RESUMO
Introduction: Current SARS-CoV-2 strains continue to mutate and attempt to evade the antibody response elicited by previous exposures and vaccinations. In September of 2022, the first updated SARS-CoV-2 vaccines, designed to create immune responses specific for the variants circulating in 2022, were approved. These new vaccines, known commonly as the bivalent boost(er), include mRNA that encodes both the original Wuhan-Hu-1 spike protein as well as the spike protein specific to the Omicron BA.4 and BA.5 variants. Methods: We recruited volunteers from University of Massachusetts student, faculty and staff members to provide samples of blood and saliva at four different time points, including pre-boost and three times post boost and analyzed samples for antibody production as well as neutralization of virus. Results: Our data provide a comprehensive analysis of the antibody response following a single dose of the bivalent boost over a 6-month period and support previous findings that the response induced after the bivalent boost does not create a strong BA.4/BA.5-specific antibody response. Conclusion: We found no evidence of a specific anti-BA.4/BA.5 response developing over time, including in a sub-population of individuals who become infected after a single dose of the bivalent booster. Additionally, we present data that support the use of saliva samples as a reliable alternative to blood for antibody detection against specific SARS-CoV-2 antigens.
Assuntos
Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Imunização Secundária , SARS-CoV-2 , Saliva , Glicoproteína da Espícula de Coronavírus , Humanos , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/prevenção & controle , Saliva/imunologia , Saliva/virologia , Vacinas contra COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Masculino , Feminino , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Pessoa de Meia-Idade , Formação de Anticorpos/imunologia , Adulto JovemRESUMO
Graft-versus-host disease (GVHD) is a primary and often lethal complication of allogenic hematopoietic stem cell transplantation (HSCT). Prophylactic regimens for GVHD are given as standard pretransplantation therapy; however, up to 50% of these patients develop acute GVHD (aGVHD) and require additional immunosuppressive intervention. Using a mouse GVHD model, we previously showed that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GVHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of A2neg human peripheral blood mononuclear cells (PBMCs). Because we could not inject human donors with EPS, we transferred EPS-pretreated dendritic cells (DCs) to inhibit aGVHD. We derived these DCs from CD34+ human cord blood cells, treated them with EPS, and then injected them together with PBMCs into the NSG-HLA-A2 mice. We found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived. This DC cell therapy could be readily translatable to humans, because we can generate large numbers of human EPS-DCs and use them as an "off the shelf" treatment for patients undergoing HSCT.
Assuntos
Doença Enxerto-Hospedeiro , Antígeno HLA-A2 , Animais , Humanos , Transplante Homólogo/efeitos adversos , Leucócitos Mononucleares , Doença Enxerto-Hospedeiro/prevenção & controle , Modelos Animais de Doenças , Células DendríticasRESUMO
Introduction: Triple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs. Methods: We investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo. Results: We confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo. Discussion: Our data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.
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Sulindaco , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Sulindaco/farmacologia , Sulindaco/uso terapêutico , Secretases da Proteína Precursora do Amiloide , Neoplasias de Mama Triplo Negativas/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Modelos Animais de DoençasRESUMO
Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.
Assuntos
Plasticidade Celular , Interleucina-6 , Proteína Quinase C-theta/metabolismo , Interleucina-6/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T Reguladores/metabolismo , Diferenciação Celular , Isoformas de Proteínas/metabolismo , Células Th17/metabolismoRESUMO
A critical feature of cancer is the ability to induce immunosuppression and evade immune responses. Tumor-induced immunosuppression diminishes the effectiveness of endogenous immune responses and decreases the efficacy of cancer immunotherapy. In this study, we describe a new immunosuppressive pathway in which adenosine promotes Casitas B-lineage lymphoma b (Cbl-b)-mediated Notch1 degradation, causing suppression of CD8+ T-cells effector functions. Genetic knockout and pharmacological inhibition of Cbl-b prevents Notch1 degradation in response to adenosine and reactivates its signaling. Reactivation of Notch1 results in enhanced CD8+ T-cell effector functions, anti-cancer response and resistance to immunosuppression. Our work provides evidence that targeting the Cbl-b-Notch1 axis is a novel promising strategy for cancer immunotherapy.
