RESUMO
As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.
Assuntos
Amiloide/metabolismo , Mieloma Múltiplo/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos Associados a Tumor/metabolismo , Microglobulina beta-2/metabolismo , Animais , Células Cultivadas , Humanos , Inflamação/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lisossomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fagocitose/imunologia , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Microglobulina beta-2/genéticaRESUMO
BACKGROUND: Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission. METHODS: We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded. RESULTS: The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization. CONCLUSIONS: In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).
Assuntos
Antígenos CD19 , Imunoterapia Adotiva , Lúpus Eritematoso Sistêmico , Agonistas Mieloablativos , Miosite , Escleroderma Sistêmico , Humanos , Antígenos CD19/administração & dosagem , Síndrome da Liberação de Citocina/etiologia , Seguimentos , Lúpus Eritematoso Sistêmico/terapia , Miosite/terapia , Escleroderma Sistêmico/terapia , Agonistas Mieloablativos/administração & dosagem , Ciclofosfamida/administração & dosagem , Infecções/etiologia , Resultado do TratamentoRESUMO
Despite the tremendous progress in the clinical management of autoimmune diseases, many patients do not respond to the currently used treatments. Autoreactive B cells play a key role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. B-cell-depleting monoclonal antibodies, such as rituximab, have poor therapeutic efficacy in autoimmune diseases, mainly due to the persistence of autoreactive B cells in lymphatic organs and inflamed tissues. The adoptive transfer of T cells engineered to target tumour cells via chimeric antigen receptors (CARs) has emerged as an effective treatment modality in B-cell malignancies. In the last 2 years treatment with autologous CAR T cells directed against the CD19 antigen has been introduced in therapy of autoimmune disease. CD19 CAR T cells induced a rapid and sustained depletion of circulating B cells, as well as in a complete clinical and serological remission of refractory systemic lupus erythematosus and dermatomyositis. In this paper, we discuss the evolving strategies for targeting autoreactive B cells via CAR T cells, which might be used for targeted therapy in autoimmune diseases.
Assuntos
Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/efeitos adversos , Doenças Autoimunes/terapia , Linfócitos T , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Rituximab/uso terapêutico , Receptores de Antígenos Quiméricos/uso terapêutico , Antígenos CD19 , Receptores de Antígenos de Linfócitos TRESUMO
OBJECTIVES: CD19-targeting chimeric antigen receptor (CAR) T-cell therapy can induce long-term drug-free remission in patients with autoimmune diseases (AIDs). The efficacy of CD19-CAR T-cell therapy is presumably based on deep tissue depletion of B cells; however, such effect has not been proven in humans in vivo. METHODS: Sequential ultrasound-guided inguinal lymph node biopsies were performed at baseline and after CD19-CAR T-cell therapy in patients with AIDs. Results were compared with lymph node biopsies from rituximab (RTX)-treated AID patients with absence of peripheral B cells. Conventional and immunohistochemistry staining were performed on lymph node tissue to assess architecture as well the number of B cells, follicular dendritic cells (FDCs), plasma cells, T cells and macrophages. RESULTS: Sequential lymph node biopsies were analysed from five patients with AID before and after CD19-CAR T-cell therapy and from five patients with AID after RTX treatment. In addition, non-lymphoid organ biopsies (colon, kidney and gallbladder) from three additional patients with AID after CD19-CAR T-cell therapy were analysed. CD19+ and CD20+ B cells were completely depleted in the lymph nodes after CD19-CAR T-cell therapy, but not after RTX treatment. Plasma cells, T cells and macrophages in the lymph nodes remained unchanged. Follicular structures were disrupted and FDCs were depleted in the lymph nodes after CD19-CAR T-cell therapy, but not after RTX. Non-lymphoid organs were completely depleted of B cells. DISCUSSION: This study demonstrates complete B-cell depletion in secondary lymphoid tissues of patients with AIDs following CD19-CAR T-cell therapy combined with standard lymphodepleting therapy.
RESUMO
Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRß) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.
