RESUMEN
Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.
Asunto(s)
Antineoplásicos , Ligando de CD40 , Humanos , Linfocitos T CD4-Positivos , Linfocitos T Colaboradores-Inductores , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: Human immunodeficiency virus-exposed uninfected (HEU) infants are a rapidly expanding population in sub-Saharan Africa and are highly susceptible to encapsulated bacterial disease in the first year of life. The mechanism of this increased risk is still poorly understood. We investigated whether human immunodeficiency virus (HIV)-exposure dysregulates HEU immunity, vaccine-antibody production, and human herpes virus amplify this effect. METHODS: Thirty-four HIV-infected and 44 HIV-uninfected pregnant women were recruited into the birth cohort and observed up to 6 weeks of age; and then a subsequent 43 HIV-infected and 61 HIV-uninfected mother-infant pairs were recruited into a longitudinal infant cohort at either: 5-7 to 14-15; or 14-15 to 18-23 weeks of age. We compared monocyte function, innate and adaptive immune cell phenotype, and vaccine-induced antibody responses between HEU and HIV-unexposed uninfected (HU) infants. RESULTS: We demonstrate (1) altered monocyte phagosomal function and B-cell subset homeostasis and (2) lower vaccine-induced anti-Haemophilus influenzae type b (Hib) and anti-tetanus toxoid immunoglobulin G titers in HEU compared with HU infants. Human herpes virus infection was similar between HEU and HU infants. CONCLUSIONS: In the era of antiretroviral therapy-mediated viral suppression, HIV exposure may dysregulate monocyte and B-cell function, during the vulnerable period of immune maturation. This may contribute to the high rates of invasive bacterial disease and pneumonia in HEU infants.
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Infecciones por VIH , Monocitos , Femenino , VIH , Humanos , Inmunoglobulina G , Lactante , Fenotipo , Embarazo , Toxoide TetánicoRESUMEN
Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.
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Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Escamosas/terapia , Neoplasias Hematológicas/terapia , Inmunoterapia Adoptiva/métodos , Melanoma/terapia , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Sarcoma Sinovial/terapia , Factor de Crecimiento Transformador beta/metabolismo , Antígenos de Neoplasias/inmunología , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Ingeniería Genética , Antígeno HLA-A2/metabolismo , Neoplasias Hematológicas/inmunología , Humanos , Tolerancia Inmunológica , Melanoma/inmunología , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Sarcoma Sinovial/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Microambiente TumoralRESUMEN
Immune senescence is a significant contributor to health problems in the developed world and may be accelerated by chronic viral infections. To date, there have been few studies of immune function in healthy older people in sub-Saharan Africa. We assessed T cell and B cell phenotypes and immune responses to CMV, EBV, and influenza virus in Malawians aged 20-69 y. Notably, the proportion of naive (CCR7+CD45RA+) CD4 and CD8 T cells was only 14% of the lymphoid repertoire even in donors aged under 30 y but did not decrease further with age. A small increase in the late differentiated (CD27-CD28-) CD8 T cell subpopulation was observed in older donors but the CD4/CD8 T cell ratio remained stable in all age groups. Interestingly, the regulatory (CD25hiFOXP3hi) T cell subpopulation was small in all age groups, and we observed no age-associated accumulation of cells expressing the senescence- and exhaustion-associated markers CD57 and PD-1. We assessed functional T cell responses to mitogenic and viral antigenic stimulation by the expression of CD154, IFN-γ, TNF-α, IL-2, and IL-17 and proliferation. All responses were robust across the life course, although we observed an age-associated shift from IFN-γ to TNF-α in the response to EBV. In summary, we found the naive T cell subpopulation of young adult Malawians was smaller than in their contemporaries in high-income settings but remains stable thereafter and that lymphocyte function is retained across the life course. These observations indicate that studies of the genetic and environmental factors influencing immune function in different environments may provide insights into minimizing immune ageing.
