RESUMO
BACKGROUND: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. METHODS: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. RESULTS: Using a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. CONCLUSIONS: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.
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Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Docetaxel/farmacologia , Apoptose , Leucócitos Mononucleares/metabolismo , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Mitose , Resistencia a Medicamentos Antineoplásicos , Microambiente Tumoral , Receptores CXCR4/genéticaRESUMO
In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4(+) T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4(+) T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Centro Germinativo/citologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Centro Germinativo/imunologia , Humanos , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Camundongos , Mutação/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Receptores CCR7/metabolismo , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismo , Transcrição Gênica/genética , Regulação para CimaAssuntos
Produtos Biológicos/administração & dosagem , Linfoma Difuso de Grandes Células B , Proteínas de Neoplasias/imunologia , Evasão Tumoral/efeitos dos fármacos , Adulto , Antígenos CD19/imunologia , Produtos Biológicos/efeitos adversos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Recidiva , Evasão Tumoral/genéticaRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has been clinically proven to efficiently combat haematological malignancies. However, continuous efforts are required to increase the specificity of CAR T-cells against tumour versus normal tissues, and are essential to improve their antitumour activity in solid tumours. This review summarises the structure of major CAR designs, and strategies to overcome immunosuppressive tumour microenvironment, and reduce toxicities. Along with reviewing currently available techniques that allow the elimination of CAR T-cells after they fulfil their desired functions, using suicide genes, drug elimination strategies are also introduced. A better understanding of the strengths and pitfalls of CAR T-cell therapy will provide fundamental knowledge for the improvement of engineered T-cell therapy in the near future.
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The microenvironment of human follicular lymphoma (FL), an incurable B cell non-Hodgkin's lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular Th cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17 but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17, but not CCL22, in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4-producing T cells rather than IFN-γ-producing T cells, and Abs to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4-producing T cells, which, in turn, may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk among TFH, tumor cells, and Tregs in FL, and offer potential targets for development of therapeutic strategies to overcome immune evasion.
Assuntos
Linfoma Folicular/imunologia , Receptor Cross-Talk/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Western Blotting , Separação Celular , Quimiocina CCL17/imunologia , Quimiocina CCL17/metabolismo , Quimiocina CCL22/imunologia , Quimiocina CCL22/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfoma Folicular/metabolismo , Linfoma Folicular/mortalidade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Endogenous or iatrogenic antitumour immune responses can improve the course of follicular lymphoma, but might be diminished by immune checkpoints in the tumour microenvironment. These checkpoints might include effects of programmed cell death 1 (PD1), a co-inhibitory receptor that impairs T-cell function and is highly expressed on intratumoral T cells. We did this phase 2 trial to investigate the activity of pidilizumab, a humanised anti-PD1 monoclonal antibody, with rituximab in patients with relapsed follicular lymphoma. METHODS: We did this open-label, non-randomised trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Adult (≥18 years) patients with rituximab-sensitive follicular lymphoma relapsing after one to four previous therapies were eligible. Pidilizumab was administered at 3 mg/kg intravenously every 4 weeks for four infusions, plus eight optional infusions every 4 weeks for patients with stable disease or better. Starting 17 days after the first infusion of pidilizumab, rituximab was given at 375 mg/m(2) intravenously weekly for 4 weeks. The primary endpoint was the proportion of patients who achieved an objective response (complete response plus partial response according to Revised Response Criteria for Malignant Lymphoma). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00904722. FINDINGS: We enrolled 32 patients between Jan 13, 2010, and Jan 20, 2012. Median follow-up was 15.4 months (IQR 10.1-21.0). The combination of pidilizumab and rituximab was well tolerated, with no autoimmune or treatment-related adverse events of grade 3 or 4. The most common adverse events of grade 1 were anaemia (14 patients) and fatigue (13 patients), and the most common adverse event of grade 2 was respiratory infection (five patients). Of the 29 patients evaluable for activity, 19 (66%) achieved an objective response: complete responses were noted in 15 (52%) patients and partial responses in four (14%). INTERPRETATION: The combination of pidilizumab plus rituximab is well tolerated and active in patients with relapsed follicular lymphoma. Our results suggest that immune checkpoint blockade is worthy of further study in follicular lymphoma. FUNDING: National Institutes of Health, Leukemia and Lymphoma Society, Cure Tech, and University of Texas MD Anderson Cancer Center.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Folicular/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Murinos/administração & dosagem , Feminino , Humanos , Linfoma Folicular/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva , RituximabRESUMO