Assuntos
Infecções por Vírus Epstein-Barr , Transplante de Células-Tronco Hematopoéticas , Transferência Adotiva , Epitopos , Herpesvirus Humano 4/fisiologia , Humanos , Peptídeo T , Peptídeos , Linfócitos TRESUMO
Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.
RESUMO
Autoreactive Bcells play a key role in the pathogenesis of autoimmune diseases, such systemic lupus erythematosus (SLE). An efficient depletion of Bcells therefore plays a special role in autoimmune diseases, especially in cases with a severe course of the disease. Treatment with chimeric antigen receptor (CAR) Tcells, which was originally developed for the treatment of Bcell lymphomas and leukemias, provides the possibility to deplete Bcells even in deep tissues. The initial results from case series with this procedure for SLE, myositis and systemic sclerosis were very positive. This review article gives an overview of the course, mechanism of action, results so far and the research agenda of CAR Tcell therapy in autoimmune diseases.
Assuntos
Imunoterapia Adotiva , Humanos , Imunoterapia Adotiva/métodos , Doenças Reumáticas/terapia , Doenças Reumáticas/imunologia , Resultado do Tratamento , Linfócitos T/imunologia , Doenças Autoimunes/terapia , Doenças Autoimunes/imunologia , Medicina Baseada em Evidências , Receptores de Antígenos Quiméricos/imunologia , Reumatologia/tendênciasRESUMO
Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) â BALB/c (H-2d) and LP/J (H-2b) â C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Piranos/farmacologia , Compostos de Pirvínio/farmacologia , Escleroderma Sistêmico/prevenção & controle , Sulfonas/farmacologia , Triazóis/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Escleroderma Sistêmico/etiologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologiaRESUMO
Hematotoxicity represents a frequent chimeric antigen receptor (CAR) T-cell-related adverse event and remains poorly understood. In this multicenter analysis, we studied patterns of hematopoietic reconstitution and evaluated potential predictive markers in 258 patients receiving axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) for relapsed/refractory large B-cell lymphoma. We observed profound (absolute neutrophil count [ANC] <100 cells per µL) neutropenia in 72% of patients and prolonged (21 days or longer) neutropenia in 64% of patients. The median duration of severe neutropenia (ANC < 500 cells per µL) was 9 days. We aimed to identify predictive biomarkers of hematotoxicity using the duration of severe neutropenia until day +60 as the primary end point. In the training cohort (n = 58), we observed a significant correlation with baseline thrombocytopenia (r = -0.43; P = .001) and hyperferritinemia (r = 0.54; P < .0001) on univariate and multivariate analysis. Incidence and severity of cytokine-release syndrome, immune effector cell-associated neurotoxicity syndrome, and peak cytokine levels were not associated with the primary end point. We created the CAR-HEMATOTOX model, which included markers associated with hematopoietic reserve (eg, platelet count, hemoglobin, and ANC) and baseline inflammation (eg, C-reactive protein and ferritin). This model was validated in independent cohorts, one from Europe (n = 91) and one from the United States (n = 109) and discriminated patients with severe neutropenia ≥14 days to <14 days (pooled validation: area under the curve, 0.89; sensitivity, 89%; specificity, 68%). A high CAR-HEMATOTOX score resulted in a longer duration of neutropenia (12 vs 5.5 days; P < .001) and a higher incidence of severe thrombocytopenia (87% vs 34%; P < .001) and anemia (96% vs 40%; P < .001). The score implicates bone marrow reserve and inflammation prior to CAR T-cell therapy as key features associated with delayed cytopenia and will be useful for risk-adapted management of hematotoxicity.
Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Produtos Biológicos/efeitos adversos , Doenças Hematológicas/etiologia , Imunoterapia Adotiva/efeitos adversos , Linfoma Difuso de Grandes Células B/terapia , Receptores de Antígenos de Linfócitos T , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/etiologia , Antineoplásicos Imunológicos/uso terapêutico , Produtos Biológicos/uso terapêutico , Síndrome da Liberação de Citocina/etiologia , Humanos , Incidência , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/terapia , Síndromes Neurotóxicas/etiologia , Neutropenia/etiologia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Estudos Retrospectivos , Trombocitopenia/etiologia , Adulto JovemAssuntos
Doenças Autoimunes , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Doenças Autoimunes/terapia , Doenças Autoimunes/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/terapia , Receptores de Antígenos de Linfócitos T/uso terapêutico , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologiaRESUMO
COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Pulmão/imunologia , Ativação Linfocitária , Receptores CCR4/imunologia , SARS-CoV-2/imunologia , Regulação para Cima/imunologia , Adulto , Linfócitos T CD8-Positivos/patologia , COVID-19/patologia , Feminino , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Treatment with convalescent plasma has been shown to be safe in coronavirus disease in 2019 (COVID-19) infection, although efficacy reported in immunocompetent patients varies. Nevertheless, neutralizing antibodies are a key requisite in the fight against viral infections. Patients depleted of antibody-producing B cells, such as those treated with rituximab (anti-CD20) for hematological malignancies, lack a fundamental part of their adaptive immunity. Treatment with convalescent plasma appears to be of general benefit in this particularly vulnerable cohort. We analyzed clinical course and inflammation markers of three B-cell-depleted patients suffering from COVID-19 who were treated with convalescent plasma. In addition, we measured serum antibody levels as well as peripheral blood CD38/HLA-DR-positive T-cells ex vivo and CD137-positive T-cells after in vitro stimulation with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides in these patients. We observed that therapy with convalescent plasma was effective in all three patients and analysis of CD137-positive T-cells after stimulation with SARS-CoV-2 peptides showed an increase in peptide-specific T-cells after application of convalescent plasma. In conclusion, we here demonstrate efficacy of convalescent plasma therapy in three B-cell-depleted patients and present data that suggest that while application of convalescent plasma elevates systemic antibody levels only transiently, it may also boost specific T-cell responses.
Assuntos
Anticorpos Antivirais/sangue , Linfócitos B/imunologia , COVID-19/terapia , Linfócitos T/imunologia , Adolescente , Idoso , Anticorpos Neutralizantes/sangue , Linfócitos B/citologia , Humanos , Imunidade Celular/imunologia , Imunização Passiva/métodos , Contagem de Linfócitos , Depleção Linfocítica , Linfoma de Células B/tratamento farmacológico , Linfoma de Célula do Manto/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Rituximab/efeitos adversos , SARS-CoV-2/imunologia , Resultado do Tratamento , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Soroterapia para COVID-19RESUMO
Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-ß (PKCß) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCß on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCß that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCß results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCß-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCß and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.
Assuntos
Células da Medula Óssea/metabolismo , Glucose/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína Quinase C beta/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Proteína Quinase C beta/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralizing antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR-αß sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , COVID-19/imunologia , COVID-19/terapia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , COVID-19/virologia , Células Clonais/imunologia , Células Clonais/virologia , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Citocinas/biossíntese , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imunização Passiva , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Soroterapia para COVID-19RESUMO
Many proteins are still routinely expressed prokaryotically in Escherichia coli, some because they are toxic to eukaryotes. Immunotoxins, which are fusion proteins of a targeting moiety and a truncated Pseudomonas exotoxin A, kill target cells by arresting protein synthesis. Thus, immunotoxins must be expressed in E. coli. Proteins expressed in E. coli are contaminated by endotoxin (also called lipopolysaccharides (LPS)). LPS binds to toll-like receptors, inducing up to life-threatening systemic inflammation in mammals. Therefore, accepted LPS limits for therapeutics as well as for substances used in immunological studies in animals are very low. Here, we report the use of Triton X-114 and polyamine-based wash strategies, which only in combination achieved LPS-contamination well below FDA limits. Resulting LPS-reduced immunotoxins were purer and up to 2.4-fold more active in vitro. Increased activity was associated with a 2.4-fold increase in affinity on cell surface expressed target antigen. The combination method maintained enzymatic function, protein stability, and in vivo efficacy and was effective for Fab as well as dsFv formats. With some modifications, the principle of this novel combination may be applied to any chromatography-based purification process.
