RESUMEN
Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)-licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov Variations in the immunosuppressive capacity between IFN-γ-licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ-licensed MSCs depended on the αV/α5 integrins when cultured on fibrin and on the α2/ß1 integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of αV/α5 integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α2/ß1 integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ-licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.
Asunto(s)
Colágeno/metabolismo , Fibrina/metabolismo , Terapia de Inmunosupresión , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Integrina alfa2beta1/metabolismo , Interferón gamma/farmacología , Células Madre Mesenquimatosas/citología , Antivirales/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Colágeno/química , Fibrina/química , Humanos , Hidrogeles/química , Hidrogeles/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismoRESUMEN
Immune checkpoint inhibitor (ICI) therapy has revolutionized anti-cancer treatment for many late-stage cancer patients. However, ICI therapy has thus far demonstrated limited efficacy for most patients, and it remains unclear why this is so. Interleukin 10 (IL-10) is a cytokine that has been recognized as a central player in cancer biology with its ability to inhibit anti-tumor T cell responses. Recent studies suggest that IL-10 might also exert some intrinsic anti-tumor T cell responses, and clinical studies using recombinant IL-10 alone or in combination with ICI are underway. This paradoxical effect of IL-10 and its underlying mechanisms impacting ICI-modulated T cell responses remain poorly understood. In this study, using an in vitro mixed lymphocyte reaction assay, we found that treatment with ICIs such as the anti-programmed cell death receptor-1 (PD-1) mAb nivolumab elicits a strong expression of IL-10. While neutralization of IL-10 signaling with an anti-IL-10 specific mAb significantly decreases the production of IFN-γ by T cells in a cohort of donor cells, the opposite effect was observed in other donor cells. Similarly, neutralization of IL-10 signaling significantly decreases the expression of T cell activation markers Ki67 and CD25, as well as the production of Granzyme B in a cohort of donor cells, whereas the opposite effect was observed in others. Furthermore, we found that nivolumab and IL-10 differentially modulate the signal transducer and activator of transcription 3 (STAT3) and AKT serine-threonine kinase pathways. Finally, we found that nivolumab activates the mitogen-activated protein kinase (MAPK) pathway, which in turn is responsible for the observed induction of IL-10 production by nivolumab. These findings provide new insights into the mechanisms underlying anti-PD-1-modulated T cell responses by IL-10, which could lead to the discovery of novel combination treatments that target IL-10 and immune checkpoint molecules.
Asunto(s)
Interleucina-10/inmunología , Activación de Linfocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nivolumab/farmacología , Linfocitos T/inmunología , Humanos , Interferón gamma/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Antígeno Ki-67/inmunología , Sistema de Señalización de MAP Quinasas/inmunologíaRESUMEN
The absence of reliable, robust, and non-invasive biomarkers for anti- Programmed cell death protein 1 (PD-1) immunotherapy is an urgent unmet medical need for the treatment of cancer patients. No predictive biomarkers have been established based on the direct assessment of T cell functions, the primary mechanism of action of anti-PD-1 therapy. In this study, we established a model system to test T cell functions modulated by Nivolumab using anti-CD3 monoclonal antibody (mAb)-stimulated peripheral blood mononuclear cells (PBMCs), and characterized T cell functions primarily based on the knowledge gained from retrospective observations of patients treated with anti-PD-1 immunotherapy. During a comprehensive cytokine profile assessment to identify potential biomarkers, we found that Nivolumab increases expression of T helper type 1 (Th1) associated cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a subset of donors. Furthermore, Nivolumab increases production of Th2, Th9, and Th17 associated cytokines, as well as many proinflammatory cytokines such as IL-6 in a subset of donors. Conversely, Nivolumab treatment has no impact on T cell proliferation, expression of CD25, CD69, or Granzyme B, and only modestly increases in the expansion of regulatory T cells. Our results suggest that assessment of cytokine production using a simple PBMC-based T cell functional assay could be used as a potential predictive marker for anti-PD-1 immunotherapy.
