RESUMO
Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor ß (TGF-ß)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.
Assuntos
Imunidade Adaptativa , Proteínas Adaptadoras de Sinalização CARD/genética , Senescência Celular/fisiologia , Guanilato Ciclase/genética , Linfoma Difuso de Grandes Células B/imunologia , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Neoplasias/genética , Linfócitos T Citotóxicos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígenos CD79/genética , Linhagem Celular Tumoral , Quimiotaxia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Genes myc , Humanos , Inibidores de Checkpoint Imunológico , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , NF-kappa B/genética , NF-kappa B/metabolismo , Mutação Puntual , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , TranscriptomaRESUMO
Adoptive T cell-receptor therapy (ACT) could represent a promising approach in the targeted treatment of epithelial ovarian cancer (EOC). However, the identification of suitable tumor-associated antigens (TAAs) as targets is challenging. We identified and prioritized TAAs for ACT and other immunotherapeutic interventions in EOC. A comprehensive list of pre-described TAAs was created and candidates were prioritized, using predefined weighted criteria. Highly ranked TAAs were immunohistochemically stained in a tissue microarray of 58 EOC samples to identify associations of TAA expression with grade, stage, response to platinum, and prognosis. Preselection based on expression data resulted in 38 TAAs, which were prioritized. Along with already published Cyclin A1, the TAAs KIF20A, CT45, and LY6K emerged as most promising targets, with high expression in EOC samples and several identified peptides in ligandome analysis. Expression of these TAAs showed prognostic relevance independent of molecular subtypes. By using a systematic vetting algorithm, we identified KIF20A, CT45, and LY6K to be promising candidates for immunotherapy in EOC. Results are supported by IHC and HLA-ligandome data. The described method might be helpful for the prioritization of TAAs in other tumor entities.
Assuntos
Autoantígenos , Neoplasias Ovarianas , Humanos , Feminino , Carcinoma Epitelial do Ovário/terapia , Autoantígenos/uso terapêutico , Antígenos de Neoplasias , Neoplasias Ovarianas/metabolismo , Terapia Baseada em Transplante de Células e TecidosRESUMO
Adoptive T cell therapy (ATT) has revolutionized the treatment of cancer patients. A sufficient number of functional T cells are indispensable for ATT efficacy; however, several ATT dropouts have been reported due to T cell expansion failure or lack of T cell persistence in vivo. With the aim of providing ATT also to those patients experiencing insufficient T cell manufacturing via standard protocol, we evaluated if minimally manipulative prolongation of in vitro expansion (long-term [LT] >3 weeks with IL-7 and IL-15 cytokines) could result in enhanced T cell yield with preserved T cell functionality. The extended expansion resulted in a 39-fold increase of murine CD8+ T central memory cells (Tcm). LT expanded CD8+ and CD4+ Tcm cells retained a gene expression profile related to Tcm and T memory stem cells (Tscm). In vivo transfer of LT expanded Tcm revealed persistence and antitumor capacity. We confirmed our in vitro findings on human T cells, on healthy donors and diffuse large B cell lymphoma patients, undergoing salvage therapy. Our study demonstrates the feasibility of an extended T cell expansion as a practicable alternative for patients with insufficient numbers of T cells after the standard manufacturing process thereby increasing ATT accessibility.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfoma Difuso de Grandes Células B/terapia , Linfócitos T/citologia , Linfócitos T/transplante , Animais , Estudos de Casos e Controles , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Memória Imunológica , Imunoterapia Adotiva , Interleucina-15/farmacologia , Interleucina-7/farmacologia , Masculino , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410-420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.
