RESUMEN
Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. JClinInvest 111: 639-647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)-expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti-low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells' capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Leucemia Mieloide Aguda/inmunología , Mieloblastina/inmunología , Proteínas de Neoplasias/inmunología , Neutrófilos/inmunología , Adulto , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Neutrófilos/patologíaRESUMEN
Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.
Asunto(s)
Absorción Fisiológica , Neoplasias de la Mama/metabolismo , Reactividad Cruzada , Elastasa de Leucocito/metabolismo , Proteínas de Neoplasias/metabolismo , Neuropilina-1/metabolismo , Secuencias de Aminoácidos , Anticuerpos Bloqueadores/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Humanos , Cinética , Elastasa de Leucocito/química , Elastasa de Leucocito/inmunología , Ligandos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neuropilina-1/antagonistas & inhibidores , Neuropilina-1/química , Neuropilina-1/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
BACKGROUND: Emerging evidence suggests anti-cancer immunity is involved in the therapeutic effect induced by oncolytic viruses. Here we investigate the effect of Delta-24-RGD oncolytic adenovirus on innate and adaptive anti-glioma immunity. DESIGN: Mouse GL261-glioma model was set up in immunocompetent C57BL/6 mouse for Delta-24-RGD treatment. The changes of the immune cell populations were analyzed by immunohistochemistry and flow cytometry. The anti-glioma immunity was evaluated with functional study of the splenocytes isolated from the mice. The efficacy of the virotherapy was assessed with animal survival analysis. The direct effect of the virus on the tumor-associated antigen presentation to CD8+ T cells was analyzed with an in vitro ovalbumin (OVA) modeling system. RESULTS: Delta-24-RGD induced cytotoxic effect in mouse glioma cells. Viral treatment in GL261-glioma bearing mice caused infiltration of innate and adaptive immune cells, instigating a Th1 immunity at the tumor site which resulted in specific anti-glioma immunity, shrunken tumor and prolonged animal survival. Importantly, viral infection and IFNγ increased the presentation of OVA antigen in OVA-expressing cells to CD8+ T-cell hybridoma B3Z cells, which is blocked by brefeldin A and proteasome inhibitors, indicating the activity is through the biosynthesis and proteasome pathway. CONCLUSIONS: Our results demonstrate that Delta-24-RGD induces anti-glioma immunity and offers the first evidence that viral infection directly enhances presentation of tumor-associated antigens to immune cells.