Assuntos
Linfoma , Neoplasias , Adenosina , Linfócitos T CD8-Positivos , Humanos , Imunoterapia , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMO
Targeted delivery of chemotherapeutic drugs can improve their therapeutic efficiency by localizing their toxic effects at the diseased site. This is often achieved either by direct conjugation of drugs to antibodies targeting overexpressed receptors on cancer cells (antibody-drug conjugates/ADCs) or by conjugating antibodies to nanoparticles bearing drugs (antibody-nanoparticle conjugates/ANCs). Here, we report a platform for utilizing hinge cysteines on antigen-binding fragment (Fab') of an anti-CD4 antibody for site-specific conjugation to nanoparticles giving rise to anti-CD4 Fab'-nanoparticle conjugates (Fab'-NCs). We demonstrate a convenient route for obtaining functional anti-CD4 Fab' from full-length antibody and examine the targeted delivery efficiencies of anti-CD4 Fab'-NCs vs ANCs for selective delivery to CD4high mT-ALL cells. Our results indicate that higher avidity of full-length anti-CD4 antibody, i.e., protein alone translated to higher binding ability to CD4high mT-ALL cells in comparison with anti-CD4 Fab' alone. However, the targeted delivery efficiency of anti-CD4 Fab'-NCs was comparable to ANCs indicating that the avidity of Fab' is restored in a nanoparticle-conjugate format. Fab'-NCs are equally capable of achieving targeted drug delivery to CD4high T-cells as ANCs and are a versatile alternative to ANCs by offering site-selective modification strategy while retaining their advantages.
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Imunoconjugados , Nanopartículas , Anticorpos Monoclonais , Linfócitos T CD4-Positivos , Fragmentos Fab das ImunoglobulinasRESUMO
BACKGROUND: The current approach to manufacture cold-stored platelets (CSP) replicates that of room temperature-stored platelets (RSP). However, this production method is associated with aggregate formation in CSP, a major pitfall that leads to significant wastage. We hypothesized that isolating platelets from whole blood as platelet-rich plasma (PRP) and storing them at a lower concentration reduces aggregates and that conventional bedside transfusion filtration removes CSP aggregates. METHODS: We collected platelets from healthy humans by apheresis (AP) and by phlebotomy, from which we generated platelet-rich plasma (PRP). We split each AP and PRP platelets into two equal aliquots, storing one at 22°C (RT-PRP and RT-AP) and the other at 4°C (4C-PRP and 4C-AP). We evaluated platelets on day 0 and day 7 of storage. After storage, we measured platelet counts, aggregates, and other key characteristics before and after filtration by a bedside filter. RESULTS: After storage, the 4C-AP platelet counts decreased significantly. 4C-PRP preserved glucose better and prevented a significant increase in lactate contrary to 4C-AP. Filtration led to significantly lower platelet counts in both 4C-PRP and 4C-AP but not in their RT counterparts. Post filtration, we observed 50% fewer aggregates only in 4C-AP, whereas 4C-PRP showed an unexpected but significant increase in aggregates. Testing confirmed activation during storage but filtration did not further activate platelets. CONCLUSION: We provide evidence that 4C-PRP is an alternative to 4C-AP and that bedside filters reduce aggregates from 4C-AP. Further studies are needed to evaluate the hemostatic potential of 4C-PRP and the management of aggregates.
Assuntos
Remoção de Componentes Sanguíneos , Plasma Rico em Plaquetas , Remoção de Componentes Sanguíneos/métodos , Plaquetas/fisiologia , Preservação de Sangue/métodos , Temperatura Baixa , HumanosRESUMO
Platelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature-stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP; however, their rate of aggregation in response to the GPVI agonist collagen was significantly lower. In a mouse model of platelet transfusion, we found a significantly lower response rate to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.
Assuntos
Plaquetas , Preservação de Sangue , Animais , Criopreservação , Humanos , Camundongos , Transfusão de Plaquetas , TemperaturaRESUMO
We report here on protein-antibody conjugates (PACs) that are used for antibody-directed delivery of protein therapeutics to specific cells. PACs have the potential to judiciously combine the merits of two prolific therapeutic approaches-biologics and antibody-drug conjugates. We utilize spherical polymer brushes to construct PACs using the combination of two simple and efficient functionally orthogonal click chemistries. In addition to the synthesis and characterization of these nanoparticles, we demonstrate that PACs are indeed capable of specifically targeting cells based on the presence of target antigen on the cell surface to deliver proteins. The potentially broad adaptability of PACs opens up new opportunities for targeted biologics in therapeutics and sensing.