Assuntos
Apresentação de Antígeno/imunologia , Ciclina A1/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/imunologia , Neoplasias Ovarianas/imunologia , Linfócitos T Citotóxicos/imunologia , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Neoplasias Ovarianas/patologia , Fragmentos de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Mutational signatures are specific patterns of somatic mutations introduced into the genome by oncogenic processes. Several mutational signatures have been identified and quantified from multiple cancer studies, and some of them have been linked to known oncogenic processes. Identification of the processes contributing to mutations observed in a sample is potentially informative to understand the cancer etiology. RESULTS: We present here SigsPack, a Bioconductor package to estimate a sample's exposure to mutational processes described by a set of mutational signatures. The package also provides functions to estimate stability of these exposures, using bootstrapping. The performance of exposure and exposure stability estimations have been validated using synthetic and real data. Finally, the package provides tools to normalize the mutation frequencies with respect to the tri-nucleotide contents of the regions probed in the experiment. The importance of this effect is illustrated in an example. CONCLUSION: SigsPack provides a complete set of tools for individual sample exposure estimation, and for mutation catalogue & mutational signatures normalization.
Assuntos
Carcinogênese/genética , Genoma Humano , Mutação , Proteínas de Neoplasias/genética , Neoplasias/genética , Software , Carcinogênese/patologia , Análise Mutacional de DNA , Humanos , Taxa de Mutação , Neoplasias/patologiaRESUMO
T-box transcription factors, T-box expressed in T cells (T-bet) encoded by Tbx21 and Eomesodermin (Eomes), drive the differentiation of effector/memory T cell lineages and NK cells. The aim of the study was to determine the prognostic influence of the expression of these transcription factors in peripheral blood (pB) in a cohort of 41 metastatic (m) RCC patients before receiving sorafenib treatment and to analyze their association with the immunophenotype in pB. In contrast to Tbx21, in the multivariate analysis including clinical features, Eomes mRNA expression was identified as an independent good prognostic factor for progression-free survival (PFS, p = 0.042) and overall survival (OS, p = 0.001) in addition to a favorable ECOG performance status (p = 0.01 and p = 0.008, respectively). Eomes expression correlated positively not only with expression of Tbx21 and TGFß1 mRNA, but also with mRNA expression of the activation marker ICOS, and with in vivo activated HLA-DR(+) T cells. Eomes expression was negatively associated with TNFα-producing T cells. On protein level, Eomes was mainly expressed by CD56(+)CD3(-) NK cells in pB. In conclusion, we identified a higher Eomes mRNA expression as an independent good prognostic factor for OS and PFS in mRCC patients treated with sorafenib.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Proteínas com Domínio T/genética , Adulto , Idoso , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Feminino , Humanos , Imunofenotipagem , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
We analysed mRNA levels of interferon response genes (ISG15, STAT1, CXCL10) of inhibitors of the JAK/STAT pathway (STAT3, SOCS1, SOCS3) and of cytokines (TNFα, IL10, TGFß1) in peripheral blood of 91 stage III melanoma patients enrolled in EORTC 18991 trial to find biomarkers indicative for disease stage and predictive for efficacy of pegylated interferon alpha-2b (PEG-IFNα-2b) therapy. mRNA levels were analysed at baseline and after 6 months. Univariate and multivariate analyses were performed to estimate the prognostic and predictive role of mRNA levels for distant metastasis-free survival (DMFS) and relapse-free survival (RFS). Compared to healthy controls, melanoma patients showed significantly higher TGFß1 mRNA levels. In a multivariate model, increasing SOCS1 and SOCS3 mRNA levels were associated with worse RFS (P = 0.02 and P = 0.04, respectively) and DMFS (P = 0.05 and P = 0.05, respectively) due to negative correlation between, respectively, SOCS1/SOCS3 mRNA levels and ulceration or Breslow thickness. No impact of PEG-IFNα-2b on mRNA levels was observed except for ISG15 mRNA levels, which decreased in the treatment arm (P = 0.001). It seems that patients with a decrease >60 % of ISG15 mRNA levels during 6 months PEG-IFNα-2b had inferior outcome.
