RESUMEN
The mechanisms driving metabolic reprogramming during B cell activation are unclear, particularly roles for enzymatic pathways involved in lipid remodeling. We found that murine B cell activation with lipopolysaccharide (LPS) led to a 1.6-fold increase in total lipids that included higher levels of phosphatidylethanolamine (PE) and plasmenyl PE. Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase involved in the synthesis of both PE and plasmenyl PE, and SELENOI expression was also upregulated during activation. Selenoi knockout (KO) B cells exhibited decreased levels of plasmenyl PE, which plays an important antioxidant role. Lipid peroxidation was measured and found to increase â¼2-fold in KO vs. wild-type (WT) B cells. Cell death was not impacted by KO in LPS-treated B cells and proliferation was only slightly reduced, but differentiation into CD138 + Blimp-1+ plasma B cells was decreased â¼2-fold. This led to examination of B cell receptors important for differentiation that recognize the ligand B cell activating factor, and levels of TACI (transmembrane activator, calcium-modulator, and cytophilin ligand interactor) (CD267) were significantly decreased on KO B cells compared with WT control cells. Vaccination with ovalbumin/adjuvant led to decreased ovalbumin-specific immunoglobulin M (IgM) levels in sera of KO mice compared with WT mice. Real-time polymerase chain reaction analyses revealed a decreased switch from surface to secreted IgM in spleens of KO mice induced by vaccination or LP-BM5 retrovirus infection. Overall, these findings detail the lipidomic response of B cells to LPS activation and reveal the importance of upregulated SELENOI for promoting differentiation into IgM-secreting plasma B cells.
Asunto(s)
Linfocitos B , Diferenciación Celular , Inmunoglobulina M , Lipopolisacáridos , Activación de Linfocitos , Selenoproteínas , Animales , Lipopolisacáridos/farmacología , Inmunoglobulina M/sangre , Inmunoglobulina M/metabolismo , Ratones , Selenoproteínas/metabolismo , Selenoproteínas/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Ratones Noqueados , Células Plasmáticas/metabolismo , Células Plasmáticas/inmunología , Lipidómica , Regulación hacia Arriba , Ratones Endogámicos C57BLRESUMEN
The mechanisms by which myeloid-derived suppressor cells (MDSCs) mediate inhibition prominently include the production of reactive nitrogen species, in particular those generated by inducible nitric oxide synthase (iNOS), and reactive oxygen species. LP-BM5 murine retroviral infection results in a profound immunodeficiency, known as murine AIDS, as well as in increased numbers and activity of monocytic-type MDSCs (M-MDSCs) that suppress both T and B cell responses. While M-MDSCs suppress T cells ex vivo in a fully iNOS/NO-dependent manner, M-MDSC suppression of B cell responses is only partially due to iNOS/NO. This study preliminarily explored the role of two redox-modulating compounds in inhibiting the M-MDSC suppressive activity in LP-BM5 infection. The tested molecules were: I-152 consisting in a conjugate of N-acetyl-cysteine (NAC) and S-acetyl-cysteamine (SMEA) and C4-GSH that is the n-butanoyl glutathione (GSH) derivative. The results show that both molecules, tested in a concentration range between 3 and 20 mM, blocked the M-MDSC suppression of activated B and T cells ex vivo and restored their proliferative capacity in vivo. Ex vivo I-152 blockade of M-MDSC suppressiveness was more significant for T cell (about 70%) while M-MDSC blockade by C4-GSH was preferential for B cell responsiveness (about 60%), which was also confirmed by in vivo investigation. Beyond insights into redox-dependent suppressive effector mechanism(s) of M-MDSCs in LP-BM5 infection, these findings may ultimately be important to identify new immunotherapeutics against infectious diseases.
RESUMEN
Monocytic (CD11b(+)Ly6G(±/Lo)Ly6C(+)) myeloid derived suppressor cells (M-MDSCs) expand following murine retroviral LP-BM5 infection and suppress ex vivo polyclonal T-cell and B-cell responses. M-MDSCs 3 weeks post LP-BM5 infection have decreased suppression of T-cell, but not B-cell, responses and alterations in the degree of iNOS/NO dependence of suppression. M-MDSCs from LP-BM5 infected mice were sorted into four quadrant populations (Ly6C/CD11b density): all quadrants suppressed B-cell responses, but only M-MDSCs expressing the highest levels of Ly6C and CD11b (Q2) significantly suppressed T-cell responses. Further subdivision of this Q2 population revealed the Ly6C(+/Hi) M-MDSC subpopulation as the most suppressive, inhibiting T- and B-cell responses in a full, or partially, iNOS/NO-dependent manner, respectively. In contrast, the lower/moderate levels of suppression by the Ly6C(+/Lo) and Ly6C(+/Mid) M-MDSC Q2 subpopulations, whether versus T- and/or B-cells, displayed little/no iNOS dependency for suppression. These results highlight differential phenotypic and functional immunosuppressive M-MDSC subsets in a retroviral immunodeficiency model.