Immunotherapeutic strategies are promising approaches for the treatment of follicular lymphoma (FL). However, their efficacy may be limited by immunosuppressive elements in the immune system and tumor microenvironment. Therefore, strategies to reverse the effects of the immunosuppressive elements are needed. We observed that regulatory T cells (Tregs) were increased in the peripheral blood at diagnosis and persisted in high numbers after induction of clinical remission with a cyclophosphamide and doxorubicin-containing chemotherapy regimen in FL patients. High levels of peripheral blood Tregs prior to therapy were associated with decreased progression-free survival in FL patients treated with either chemotherapy or combination immunotherapy that targeted CD20 and PD-1 with monoclonal antibodies rituximab and pidilizumab, respectively. Intratumoral and peripheral blood Tregs potently suppressed autologous antitumor effector T cells in FL. However, the effects of FL Tregs could be reversed by triggering Toll-like receptors (TLR) with TLR ligands Pam3 CSK4 (TLR 1/2), flagellin (TLR 5), and CpG-B (TLR 9), and/or OX40. The TLR ligands synergized with each other as well as OX40 signaling to inhibit Tregs. Furthermore, they restored the function of FL tumor-specific effector T cells. Our results suggest that a state of tolerance exists in FL patients at diagnosis and after induction of clinical remission, and agents that activate TLRs 1/2, 5, and 9, and OX40 may serve as adjuvants to enhance the efficacy of antitumor immunotherapeutic strategies and preventive vaccines against infectious diseases in these patients.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/metabolismo , Receptores OX40/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Adulto , Idoso , Antígenos CD20/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Separação Celular , Ciclofosfamida/farmacologia , Intervalo Livre de Doença , Doxorrubicina/farmacologia , Feminino , Citometria de Fluxo , Humanos , Imunossupressores/farmacologia , Imunoterapia/métodos , Interleucina-10/metabolismo , Ligantes , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Indução de Remissão , Linfócitos T Reguladores/citologia , Resultado do Tratamento , Adulto JovemRESUMO
Immunotherapy with therapeutic idiotype vaccines offers promise for treatment of B-cell malignancies. However, identification of novel immunogenic lymphoma-associated antigens that are universally expressed is necessary to overcome the barriers of patient-specific idiotype vaccines. Here, we determined whether T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene could be a target for immunotherapy of B-cell malignancies. We show that TCL1 mRNA and protein are selectively expressed in normal B cells but markedly hyperexpressed in multiple human B-cell lymphomas, including follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, and splenic marginal zone B-cell lymphoma. We demonstrated that TCL1-specific CD8(+) T cells can be generated from HLA-A*0201 (HLA-A2)(+) normal donors and identified TCL1(71-78) (LLPIMWQL) as the minimal epitope recognized by these T cells. More importantly, TCL1(71-78) peptide-specific T cells were present in the peripheral blood and tumor-infiltrating lymphocytes of lymphoma patients, could be expanded in vitro, and lysed autologous tumor cells but not normal B cells in an HLA-A2-restricted manner. Our results suggest that TCL1 is naturally processed and presented on the surface of lymphoma cells for recognition by cytotoxic T cells and can serve as a novel target for development of immunotherapeutic strategies against common B-cell lymphomas.
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Antígenos de Neoplasias/imunologia , Imunoterapia/métodos , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Proteínas Proto-Oncogênicas/imunologia , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Regulação Neoplásica da Expressão Gênica , Antígeno HLA-A2/imunologia , Humanos , Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Células Tumorais CultivadasRESUMO
Background: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. Methods: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4expression. Three-dimensional co-culture (tumor cells/ breast cancer-associated fibroblasts / human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effect of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. Results: Using multiple cell lines and patient breast cancer samples we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated that the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using multiple trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Conclusions: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.
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BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas. METHODS: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models. RESULTS: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion. CONCLUSION: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.
