RESUMEN
CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos , Reactividad Cruzada , Infecciones por Citomegalovirus , Animales , Linfocitos T CD8-positivos/inmunología , Ratones , Reactividad Cruzada/inmunología , Presentación de Antígeno/inmunología , Infecciones por Citomegalovirus/inmunología , Muromegalovirus/inmunología , Apoptosis , Citomegalovirus/inmunología , Células Presentadoras de Antígenos/inmunología , Ratones Endogámicos C57BL , Antígenos Virales/inmunologíaRESUMEN
Human cytomegalovirus (HCMV) is the most common congenital infection. Several HCMV vaccines are in development, but none have yet been approved. An understanding of the kinetics of CMV replication and transmission may inform the rational design of vaccines to prevent this infection. The salivary glands (SG) are an important site of sustained CMV replication following primary infection and during viral reactivation from latency. As such, the strength of the immune response in the SG likely influences viral dissemination within and between hosts. To study the relationship between the immune response and viral replication in the SG, and viral dissemination from the SG to other tissues, mice were infected with low doses of murine CMV (MCMV). Following intra-SG inoculation, we characterized the viral and immunological dynamics in the SG, blood, and spleen, and identified organ-specific immune correlates of protection. Using these data, we constructed compartmental mathematical models of MCMV infection. Model fitting to data and analysis indicate the importance of cellular immune responses in different organs and point to a threshold of infection within the SG necessary for the establishment and spread of infection.
Asunto(s)
Muromegalovirus , Glándulas Salivales , Animales , Glándulas Salivales/virología , Glándulas Salivales/inmunología , Ratones , Muromegalovirus/inmunología , Muromegalovirus/fisiología , Replicación Viral/fisiología , Cinética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Infecciones por Citomegalovirus/transmisión , Biología ComputacionalRESUMEN
Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have contributed to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMVs) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant MCMV vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, spike-specific humoral and cellular immunity was not only maintained but also even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two variants of concern (VOCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19.
Asunto(s)
Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Ratones , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Muromegalovirus/inmunología , Muromegalovirus/genética , Femenino , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ratones Endogámicos BALB C , Humanos , Vectores Genéticos , Inmunidad Celular , Inmunidad Humoral , Modelos Animales de EnfermedadRESUMEN
CMV drives the accumulation of virus-specific, highly differentiated CD8 memory T cells (memory inflation [MI]). In mice, MI was shown to directly correlate with the CMV infection dose, yet the CMV-associated CD8 MI plateaus over time. It is unclear how MI is regulated with aging. We infected young mice with 102, 104, and 106 PFU of murine CMV and confirmed that MI magnitude was directly proportional to the infectious dose, reaching a setpoint by midlife. By old age, MI subsided, most prominently in mice infected with 106 PFU, and reached statistical parity between groups in 26-mo-old mice. This corresponded to an age-related loss in lymphatic endothelial cells in lymph nodes, recently shown to be sufficient to drive MI in mice. We propose that MI size and persistence over the lifespan is controlled by the size of the lymphatic endothelial cell niche, whose shrinking leads to reduced MI with aging.
Asunto(s)
Linfocitos T CD8-positivos , Memoria Inmunológica , Muromegalovirus , Latencia del Virus , Animales , Ratones , Linfocitos T CD8-positivos/inmunología , Latencia del Virus/inmunología , Memoria Inmunológica/inmunología , Muromegalovirus/inmunología , Envejecimiento/inmunología , Ratones Endogámicos C57BL , Células T de Memoria/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Herpesviridae/inmunología , Células Endoteliales/inmunología , Células Endoteliales/virología , Citomegalovirus/inmunología , Citomegalovirus/fisiología , Ganglios Linfáticos/inmunologíaRESUMEN
M.R2k/b mice are identical to the MA/My parent strain aside from a 5.58-Mb C57L-derived region on chromosome 17 (Cmv5s) that causes increased susceptibility to acute murine CMV (MCMV) infection and the development of significant spleen tissue damage. Spleen pathology begins at the marginal zone (MZ), apparent by 2 d postinfection (dpi), and progresses throughout the red pulp by 4 dpi. To better understand how M.R2k/b mice respond to infection and how Cmv5s contributes to tissue damage in the spleen, we assessed the regulation of myeloid cells and inflammation during acute MCMV infection in MA/My and M.R2k/b mice. We found that Cmv5s drove increased neutrophil accumulation and cell death at the MZ, which corresponded with evidence of localized oxidative stress and increased overall spleen IL-6 and TGF-ß1 early during infection. Further assessment of MCMV infection dynamics at the early MZ revealed infected SIGNR1+ MZ macrophages as the first apparent cell type lost during infection in these mice and the likely target of early neutrophil recruitment. Spleen macrophages were also identified as the mediators of differential spleen IL-6 and TGF-ß1 between MA/My and M.R2k/b mice. Interrogation of MCMV progression past 2 dpi revealed substantial M.R2k/b F480+ red pulp macrophage loss along with buildup of oxidative stress and MZ macrophage debris that was not neutrophil dependent. Together we identify Cmv5s-driven macrophage loss and inflammation during acute MCMV infection corresponding with the spatial and temporal development of spleen tissue damage.