Assuntos
Antineoplásicos/uso terapêutico , Citocinas/genética , Interferon-alfa/uso terapêutico , Melanoma/tratamento farmacológico , Polietilenoglicóis/uso terapêutico , RNA Mensageiro/sangue , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Biomarcadores Tumorais/genética , Quimioterapia Adjuvante , Citocinas/sangue , Intervalo Livre de Doença , Feminino , Expressão Gênica , Humanos , Interferon alfa-2 , Masculino , Melanoma/sangue , Melanoma/genética , Melanoma/mortalidade , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Neoplasias Cutâneas/mortalidade , Resultado do TratamentoRESUMO
BACKGROUND: Clinically effective T-cell responses can be elicited by single peptide vaccination with Wilms' tumor 1 (WT1) epitope 126-134 in patients with acute myeloid leukemia (AML). We recently showed that a predominant T-cell receptor (TCR) ß chain was associated with vaccine-induced complete remission in an AML patient (patient 1). In this study, we address the question of whether this predominant clone or the accompanying Vß11 restriction could be found in other AML patients vaccinated with the same WT1 peptide. MATERIALS AND METHODS: For assessment of Vß usage, cytotoxic T lymphocytes (CTLs) from four vaccinated patients were divided into specific and non-specific by epitope-specific enrichment. Vß families were quantified in both fractions using reverse transcribed quantitative PCR. Vß11-positive 'complementary determining region 3' (CDR3) sequences were amplified from these samples, from bone marrow samples of 17 other vaccination patients, and from peripheral blood of six healthy controls, cloned and sequenced. RESULTS: We observed a clear bias towards Vß11 usage of the WT1-specific CTL populations in all four patients. The predominant CDR3ß amino acid (AA) sequence of patient 1 was detected in two other patients. CDR3ß loops with closely related AA sequences were only found in patient 1. There were no CDR3ß AA sequences with side chains of identical chemical properties detected in any patient. CONCLUSION: We provide the first data addressing TCR Vß chain usage in WT1-specific T-cell populations after HLA A*0201-restricted single peptide vaccination. We demonstrate both shared Vß restriction and the sharing of a TCR ß transcript with proven clinical impact in one patient.
Assuntos
Vacinas Anticâncer/imunologia , Leucemia Mieloide/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas WT1/imunologia , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Vacinas Anticâncer/administração & dosagem , Células Clonais/imunologia , Células Clonais/metabolismo , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Leucemia Mieloide/terapia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Indução de Remissão , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Proteínas WT1/química , Adulto JovemRESUMO
The novel AKT inhibitor perifosine possesses myelopoiesis-stimulating effects in rodents. We studied the in vitro effects of the novel agents perifosine, bortezomib and lenalidomide in addition to adriamycin against normal human hematopoietic progenitor cells (HPC) using different clonogenic and non-clonogenic assays. All agents inhibited colony-forming unit (CFU) formation, perifosine inhibiting mainly CFU-granulocyte/macrophage formation and the other agents burst-forming unit-erythroid formation. Perifosine combined with lenalidomide or adriamycin tended to act antagonistically in suppressing CFU formation. Despite their inhibition of CFU formation, perifosine, bortezomib and lenalidomide induced only slight or moderate cytotoxicity in CD34(+) selected HPC, as assessed using different assays such as flow cytometry-based detection of activated caspases and immunohistochemistry studies (e.g., Ki-67 staining). In contrast to its myelopoiesis-stimulating effects in rodents, perifosine--like bortezomib and lenalidomide--suppresses the clonogenic potential of HPC from healthy donors in vitro and thus probably plays no role in preventing neutropenia or in shorting its duration after intensive chemotherapy. However, all these novel agents typically induce only slight or moderate suppression of the clonogenic potential or loss of viability of normal HPC at clinically achievable plasma concentrations, assuming that hematoxicity is manageable and functional HPC can be collected after treatment with these compounds.