Assuntos
Anticorpos/química , Fulerenos/química , Receptor ErbB-2/química , Linhagem Celular Tumoral , Humanos , Nanopartículas/química , Tamanho da PartículaRESUMO
Notch signalling is involved in many aspects of cancer biology, including angiogenesis, tumour immunity and the maintenance of cancer stem-like cells. In addition, Notch can function as an oncogene and a tumour suppressor in different cancers and in different cell populations within the same tumour. Despite promising preclinical results and early-phase clinical trials, the goal of developing safe, effective, tumour-selective Notch-targeting agents for clinical use remains elusive. However, our continually improving understanding of Notch signalling in specific cancers, individual cancer cases and different cell populations, as well as crosstalk between pathways, is aiding the discovery and development of novel investigational Notch-targeted therapeutics.
Assuntos
Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Neoplasias/metabolismo , Receptores Notch/metabolismo , Animais , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/tendências , Humanos , Imunoterapia/métodos , Imunoterapia/tendências , Neoplasias/imunologia , Neoplasias/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Receptores Notch/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Despite recent advancements in cancer immunotherapy, accurate monitoring of its efficacy is challenging due to heterogeneous immune responses. Conventional imaging techniques lack the sensitivity and specificity for early response assessment. In this study, we designed a granzyme B (GrB) nanoreporter (GNR) that can deliver an immune checkpoint inhibitor to the tumor and track time-sensitive GrB activity as a direct way to monitor initiation of effective immune responses. Anti-programmed death-ligand 1 (PD-L1) antibody-conjugated GNRs inhibited PD-1/PD-L1 interactions efficiently and induced T cell-mediated GrB release that can be imaged using activatable imaging probe. GNRs enabled real-time immunotherapy response monitoring in a tumor-bearing mice model and distinguished between highly responsive and poorly responsive tumors. Furthermore, increasing doses resulted in a better response and enhanced sensitivity in poorly responsive tumors. These findings indicate that GNR has the potential to serve as a tool for sensitive and noninvasive evaluation of immunotherapy efficacy.
Assuntos
Antígeno B7-H1 , Neoplasias , Animais , Linhagem Celular Tumoral , Granzimas , Fatores Imunológicos , Imunoterapia/métodos , Camundongos , Neoplasias/terapia , Linfócitos TRESUMO
Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation.
Assuntos
Transferência Adotiva/métodos , Anticorpos/imunologia , Anticorpos/farmacologia , Peptídeos Penetradores de Células , Doença Enxerto-Hospedeiro/terapia , Tolerância Imunológica/efeitos dos fármacos , Líquido Intracelular/imunologia , Proteína Quinase C-theta/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Humanos , Tolerância Imunológica/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptor de Morte Celular Programada 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resultado do TratamentoRESUMO
T cell receptor signaling, together with cytokine-induced signals, can differentially regulate RNA processing to influence T helper versus regulatory T cell fate. Protein kinase C family members have been shown to function in alternative splicing and RNA processing in various cell types. T cell-specific protein kinase C theta, a molecular regulator of T cell receptor downstream signaling, has been shown to phosphorylate splicing factors and affect post-transcriptional control of T cell gene expression. In this study, we explored how using a synthetic cell-penetrating peptide mimic for intracellular anti-protein kinase C theta delivery fine-tunes differentiation of induced regulatory T cells through its differential effects on RNA processing. We identified protein kinase C theta signaling as a critical modulator of two key RNA regulatory factors, heterogeneous nuclear ribonucleoprotein L (hnRNPL) and protein-l-isoaspartate O-methyltransferase-1 (PCMT1), and loss of protein kinase C theta function initiated a "switch" in post-transcriptional organization in induced regulatory T cells. More interestingly, we discovered that protein-l-isoaspartate O- methyltransferase-1 acts as an instability factor in induced regulatory T cells, by methylating the forkhead box P3 (FOXP3) promoter. Targeting protein-l-isoaspartate O-methyltransferase-1 using a cell-penetrating antibody revealed an efficient means of modulating RNA processing to confer a stable regulatory T cell phenotype.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/genética , Proteína Quinase C-theta/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Fatores de Transcrição Forkhead/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Estabilidade Proteica , Transdução de SinaisRESUMO
BACKGROUND: γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS: For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aß and Aß-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT: These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION: Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term ß-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.