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Linfoma de Células B , Receptores de Antígenos Quiméricos , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Linfoma de Células B/tratamento farmacológico , Linfócitos T , Anticorpos Monoclonais/metabolismoRESUMO
PD-1 blockade enhances the function of antitumor T cells and antibody-dependent, cell-mediated cytotoxicity (ADCC) of NK cells. In a single-center, open-label, phase 2 trial, we tested the combination of pembrolizumab, an anti-PD-1 monoclonal antibody, and rituximab, an anti-CD20 monoclonal antibody that induces ADCC, in 30 patients with follicular lymphoma (FL) with rituximab-sensitive disease who had relapsed after ≥1 prior therapy. Pembrolizumab was administered at 200 mg IV every 3 weeks for up to 16 cycles, and rituximab was given at 375 mg/m2 IV weekly for 4 weeks in cycle 1 only. The most common grade 3/4 adverse events (AEs) were liver enzyme abnormalities (3%), diarrhea (3%), nausea (3%), aseptic meningitis (3%), and pancreatitis (3%). Low-grade immune-related AEs were reported in 80% of patients, including diarrhea (43%), liver enzyme abnormalities (33%), thyroid dysfunction (27%), and rash (23%). Grade 3 or 4 immune-related AEs occurred in 13% of the patients. Treatment-related AEs led to discontinuation in 6 (20%) patients. The overall response rate (primary end point) was 67%, and the complete response (CR) rate was 50%. Median progression-free survival (PFS) was 12.6 months (95% confidence interval, 8.2-27.6), the 3-year overall survival rate was 97%, and 23% of patients were in remission at a median follow-up of 35 months. The presence of a high CD8+ T-effector score at baseline in the tumor was associated with induction of a CR and improved PFS. In this single-arm, phase 2 study, the combination of pembrolizumab and rituximab demonstrates favorable efficacy and safety profile in relapsed FL. This trial is registered at www.clinicaltrials.gov as #NCT02446457.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma Folicular , Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Diarreia/induzido quimicamente , Humanos , Linfoma Folicular/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Rituximab/uso terapêuticoRESUMO
Three-dimensional structures of beta(2)-microglobulin (beta2m) from chicken and various mammals have been described previously, but aside from genomic sequences, very little is known about the three-dimensional structures of beta2m in species other than warm-blooded vertebrates. Here, we present the first three-dimensional structure of beta2m from bony fish grass carp (Ctid-beta2m), resolved at 2.1 A. The key structural differences between this new structure and previously published structures are two new hydrogen bonds at positions Ile(37) and Glu(38) in strand C and Lys(66) in strand E, and a hydrophobic pocket around the center of the protein found in Ctid-beta2m. Importantly, Ctid-beta2m has a short D strand and a long loop between stands C and D, rather than the flexible region found in other beta2m structures that serves as a putative binding region for the major histocompatibility complex heavy chain. Comparing the Ctid-beta2m structure with those of bovine and human beta2ms, the Calpha root mean square deviation of the latter are 1.3 A and 1.8 A, respectively. Compared with the constant domains of Lamprey T cell receptor-like receptor (Lamp-TCRLC) and Amphioxus V and C domain-bearing protein (Amphi-VCPC), Ctid-beta2m exhibits very different topology. The three-dimensional structures of domains predicted from Amphi-VCPC/Lamp-TCRLC are distinctly lacking in strand A of beta2ms. There are 18 amino acids at the N terminus of Amphi-VCPC that may have evolved into strand A of beta2ms. A mutation in the BC loops of Amphi-VCPC may have led to the novel topology found in beta2m. Based on these results, Ctid-beta2m may well reflect evolutionary characteristics of ancestral C set molecules.
Assuntos
Carpas/metabolismo , Evolução Molecular , Regiões Constantes de Imunoglobulina/química , Família Multigênica/genética , Microglobulina beta-2/química , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Lampreias , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged with highly contagious and life-threatening characteristics in 2002, remains a potential risk for future outbreaks. Membrane (M) and envelope (E) proteins are major structural proteins of the SARS-CoV. The M protein has been determined as a protective antigen in humoral responses. However, its potential roles in stimulating cellular immunity remain elusive. METHODS: In this study, a panel of peptides derived from M and E proteins were tested by in vitro refolding, T2 cell-binding assays, and responses stimulated by cytotoxic T-lymphocyte (CTL) epitopes in HLA-A2.1/K(b) transgenic mice and human peripheral blood mononuclear cells (PBMCs). RESULTS: A nonameric epitope Mn2 and a decameric epitope Md3 derived from the M protein were identified and used for the evaluation of M protein-specific immunity. Responses stimulated by M protein-specific CTL epitopes have been found in the PBMCs of donors who had recovered from SARS infection. Additionally, the transmembrane domain of the M protein may contain a T cell epitope cluster revealed by the immunogenic and structural analysis of a panel of truncated peptides overlapping with Mn2 and Md3. CONCLUSIONS: The M protein of SARS-CoV holds dominant cellular immunogenicity. This, together with previous reports of a strong humoral response against the M protein, may help to further explain the immunogenicity of SARS and serves as potential targets for SARS-CoV vaccine design.