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Subgrupos de Linfocitos T/inmunología , Adulto , Factores de Edad , Anciano , Diferenciación Celular , Citomegalovirus/inmunología , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Memoria Inmunológica , Gripe Humana/inmunología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/citologíaRESUMEN
Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. In contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterized by anti-EBV IgM antibody positivity, high loads of circulating latently infected B cells, and a marked lymphocytosis caused by hyperexpansion of EBV-specific CD8+ T cells plus a milder expansion of CD56dim NKG2A+ KIR- natural killer (NK) cells. How the two situations compare is unclear due to the paucity of studies on clinically silent infection. Here we describe five prospectively studied patients with asymptomatic infections identified in a seroepidemiologic survey of university entrants. In each case, the key blood sample had high cell-associated viral loads without a marked CD8 lymphocytosis or NK cell disturbance like those seen in patients during the acute phase of IM. Two of the cases with the highest viral loads showed a coincident expansion of activated EBV-specific CD8+ T cells, but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two cases with slightly lower loads, in whom serology suggests the infection may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another case with a higher viral load, in which T and NK cell responses were undetectable in the primary blood sample in which infection was detected, EBV-specific T cell responses did not appear until several months later, by which time the viral loads in the blood had already fallen. Thus, some patients with asymptomatic primary infections have very high circulating viral loads similar to those in patients during the acute phase of IM and a cell-mediated immune response that is qualitatively similar to that in IM patients but of a lower magnitude. However, other patients may have quite different immune responses that ultimately could reveal novel mechanisms of host control.IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat, and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness in which patients have high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural killer (NK) cells. Here we show that in five cases of asymptomatic infection, viral loads in the blood were as high as those in patients during the acute phase of IM, whereas the cell-mediated responses, even when they resembled those in patients during the acute phase of IM in timing and quality, were never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se but of the hyperactivated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between the viral load and the response kinetics, emphasizing how much there is still to learn about primary EBV infection.
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Infecciones Asintomáticas/epidemiología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Virus de Epstein-Barr/virología , Células Asesinas Naturales/inmunología , Adulto , Anticuerpos Antivirales/sangre , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/inmunología , Femenino , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Masculino , Pronóstico , Estudios Prospectivos , Reino Unido/epidemiología , Carga Viral , Adulto JovenRESUMEN
Cytomegalovirus (CMV) infection elicits a strong T-cell immune response, which increases further during aging in a process termed "memory inflation." CMV downregulates the expression of HLA-A and HLA-B on the surface of infected cells to limit presentation of viral peptides to T-cells although HLA-C is relatively spared as it also engages with inhibitory killer immunoglobulin receptor receptors and therefore reduces lysis by natural killer cells. We investigated the magnitude and functional properties of CMV-specific CD8+ T-cells specific for 10 peptides restricted by HLA-C in a cohort of 53 donors between the age of 23 and 91 years. This was achieved via peptide stimulation of PBMCs followed by multicolor flow cytometry. Three peptides, derived from proteins generated in the immediate-early period of viral replication and restricted by HLA-Cw*0702, elicited strong immune responses, which increased substantially with age such that the average aggregate response represented 37% of the CD8+ T-cell pool within donors above 70 years of age. Remarkably, a single response represented 70% of the total CD8+ T-cell pool within a 91-year-old donor. HLA-Cw*0702-restricted CD8+ T-cell responses were immunodominant over HLA-A and HLA-B-restricted CMV-specific responses and did not show features of exhaustion such as PD-1 or CD39 expression. Indeed, such CTL exhibit a polyfunctional cytokine profile with co-expression of IFN-γ and TNF-α and a strong cytotoxic phenotype with intracellular expression of perforin and granzymeB. Functionally, HLA-Cw*0702-restricted CTL show exceptionally high avidity for cognate peptide-HLA and demonstrate very early and efficient recognition of virally infected cells. These observations indicate that CD8+ T-cells restricted by HLA-C play an important role in the control of persistent CMV infection and could represent a novel opportunity for CD8+ T-cell therapy of viral infection within immunosuppressed patients. In addition, the findings provide further evidence for the importance of HLA-C-restricted T-cells in the control of chronic viral infection.