Assuntos
Ácidos Borônicos/farmacologia , Saúde , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Pirazinas/farmacologia , Talidomida/análogos & derivados , Doadores de Tecidos , Anexina A5/metabolismo , Antígenos CD34/metabolismo , Bortezomib , Caspases/metabolismo , Ensaio de Unidades Formadoras de Colônias , Doxorrubicina/farmacologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Humanos , Imuno-Histoquímica , Lenalidomida , Fosforilcolina/farmacologia , Coloração e Rotulagem , Talidomida/farmacologia , Azul Tripano/metabolismoRESUMO
The novel AKT inhibitor perifosine, a synthetic alkylphospholipid, is currently being investigated in clinical trials for the treatment of different hematological and oncological malignancies. The in vitro cytotoxicity of perifosine, bortezomib and lenalidomide against 6 cell lines derived from hematological malignancies was investigated using trypan blue staining, flow cytometry-based detection of activated caspases, Annexin V assays, immunohistochemistry studies (KI-67 and caspase-3 staining) and the immature-myeloid-information (IMI) technique. Perifosine and bortezomib induced concentration- and time-dependent cytotoxicity in all cell lines tested. Perifosine together with bortezomib largely exerted additive or synergistic effects with combination indices ranging from 1.13 to 0.22 for combined efficacies of 25% to 75% after 24-hour incubation. Lenalidomide-triggered cytotoxicity was low in all cell lines tested with any assay (less than 10% compared to the negative control). Finally, perifosine, but not bortezomib or lenalidomide, significantly increased the number of cells detected in the IMI channel. Perifosine and bortezomib- but not lenalidomide- trigger substantial cytotoxicity by caspase activation and mainly act additively or synergistically. The IMI technique might be a useful tool for studying cytotoxicity of agents like perifosine that interact mainly with the cellular membrane.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácidos Borônicos/farmacologia , Linfoma/patologia , Mieloma Múltiplo/patologia , Fosforilcolina/análogos & derivados , Pirazinas/farmacologia , Talidomida/análogos & derivados , Apoptose/efeitos dos fármacos , Bortezomib , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Citometria de Fluxo , Células HL-60 , Humanos , Imuno-Histoquímica , Concentração Inibidora 50 , Células K562 , Antígeno Ki-67/metabolismo , Lenalidomida , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Linfoma/metabolismo , Mieloma Múltiplo/metabolismo , Fosforilcolina/farmacologia , Talidomida/farmacologia , Fatores de TempoRESUMO
(1) Background: Mutation-specific T cell receptor (TCR)-based adoptive T cell therapy represents a truly tumor-specific immunotherapeutic strategy. However, isolating neoepitope-specific TCRs remains a challenge. (2) Methods: We investigated, side by side, different TCR repertoires-patients' peripheral lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs), PBLs of healthy donors, and a humanized mouse model-to isolate neoepitope-specific TCRs against eight neoepitope candidates from a colon cancer and an ovarian cancer patient. Neoepitope candidates were used to stimulate T cells from different repertoires in vitro to generate neoepitope-specific T cells and isolate the specific TCRs. (3) Results: We isolated six TCRs from healthy donors, directed against four neoepitope candidates and one TCR from the murine T cell repertoire. Endogenous processing of one neoepitope, for which we isolated one TCR from both human and mouse-derived repertoires, could be shown. No neoepitope-specific TCR could be generated from the patients' own repertoire. (4) Conclusion: Our data indicate that successful isolation of neoepitope-specific TCRs depends on various factors such as the heathy donor's TCR repertoire or the presence of a tumor microenvironment allowing neoepitope-specific immune responses of the host. We show the advantage and feasibility of using healthy donor repertoires and humanized mouse TCR repertoires to generate mutation-specific TCRs with different specificities, especially in a setting when the availability of patient material is limited.
