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1.
Biopreserv Biobank ; 21(2): 166-175, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35771982

RESUMO

Biobanking during the COVID-19 pandemic presented unique challenges regarding patient enrollment, sample collection, and experimental analysis. This report details the ways in which we rapidly overcame those challenges to create a robust database of clinical information and patient samples while maintaining clinician and researcher safety. We developed a pipeline using REDCap (Research Electronic Data Capture) to coordinate electronic informed consent, sample collection, immunological assay execution, and data analysis for biobanking samples from patients with COVID-19. We then integrated immunological assay data with clinical data extracted from the electronic health record to link study parameters with clinical readouts. Of the 193 inpatients who participated in this study, 138 consented electronically and 56 provided paper consent. We collected and banked blood samples to measure circulating cytokines and chemokines, peripheral immune cell composition and activation status, anti-COVID-19 antibodies, and germline gene polymorphisms. In addition, we collected DNA and RNA from nasopharyngeal swabs to assess viral titer and microbiome composition by 16S sequencing. The rapid spread and contagious nature of COVID-19 required special considerations and innovative solutions to biobank samples quickly while protecting researchers and clinicians. Overall, this workflow and computational pipeline allowed for comprehensive immune profiling of 193 inpatients infected with COVID-19, as well as 89 outpatients, 157 patients receiving curbside COVID-19 testing, and 86 healthy controls. We describe a novel electronic framework for biobanking and analyzing patient samples during COVID-19, and present insights and strategies that can be applied more broadly to other biobank studies.


Assuntos
COVID-19 , Humanos , COVID-19/epidemiologia , Bancos de Espécimes Biológicos , Teste para COVID-19 , Pandemias , Consentimento Livre e Esclarecido , Bases de Dados Factuais
2.
Sci Adv ; 8(40): eabn3777, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36206332

RESUMO

Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation, and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme 2 (ACE2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized patients with COVID-19 developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or receptor-binding domain (RBD), to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Angiotensina II , Autoanticorpos , Pressão Sanguínea , Epitopos/metabolismo , Humanos , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , SARS-CoV-2 , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus
3.
Blood Adv ; 6(24): 6249-6262, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-35977099

RESUMO

Clinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung but also in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers (GCs). It strongly correlated with the presence of intralymphatic NETs. In mice, tumor necrosis factor α induced intralymphatic fibrin clots; this could be inhibited by DNase I, which degrades NETs. In vitro, TNF-α induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8, among other neutrophil-recruiting factors, as well as thrombomodulin downregulation; in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of Myeloperoxidase-DNA (MPO-DNA, a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. Patients with high MPO-DNA but low D-dimer levels generated poor antiviral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of GCs necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation affects adaptive immune responses.


Assuntos
COVID-19 , Armadilhas Extracelulares , Trombose , Camundongos , Animais , Trombose/metabolismo , Pulmão/metabolismo , DNA/metabolismo , Linfonodos
4.
J Immunother Cancer ; 10(6)2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35732350

RESUMO

BACKGROUND: Patients with cancer were excluded from phase 3 COVID-19 vaccine trials, and the immunogenicity and side effect profiles of these vaccines in this population is not well understood. Patients with cancer can be immunocompromised from chemotherapy, corticosteroids, or the cancer itself, which may affect cellular and/or humoral responses to vaccination. PD-1 is expressed on T effector cells, T follicular helper cells and B cells, leading us to hypothesize that anti-PD-1 immunotherapies may augment antibody or T cell generation after vaccination. METHODS: Antibodies to the SARS-CoV-2 receptor binding domain (RBD) and spike protein were assessed in patients with cancer (n=118) and healthy donors (HD, n=22) after 1, 2 or 3 mRNA vaccine doses. CD4+ and CD8+ T cell reactivity to wild-type (WT) or B.1.617.2 (delta) spike peptides was measured by intracellular cytokine staining. RESULTS: Oncology patients without prior COVID-19 infections receiving immunotherapy (n=36), chemotherapy (n=15), chemoimmunotherapy (n=6), endocrine or targeted therapies (n=6) and those not on active treatment (n=26) had similar RBD and Spike IgG antibody titers to HDs after two vaccinations. Contrary to our hypothesis, PD-1 blockade did not augment antibody titers or T cell responses. Patients receiving B-cell directed therapies (n=14) including anti-CD20 antibodies and multiple myeloma therapies had decreased antibody titers, and 9/14 of these patients were seronegative for RBD antibodies. No differences were observed in WT spike-reactive CD4+ and CD8+ T cell generation between treatment groups. 11/13 evaluable patients seronegative for RBD had a detectable WT spike-reactive CD4+ T cell response. T cells cross-reactive against the B.1.617.2 variant spike peptides were detected in 31/59 participants. Two patients with prior immune checkpoint inhibitor-related adrenal insufficiency had symptomatic hypoadrenalism after vaccination. CONCLUSIONS: COVID-19 vaccinations are safe and immunogenic in patients with solid tumors, who developed similar antibody and T cell responses compared with HDs. Patients on B-cell directed therapies may fail to generate RBD antibodies after vaccination and should be considered for prophylactic antibody treatments. Many seronegative patients do develop a T cell response, which may have an anti-viral effect. Patients with pre-existing adrenal insufficiency may need to take stress dose steroids during vaccination to avoid adrenal crisis.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Neoplasias , Insuficiência Adrenal/complicações , Anticorpos Antivirais/sangue , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , SARS-CoV-2 , Linfócitos T/imunologia , Vacinação , Vacinas Sintéticas , Vacinas de mRNA/imunologia
5.
Res Sq ; 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34845442