Asunto(s)
Inflamación , Macrófagos , Muromegalovirus , Bazo , Animales , Ratones , Macrófagos/inmunología , Inflamación/inmunología , Muromegalovirus/inmunología , Bazo/inmunología , Bazo/patología , Bazo/virología , Modelos Animales de Enfermedad , Neutrófilos/inmunología , Ratones Endogámicos C57BL , Infecciones por Herpesviridae/inmunología , Enfermedad Aguda , Susceptibilidad a Enfermedades/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/genética , Predisposición Genética a la EnfermedadRESUMEN
The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αß T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αß T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αß CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.
Asunto(s)
Infecciones por Herpesviridae , Memoria Inmunológica , Células T de Memoria , Muromegalovirus , Receptores de Antígenos de Linfocitos T gamma-delta , Animales , Ratones , Muromegalovirus/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Infecciones por Herpesviridae/inmunología , Memoria Inmunológica/inmunología , Células T de Memoria/inmunología , Reinfección/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Citomegalovirus/inmunologíaRESUMEN
Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups.
Asunto(s)
Muromegalovirus , Linfocitos T , Animales , Linfocitos T/inmunología , Ratones , Muromegalovirus/inmunología , Muromegalovirus/fisiología , Humanos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Línea Celular , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.
Asunto(s)
Diferenciación Celular , Células Asesinas Naturales , Células Asesinas Naturales/inmunología , Animales , Ratones , Humanos , Diferenciación Celular/inmunología , Ratones Endogámicos C57BL , Inmunidad Innata , Antígeno CD56/metabolismo , Muromegalovirus/inmunología , Linaje de la Célula/inmunología , Interferón gamma/metabolismo , Interferón gamma/inmunología , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/inmunología , Ratones Noqueados , Células CultivadasRESUMEN
Human cytomegalovirus (hCMV) is a ubiquitous facultative pathogen, which establishes a characteristic latent and reactivating lifelong infection in immunocompetent hosts. Murine CMV (mCMV) infection is widely used as an experimental model of hCMV infection, employed to investigate the causal nature and extent of CMV's contribution to inflammatory, immunological, and health disturbances in humans. Therefore, mimicking natural human infection in mice would be advantageous to hCMV research. To assess the role of route and age at infection in modeling hCMV in mice, we infected prepubescent and young sexually mature C57BL/6 (B6) mice intranasally (i.n., a likely physiological route in humans) and intraperitoneally (i.p., a frequently used experimental route, possibly akin to transplant-mediated infection). In our hands, both routes led to comparable early viral loads and tissue spreads. However, they yielded differential profiles of innate and adaptive systemic immune activation. Specifically, the younger, prepubescent mice exhibited the strongest natural killer cell activation in the blood in response to i.p. infection. Further, the i.p. infected animals (particularly those infected at 12 weeks) exhibited larger anti-mCMV IgG and greater expansion of circulating CD8+ T cells specific for both acute (non-inflationary) and latent phase (inflationary) mCMV epitopes. By contrast, tissue immune responses were comparable between i.n. and i.p. groups. Our results illustrate a distinction in the bloodborne immune response profiles across infection routes and ages and are discussed in light of physiological parameters of interaction between CMV, immunity, inflammation, and health over the lifespan. IMPORTANCE: The majority of experiments modeling human cytomegalovirus (hCMV) infection in mice have been carried out using intraperitoneal infection in sexually mature adult mice, which stands in contrast to the large number of humans being infected with human CMV at a young age, most likely via bodily fluids through the nasopharyngeal/oral route. This study examined the impact of the choice of age and route of infection in modeling CMV infection in mice. By comparing young, prepubescent to older sexually mature counterparts, infected either via the intranasal or intraperitoneal route, we discovered substantial differences in deployment and response intensity of different arms of the immune system in systemic control of the virus; tissue responses, by contrast, appeared similar between ages and infection routes.