Assuntos
Aminas/química , Imunotoxinas/isolamento & purificação , Lipopolissacarídeos/toxicidade , Octoxinol/química , Animais , Humanos , Imunotoxinas/toxicidade , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidadeRESUMO
Mesenchymal stromal cells (MSCs) are characterized by their multipotency, regenerative potential, and immunoregulatory properties. Nowadays, MSCs represent a promising cell-therapeutic option for hyperinflammatory conditions such as graft-vs-host disease following allogeneic hematopoietic stem cell transplantation. A better understanding of their biology is a prerequisite for improving their treatment efficacy. Emerging evidence suggests that immunosuppressive properties are not constitutively active in MSCs. Instead, microenvironmental inflammatory stimuli such as the cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α license MSCs to acquire a tolerance-promoting phenotype. The immunological checkpoint molecule programmed death-ligand 1 (PD-L1) is an important regulator of T-cell responses. Binding of PD-L1 to the programmed cell death protein 1 (PD-1) receptor on T-cells suppresses their activation, proliferation, and induces apoptosis. Previous studies have revealed that cell surface expression and secretion of PD-L1 are part of the MSCs' immunomodulatory armamentarium. Here, we report that inflammatory licensing leads to an enhanced PD-L1 cell surface expression and secretion, which are both accompanied by an increased posttranslational protein N-glycosylation. These post-translational modifications have been shown to be critical for key biological processes such as cell trafficking, receptor signaling, and immunohomeostasis. In fact, promoting N-glycosylation in MSCs yielded increased PD-L1 levels. We report for the first time that PD-L1 N-glycosylation plays a decisive role for its transport to the MSCs' cell surface and its subsequent secretion (in response to proinflammatory trigger). Our data offer insights into a novel regulatory mechanism with the potential to be exploited as a means to foster the immunosuppressive potency of human MSCs.
Assuntos
Antígeno B7-H1/metabolismo , Células-Tronco Mesenquimais/metabolismo , Glicosilação , Humanos , Regulação para CimaRESUMO
BACKGROUND: Protein-losing enteropathy (PLE) is a severe complication of the Fontan circulation. There is increasing discussion about whether lymphatic dysregulation is involved as pathomechanism of PLE. This investigation focuses on the interplay between alteration of lymphatic cells and immunologic pathway alterations. METHODS: Micro-ribonucleic acid (miRNA) expression profiling was performed in 49 patients (n = 10 Fontan patients with PLE, n = 30 Fontan patients without PLE, and n = 9 patients with dextro-transposition of the great arteries (dTGA). miRNA pathway analysis was performed to identify significantly enriched pathways. To determine lymphocyte populations and subtypes multiparameter flow cytometry was used. RESULTS: miRNAs pathway analysis of Fontan patients with PLE revealed 20 significantly changed networks of which four of the ten largest were associated with immunologic processes. This finding is supported by significant T cell deficiency with decreased CD4+ count (p = 0.0002), altered CD4 +/CD8+ ratio, and significantly modified CD4+ (p < 0.0001) and CD8+ (p = 0.0002) T cell differentiation toward effector and terminal differentiated T cells in Fontan patients with PLE. Analyses of CD4+ T cell subsets demonstrated significantly increased frequencies of CD4+ CD25+ CD127- regulatory T cells (Treg) in Fontan patients with PLE (p = 0.0011). CONCLUSION: PLE in Fontan patients is associated with severe lymphopenia, T cell deficiency, significant alterations of T cell differentiation, and increased Treg frequency reflecting an immune status of chronic inflammation and shortened protection against pathogens and autoimmunity. These cellular alterations seemed to be dysregulated by several miRNA controlled immunological pathways.