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Epitopos de Linfócito T/imunologia , Imunidade Celular , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte , Proteínas M de Coronavírus , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Estrutura Quaternária de Proteína , Vacinas de DNA/imunologia , Proteínas da Matriz Viral/química , Vacinas Virais/imunologiaRESUMO
Nectin-2 belongs to a family of immunoglobulin-like cell adhesion molecules that are characterized by the presence of three immunoglobulin-like domains (V, C2 and C2) in the extracellular region. The V domain plays important roles in cell adhesion, NK cell activation and the entry of some herpesvirus. In this study, the V domain of human nectin-2 was expressed in Escherichia coli in the form of inclusion bodies, which were subsequently denatured and refolded. The soluble protein was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 1.85 A resolution and belonged to space group P2(1), with unit-cell parameters a = 52.3, b = 43.9, c = 56.1 A, beta = 118.2 degrees .
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Moléculas de Adesão Celular/química , Difração de Raios X , Sequência de Aminoácidos , Moléculas de Adesão Celular/genética , Clonagem Molecular , Cristalização , Coleta de Dados , Dimerização , Escherichia coli/genética , Vetores Genéticos , Humanos , Corpos de Inclusão/química , Dados de Sequência Molecular , Peso Molecular , Nectinas , Plasmídeos , Desnaturação Proteica , Renaturação Proteica , Estrutura Terciária de Proteína , Solubilidade , Estatística como Assunto , Temperatura , Transformação BacterianaRESUMO
CXCR5 mediates homing of both B and follicular helper T (TFH) cells into follicles of secondary lymphoid organs. We found that CXCR5+CD8+ T cells are present in human tonsils and follicular lymphoma, inhibit TFH-mediated B cell differentiation, and exhibit strong cytotoxic activity. Consistent with these findings, adoptive transfer of CXCR5+CD8+ T cells into an animal model of lymphoma resulted in significantly greater antitumor activity than CXCR5-CD8+ T cells. Furthermore, RNA-Seq-based transcriptional profiling revealed 77 differentially expressed genes unique to CXCR5+CD8+ T cells. Among these, a signature comprised of 33 upregulated genes correlated with improved survival in follicular lymphoma patients. We also showed that CXCR5+CD8+ T cells could be induced and expanded ex vivo using IL-23 plus TGF-ß, suggesting a possible strategy to generate these cells for clinical application. In summary, our study identified CXCR5+CD8+ T cells as a distinct T cell subset with ability to suppress TFH-mediated B cell differentiation, exert strong antitumor activity, and confer favorable prognosis in follicular lymphoma patients.
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Linfócitos T CD8-Positivos/citologia , Receptores CXCR5/metabolismo , Subpopulações de Linfócitos T/citologia , Transferência Adotiva , Animais , Linfócitos B/citologia , Diferenciação Celular , Técnicas de Cocultura , Feminino , Centro Germinativo/imunologia , Humanos , Leucócitos Mononucleares/citologia , Ativação Linfocitária , Linfoma Folicular/imunologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Tonsila Palatina/citologia , Receptores de Antígenos de Linfócitos T/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: There is increasing evidence to suggest an immunomodulation function both within the intestines and systemically upon consuming probiotic species. We recently isolated a novel LAB, Lactobacillus caseiZhang (LcZhang) from koumiss. LcZhang exhibited favorable probiotic properties, such as acid resistance, bile resistance, gastrointestinal (GI) colonization ability, etc. In order to examine the immunomodulatory qualities of LcZhang, we administered LcZhang to healthy mice with varying doses of either live or heat-killed LcZhang and measured various parameters of the host immune response. RESULTS: The study was performed in four separate experiments via oral administration of live and heat-killed LcZhang to BALB/c mice for several consecutive days. We investigated the immunomodulating capacity of LcZhang in vivo by analyzing the profile of cytokines, T cell subpopulations, and immunoglobulin concentrations induced in blood serum and intestinal fluid in BALB/c mice. Only live bacteria elicited a wide range of immune responses, which include the increased production of interferon-gamma (IFN-gamma), and depression of tumor necrosis factor-alpha (TNF-alpha) levels. In addition, interleukin-2 (IL-2) and IL-2 receptor gene transcription increased significantly, but the proportion of T cell subsets appeared to be unaffected. We also observed that LcZhang was capable of inducing gut mucosal responses by enhancing the production of secretory Immunoglobulin A (sIgA) as well influencing the systemic immunity via the cytokines released to the circulating blood. CONCLUSION: The present work shows that the dose-dependent administration of LcZhang is capable of influencing immune responses, implying that it may be a valuable strain for probiotic use in humans.
