RESUMEN
DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.
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Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.
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COVID-19 , SARS-CoV-2 , Humanos , Femenino , Animales , Ratones , Anticuerpos ampliamente neutralizantes , Leucocitos Mononucleares , Anticuerpos Antivirales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Epítopos , Glicoproteína de la Espiga del Coronavirus/genética , Pruebas de NeutralizaciónRESUMEN
Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.
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COVID-19 , SARS-CoV-2 , Australia , COVID-19/diagnóstico , Humanos , Pandemias , SARS-CoV-2/genéticaRESUMEN
Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.
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Betacoronavirus/fisiología , Vacunas contra la COVID-19/inmunología , Infecciones por Coronavirus/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Secuencia Conservada/genética , Evolución Molecular , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Unión Proteica , Dominios Proteicos/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Desarrollo de VacunasRESUMEN
Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Especificidad de Anticuerpos , Reacciones Cruzadas , Humanos , Mutagénesis Sitio-DirigidaRESUMEN
Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antígeno B7-H1/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Diferenciación Celular/genética , Humanos , Neoplasias Pulmonares/genética , MutaciónRESUMEN
Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.
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Carcinoma de Células Escamosas/inmunología , Vigilancia Inmunológica/inmunología , Neoplasias Pulmonares/inmunología , HumanosRESUMEN
PURPOSE: Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy. EXPERIMENTAL DESIGN: We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival. RESULTS: We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4eff):Treg ratio and increased frequency of PD-1-expressing CD4eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction. CONCLUSIONS: BM-infiltrating T-cell subsets, specifically Tregs and PD-1-expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies.
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Médula Ósea/patología , Linfocitos Infiltrantes de Tumor/inmunología , Mieloma Múltiple/etiología , Mieloma Múltiple/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T Reguladores/inmunología , Biomarcadores de Tumor , Estudios de Casos y Controles , Citocinas/metabolismo , Femenino , Humanos , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Masculino , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/mortalidad , Pronóstico , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patologíaRESUMEN
Intratumoral regulatory T cell (Treg) abundance associates with diminished anti-tumor immunity and poor prognosis in human cancers. Recent work demonstrates that CD25, the high affinity receptor subunit for IL-2, is a selective target for Treg depletion in mouse and human malignancies; however, anti-human CD25 antibodies have failed to deliver clinical responses against solid tumors due to bystander IL-2 receptor signaling blockade on effector T cells, which limits their anti-tumor activity. Here we demonstrate potent single-agent activity of anti-CD25 antibodies optimized to deplete Tregs whilst preserving IL-2-STAT5 signaling on effector T cells, and demonstrate synergy with immune checkpoint blockade in vivo. Pre-clinical evaluation of an anti-human CD25 (RG6292) antibody with equivalent features demonstrates, in both non-human primates and humanized mouse models, efficient Treg depletion with no overt immune-related toxicities. Our data supports the clinical development of RG6292 and evaluation of novel combination therapies incorporating non-IL-2 blocking anti-CD25 antibodies in clinical studies.
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Interleucina-2 , Neoplasias , Animales , Anticuerpos Monoclonales/farmacología , Interleucina-2/farmacología , Ratones , Transducción de Señal , Linfocitos T ReguladoresRESUMEN
The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.
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Antígenos de Neoplasias/inmunología , Evolución Molecular , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Escape del Tumor/inmunología , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pulmonares/genética , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Pronóstico , Microambiente Tumoral/inmunologíaRESUMEN
With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches.
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Antineoplásicos Inmunológicos/administración & dosificación , Melanoma/tratamiento farmacológico , Polimorfismo de Nucleótido Simple , Receptores de IgG/genética , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos Inmunológicos/farmacología , Antígeno CTLA-4/antagonistas & inhibidores , Línea Celular Tumoral , Femenino , Humanos , Ipilimumab/administración & dosificación , Ipilimumab/farmacología , Melanoma/genética , Melanoma/inmunología , Ratones , Receptores de IgG/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology.
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Anticuerpos Monoclonales/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral , Citometría de Flujo , Humanos , Inmunoterapia/métodos , Células K562 , Estimación de Kaplan-Meier , Depleción Linfocítica , Ratones , Neoplasias/patología , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica/inmunología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c-MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3-CD11c-MHCII-PU.1lo monocytes differentiated into FcγRIII+PD-L2-CD209a-iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/- mice had reduced Flt3+CD11c-MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.
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Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Traslado Adoptivo , Animales , Antígenos Ly/inmunología , Separación Celular , Células Dendríticas/citología , Citometría de Flujo , Macrófagos/citología , Ratones , Monocitos/citología , Óxido Nítrico Sintasa de Tipo II/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la PolimerasaRESUMEN
As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens.