Asunto(s)
Inmunidad Adaptativa , Infecciones por Citomegalovirus , Inmunidad Innata , Muromegalovirus , Animales , Femenino , Humanos , Ratones , Factores de Edad , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Modelos Animales de Enfermedad , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Células Asesinas Naturales/inmunología , Ratones Endogámicos C57BL , Muromegalovirus/inmunología , Carga ViralRESUMEN
CD4+ T cells are key to controlling cytomegalovirus infections. Salivary gland infection by murine cytomegalovirus (MCMV) provides a way to identify mechanisms. CD11c+ dendritic cells (DC) disseminate MCMV to the salivary glands, where they transfer infection to acinar cells. Antiviral CD4+ T cells are often considered to be directly cytotoxic for cells expressing major histocompatibility complex class II (MHCII). However, persistently infected salivary gland acinar cells are MHCII- and are presumably inaccessible to direct CD4 T cell recognition. Here, we show that CD4+ T cell depletion amplified infection of MHCII- acinar cells but not MHCII+ cells. MCMV-infected mice with disrupted MHCII on CD11c+ cells showed increased MHCII- acinar infection; antiviral CD4+ T cells were still primed, but their recruitment to the salivary glands was reduced, suggesting that engagement with local MHCII+ DC is important for antiviral protection. As MCMV downregulates MHCII on infected DC, the DC participating in CD4 protection may thus be uninfected. NK cells and gamma interferon (IFN-γ) may also contribute to CD4+ T cell-dependent virus control: CD4 T cell depletion reduced NK cell recruitment to the salivary glands, and both NK cell and IFN-γ depletion equalized infection between MHCII-disrupted and control mice. Taken together, these results suggest that CD4+ T cells protect indirectly against infected acinar cells in the salivary gland via DC engagement, requiring the recruitment of NK cells and the action of IFN-γ. Congruence of these results with an established CD4+ T cell/NK cell axis of gammaherpesvirus infection control suggests a common mode of defense against evasive viruses. IMPORTANCE Cytomegalovirus infections commonly cause problems in immunocompromised patients and in pregnancy. We lack effective vaccines. CD4+ T cells play an important role in normal infection control, yet how they act has been unknown. Using murine cytomegalovirus as an accessible model, we show that CD4+ T cells are unlikely to recognize infected cells directly. We propose that CD4+ T cells interact with uninfected cells that present viral antigens and recruit other immune cells to attack infected targets. These data present a new outlook on understanding how CD4+ T cell-directed control protects against persistent cytomegalovirus infection.