Assuntos
Produtos Fermentados do Leite/imunologia , Produtos Fermentados do Leite/microbiologia , Lacticaseibacillus casei/imunologia , Lacticaseibacillus casei/isolamento & purificação , Probióticos/administração & dosagem , Animais , China , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Interferon gama/biossíntese , Interleucina-2/genética , Camundongos , Camundongos Endogâmicos BALB C , Probióticos/farmacologia , Receptores de Interleucina-2/efeitos dos fármacos , Receptores de Interleucina-2/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 A and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 A resolution.
Assuntos
Proteínas de Bactérias/química , Streptococcus suis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Beta(2)-microglobulin (beta(2)m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of beta(2)m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of beta2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) beta(2)m (Ctid-beta(2)m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 A. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 A. The Matthews coefficient and the solvent content were calculated to be 2.56 A Da(-1) and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human beta(2)m as a model.
Assuntos
Carpas/metabolismo , Expressão Gênica , Microglobulina beta-2/sangue , Microglobulina beta-2/metabolismo , Animais , Carpas/genética , Cromatografia em Gel , Cristalização , Difração de Raios X , Microglobulina beta-2/genética , Microglobulina beta-2/isolamento & purificaçãoRESUMO
Mesenchymal stem or stromal cells (MSCs) are non-hematopoietic stem cells that facilitate tissue regeneration through mechanisms involving self-renewal and differentiation, supporting angiogenesis and tissue cell survival, and limiting inflammation. MSCs were originally identified and expanded in long-term cultures of cells from bone marrow and other organs; and their native identity was recently confined into pericytes and adventitial cells in vascularized tissue. The multipotency, as well as the trophic and immunosuppressive effects, of MSCs have prompted the rapid development of clinical applications for many diseases involving tissue inflammation and immune disorders, including inflammatory bowel disease. Although standard criteria have been established to define MSCs, their therapeutic efficacy has varied significantly among studies due to their natural heterogenicity. Thus, understanding the biological and immunological features of MSCs is critical to standardize and optimize MSCs-based therapy. In this review, we highlight the cellular and molecular mechanisms involved in MSCs-mediated tissue repair and immunosuppression. We also provide an update on the current development of MSCs-based clinical trials, with a detailed discussion of MSC-based cell therapy in inflammatory bowel disease.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Doenças Inflamatórias Intestinais/terapia , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Humanos , Terapia de Imunossupressão , Células-Tronco Mesenquimais/citologiaRESUMO
Despite the potent ability of dendritic cells (DCs) to stimulate lymphocyte responses and host immunity, granulocyte-macrophage colony-stimulating factor-derived DCs (GM-DCs) used as antitumor vaccines have demonstrated relatively modest success in cancer immunotherapy. We found that injecting GM-DCs into melanoma tumors in mice, or culturing GM-DCs with melanoma-secreted cytokines or melanoma-conditioned medium, rapidly suppressed DC-intrinsic expression of the gene encoding inhibitor of differentiation 2 (ID2), a transcriptional regulator. Melanoma-associated cytokines repressed Id2 transcription in murine DCs through the activation of signal transducer and activator of transcription 3 (STAT3). Enforced expression of ID2 in GM-DCs (ID2-GM-DCs) suppressed their production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Vaccination with ID2-GM-DCs slowed the progression of melanoma tumors and enhanced animal survival, which was associated with an increased abundance of tumor-infiltrating interferon-γ-positive CD4(+) effector and CD8(+) cytotoxic T cells and a decreased number of tumor-infiltrating regulatory CD4(+) T cells. The efficacy of the ID2-GM-DC vaccine was improved by combinatorial treatment with a blocking antibody to programmed cell death protein-1 (PD-1), a current immunotherapy that overcomes suppressive immune checkpoint signaling. Collectively, our data reveal a previously unrecognized STAT3-mediated immunosuppressive mechanism in DCs and indicate that DC-intrinsic ID2 promotes tumor immunity by modulating tumor-associated CD4(+) T cell responses. Thus, inhibiting STAT3 or overexpressing ID2 selectively in DCs may improve the efficiency of DC vaccines in cancer therapy.