Asunto(s)
Linfocitos B/inmunología , Células Mieloides/inmunología , Células Mieloides/virología , Retroviridae/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Linfocitos B/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Expresión Génica , Inmunomodulación , Inmunofenotipificación , Ratones , Células Mieloides/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Linfocitos T/metabolismoRESUMEN
Inhibition of T-cell responses in tumor microenvironments by myeloid-derived suppressor cells (MDSCs) is widely accepted. We demonstrated augmentation of monocytic MDSCs whose suppression of not only T-cell, but also B-cell, responsiveness paralleled the immunodeficiency during LP-BM5 retrovirus infection. MDSCs inhibited T cells by inducible nitric oxide synthase (iNOS)/nitric oxide (NO), but uniquely, inhibition of B cells was ~50% dependent each on iNOS/NO and the MDSC-expressed negative-checkpoint regulator VISTA. Blockade with a combination of iNOS/NO and VISTA caused additive or synergistic abrogation of MDSC-mediated suppression of B-cell responsiveness.
Asunto(s)
Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/inmunología , Proteínas de la Membrana/inmunología , Monocitos/inmunología , Infecciones por Retroviridae/inmunología , Animales , Linfocitos B/patología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Proteínas de la Membrana/genética , Ratones , Monocitos/patología , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Presentación de Antígeno , Proteínas Bacterianas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Pseudomonas aeruginosa/metabolismo , Ubiquitinación , Factores de Virulencia/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/inmunología , Ubiquitina Tiolesterasa/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease settings, especially in many tumor systems. Compared to their involvement in tumor microenvironments, however, MDSCs have been less well studied in their responses to infectious disease processes, in particular to retroviruses that induce immunodeficiency. Here, we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired immunodeficiency. These MDSCs express a cell surface marker signature (CD11b(+) Gr-1(+) Ly6C(+)) characteristic of monocyte-type MDSCs. Such MDSCs profoundly inhibit immune responsiveness by a cell dose- and substantially inducible nitric oxide synthase (iNOS)-dependent mechanism that is independent of arginase activity, PD-1-PD-L1 expression, and interleukin 10 (IL-10) production. These MDSCs display levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-infected wild-type (w.t.) versus knockout mouse strains that are differentially susceptible to pathogenesis. These MDSCs suppressed not only T-cell but also B-cell responses, which are an understudied target for MDSC inhibition. The MDSC immunosuppression of B-cell responses was confirmed by the use of purified B responder cells, multiple B-cell stimuli, and independent assays measuring B-cell expansion. Retroviral load measurements indicated that the suppressive Ly6G(low/±) Ly6C(+) CD11b(+)-enriched MDSC subset was positive for LP-BM5, albeit at a significantly lower level than that of nonfractionated splenocytes from LP-BM5-infected mice. These results, including the strong direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression and, as this broadly suppressive function mirrors that of the LP-BM5-induced disease syndrome, support a possible pathogenic effector role for these retrovirus-induced MDSCs.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Linfocitos B/inmunología , Tolerancia Inmunológica , Monocitos/inmunología , Retroviridae/patogenicidad , Linfocitos T/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Animales , Antígenos Ly/análisis , Antígeno CD11b/análisis , Inmunofenotipificación , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Quimiocina/análisis , Retroviridae/inmunologíaRESUMEN
CD8 T-cell responses constitute a key host defense mechanism against tumor cells and a variety of viral infections, including retroviral infections that lead to acquired immunodeficiency. However, both for tumor cells and for many viral infections, there can be a relative paucity of immunodominant protective CD8 T-cell responses. For retroviruses, their rapid and error-prone replication, coupled with initial CD8 T-cell immunoselection of epitope-variant, retroviral quasi-species, are major impediments to sustaining a protective CD8 T-cell response. To approach this limitation of functional CD8 T-cell epitopes, here we further characterize an underappreciated source of additional T-cell epitopes: cryptic determinants, in particular those encoded in unconventional, alternative reading frames (ARFs). By use of the CD8 T-cell epitope, SYNTGRFPPL, which we have defined as encoded by the +1NT ARF of the gag gene of the LP-BM5 retrovirus that causes murine AIDS, we further characterize the regulation of ARF-epitope expression. Specifically, we examine the translation initiation requirements for production of sufficient epitope for effector CD8 T-cell recognition. Such translation must arise from rare frame-shifting events, making it crucial to understand any other constraints on epitope production, and therefore on the ability of the anti-Kd/SYNTGRFPPL CD8 T cells to protect from LP-BM5 pathogenesis and retroviral load, as we have previously shown. The data herein demonstrate that ARF epitope production depends entirely on conventional AUG-initiated translation, and that the more proximal in-frame ARF AUG is most important. However, maximal epitope production for protective CD8 T-cell lytic function also requires synergy of this initiation codon with a counterpart conventional AUG codon upstream in the same ARF (ORF 2), and with the classic ORF 1 AUG that initiates conventional gag polyprotein translation. These results have implications for the design of ARF-epitope-based vaccines, both to counter retroviral pathogenesis, as well as potentially more broadly, including in tumor systems.