Asunto(s)
Linfocitos T CD4-Positivos , Infecciones por Citomegalovirus , Muromegalovirus , Animales , Antivirales , Linfocitos T CD4-Positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Humanos , Interferón gamma , Ratones , Muromegalovirus/inmunologíaRESUMEN
NK cells are important mediators of viral control with the capacity to form adaptive immune features following infection. However, studies of infection-induced adaptive NK cells require adoptive cell transfer to lower the precursor frequency of "Ag-specific" NK cells, potentially limiting the diversity of the NK cell response. In seeking an unmanipulated model to probe the adaptive NK cells, we interrogated a wide range of Collaborative Cross (CC) inbred mice, inbred mouse strains that exhibit broad genetic diversity across strains. Our assessment identified and validated a putative "ideal" CC strain, CC006, which does not require manipulation to generate and maintain adaptive NK cells. Critically, CC006 mice, in contrast to C57BL/6 mice, are capable of developing enhanced NK cell-mediated protective responses to murine CMV infection following m157-mediated vaccination. This work both furthers our understanding of adaptive NK cells and demonstrates the utility of CC mice in the development and interrogation of immunologic models.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Muromegalovirus/inmunología , Traslado Adoptivo , Animales , Femenino , Infecciones por Herpesviridae/patología , Células Asesinas Naturales/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
The potential of memory T cells to provide protection against reinfection is beyond question. Yet, it remains debated whether long-term T cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8+ T cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T cells, depending on factors such as exposure to cognate Ag. CMV infection induces not only conventional, contracting T cell responses, but also inflationary CD8+ T cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8+ T cells. Using in vivo deuterium labeling and mathematical modeling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8+ T cells are very similar to those of bulk memory-phenotype CD8+ T cells. Even CMV-specific inflationary CD8+ T cell responses that differ 3-fold in size were found to turn over at similar rates.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Interacciones Huésped-Patógeno/inmunología , Memoria Inmunológica , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Muromegalovirus/inmunología , Algoritmos , Animales , Biomarcadores , Linfocitos T CD8-positivos/metabolismo , Infecciones por Citomegalovirus/virología , Epítopos de Linfocito T/inmunología , Femenino , Inmunofenotipificación , Ratones , Modelos Teóricos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Vaccination against the betaherpesvirus, human cytomegalovirus (HCMV) is a public health goal. However, HCMV has proved difficult to vaccinate against. Vaccination against single HCMV determinants has not worked, suggesting that immunity to a wider antigenic profile may be required. Live attenuated vaccines provide the best prospects for protection, but the question remains as to how to balance vaccine virulence with safety. Animal models of HCMV infection provide insights into identifying targets for virus attenuation and understanding how host immunity blocks natural, mucosal infection. Here, we evaluated the vaccine potential of a mouse cytomegalovirus (MCMV) vaccine deleted of a viral G protein-coupled receptor (GPCR), designated M33, that renders it attenuated for systemic spread. A single noninvasive olfactory ΔM33 MCMV vaccine replicated locally, but as a result of the loss of the M33 GPCR, it failed to spread systemically and was attenuated for latent infection. Vaccination did not prevent host entry of a superinfecting MCMV but spread from the mucosa was blocked. This approach to vaccine design may provide a viable alternative for a safe and effective betaherpesvirus vaccine. IMPORTANCE Human cytomegalovirus (HCMV) is the most common cause of congenital infection for which a vaccine is not yet available. Subunit vaccine candidates have failed to achieve licensure. A live HCMV vaccine may prove more efficacious, but it faces safety hurdles which include its propensity to persist and to establish latency. Understanding how pathogens infect guide rational vaccine design. However, HCMV infections are asymptomatic and thus difficult to capture. Animal models of experimental infection provide insight. Here, we investigated the vaccine potential of a mouse cytomegalovirus (MCMV) attenuated for systemic spread and latency. We used olfactory vaccination and virus challenge to mimic its natural acquisition. We provide proof of concept that a single olfactory MCMV that is deficient in systemic spread can protect against wild-type MCMV superinfection and dissemination. This approach of deleting functional counterpart genes in HCMV may provide safe and effective vaccination against congenital HCMV disease.
