tumoral , Animais , Microambiente Tumoral/imunologia , Camundongos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular Tumoral , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Proteínas Repressoras/genética , Proteína do Gene 3 de Ativação de Linfócitos , Humanos , Progressão da Doença , Linfócitos T CD8-Positivos/imunologia , Receptor de Morte Celular Programada 1 , Feminino , Papillomavirus Humano 16/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/virologiaRESUMO
The curative treatment of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC), utilizes radiation. The outcomes for HPV/p16-negative HNSCC are significantly worse than HPV/p16-positive tumors, with increased radiation resistance leading to worse locoregional recurrence (LRR) and ultimately death. This study analyzed the relationship between immune function and outcomes following radiation in HPV/p16-negative tumors to identify mechanisms of radiation resistance and prognostic immune biomarkers. A discovery cohort of 94 patients with HNSCC treated uniformly with surgery and adjuvant radiation and a validation cohort of 97 similarly treated patients were utilized. Tumor immune infiltrates were derived from RNAseq gene expression. The immune cell types significantly associated with outcomes in the discovery cohort were examined in the independent validation cohort. A positive association between high Th2 infiltration and LRR was identified in the discovery cohort and validated in the validation cohort. Tumor mutations in CREBBP/EP300 and CASP8 were significantly associated with Th2 infiltration. A pathway analysis of genes correlated with Th2 cells revealed the potential repression of the antitumor immune response and the activation of BRCA1-associated DNA damage repair in multiple cohorts. The Th2 infiltrates were enriched in the HPV/p16-negative HNSCC tumors and associated with LRR and mutations in CASP8, CREBBP/EP300, and pathways previously shown to impact the response to radiation.
RESUMO
The SET domain is found in histone methyltransferases and other lysine methyltransferases. SET domain-containing proteins such as MLL1 play a critical role in leukemogenesis, while others such as SETD2 may function as a tumor suppressor in breast cancer and renal cell carcinoma. We recently discovered that SETD3, a well-conserved SET domain-containing protein, was involved in a translocation to the immunoglobulin lambda light chain locus in one of the non-homologous end-joining/p53-deficient peripheral B-cell lymphomas. We showed that a truncated mRNA lacking the SET domain sequences in Setd3 gene was highly expressed in the lymphoma. Furthermore, we found that the truncated SET-less protein displayed oncogenic potential while the full length SETD3 protein did not. Finally, SETD3 exhibits histone methyltransferases activity on nucleosomal histone 3 in a SET-domain dependent manner. We propose that this newly identified Setd3 gene may play an important role in carcinogenesis.