Assuntos
Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Células HEK293 , Humanos , Presenilina-2/genética , Transdução de SinaisRESUMO
CD4+ T lymphocytes play an important role in controlling many malignancies. The modulation of CD4+ T cells through immunomodulatory or cytotoxic drugs could change the course of disease progression for disorders such as autoimmunity, immunodeficiency, and cancer. Here, we demonstrate that anti-CD4 conjugated polymeric nanogels can deliver a small molecule cargo to primary CD4+ T cells and a CD4high T cell lymphoma. The antibody conjugation not only increased the uptake efficiency of the nanogel (NG) by CD4+ T cells but also decreased the non-specific uptake of the NG by CD4- lymphocytes. For T lymphoma cell lines, the mertansine-loaded conjugate displayed a dose-dependent cell growth inhibition at 17 ng/mL antibody concentration. On the other hand, antibody-drug conjugate (ADC)-type formulation of the anti-CD4 reached similar levels of cell growth inhibition only at the significantly higher concentration of 1.8 µg/mL. NG and antibody conjugates have the advantage of carrying a large payload to a defined target in a more efficient manner as it needs far less antibody to achieve a similar outcome.
Assuntos
Antineoplásicos , Imunoconjugados , Maitansina , Linfócitos T CD4-Positivos , NanogéisRESUMO
Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Notch ligands such as Delta-like ligands (DLL) 1, 3, 4, and JAG1, 2 can impact Notch signaling positively or negatively, by trans-activation or cis-inhibition. Trans and cis interactions are receptor-ligand interaction on two adjacent cells and interaction on the same cell, respectively. The former sends an activation signal and the later, a signal for inhibition of Notch. However, earlier reports suggested that Notch is activated in the absence of Notch ligand-expressing APCs in a purified population of CD4 T cells. Thus, the role of ligands in Notch activation, in a purified population of CD4 T cells, remains obscure. In this study, we demonstrate that mature CD4 T cells are capable of expressing Notch ligands on their surface very early upon activation with soluble antibodies against CD3 and CD28. Moreover, signaling solely through CD28 induces Notch ligand expression and CD3 signaling inhibits ligand expression, in contrast to Notch which is induced by CD3 signaling. Additionally, by using decoys, mimicking the Notch extracellular domain, we demonstrated that DLL1, DLL4, and JAG1, expressed on the T cells, can cis-interact with the Notch receptor and inhibit activation of Notch. Thus, our data indicate a novel mechanism of the regulation of Notch ligand expression on CD4 T cells and its impact on activated Notch.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Jagged-1/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/imunologia , Animais , Anticorpos/farmacologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Feminino , Células HEK293 , Humanos , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular ß-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.
Assuntos
Presenilina-1/fisiologia , Presenilina-2/fisiologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Hidrólise , Camundongos , Presenilina-1/genética , Presenilina-2/genética , Especificidade por SubstratoRESUMO
Acute graft-versus-host disease is a frequent complication associated with allogeneic hematopoietic stem cell transplantation. Patients that become refractory to initial steroid treatment have a poor prognosis. apceth-201 consists of human allogeneic mesenchymal stromal cells, engineered by lentiviral transduction to express the protease inhibitor alpha-1 antitrypsin, to augment the anti-inflammatory potential of the mesenchymal stromal cells. We show that apceth-201 mesenchymal stromal cells efficiently suppress T cell proliferation and polarize macrophages to an anti-inflammatory M2 type, in vitro. To assess the in vivo efficacy of apceth-201, it was tested in two different mouse models of acute graft-versus-host disease. Control animals in a humanized model succumbed quickly to disease, whereas median survival was doubled in apceth-201-treated animals. The product was also tested in a graft-versus-host disease model system that closely mimics haploidentical hematopoietic stem cell transplantation, an approach that is now being evaluated for use in the clinic. Control animals succumbed quickly to disease, whereas treatment with apceth-201 resulted in long-term survival of 57% of the animals. Within 25 days after the second injection, clinical scores returned to baseline in responding animals, indicating complete resolution of graft-versus-host disease. These promising data have led to planning of a phase I study using apceth-201.