RESUMEN
Cytomegalovirus (CMV) infection elicits a very strong and sustained intravascular T cell immune response which may contribute towards development of accelerated immune senescence and vascular disease in older people. Virus-specific CD8+ T cell responses have been investigated extensively through the use of HLA-peptide tetramers but much less is known regarding CMV-specific CD4+ T cells. We used a range of HLA class II-peptide tetramers to investigate the phenotypic and transcriptional profile of CMV-specific CD4+ T cells within healthy donors. We show that such cells comprise an average of 0.45% of the CD4+ T cell pool and can reach up to 24% in some individuals (range 0.01-24%). CMV-specific CD4+ T cells display a highly differentiated effector memory phenotype and express a range of cytokines, dominated by dual TNF-α and IFN-γ expression, although substantial populations which express IL-4 were seen in some donors. Microarray analysis and phenotypic expression revealed a profile of unique features. These include the expression of CX3CR1, which would direct cells towards fractalkine on activated endothelium, and the ß2-adrenergic receptor, which could permit rapid response to stress. CMV-specific CD4+ T cells display an intense cytotoxic profile with high level expression of granzyme B and perforin, a pattern which increases further during aging. In addition CMV-specific CD4+ T cells demonstrate strong cytotoxic activity against antigen-loaded target cells when isolated directly ex vivo. PD-1 expression is present on 47% of cells but both the intensity and distribution of the inhibitory receptor is reduced in older people. These findings reveal the marked accumulation and unique phenotype of CMV-specific CD4+ T cells and indicate how such T cells may contribute to the vascular complications associated with CMV in older people.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Endotelio Vascular/inmunología , Inmunidad Celular , Adulto , Anciano , Anciano de 80 o más Años , Infecciones por Citomegalovirus/patología , Endotelio Vascular/patología , Endotelio Vascular/virología , Femenino , Humanos , Interferón gamma/inmunología , Interleucina-4/inmunología , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Human cytomegalovirus (HCMV) is a widely prevalent herpes virus which establishes a state of chronic infection. The establishment of CMV-specific immunity controls viral reactivation and leads to the accumulation of very large numbers of virus-specific T cells which come to dominate the immune repertoire. There is concern that this may reduce the immune response to heterologous infections and HCMV infection has been associated with reduced survival in elderly people. Patients with chronic lymphocytic leukemia (B-CLL) suffer from a state of immune suppression but have a paradoxical increase in the magnitude of the CMV-specific T cell and humoral immune response. As such, there is now considerable interest in how CMV infection impacts on the clinical outcome of patients with B-CLL. Utilizing a large prospective cohort of patients with B-CLL (n = 347) we evaluated the relationship between HCMV seropositivity and patient outcome. HCMV seropositive patients had significantly worse overall survival than HCMV negative patients in univariate analysis (HR = 2.28, 95% CI: 1.34-3.88; P = 0.002). However, CMV seropositive patients were 4 years older than seronegative donors and this survival difference was lost in multivariate modeling adjusted for age and other validated prognostic markers (P = 0.34). No significant difference was found in multivariate modeling between HCMV positive and negative patients in relation to the time to first treatment (HR = 1.12, 95% CI: 0.68-1.84; P = 0.65). These findings in a second independent cohort of 236 B-CLL patients were validated. In conclusion no evidence that HCMV impacts on the clinical outcome of patients with B-CLL was found. Am. J. Hematol. 91:776-781, 2016. © 2016 Wiley Periodicals, Inc.
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Leucemia Linfocítica Crónica de Células B/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/mortalidad , Femenino , Humanos , Inmunidad , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Tasa de Supervivencia , Tiempo de Tratamiento , Resultado del Tratamiento , Adulto JovenRESUMEN
Cytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality after allogeneic hematopoietic stem cell transplant. T-cell immunity is critical for control of CMV infection, and correction of the immune deficiency induced by transplant is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T- cell therapy exclude patients with graft-versus-host disease (GVHD) from receiving systemic corticosteroid therapy, which impairs cellular immunity. This group of patients remains the highest clinical risk group for recurrent and problematic infections. Here, we address this unmet clinical need by genetic disruption of the glucocorticoid receptor (GR) gene using electroporation of transcription activator-like effector nuclease (TALEN) messenger RNA. We demonstrate efficient inactivation of the GR gene without off-target activity in Streptamer-selected CMV-specific CD8(+) T cells (HLA-A02/NLV peptide), conferring resistance to glucocorticoids. TALEN-modified CMV-specific T cells retained specific killing of target cells pulsed with the CMV peptide NLV in the presence of dexamethasone (DEX). Inactivation of the GR gene also conferred resistance to DEX in a xenogeneic GVHD model in sublethally irradiated NOD-scid IL2rγ(null) mice. This proof of concept provides the rationale for the development of clinical protocols for producing and administering high-purity genetically engineered virus-specific T cells that are resistant to the suppressive effects of corticosteroids.