Asunto(s)
Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/inmunología , Citomegalovirus/inmunología , Muromegalovirus/inmunología , Mucosa Olfatoria/virología , Sobreinfección/prevención & control , Sobreinfección/virología , Animales , Infecciones por Citomegalovirus/inmunología , Vacunas contra Citomegalovirus/administración & dosificación , Femenino , Inmunidad Innata , Ratones , Ratones Endogámicos BALB C , Nariz/virología , Prueba de Estudio Conceptual , Vacunación/métodos , Vacunas AtenuadasRESUMEN
The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1ßR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Muromegalovirus/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Animales , Proliferación Celular/fisiología , Citocinas/inmunología , Femenino , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Receptores de Células Asesinas Naturales/inmunología , Transducción de Señal/inmunología , Transcriptoma/inmunologíaRESUMEN
Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo. Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra Citomegalovirus/inmunología , Memoria Inmunológica , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Fenotipo , Animales , Vacunas contra el Cáncer/inmunología , Biología Computacional/métodos , Citomegalovirus/inmunología , Citotoxicidad Inmunológica , Perfilación de la Expresión Génica , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Inmunofenotipificación , Activación de Linfocitos/inmunología , Ratones , Muromegalovirus/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , TranscriptomaRESUMEN
Natural killer (NK) cells are the predominant innate lymphocytes that provide early defense against infections. In the inflammatory milieu, NK cells modify their metabolism to support high energy demands required for their proliferation, activation, and functional plasticity. This metabolic reprogramming is usually accompanied by the upregulation of nutrient transporter expression on the cell surface, leading to increased nutrient uptake required for intense proliferation. The interleukin-1 family members of inflammatory cytokines are critical in activating NK cells during infection; however, their underlying mechanism in NK cell metabolism is not fully elucidated. Previously, we have shown that IL-18 upregulates the expression of solute carrier transmembrane proteins and thereby induces a robust metabolic boost in NK cells. Unexpectedly, we found that IL-18 signaling is dispensable during viral infection in vivo, while the upregulation of nutrient transporters is primarily MyD88-dependent. NK cells from Myd88-/- mice displayed significantly reduced surface expression of nutrient receptors and mTOR activity during MCMV infection. We also identified that IL-33, another cytokine employing MyD88 signaling, induces the expression of nutrient transporters but requires a pre-exposure to IL-12. Moreover, signaling through the NK cell activating receptor, Ly49H, can also promote the expression of nutrient transporters. Collectively, our findings revealed multiple pathways that can induce the expression of nutrient transporters on NK cells while highlighting the imperative role of MyD88 in NK cell metabolism during infection.
Asunto(s)
Infecciones por Herpesviridae/etiología , Infecciones por Herpesviridae/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Muromegalovirus/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Nutrientes/metabolismo , Animales , Biomarcadores , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Metabolismo Energético , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Transducción de SeñalRESUMEN
Interleukin-1α (IL-1α) is an alarmin involved in the recruitment of macrophages and neutrophils during tissue inflammation. IL-1α can undergo cleavage by proteases, such as calpain-1, that enhances IL-1α binding to its receptor, although proteolytic cleavage is not necessary for biological activity. Macrophages and neutrophils are involved in the retinal inflammation associated with development of AIDS-related human cytomegalovirus (HCMV) retinitis. We therefore performed studies to test the hypothesis that IL-1α gene expression is stimulated intraocularly during retinitis development using two mouse models of murine cytomegalovirus (MCMV) retinitis that differ in method of immunosuppression, one by retrovirus-induced immunosuppression (MAIDS) and the other by corticosteroid-induced immunosuppression. MCMV-infected eyes of groups of retinitis-susceptible mice with MAIDS of 10 weeks duration (MAIDS-10 mice) and retinitis-susceptible corticosteroid-treated mice showed significant stimulation of IL-1α mRNA. Western blot analysis confirmed IL-1α protein production within the MCMV-infected eyes of MAIDS-10 mice. Whereas significant intraocular calpain-1 mRNA and protein production were also observed within MCMV-infected eyes of MAIDS-10 mice, the MCMV-infected eyes of retinitis-susceptible corticosteroid-treated mice showed a pattern of mRNA synthesis equivalent to that found within the MCMV-infected eyes of healthy mice that fail to develop retinitis. Our findings suggest a role for the alarmin IL-1α in the pathogenesis of MCMV retinitis in immunosuppressed mice. These findings may extend to the pathogenesis of HCMV retinitis in patients with AIDS or other forms of immunosuppression.
Asunto(s)
Retinitis por Citomegalovirus/inmunología , Interleucina-1alfa/inmunología , Síndrome de Inmunodeficiencia Adquirida del Murino/inmunología , Muromegalovirus/inmunología , Retina/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Tolerancia Inmunológica/inmunología , Terapia de Inmunosupresión/métodos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/inmunologíaRESUMEN
Natural killer (NK) cells are cytotoxic lymphocytes capable of rapid cytotoxicity, cytokine secretion, and clonal expansion. To sustain such energetically demanding processes, NK cells must increase their metabolic capacity upon activation. However, little is known about the metabolic requirements specific to NK cells in vivo. To gain greater insight, we investigated the role of aerobic glycolysis in NK cell function and demonstrate that their glycolytic rate increases rapidly following viral infection and inflammation, prior to that of CD8+ T cells. NK cell-specific deletion of lactate dehydrogenase A (LDHA) reveals that activated NK cells rely on this enzyme for both effector function and clonal proliferation, with the latter being shared with T cells. As a result, LDHA-deficient NK cells are defective in their anti-viral and anti-tumor protection. These findings suggest that aerobic glycolysis is a hallmark of NK cell activation that is key to their function.