RESUMO
BACKGROUND: Inhibitors of the cyclin-dependent kinases 4 and 6 (CDK4/6i) have significantly improved clinical outcomes in patients with advanced hormone receptor-positive (HR+) breast cancer and have demonstrated favourable antitumour immune responses in preclinical studies. METHODS: Here, we investigated peripheral immune responses to ribociclib in patients with metastatic HR+ breast cancer as a preplanned exploratory subanalysis of the RIBECCA trial (NCT03096847). Peripheral blood mononuclear cells were subjected to immune cell profiling, gene expression analysis of immune-related signatures, and deep T cell receptor profiling before treatment started and after 12 weeks of treatment with ribociclib. RESULTS: Gene expression analysis revealed an upregulation of signatures associated with an activated adaptive immune system and a decrease in immunosuppressive cytokine signalling during treatment with ribociclib. Profiling of peripheral immune cell subpopulations showed a decrease in Treg cell frequencies, which was associated with treatment response. Furthermore, induction of CD4+ naive T cells could be seen, whereas effector and memory T cell populations remained largely unchanged. Correspondingly, T cell repertoire diversity remained mostly unchanged during treatment, although an increase in clonality could be observed in single patients. CONCLUSIONS: We show that treatment with ribociclib has significant effects on the peripheral innate and adaptive immune response in patients with HR+ breast cancer. Our data suggest that these effects lead to an activation of an already existing immune response rather than a de novo induction and make a strong case for future combination strategies of CDK4/6i with immunotherapies to enhance the adaptive immune response in HR+ breast cancer.
Assuntos
Neoplasias da Mama , Aminopiridinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/patologia , Feminino , Humanos , Imunidade , Leucócitos Mononucleares/metabolismo , Purinas , Receptor ErbB-2/metabolismoRESUMO
Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell-mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell-mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.
Assuntos
Apresentação de Antígeno , Neoplasias , Antígenos de Histocompatibilidade Classe I , Humanos , Evasão da Resposta Imune , Neoplasias/patologia , SumoilaçãoRESUMO
This study investigated the immunogenicity of Wilms tumor gene product 1 (WT1)-peptide vaccination in WT1-expressing acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients without curative treatment option. Vaccination consisted of granulocyte-macrophage colony-stimulating factor subcutaneously days 1 to 4, and WT1.126-134 peptide and 1 mg keyhole limpet hemocyanin on day 3. The initial 9 patients received 4 vaccinations biweekly, then monthly, and the subsequent 10 patients received continual biweekly vaccination. Seventeen AML patients and 2 refractory anemia with excess blasts patients received a median of 11 vaccinations. Treatment was well tolerated. Objective responses in AML patients were 10 stable diseases (SDs) including 4 SDs with more than 50% blast reduction and 2 with hematologic improvement. An additional 4 patients had clinical benefit after initial progression, including 1 complete remission and 3 SDs. WT1 mRNA levels decreased at least 3-fold from baseline in 35% of patients. In 8 of 18 patients, WT1-tetramer(+) T cells increased in blood and in 8 of 17 patients in bone marrow, with a median frequency in bone marrow of 0.18% at baseline and 0.41% in week 18. This WT1 vaccination study provides immunologic, molecular, and preliminary evidence of potential clinical efficacy in AML patients, warranting further investigations.