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Traslado Adoptivo/métodos , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Técnicas de Silenciamiento del Gen/métodos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Receptores de Glucocorticoides/genética , Animales , Infecciones por Citomegalovirus/prevención & control , Electroporación , Endonucleasas/genética , Enfermedad Injerto contra Huésped , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , ARN Mensajero , TransfecciónRESUMEN
CMV infection is a significant cause of morbidity and mortality in immunocompromised individuals, and the development of a vaccine is of high priority. Glycoprotein B (gB) is a leading vaccine candidate but the glycoprotein H (gH) pentameric complex is now recognized as the major target for neutralizing Abs. However, little is known about the T cell immune response against gH and glycoprotein L (gL) and this is likely to be an important attribute for vaccine immunogenicity. In this study, we examine and contrast the magnitude and phenotype of the T cell immune response against gB, gH, and gL within healthy donors. gB-specific CD4(+) T cells were found in 95% of donors, and 29 epitopes were defined with gB-specific response sizes ranging from 0.02 to 2.88% of the CD4(+) T cell pool. In contrast, only 20% of donors exhibited a T cell response against gH or gL. Additionally, gB-specific CD4(+) T cells exhibited a more cytotoxic phenotype, with high levels of granzyme B expression. Glycoproteins were effectively presented following delivery to APCs but only gB-derived epitopes were presented following endogenous synthesis. gB expression was observed exclusively within vesicular structures colocalizing with HLA-DM whereas gH was distributed evenly throughout the cytoplasm. Grafting of the C-terminal domain from gB onto gH could not transfer this pattern of presentation. These results reveal that gB is a uniquely immunogenic CMV glycoprotein and this is likely to reflect its unique pattern of endogenous Ag presentation. Consideration may be required toward mechanisms that boost cellular immunity to gH and gL within future subunit vaccines.
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Linfocitos T CD4-Positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Epítopos de Linfocito T/inmunología , Inmunidad Celular , Proteínas del Envoltorio Viral/inmunología , Adulto , Linfocitos T CD4-Positivos/patología , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/patología , Vacunas contra Citomegalovirus/genética , Vacunas contra Citomegalovirus/inmunología , Epítopos de Linfocito T/genética , Femenino , Granzimas , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Envoltorio Viral/genéticaAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Péptidos/inmunología , Trasplante de Células Madre , Activación Viral/inmunología , Linfocitos T CD4-Positivos/metabolismo , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase II/química , Humanos , Péptidos/química , Multimerización de Proteína , Trasplante de Células Madre/efectos adversos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Trasplante HomólogoRESUMEN
Cytomegalovirus (CMV) infects the majority of the global population and leads to the development of a strong virus-specific immune response. The CMV-specific CD4+ and CD8+ T cell immune response can comprise between 10 and 50% of the T cell pool within peripheral blood and there is concern that this may impair immunity to other pathogens. Elderly individuals with the highest magnitude of CMV-specific immune response have been demonstrated to be at increased risk of mortality and there is increasing interest in interventions that may serve to moderate this. Acyclovir is an anti-viral drug with activity against a range of herpes viruses and is used as long term treatment to suppress reactivation of herpes simplex virus. We studied the immune response to CMV in patients who were taking acyclovir to assess if therapy could be used to suppress the CMV-specific immune response. The T cell reactivity against the immunodominant late viral protein pp65 was reduced by 53% in people who were taking acyclovir. This effect was seen within one year of therapy and was observed primarily within the CD4+ response. Acyclovir treatment only modestly influenced the immune response to the IE-1 target protein. These data show that low dose acyclovir treatment has the potential to modulate components of the T cell response to CMV antigen proteins and indicate that anti-viral drugs should be further investigated as a means to reduce the magnitude of CMV-specific immune response and potentially improve overall immune function.