Asunto(s)
Glucólisis , Células Asesinas Naturales/inmunología , Lactato Deshidrogenasa 5/metabolismo , Muromegalovirus/inmunología , Neoplasias/inmunología , Aerobiosis , Animales , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Células Clonales , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Homeostasis , Ratones Endogámicos C57BL , Neoplasias/patología , Regulación hacia ArribaRESUMEN
Acute infection with murine cytomegalovirus (mCMV) is controlled by CD8+ T cells and develops into a state of latent infection, referred to as latency, which is defined by lifelong maintenance of viral genomes but absence of infectious virus in latently infected cell types. Latency is associated with an increase in numbers of viral epitope-specific CD8+ T cells over time, a phenomenon known as "memory inflation" (MI). The "inflationary" subset of CD8+ T cells has been phenotyped as KLRG1+CD62L- effector-memory T cells (iTEM). It is agreed upon that proliferation of iTEM requires repeated episodes of antigen presentation, which implies that antigen-encoding viral genes must be transcribed during latency. Evidence for this has been provided previously for the genes encoding the MI-driving antigenic peptides IE1-YPHFMPTNL and m164-AGPPRYSRI of mCMV in the H-2d haplotype. There exist two competing hypotheses for explaining MI-driving viral transcription. The "reactivation hypothesis" proposes frequent events of productive virus reactivation from latency. Reactivation involves a coordinated gene expression cascade from immediate-early (IE) to early (E) and late phase (L) transcripts, eventually leading to assembly and release of infectious virus. In contrast, the "stochastic transcription hypothesis" proposes that viral genes become transiently de-silenced in latent viral genomes in a stochastic fashion, not following the canonical IE-E-L temporal cascade of reactivation. The reactivation hypothesis, however, is incompatible with the finding that productive virus reactivation is exceedingly rare in immunocompetent mice and observed only under conditions of compromised immunity. In addition, the reactivation hypothesis fails to explain why immune evasion genes, which are regularly expressed during reactivation in the same cells in which epitope-encoding genes are expressed, do not prevent antigen presentation and thus MI. Here we show that IE, E, and L genes are transcribed during latency, though stochastically, not following the IE-E-L temporal cascade. Importantly, transcripts that encode MI-driving antigenic peptides rarely coincide with those that encode immune evasion proteins. As immune evasion can operate only in cis, that is, in a cell that simultaneously expresses antigenic peptides, the stochastic transcription hypothesis explains why immune evasion is not operative in latently infected cells and, therefore, does not interfere with MI.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por Herpesviridae/virología , Evasión Inmune , Memoria Inmunológica , Infección Latente/virología , Pulmón/virología , Muromegalovirus/patogenicidad , Activación Viral , Latencia del Virus , Animales , Antígenos Virales/genética , Antígenos Virales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/metabolismo , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Infección Latente/inmunología , Infección Latente/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ratones Endogámicos BALB C , Modelos Inmunológicos , Muromegalovirus/genética , Muromegalovirus/inmunología , Fenotipo , Procesos Estocásticos , Factores de Tiempo , Transcripción GenéticaRESUMEN
Cytokine signaling via signal transducer and activator of transcription (STAT) proteins is crucial for optimal antiviral responses of natural killer (NK) cells. However, the pleiotropic effects of both cytokine and STAT signaling preclude the ability to precisely attribute molecular changes to specific cytokine-STAT modules. Here, we employed a multi-omics approach to deconstruct and rebuild the complex interaction of multiple cytokine signaling pathways in NK cells. Proinflammatory cytokines and homeostatic cytokines formed a cooperative axis to commonly regulate global gene expression and to further repress expression induced by type I interferon signaling. These cytokines mediated distinct modes of epigenetic regulation via STAT proteins, and collective signaling best recapitulated global antiviral responses. The most dynamically responsive genes were conserved across humans and mice, which included a cytokine-STAT-induced cross-regulatory program. Thus, an intricate crosstalk exists between cytokine signaling pathways, which governs NK cell responses.