RESUMO

The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.

6.
medRxiv ; 2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34751272

RESUMO

Patients infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can experience life-threatening respiratory distress, blood pressure dysregulation and thrombosis. This is thought to be associated with an impaired activity of angiotensin-converting enzyme-2 (ACE-2), which is the main entry receptor of SARS-CoV-2 and which also tightly regulates blood pressure by converting the vasoconstrictive peptide angiotensin II (AngII) to a vasopressor peptide. Here, we show that a significant proportion of hospitalized COVID-19 patients developed autoantibodies against AngII, whose presence correlates with lower blood oxygenation, blood pressure dysregulation, and overall higher disease severity. Anti-AngII antibodies can develop upon specific immune reaction to the SARS-CoV-2 proteins Spike or RBD, to which they can cross-bind, suggesting some epitope mimicry between AngII and Spike/RBD. These results provide important insights on how an immune reaction against SARS-CoV-2 can impair blood pressure regulation.

8.
Br J Haematol ; 185(4): 679-690, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30828801

RESUMO

Acute myeloid leukaemia (AML) is a lethal haematological malignancy characterized by an immunosuppressive milieu in the tumour microenvironment (TME) that fosters disease growth and therapeutic resistance. Hypomethylating agents (HMAs) demonstrate clinical efficacy in AML patients and exert immunomodulatory activities. In the present study, we show that guadecitabine augments both antigen processing and presentation, resulting in increased AML susceptibility to T cell-mediated killing. Exposure to HMA results in the activation of the endogenous retroviral pathway with concomitant downstream amplification of critical mediators of inflammation. In an immunocompetent murine leukaemia model, guadecitabine negatively regulates inhibitory accessory cells in the TME by decreasing PD-1 (also termed PDCD1) expressing T cells and reducing AML-mediated expansion of myeloid-derived suppressor cells. Therapy with guadecitabine results in enhanced leukaemia-specific immunity, as manifested by increased CD4 and CD8 cells targeting syngeneic leukaemia cells. We have previously reported that vaccination with AML/dendritic cell fusions elicits the expansion of leukaemia-specific T cells and protects against disease relapse. In the present study, we demonstrate that vaccination in conjunction with HMA therapy results in enhanced anti-leukaemia immunity and survival. The combination of a novel personalized dendritic cell/AML fusion vaccine and an HMA has therapeutic potential, and a clinical trial investigating this combination is planned.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Azacitidina/análogos & derivados , Vacinas Anticâncer/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Microambiente Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/imunologia , Azacitidina/imunologia , Azacitidina/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/imunologia , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/metabolismo , Retroviridae/imunologia , Ativação Viral/imunologia
9.
Leukemia ; 33(1): 132-147, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29946193

RESUMO

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.


Assuntos
Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/imunologia , Linfoma Difuso de Grandes Células B/imunologia , MicroRNAs/genética , Proteínas Virais/metabolismo , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/virologia , Prognóstico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Células Tumorais Cultivadas , Proteínas Virais/genética
10.
J Immunother ; 40(9): 315-322, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28961609

RESUMO

Multiple myeloma (MM) is characterized by progressive immune dysregulation, loss of myeloma-specific immunity, and an immunosuppressive milieu that fosters disease growth and immune escape. Accordingly, cancer vaccines that reverse tumor-associated immune suppression represent a promising therapeutic avenue of investigation. We examined the potential of an allogeneic cellular vaccine to generate immune responses against MM tumor cells. The DCOne vaccine is comprised of a human myeloid leukemia cell line differentiated into a fully functional dendritic cell, expressing a range of tumor-associated antigens that are also known targets in MM. We found that the myeloma-specific antigens expressed by the DCOne vaccine can traffic via extracellular vesicles to surrounding antigen-presenting cells, thus stimulating autologous T-cell responses. Indeed, coculture of peripheral blood mononuclear cells from patients with MM with the DCOne vaccine resulted in the expansion of activated CD8 T cells expressing interferon-γ and perforin, with no significant change in the percentage of CD4 T cells producing interleukin-10. Further, coculture of patient's tumor cells with peripheral blood mononuclear cells and DCOne induced cytotoxic T-lymphocyte-mediated killing of autologous MM cells. These findings demonstrate that the allogeneic DCOne vaccine can induce T-cell activation and myeloma-specific immunity via cross presentation of antigens by native antigen-presenting cells.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vesículas Extracelulares/imunologia , Imunoterapia Adotiva/métodos , Mieloma Múltiplo/terapia , Vacinas Anticâncer , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Apresentação Cruzada , Citotoxicidade Imunológica , Células Dendríticas/transplante , Humanos , Interferon gama/metabolismo , Isoantígenos/imunologia , Ativação Linfocitária , Mieloma Múltiplo/imunologia , Perforina/metabolismo , Microambiente Tumoral
11.
Cancer Discov ; 7(7): 750-765, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28274958

