RESUMEN
Natural killer (NK) cells are innate lymphocytes that play an important role in control of viral infections. We recently showed that intranasal infection of mice with influenza virus induced the accumulation of NK cells in the airways. NK cells however did not proliferate in the airways or in the draining lymph node, but in the bone marrow mainly. As also monocyte-precursors undergo vigorous proliferation in the bone marrow (BM) during infections and then egress CCR2-dependently, we decided to determine the role of CCR2 in NK cell migration during intranasal influenza virus infection. We show that a unique population of NK cells in the BM expressed CCR2 and that monocyte chemotactic protein-1 (MCP-1), one of the CCR2 ligands, was produced in the airways of influenza virus infected mice. Analysis of BM chimeric mice reconstituted with a mix of wild-type (wt) and CCR2-deficient BM cells showed that upon influenza virus infection, a significantly lower proportion of CCR2-deficient than wt NK cells was recovered from the bronchoalveolar lavage (BAL). Taken together, our data demonstrate that during influenza virus infection a proportion of NK cells migrate in a CCR2-dependent fashion.
Asunto(s)
Movimiento Celular/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Receptores CCR2/metabolismo , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Movimiento Celular/genética , Quimiocina CCL2/metabolismo , Inmunofenotipificación , Pulmón/citología , Pulmón/inmunología , Ratones , Ratones Noqueados , Infecciones por Orthomyxoviridae/genética , Receptores CCR2/genética , Bazo/citología , Bazo/inmunologíaRESUMEN
Recently we have shown that liposomes can be used as artificial microbes for the production and delivery of DNA-encoded antigens. These so-called antigen-expressing immunostimulatory liposomes (AnExILs) were superior in inducing antigen-specific antibodies compared to conventional liposomal protein or DNA vaccines when tested in mice after i.m. immunization. In this study, we investigated the capacity of AnExILs to induce T-cell responses. By using a plasmid vector encoding a model antigen under control of both the prokaryotic T7 and the eukaryotic CMV promoter we hypothesized that antigen production could lead to CTL activation via two distinct routes: i. production of antigens inside the AnExILs with subsequent cross-presentation after processing by APCs and ii. endogenous production of antigens after AnExIL-mediated transfection of the pDNA. Although we were not able to demonstrate transfection-mediated expression of luc-NP in mice, i.m. injection of AnExILs producing luc-NP resulted in T-cell responses against the encoded NP epitope, as determined by tetramer staining. T-cell responses were comparable to the responses obtained after i.m. injection of naked pDNA. In order to find out whether CTL activation was caused by cross-presentation of the exogenous antigens produced inside AnExILs or by endogenous antigen production from transfection with the same pDNA source a second study was initiated in which the contribution of each of these effects could be separately determined. These results demonstrate that the observed T-cell responses were not exclusively caused by cross-presentation of the AnExIL-produced antigens alone, but were rather a combination of dose-dependent antigen cross-presentation and low levels of endogenous antigen production.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos/genética , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Vacunas de ADN/administración & dosificación , Animales , Antígenos/inmunología , Femenino , Liposomas , Luciferasas/genética , Luciferasas/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Plásmidos , Linfocitos T/inmunología , Vacunas de ADN/inmunología , beta-Galactosidasa/genética , beta-Galactosidasa/inmunologíaRESUMEN
Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8(+) T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8(+) T-cells expand relatively late. Induction of CD8(+) T-cell memory against a single CD8(+) T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8(+) T-cells, covering the entire PVM-specific CD8(+) T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8(+) T-cells offer significant protection to PVM-induced disease. Thus, CD8(+) T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD8-positivos/inmunología , Virus de la Neumonía Murina/inmunología , Infecciones por Pneumovirus/inmunología , Animales , Femenino , Memoria Inmunológica , Subtipo H3N2 del Virus de la Influenza A/inmunología , Interferón gamma/inmunología , Interleucina-4/inmunología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Virus Sincitiales Respiratorios/inmunologíaRESUMEN
NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Proliferación Celular , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Traslado Adoptivo , Animales , Células de la Médula Ósea/patología , Supervivencia Celular/inmunología , Virus de la Influenza A/inmunología , Células Asesinas Naturales/patología , Trasplante de Hígado/inmunología , Trasplante de Hígado/patología , Trasplante de Pulmón/inmunología , Trasplante de Pulmón/patología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Bazo/citología , Bazo/inmunología , Bazo/trasplanteRESUMEN
Natural killer (NK) cells are part of the innate immune system and contribute to the eradication of virus infected cells and tumors. NK cells express inhibitory and activating receptors and their decision to kill a target cell is based on the balance of signals received through these receptors. MHC class I molecules are recognized by inhibitory receptors, and their presence during NK cell education influences the responsiveness of peripheral NK cells. We here demonstrate that mice with reduced MHC class I cell surface expression, due to deficiency of immunoproteasomes, have responsive NK cells in the periphery, indicating that the lower MHC class I levels do not alter NK cell education. Following adoptive transfer into wild-type (wt) recipients, immunoproteasome-deficient splenocytes are tolerated in naive but rejected in virus-infected recipients, in an NK cell dependent fashion. These results indicate that the relatively low MHC class I levels are sufficient to protect these cells from rejection by wt NK cells, but that this tolerance is broken in infection, inducing an NK cell-dependent rejection of immunoproteasome-deficient cells.
Asunto(s)
Células Asesinas Naturales/inmunología , Linfocitos/inmunología , Complejo de la Endopetidasa Proteasomal/deficiencia , Complejo de la Endopetidasa Proteasomal/inmunología , Virosis/inmunología , Traslado Adoptivo , Animales , Antígenos de Histocompatibilidad Clase I , Inmunoproteínas/inmunología , Células Asesinas Naturales/virología , Linfocitos/virología , Ratones , Bazo/patologíaRESUMEN
Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits ß5i/LMP7 and ß2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.