Asunto(s)
Aciclovir/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Fosfoproteínas/inmunología , Proteínas de la Matriz Viral/inmunología , Adulto , Anciano , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/genética , Masculino , Persona de Mediana Edad , Fosfoproteínas/genética , Proteínas de la Matriz Viral/genéticaRESUMEN
BACKGROUND & AIMS: Human cytomegalovirus infection (HCMV) is associated with an increased morbidity after liver transplantation, by facilitating allograft rejection and accelerating underlying hepatic inflammation. We hypothesized that human hepatic sinusoidal endothelial cells infected with HCMV possess the capacity to modulate allogeneic T cell recruitment and activation, thereby providing a plausible mechanism of how HCMV infection is able to enhance hepatic immune activation. METHODS: Human hepatic sinusoidal endothelial cells were isolated from explanted livers and infected with recombinant endotheliotropic HCMV. We used static and flow-based models to quantify adhesion and transendothelial migration of allogeneic T cell subsets and determine their post-migratory phenotype and function. RESULTS: HCMV infection of primary human hepatic sinusoidal endothelial cells facilitated ICAM-1 and CXCL10-dependent CD4 T cell transendothelial migration under physiological levels of shear stress. Recruited T cells were primarily non-virus-specific CXCR3(hi) effector memory T cells, which demonstrated features of LFA3-dependent Th1 activation after migration, and activated regulatory T cells, which retained a suppressive phenotype following transmigration. CONCLUSIONS: The ability of infected hepatic endothelium to recruit distinct functional CD4 T cell subsets shows how HCMV facilitates hepatic inflammation and immune activation and may simultaneously favor virus persistence.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Infecciones por Citomegalovirus/inmunología , Endotelio Vascular/metabolismo , Inmunidad Celular , Hígado/inmunología , Adhesión Celular , Movimiento Celular , Células Cultivadas , Citomegalovirus , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Endotelio Vascular/patología , Endotelio Vascular/virología , Humanos , Hígado/metabolismo , Hígado/virologíaRESUMEN
PURPOSE: Conjunctival epithelial T cells are dominated by CD3(+)CD56-TCRαß(+)CD8αß(+) lymphocytes. In this study we explored the antigen experience status, mucosal homing phenotype, cytokine expression, and viral antigen recognition of conjunctival epithelial CD8(+) T cells from healthy individuals. METHODS: Following ocular surface impression cytology, conjunctival cells were recovered by gentle agitation and analyzed by flow cytometry for cell surface markers, cytokine production (stimulated by phorbol 12-myristate 13-acetate [PMA]/ionomycin), and Epstein-Barr virus (EBV)/cytomegalovirus (CMV) immunodominant epitope recognition using major histocompatibility complex (MHC) class I peptide tetramers. RESULTS: In contrast to peripheral blood, conjunctival epithelial CD8(+) T cells were dominantly CD45RA(-)CCR7(-) effector memory cells, and the vast majority expressed the mucosal homing integrin αEß7. Conjunctival memory CD8(+) T cells maintained effector functions with the ability to secrete IFN-γ and expression of Granzyme B, although they expressed significantly reduced amounts per cell compared to peripheral blood T cells. Interestingly, herpetic virus-specific CD8(+) T cells recognizing epitopes derived from EBV and CMV could be detected in the conjunctival cells of healthy virus carriers, although they were generally at lower frequencies than in the peripheral blood of the same donor. Virus-specific conjunctival CD8(+) T cells were dominated by CD45RA(-)CCR7(-) effector memory cells that expressed αEß7. CONCLUSIONS: These data demonstrate that the majority of conjunctival epithelial CD8(+) T cells are mucosal homing αEß7(+) effector memory T cells, which can recognize viral epitopes and are capable of secreting Granzyme B and IFN-γ.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Conjuntiva/inmunología , Citocinas/metabolismo , Epítopos de Linfocito T/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Superficie/metabolismo , Antígenos Virales/inmunología , Estudios de Cohortes , Infecciones por Citomegalovirus/inmunología , Epitelio/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Membrana Mucosa/inmunología , Adulto JovenRESUMEN
Late Epstein-Barr virus infection and hypovitaminosis-D as environmental risk factors in the pathogenesis of multiple sclerosis are gaining great interest. We, therefore, tested for in-vivo interdependence between Epstein-Barr-virus (EBV)-status and 25-hydroxyvitamin D3 (25(OH)D3) -level in healthy young individuals from a United Kingdom (UK) autumn cohort. EBV-load was measured by quantitative polymerase chain reaction and 25(OH)D3 levels by isotope-dilution liquid chromatography-tandem mass spectrometry. This young, healthy UK autumn cohort showed surprisingly low levels of 25(OH)D3 (mean value: 40.5 nmol/L ± 5.02). Furthermore, we found that low 25(OH)D3 levels did not impact on EBV load and anti-EBV nuclear antigen-1 (EBNA-1) titers. However, we observed a correlation between EBV load and EBNA-1 titers. These observations should be of value in the study of the potential relationship between hypovitaminosis-D and EBV-status in the pathophysiology of multiple sclerosis.