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Adenocarcinoma/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Vigilancia Inmunológica , Neoplasias Pulmonares/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Antineoplásicos/uso terapéutico , Antígeno CTLA-4/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Puntos de Control del Ciclo Celular/inmunología , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Mutación , Receptor de Muerte Celular Programada 1/inmunología , Neoplasias Cutáneas/inmunologíaRESUMEN
Cytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality after allogeneic hematopoietic stem cell transplant. T-cell immunity is critical for control of CMV infection, and correction of the immune deficiency induced by transplant is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T- cell therapy exclude patients with graft-versus-host disease (GVHD) from receiving systemic corticosteroid therapy, which impairs cellular immunity. This group of patients remains the highest clinical risk group for recurrent and problematic infections. Here, we address this unmet clinical need by genetic disruption of the glucocorticoid receptor (GR) gene using electroporation of transcription activator-like effector nuclease (TALEN) messenger RNA. We demonstrate efficient inactivation of the GR gene without off-target activity in Streptamer-selected CMV-specific CD8(+) T cells (HLA-A02/NLV peptide), conferring resistance to glucocorticoids. TALEN-modified CMV-specific T cells retained specific killing of target cells pulsed with the CMV peptide NLV in the presence of dexamethasone (DEX). Inactivation of the GR gene also conferred resistance to DEX in a xenogeneic GVHD model in sublethally irradiated NOD-scid IL2rγ(null) mice. This proof of concept provides the rationale for the development of clinical protocols for producing and administering high-purity genetically engineered virus-specific T cells that are resistant to the suppressive effects of corticosteroids.
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Traslado Adoptivo/métodos , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Técnicas de Silenciamiento del Gen/métodos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Receptores de Glucocorticoides/genética , Animales , Infecciones por Citomegalovirus/prevención & control , Electroporación , Endonucleasas/genética , Enfermedad Injerto contra Huésped , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , ARN Mensajero , TransfecciónRESUMEN
Cytomegalovirus (CMV) remains a significant cause of morbidity after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical risk varies according to a number of factors, including recipient/donor CMV serostatus. Current dogma suggests risk is greatest in seropositive recipient (R+)/seronegative donor (D-) transplants and is exacerbated by T-cell depletion. We hypothesized that in the setting of reduced-intensity T-cell-depleted conditioning, recipient-derived CMV-specific T cells escaping deletion may contribute significantly to CMV-specific immunity and might therefore also influence chimerism status. We evaluated 105 recipients of alemtuzumab-based reduced-intensity HSCT and collated details on CMV infection episodes and T-cell chimerism. We used CMV-specific HLA multimers to enumerate CMV-specific T-cell numbers and select cells to assess chimerism status in a subset of R+/D- and R+/seropositive donor patients. We show that in R+/D- patients, CMV-specific T cells are exclusively of recipient origin, can protect against recurrent CMV infections, and significantly influence the chimerism status toward recipients. The major findings were replicated in a separate validation cohort. T-cell depletion in the R+/D- setting may actually, therefore, foster more rapid reconstitution of protective antiviral immunity by reducing graft-vs-host directed alloreactivity and the associated elimination of the recipient T-cell compartment. Finally, conversion to donor chimerism after donor lymphocytes is associated with clinically occult transition to donor-derived immunity.
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Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Trasplante de Células Madre Hematopoyéticas , Inmunidad Celular , Depleción Linfocítica , Quimera por Trasplante/inmunología , Aloinjertos , Femenino , Enfermedad Injerto contra Huésped/inmunología , Humanos , MasculinoRESUMEN
Treatment with monoclonal antibody specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma. Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression. We demonstrate, however, that the activity of anti-CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions. Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner. Our results reveal further mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.
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Antígeno CTLA-4/antagonistas & inhibidores , Depleción Linfocítica , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Melanoma/terapia , Receptores de IgG/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Bloqueadores/uso terapéutico , Antígeno CD11b/metabolismo , Antígenos CD4/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Clonales , Proteína Ligando Fas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Recuento de Linfocitos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Melanoma/prevención & control , Ratones , Ratones Endogámicos C57BL , Fagocitos/efectos de los fármacos , Fagocitos/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
The IMiDs(®) immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 µm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD(®) immunomodulatory compounds increased expression of Class I (H2-Kb), CD86, and pomalidomide also increased Class II (I-Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 µm pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8(+) T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4(+) T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/efectos de los fármacos , Talidomida/análogos & derivados , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Femenino , Factores Inmunológicos/inmunología , Factores Inmunológicos/farmacología , Lenalidomida , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Talidomida/inmunología , Talidomida/farmacologíaRESUMEN
Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma, and along with pomalidomide are being investigated in a variety of other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about the primary mode of therapeutic action in patients with cancer. This paper describes a microarray study of the in vitro and in vivo effects of these drugs, and contrasts the difference in gene profiles achieved in the two models. In the current study, Agilent whole mouse genome oligonucleotide microarrays (44 K) were used to examine alterations in gene expression of colorectal cancer cells after treatment. Venn analysis revealed a divergence of gene signature for pomalidomide and lenalidomide, which although similar in vitro, different in vivo. Several clusters of genes involved in various cellular processes such as immune response, cell signalling and cell adhesion were altered by treatment, and common to the three drugs. Notably, the expressions of linked genes within the Notch/Wnt signalling pathway, including kremen2 and dtx4, highlighted a possible novel mechanistic pathway for these drugs. This study also showed that gene signatures were not greatly divergent in the models, and recapitulated the complex nature of these drugs. Overall, these microarray studies highlighted the diversity of this class of drug, which have effects ranging from cell signalling to translation initiation.