RESUMO

Several kinase inhibitors that target aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in cancer clinical trial patients across several malignancies. Here, we show that cabozantinib rapidly eradicates invasive, poorly differentiated PTEN/p53-deficient murine prostate cancer. This was associated with enhanced release of neutrophil chemotactic factors from tumor cells, including CXCL12 and HMGB1, resulting in robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor plerixafor. Collectively, these data demonstrate that cabozantinib triggers a neutrophil-mediated anticancer innate immune response, resulting in tumor clearance.Significance: This study is the first to demonstrate that a tyrosine kinase inhibitor can activate neutrophil-mediated antitumor innate immunity, resulting in invasive cancer clearance. Cancer Discov; 7(7); 750-65. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.


Assuntos
Anilidas/administração & dosagem , Quimiocina CXCL12/antagonistas & inibidores , Proteína HMGB1/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/tratamento farmacológico , Piridinas/administração & dosagem , Proteína Supressora de Tumor p53/genética , Animais , Benzilaminas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/genética , Ciclamos , Proteína HMGB1/genética , Compostos Heterocíclicos/administração & dosagem , Humanos , Imunidade Inata/efeitos dos fármacos , Masculino , Camundongos , Neutrófilos/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Microambiente Tumoral/genética
12.
Br J Haematol ; 176(6): 929-938, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28107546

RESUMO

Multiple myeloma (MM) is a lethal haematological malignancy that arises in the context of a tumour microenvironment that promotes resistance to apoptosis and immune escape. In the present study, we demonstrate that co-culture of MM cells with stromal cells results in increased resistance to cytotoxic and biological agents as manifested by decreased rates of cell death following exposure to alkylating agents and the proteosome inhibitor, bortezomib. To identify the mechanism of increased resistance, we examined the effect of the co-culture of MM cells with stroma cells, on expression of the MUC1 oncogene, known to confer tumour cells with resistance to apoptosis and necrosis. Co-culture of stroma with MM cells resulted in increased MUC1 expression by tumour cells. The effect of stromal cell co-culture on MUC1 expression was not dependent on cell contact and was therefore thought to be due to soluble factors secreted by the stromal cells into the microenvironment. We demonstrated that MUC1 expression was mediated by interleukin-6 and subsequent up-regulation of the JAK-STAT pathway. Interestingly, the effect of stromal cell co-culture on tumour resistance was partially reversed by silencing of MUC1 in MM cells, consistent with the potential role of MUC1 in mediating resistance to cytotoxic-based therapies.


Assuntos
Medula Óssea/metabolismo , Medula Óssea/patologia , Comunicação Celular , Mucina-1/biossíntese , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Células Estromais/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Humanos , Janus Quinase 2/metabolismo , Mucina-1/genética , Mieloma Múltiplo/genética , Inibidores de Proteassoma/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Hum Vaccin Immunother ; 10(11): 3125-31, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25625926

RESUMO

The potential for the immune system to target hematological malignancies is demonstrated in the allogeneic transplant setting, where durable responses can be achieved. However, allogeneic transplantation is associated with significant morbidity and mortality related to graft versus host disease. Cancer immunotherapy has the capacity to direct a specific cytotoxic immune response against cancer cells, particularly residual cancer cells, in order to reduce the likelihood of disease relapse in a more targeted and tolerated manner. Ex vivo dendritic cells can be primed in various ways to present tumor associated antigen to the immune system, in the context of co-stimulatory molecules, eliciting a tumor specific cytotoxic response in patients. Several approaches to prime dendritic cells and overcome the immunosuppressive microenvironment have been evaluated in pre-clinical and early clinical trials with promising results. In this review, we summarize the clinical data evaluating dendritic cell based vaccines for the treatment of hematological malignancies.


Assuntos
Vacinas Anticâncer/uso terapêutico , Células Dendríticas/transplante , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Ensaios Clínicos como Assunto , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Neoplasias Hematológicas/imunologia , Humanos , Transplante Homólogo , Vacinação
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