Assuntos
Anemia Refratária com Excesso de Blastos/terapia , Vacinas Anticâncer/uso terapêutico , Imunoterapia Ativa , Leucemia Mieloide/terapia , Fragmentos de Peptídeos/imunologia , Proteínas WT1/imunologia , Doença Aguda , Adjuvantes Imunológicos/administração & dosagem , Idoso , Idoso de 80 Anos ou mais , Anemia Refratária com Excesso de Blastos/genética , Anemia Refratária com Excesso de Blastos/imunologia , Anemia Refratária com Excesso de Blastos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Eritema/etiologia , Feminino , Genes do Tumor de Wilms , Humanos , Esquemas de Imunização , Imunoterapia Ativa/efeitos adversos , Injeções Intradérmicas , Injeções Subcutâneas , Leucemia Mieloide/genética , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/efeitos adversos , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Terapia de Salvação , Especificidade do Receptor de Antígeno de Linfócitos T , Proteínas WT1/administração & dosagem , Proteínas WT1/efeitos adversosRESUMO
BACKGROUND: Adoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC-peptide presentation. METHODS: Here we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas. RESULTS: Generated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity. CONCLUSION: Taken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Linfoma de Células B/tratamento farmacológico , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Humanos , Linfoma de Células B/imunologia , MutaçãoRESUMO
In an earlier study, we described a patient who developed an anti-tyrosinase T-cell response leading to long-term tumor control. Here we analyzed this response with regard to T-cell receptor (TCR) Vbeta family usage and clonality in order to further elucidate the nature of the T cell response in this patient. For identification of expanded specific cytotoxic T-cell (CTL) clones, tetramer enrichment of tyrosinase reactive T-cells was followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR Vbeta-families and sequencing of family Vbeta4 elevated in the enriched fraction. The predominant specific clone was quantified by clonotypic qRT PCR in multiple samples from blood, bone marrow, and tumor tissue. FACS analyses with staining of TYR.A2 and TCR Vbeta4 were performed. Epitope specific enrichment revealed an isolated increase of Vbeta-family 4. FACS analysis showed a shift of specific CTLs to Vbeta-family 4 during tumor regression with a maximum of 80% of all TYR.A2 specific cells belonging to this family. Sequencing revealed a single predominant clone against polyclonal background coding for identical CDR3 loops. The predominant clone was highly expressed in bone marrow and tumor tissue, and was detectable in blood over a period of ten years. Considering the results of previous studies showing a specific effector phenotype in blood and a specific memory compartment in bone marrow of this patient, this data implicate the predominant clone featured all attributes of a sufficient CTL response including homing capacity and memory formation resulting in long term clonal persistence and tumor control.
Assuntos
Células Clonais , Melanoma/imunologia , Melanoma/secundário , Monofenol Mono-Oxigenase , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T , Células Clonais/imunologia , Feminino , Citometria de Fluxo , Humanos , Melanoma/enzimologia , Monofenol Mono-Oxigenase/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/enzimologia , Linfócitos T/enzimologia , Fatores de TempoRESUMO
BACKGROUND: Efficacy of cancer vaccines may be limited due to immune escape mechanisms like loss or mutation of target antigens. Here, we analyzed 10 HLA-A2 positive patients with acute myeloid leukemia (AML) for loss or mutations of the WT1 epitope or epitope flanking sequences that may abolish proper T cell recognition or epitope presentation. METHODS: All patients had been enrolled in a WT1 peptide phase II vaccination trial (NCT00153582) and ultimately progressed despite induction of a WT1 specific T cell response. Blood and bone marrow samples prior to vaccination and during progression were analyzed for mRNA expression level of WT1. Base exchanges within the epitope sequence or flanking regions (10 amino acids N- and C-terminal of the epitope) were assessed with melting point analysis and sequencing. HLA class I expression and WT1 protein expression was analyzed by flow cytometry. RESULTS: Only in one patient, downregulation of WT1 mRNA by 1 log and loss of WT1 detection on protein level at time of disease progression was observed. No mutation leading to a base exchange within the epitope sequence or epitope flanking sequences could be detected in any patient. Further, no loss of HLA class I expression on leukemic blasts was observed. CONCLUSION: Defects in antigen presentation caused by loss or mutation of WT1 or downregulation of HLA molecules are not the major basis for escape from the immune response induced by WT1 peptide vaccination.