Asunto(s)
Biomarcadores/metabolismo , Inmunoensayo , Inmunoterapia , Leucocitos Mononucleares/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Antígenos CD/metabolismo , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Granzimas/metabolismo , Humanos , Activación de Linfocitos/efectos de los fármacos , Nivolumab/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Donantes de TejidosRESUMEN
Immune checkpoint inhibitors (ICIs) such as the anti-PD-1 antibody Nivolumab, achieve remarkable clinical efficacy in patients with late stage cancers. However, only a small subset of patients benefit from this therapy. Numerous clinical trials are underway testing whether combining ICIs with other anti-cancer therapies can increase this response rate. For example, anti-PD-1/PD-L1 therapy combined with MAP kinase inhibition using BRAF inhibitors (BRAFi) and/or MEK inhibitors (MEKi) are in development for treatment of late stage melanomas. However, the benefits and underlying mechanisms of these combinatorial therapies remain unclear. In the current study, we assess the effects of MAPK inhibition on Nivolumab-induced T cell responses. Using an in vitro mixed lymphocyte reaction assay, we demonstrate that Nivolumab-induced T cell activation is highly heterogeneous. While BRAFi inhibits Nivolumab-induced cytokine production, T cell proliferation, activation markers (CD69, CD25), and Granzyme B in a substantial proportion of donor pairs, a small subset of donor pairs shows an additive effect. MEKi alone significantly inhibits Nivolumab-induced T cell activation; the addition of BRAFi significantly enhances this inhibitory effect. Mechanistically, the effects of BRAFi and/or MEKi on Nivolumab-induced T cell activation may be due to alteration of the activation of the AKT and T cell receptor (TCR) signaling pathways. Our results suggest that MAPK inhibition may not provide a clinical benefit for most melanoma patients being treated with anti-PD-1 therapy.
RESUMEN
Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that ß-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women.
Asunto(s)
Estrógenos/farmacología , Hepacivirus/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Ocludina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Ciclopentanos/farmacología , Antagonistas de Estrógenos/farmacología , Hepacivirus/genética , Hepacivirus/fisiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Immunoblotting , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Células MCF-7 , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Éteres Fenílicos/farmacología , Proteolisis/efectos de los fármacos , Quinolinas/farmacología , Interferencia de ARN , ARN Viral/genética , ARN Viral/metabolismo , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Tamoxifeno/farmacologíaRESUMEN
BACKGROUND: Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. METHODOLOGY: Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice. CONCLUSIONS: Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice.
Asunto(s)
Envejecimiento/inmunología , Leishmania donovani/inmunología , Vacunas contra la Leishmaniasis/uso terapéutico , Leishmaniasis Visceral/inmunología , Células TH1/inmunología , Inmunidad Adaptativa , Animales , Anticuerpos Antiprotozoarios/sangre , Células Cultivadas , Técnicas de Cocultivo , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Técnicas de Inactivación de Genes , Inmunidad Innata , Leishmania donovani/genética , Leishmaniasis Visceral/parasitología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
Recent evidence suggests that dendritic cells (DCs) can regulate and amplify immune responses. Flt3 ligand (FL)-derived DC function was tested as a stimulator of allogeneic lymphocytes in vitro and in vivo. Treatment of mice with FL dramatically expanded DC number, but DCs isolated from FL-treated mice (FL DCs) were poor stimulators of allogeneic T-cell responses in vitro. Further activation of FL DCs did not restore their stimulatory ability, and FL DCs did not suppress the stimulation of the allogeneic T cells by normal DCs. FL treatment significantly increased the CD8 alpha(+) DC subset, which appeared to be the reason for their poor stimulatory capacity. These observations were confirmed in vivo using a mouse model of acute graft-versus-host disease (GVHD) wherein host DCs play a critical role. FL treatment of recipients before allogeneic bone marrow transplantation dramatically suppressed donor T-cell responses to host antigens, thereby reducing GVHD mortality (P <.01). These data represent a novel strategy that alters host DCs and reduces acute GVHD.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Trasplante de Médula Ósea/métodos , Células Dendríticas/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Proteínas de la Membrana/farmacología , Enfermedad Aguda , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Animales , Trasplante de Médula Ósea/inmunología , Antígenos CD8/análisis , División Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Dendríticas/citología , Modelos Animales de Enfermedad , Femenino , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Activación de Linfocitos/inmunología , Proteínas de la Membrana/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Tasa de Supervivencia , Linfocitos T/inmunología , Trasplante Homólogo/inmunología , Trasplante Homólogo/métodosRESUMEN
The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.