Asunto(s)
Avitaminosis/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/virología , Vitamina D/metabolismo , Adulto , Anticuerpos Antivirales/análisis , Estudios de Cohortes , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Humanos , Factores de Riesgo , Estaciones del Año , Reino Unido , Adulto JovenRESUMEN
EBV elicits primary CD8(+) T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed toward immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L Ag-specific responses have been detected regularly in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-γ ELISPOT assays using peptides spanning the two IE, six representative E, and seven representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7 L) new epitopes using memory CD8(+) T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8(+) T cell clones against these novel lytic cycle epitopes recognized lytically infected cells naturally expressing their target Ag. Surprisingly, however, clones against the most frequently recognized L Ag, the BNRF1 tegument protein, also recognized latently infected, growth-transformed cells. We infer that BNRF1 is also a latent Ag that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.
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Linfocitos T CD8-positivos/inmunología , Herpesvirus Humano 4/inmunología , Mononucleosis Infecciosa/inmunología , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/virología , Células Cultivadas , Ensayo de Immunospot Ligado a Enzimas , Antígenos HLA/metabolismo , Humanos , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Interferón gamma/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Latencia del Virus/inmunologíaRESUMEN
Cytomegalovirus (CMV) is a widely prevalent herpesvirus that is well tolerated by an immune competent host yet establishes a state of chronic infection. The virus is thought to undergo frequent subclinical episodes of reactivation which leads to an unusually large accumulation of CMV-specific CD8(+) T lymphocytes in the peripheral blood, a phenomenon termed "memory inflation." The high magnitude of the CMV T cell response has been implicated in impaired immunity to heterologous pathogens such as EBV, influenza and West Nile virus. Here, using murine CMV (MCMV), we show that memory inflation of virus-specific CD8(+) T cells is avoided if mice are infected with a replication defective virus called temperature-sensitive mutant 5 (tsm5), which carries an attenuating mutation within the DNA primase gene. Mice infected with tsm5 do generate primary T cell responses towards viral proteins but these do not amass to skew the memory repertoire of CD8(+) T cells. Therefore, attenuation of the virus replication machinery may be valuable in future CMV vaccine designs because the virus remains immunogenic but does not contribute to CMV associated T cell immune senescence.
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Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Memoria Inmunológica , Muromegalovirus/inmunología , Animales , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/inmunología , Modelos Animales de Enfermedad , Femenino , Infecciones por Herpesviridae/virología , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Mutación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Virulencia , Replicación ViralRESUMEN
Cytomegalovirus (CMV) infection increases the risk of complications after renal transplantation, but the mechanisms controlling donor-derived infection are not adequately characterized. Here, we assessed the risk of clinically significant CMV disease in donor-seropositive, recipient-seropositive (D+R+) renal transplantation and examined recipients' CMV antigen-specific cellular immune responses primed directly by donor cells. In a retrospective cohort of 569 patients administered standardized basiliximab-tacrolimus-mycophenolate-corticosteroid immunosuppressive therapy, CMV disease rates increased in D+R+ serostatus pairings compared with D-R+ pairings (hazard ratio [HR], 2.61; 95% confidence interval [CI], 1.36 to 5.01; P=0.004) and associated with increased donor-recipient HLA mismatch in the D+R+ group (HR [per class 1 mismatch], 1.43; 95% CI, 1.12 to 1.82]; P=0.02). D+R+ and D+R- transplants in which the donor and recipient differentially expressed at least one HLA class I allele were followed prospectively from the time of transplantation. During the first year after transplantation, four of eight seropositive recipients and one of three seronegative recipients displayed peripheral blood CD8+ T cell responses to CMV presented by recipient-specific HLA. Notably, no recipients mounted responses to CMV presented by donor-specific HLA, despite the detection of CMV antigen expression in all seropositive donor organs examined (n=10), suggesting that the allograft of Class I HLA-mismatched seropositive donors is inaccessible to CD8+ T cell responses. Finally, pretransplant assays of anti-CMV cellular immunity predicted post-transplant CMV replication less accurately in D+R+ pairings than in D-R+ pairings, possibly reflecting in vitro assay specificity for recipient, rather than donor, HLA. These findings are relevant to the clinical management and immunologic understanding of donor-transmitted viral infection.