Assuntos
Diferenciação Celular , Técnica de Fontan/efeitos adversos , Cardiopatias Congênitas/cirurgia , Linfopenia/imunologia , Enteropatias Perdedoras de Proteínas/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Animais , Autoimunidade , Estudos de Casos e Controles , Criança , Pré-Escolar , Bases de Dados Factuais , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Imunofenotipagem , Lactente , Linfopenia/diagnóstico , Linfopenia/genética , Linfopenia/microbiologia , Masculino , Camundongos , MicroRNAs/genética , Fenótipo , Enteropatias Perdedoras de Proteínas/diagnóstico , Enteropatias Perdedoras de Proteínas/genética , Transcriptoma , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Treatment with proteasome inhibitors like carfilzomib in patients with multiple myeloma (MM) can induce thrombotic microangiopathy (TMA) characterized by neurological symptoms, acute kidney injury, hemolysis and thrombocytopenia. Successful treatment with the monoclonal antibody eculizumab was described for these patients, but reports of ideal management and definitive treatment protocols are lacking. CASE PRESENTATION: The first case describes a 43-years-old IgG-kappa-MM patient that developed TMA during the first course of carfilzomib-lenalidomide-dexamethasone (KRd) consolidation after autologous stem cell transplantation (ASCT). In the second case, a 59-years-old IgG-kappa-MM patient showed late-onset TMA during the fourth and last cycle of elotuzumab-KRd consolidation within the DSMM XVII study of the German study group MM (DSMM; clinicalTrials.gov Identifier: NCT03948035). Concurrently, he suffered from influenza A/B infection. Both patients had a high TMA-index for a poor prognosis of TMA. Therapeutically, in both patients plasma exchange (TPE) was initiated as soon as TMA was diagnosed. In patient #1, dialysis became necessary. For both patients, only when the complement inhibitor eculizumab was administered, kidney function and blood values impressively improved. CONCLUSION: In this small case series, two patients with MM developed TMA due to carfilzomib treatment (CFZ-TMA), the second patient as a late-onset form. Even though TMA could have been elicited by influenza in the second patient and occurred after ASCT in both patients, with cases of TMA post-transplantation in MM being described, a relation of TMA and carfilzomib treatment was most likely. In both patients, treatment with eculizumab over two months efficiently treated TMA without recurrence and with both patients remaining responsive months after TMA onset. Taken together, we describe two cases of TMA in MM patients on carfilzomib-combination treatment, showing similar courses of this severe adverse reaction, with good responses to two months of eculizumab treatment.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/efeitos adversos , Microangiopatias Trombóticas/induzido quimicamente , Microangiopatias Trombóticas/tratamento farmacológico , Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/uso terapêutico , Indução de RemissãoRESUMO
Targeted immunotherapies have greatly changed treatment of patients with B cell malignancies. To further enhance immunotherapies, research increasingly focuses on the tumor microenvironment (TME), which differs considerably by organ site. However, immunocompetent mouse models of disease to study immunotherapies targeting human molecules within organ-specific TME are surprisingly rare. We developed a myc-driven, primary murine lymphoma model expressing a human-mouse chimeric CD22 (h/mCD22). Stable engraftment of three distinct h/mCD22+ lymphoma was established after subcutaneous and systemic injection. However, only systemic lymphoma showed immune infiltration that reflected human disease. In this model, myeloid cells supported lymphoma growth and showed a phenotype of myeloid-derived suppressor cells. The human CD22-targeted immunotoxin Moxetumomab was highly active against h/mCD22+ lymphoma and similarly reduced infiltration of bone marrow and spleen of all three models up to 90-fold while efficacy against lymphoma in lymph nodes varied substantially, highlighting relevance of organ-specific TME. As in human aggressive lymphoma, anti-PD-L1 as monotherapy was not efficient. However, anti-PD-L1 enhanced efficacy of Moxetumomab suggesting potential for future clinical application. The novel model system of h/mCD22+ lymphoma provides a unique platform to test targeted immunotherapies and may be amenable for other human B cell targets such as CD19 and CD20.