Assuntos
Vacinas Anticâncer/imunologia , Leucemia Mieloide Aguda , Evasão Tumoral , Vacinas de Subunidades Antigênicas/imunologia , Proteínas WT1/genética , Tumor de Wilms , Apresentação de Antígeno/imunologia , Ensaios Clínicos Fase II como Assunto , Progressão da Doença , Epitopos , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Mutação , Linfócitos T/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Proteínas WT1/imunologia , Tumor de Wilms/genética , Tumor de Wilms/imunologiaRESUMO
Several immunotherapeutic approaches rely on antigen-specific T-cells. Restrictions in the T-cell receptor (TCR) repertoire were reported as indicator of anti-tumor cytotoxic T-lymphocyte (CTL) response in various tumor entities. It is unclear yet whether a TCR restriction in peripheral blood mirrors the tumor compartment. We compared the expression of TCR Vbeta-families for the quantification of TCR repertoire alterations in blood and tissue samples from patients with colorectal carcinoma. Blood samples from patients with colorectal carcinoma and healthy volunteers and tissue samples of normal colonic mucosa and colorectal carcinoma were analyzed. Relative Vbeta-family quantification was performed based on quantitative reverse transcribed PCR. Standard deviation and average mean of the single families were determined. Two variables describing the degree of Vbeta-repertoire restriction were defined. Forty-eight blood samples and 37 tissue samples were analyzed. TCR repertoire restriction was higher in blood of tumor patients than in blood of healthy controls (p < 0.05). No difference in the degree of TCR repertoire restriction was found between carcinoma and unaffected colon tissue. We found no corresponding elevated TCR families among the different compartments blood, normal colon, and carcinoma tissue of the same patient. In conclusion, we observed a repertoire restriction in peripheral blood as well as in tumor tissue of cancer patients. However, in tumor tissue, repertoire alterations were comparable to normal mucosa, suggesting compartment-specific TCR distribution rather than alterations due to tumor-T-cell interaction questioning the presence of highly restricted clonal T-cell expansions in colorectal cancer as they have been described in other, assumingly more immunogenic tumor entities.
Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The role of the Wilms tumor 1 gene (WT1) in acute leukemias has been underscored by mutations found in acute myeloid leukemia identifying patients with inferior survival. Furthermore, aberrant expression of WT1 in acute myeloid leukemia was associated with an increased risk of relapse. No larger studies have performed a combined approach including WT1 mutation and expression analyses in acute T-lymphoblastic leukemia. DESIGN AND METHODS: We analyzed the WT1 mutations and the expression status in a total of 252 consecutive adult patients with newly diagnosed T-lymphoblastic leukemia, who were registered on the GMALL 06/99 and 07/03 protocols and had sufficient material available. The GMALL protocols included intensive chemotherapy as well as stem cell transplantation according to a risk-based model with indication for stem cell transplantation in first complete remission for early and mature T-lymphoblastic leukemia patients; patients with thymic T-lymphoblastic leukemia were allocated to a standard risk group and treated with intensive chemotherapy. RESULTS: Twenty of the 238 patients analyzed had WT1 mutations (WT1mut) in exon 7. WT1mut cases were characterized by immature features such as an early immunophenotype and higher WT1 expression. In thymic T-lymphoblastic leukemia, WT1mut patients had an inferior relapse-free survival compared to WT1 wild-type patients. T-lymphoblastic leukemia patients with aberrant WT1 expression (high or negative) showed a higher relapse rate and an inferior outcome compared to patients with intermediate WT1 expression. In the standard risk group of thymic T-lymphoblastic leukemia, aberrant WT1 expression was predictive for an inferior relapse-free survival as compared to patients with intermediate expression. In multivariate analysis, WT1 expression was of independent prognostic significance for relapse-free survival. CONCLUSIONS: WT1 mutations were associated with an inferior relapse-free survival in standard risk thymic T-lymphoblastic leukemia patients. Moreover, altered expression associated with inferior outcome also suggests a role of WT1 in T-lymphoblastic leukemia and the potential use of molecularly-based treatment stratification to improve outcome.