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Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Trasplante de Riñón/efectos adversos , Activación de Linfocitos/inmunología , Complicaciones Posoperatorias/virología , Adulto , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Citomegalovirus/aislamiento & purificación , Epítopos de Linfocito T/inmunología , Femenino , Genes MHC Clase I/inmunología , Humanos , Riñón/inmunología , Riñón/patología , Riñón/virología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Complicaciones Posoperatorias/inmunología , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
Cytomegalovirus (CMV) is a common herpesvirus infection and stimulates the expansion of very large numbers of CMV-specific T cells that reduce the CD4/CD8 ratio and suppress the number of naïve T cells. CMV infection has been associated with frailty and impaired survival. We investigated the correlates of CMV and the impact of the CMV infection on mortality within a cohort of 511 individuals aged at least 65 years who were followed up for 18 years. The mean age of the participants was 74 years of which 70% were CMV-seropositive. CMV was strongly linked to socio-economic status, and CMV infection increased the annual mortality rate by 42% (Hazard ratio = 1.42, 95% CI: 1.11-1.76 after adjusting for age, sex and baseline socio-economic and health variables) corresponding to 3.7 years lower life expectancy from age 65. Infection was associated with a near doubling of cardiovascular deaths, whereas there was no increase in mortality from other causes. These results show that CMV infection markedly increases the mortality rate in healthy older individuals due to an excess of vascular deaths. These findings may have significant implications for the study of immune senescence and if confirmed more generally could have important implications for measures to optimize the health of the elderly.
Asunto(s)
Envejecimiento , Relación CD4-CD8 , Infecciones por Citomegalovirus/mortalidad , Citomegalovirus/inmunología , Factores de Edad , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Enfermedades Cardiovasculares/mortalidad , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/inmunología , Femenino , Anciano Frágil , Humanos , Masculino , Análisis de SupervivenciaRESUMEN
Cytomegalovirus (CMV) infection leads to the development of adaptive and humoral immune responses that are among the largest for any pathogen, and intriguingly, the magnitude of the immune response increases with age, a phenomenon termed "memory inflation." Elevated CMV-specific immunity has been correlated with an increased mortality rate in elderly individuals and with impaired vaccination responses. The latent phase of CMV infection is characterized by intermittent episodes of subclinical viral reactivation and the production of immunogenic transcripts that may maintain memory inflation of virus-specific cytotoxic lymphocytes. However, the relative importance of CMV reactivation in the development of memory inflation is uncertain, as is the potential for antiviral treatment to reverse this effect. Here, we administered valaciclovir for up to 12 months in mice with established murine CMV (MCMV) infection. Treatment reduced the magnitude of the MCMV-specific CD8(+) T-lymphocyte response by 80%, and the residual MCMV tetramer-specific lymphocytes exhibited a less differentiated phenotype. In addition, latent MCMV infection suppressed the proportion of naïve CD8(+) T cells by 60% compared to antiviral-treated mice or MCMV-negative animals. Furthermore, treatment led to a reduction in influenza A viral loads following a challenge in elderly MCMV-infected animals and also reduced the differentiation of influenza virus-specific cytotoxic lymphocytes. These observations demonstrate that MCMV-specific memory inflation is maintained by viral replication and that therapeutic intervention could lead to improved immune function.