Asunto(s)
Epítopos de Linfocito T/genética , Sistemas de Lectura Abierta , Retroviridae/genética , Linfocitos T Citotóxicos/inmunología , Animales , Presentación de Antígeno , Línea Celular , Codón Iniciador , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Mutación del Sistema de Lectura , Sistema de Lectura Ribosómico , Masculino , Ratones , Biosíntesis de Proteínas , Retroviridae/inmunologíaRESUMEN
Pathology due to the immune system's response to viral infections often represents a delicate balance between inhibition of viral pathogenesis and regulation of protective immunity. In susceptible C57BL/6 (B6) mice, the murine retroviral isolate LP-BM5 induces splenomegaly, hypergammaglobulinemia, profound B- and T-cell immunodeficiency, and increased susceptibility to opportunistic pathogens and terminal B-cell lymphomas. Here, we report that B6.PD-1 (programmed death-1) and B6.IL-10 knockout mice are substantially more susceptible to LP-BM5-induced disease than wild-type B6 mice. LP-BM5-infected B6.PD-1(-/-) mice developed more severe splenomegaly, hypergammaglobulinemia, and immunodeficiency than infected B6 mice: PD-1(-/-) mice are more susceptible to lower doses of LP-BM5 and show more exaggerated disease early postinfection. LP-BM5-infected B6.IL-10(-/-) mice also develop exaggerated LP-BM5-induced disease, compared to B6 mice, without a significant change in the retroviral load. By reciprocal reconstitution experiments, comparing wild-type versus PD-1(-/-) sources of the requisite cells for LP-BM5 pathogenesis-CD4 T and B cells, PD-1(+) B cells appear to be crucial in the normal limitation of LP-BM5-induced disease in B6 mice. Also, infected B6 mice have increased CD11b(+) spleen cells that express interleukin-10 (IL-10). However, PD-1(-/-) mice, though showing an even greater expansion of CD11b(+) cells after LP-BM5 inoculation, did not show an equivalent increase in IL-10-producing cells. Thus, it appears that PD-1/PD-L interactions and IL-10 are primarily important in moderating the effects of LP-BM5-induced disease in B6 mice.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Regulación hacia Abajo , Síndromes de Inmunodeficiencia/fisiopatología , Interleucina-10/fisiología , Retroviridae/fisiología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.
Asunto(s)
Antígenos CD40/fisiología , Síndrome de Inmunodeficiencia Adquirida del Murino/etiología , Proteínas/fisiología , Animales , Sitios de Unión , Ratones , Ratones Transgénicos , Factor 6 Asociado a Receptor de TNFRESUMEN
Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus (MuLV) complex that contains a mixture of defective (BM5def) and replication-competent viruses. While the BM5def virus is the causative agent in MAIDS, the replication-competent viruses in LP-BM5, including ecotropic MuLV (BM5eco), are required for BM5def propagation and thus function as helper viruses. We describe quantitative real-time RT-PCR assays for RNA encoded by the BM5def and BM5eco components of LP-BM5. The assays were used to standardize better the input doses of LP-BM5 viruses across viral preparations and to quantify BM5def and BM5eco gag RNA levels in spleen and blood cells from MAIDS-susceptible and -insusceptible infected mice. Spleens of MAIDS-susceptible infected mice harbored approximately similar levels of BM5def gag RNA as infected spleens of mice that are insusceptible to MAIDS due to lack of CD40. In contrast, the same infected spleens of CD40-deficient mice contained substantially higher (up to 10-fold) levels of BM5eco gag RNA compared with susceptible controls. Similar to that seen in spleen, infected blood of CD40-deficient mice contained similar levels of BM5def gag as susceptible strains, but increased levels (up to threefold) of BM5eco gag RNA. The assays described below can be used to characterize better the contributions of different functional viral components of the LP-BM5 mixture to the development of MAIDS.
Asunto(s)
Virus de la Leucemia Murina/genética , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virología/métodos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Expresión Génica , Genes gag , Virus Helper/genética , Virus de la Leucemia Murina/patogenicidad , Ratones , Ratones Noqueados , Ratones Transgénicos , Síndrome de Inmunodeficiencia Adquirida del Murino/virología , ARN Viral/sangre , Bazo/virologíaRESUMEN
C57BL/6 (B6) mice infected with LP-BM5 retroviruses develop disease, including an immunodeficiency similar to AIDS. This disease, murine AIDS (MAIDS), is inhibited by in vivo anti-CD154 monoclonal antibody treatment. The similar levels of insusceptibility of CD40(-/-) and CD154(-/-) B6 mice indicate that CD154/CD40 molecular interactions are required for MAIDS. CD4(+) T and B cells, respectively, provide the CD154 and CD40 expression needed for MAIDS induction. Here, the required CD154/CD40 interaction is shown to be independent of CD80 and CD86 expression: CD80/CD86(-/-) B6 mice develop MAIDS after LP-BM5 infection.