RESUMEN
The immune response to human cytomegalovirus (HCMV) infection is characterized by the accumulation of HCMV-specific CD8(+) T cells, particularly in the elderly; such expansions may impair immune responses to other pathogens. We investigated mechanisms underlying HCMV-specific expansions in 12 young and 21 old healthy subjects (although not all analyses were performed on all subjects). Phenotypically, HCMV-pentamer(+) CD8(+) T cells were characterized by marked Vß restriction, advanced differentiation (being predominantly CD27(-) CD28(-) ), and variable CD45RO/RA expression. Although more common and larger in older subjects, expansions had similar phenotypic characteristics in the young. In one old subject, repeated studies demonstrated stability in size and Vß distribution of pentamer(+) populations over 6 years. We tested whether HCMV-specific CD8(+) T-cell expansions arose from accelerated proliferation or extended lifespan by in vivo labelling with deuterated glucose and ex vivo Ki-67 expression. Uptake of deuterated glucose was lower in pentamer(+) cells than in pentamer(-) CD8(+) CD45RO(+) or CD8(+) CD45RA(+) cells in three old subjects, consistent with reduced proliferation and extended lifespan. Similarly Ki-67 labelling showed no evidence for increased proliferation in HCMV-specific CD8(+) expansions in older subjects, although pentamer(-) CD45RA(+) cells from young donors expressed very little Ki-67. We investigated Bcl-2 and CD95 as possible anti-apoptotic mediators, but neither was associated with pentamer-positivity. To investigate whether expansion represents a compensatory response to impaired functionality, we performed two tests of functionality, peptide-stimulated proliferation and CD107 expression; both were intact in pentamer(+) cells. Our data suggest that HCMV-specific CD8(+) expansions in older subjects accumulate by extended lifespan, rather than accelerated proliferation.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/inmunología , Proliferación Celular , Infecciones por Citomegalovirus/virología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Antígeno Ki-67/biosíntesis , Antígeno Ki-67/inmunología , Fenotipo , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Adulto Joven , Receptor fas/inmunologíaRESUMEN
Human T-lymphotropic virus type 1 (HTLV-1) is a persistent CD4+ T-lymphotropic retrovirus. Most HTLV-1-infected individuals remain asymptomatic, but a proportion develop adult T cell leukemia or inflammatory disease. It is not fully understood how HTLV-1 persists despite a strong immune response or what determines the risk of HTLV-1-associated diseases. Until recently, it has been difficult to quantify lymphocyte kinetics in humans in vivo. Here, we used deuterated glucose labeling to quantify in vivo lymphocyte dynamics in HTLV-1-infected individuals. We then used these results to address four questions. (i) What is the impact of HTLV-1 infection on lymphocyte dynamics? (ii) How does HTLV-1 persist? (iii) What is the extent of HTLV-1 expression in vivo? (iv) What features of lymphocyte kinetics are associated with HTLV-1-associated myelopathy/tropical spastic paraparesis? We found that CD4+CD45RO+ and CD8+CD45RO+ T lymphocyte proliferation was elevated in HTLV-1-infected subjects compared with controls, with an extra 10(12) lymphocytes produced per year in an HTLV-1-infected subject. The in vivo proliferation rate of CD4+CD45RO+ cells also correlated with ex vivo viral expression. Finally, the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with significantly increased CD4+CD45RO+ cell proliferation. We suggest that there is persistent viral gene expression in vivo, which is necessary for the maintenance of the proviral load and determines HTLV-1-associated myelopathy/tropical spastic paraparesis risk.
Asunto(s)
Infecciones por HTLV-I/inmunología , Linfocitos T/inmunología , División Celular , Productos del Gen tax/análisis , Humanos , Antígenos Comunes de Leucocito/análisis , Activación de Linfocitos , Paraparesia Espástica Tropical/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium-enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24-hr intravenous infusion of 6,6-D(2)-glucose, CD3(-) CD16(+) NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n=5), deuterium enrichment was maximal approximately 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (+/- standard deviation) proliferation rate was 4 x 3 +/- 2 x 4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 +/- (7 x 6) x 10(6) cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6 x 9 +/- 4 x 0%/day; half-life (T((1/2))) < 10 days]. Healthy elderly subjects (n=8) had lower proliferation and production rates (P=2 x 5 +/- 1 x 0%/day and 7 x 3 +/- (3 x 7) x 10(6) cells/l/day, respectively; P=0 x 04). Similar rates were seen in patients chronically infected with human T-cell lymphotropic virus type I (HTLV-I) (P=3 x 2 +/- 1 x 9%/day). In acute infectious mononucleosis (n=5), NK cell numbers were increased but kinetics were unaffected (P=2 x 8 +/- 1 x 0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV-I infection and normalize rapidly following acute Epstein-Barr virus infection.
Asunto(s)
Envejecimiento/inmunología , Células Asesinas Naturales/inmunología , Virosis/inmunología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Enfermedad Crónica , Femenino , Infecciones por HTLV-I/inmunología , Humanos , Mononucleosis Infecciosa/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Nutritional status is highly dependent on season in countries such as The Gambia. In a rural Gambian setting, individuals born during periods of seasonal nutritional deprivation ("hungry seasons") are susceptible to mortality from infectious diseases in adult life. OBJECTIVE: We investigated the hypothesis that impaired immunocompetence in those born in the hungry season results from an underlying defect in immunologic memory, similar to the immunosenescence of old age, which is likely to be reflected in the phenotype and kinetics of T lymphocytes in young adults. DESIGN: T cell phenotype in terms of CD3, CD4, CD8, CD45RA, and CD45R0 expression and in vivo dynamics measured by stable isotope labeling of T cell subsets combined with gas chromatography-mass spectrometry and frequency of T cell receptor excision circles were measured in 25 young (18-24-y-old) Gambian men. Thirteen of these 25 men were exposed to perinatal malnutrition as defined by birth season and birth weight. RESULTS: In persons born in the hungry season with low birth weight, no differences in the proportions of memory or naive T cells were found. Kinetic analysis showed higher proliferation rates in memory (CD45R0(+)) subsets of T cells than in naïve (CD45R0(-)) cells, which is consistent with previous studies, but no evidence was found for an effect of birth weight or season on T lymphocyte proliferation and disappearance rates. No significant correlations were found between in vivo T cell kinetics and frequency of T cell receptor excision circles. Only absolute numbers of granulocytes were elevated in those born in the nutritionally deprived season. CONCLUSION: In healthy young Gambian men, T lymphocyte homeostasis is extremely robust regardless of perinatal nutritional compromise.
Asunto(s)
Peso al Nacer/inmunología , Desnutrición/inmunología , Atención Perinatal , Efectos Tardíos de la Exposición Prenatal/inmunología , Estaciones del Año , Linfocitos T/inmunología , Adolescente , Adulto , Envejecimiento , Femenino , Abastecimiento de Alimentos/estadística & datos numéricos , Gambia , Humanos , Masculino , Embarazo , Linfocitos T/citologíaRESUMEN
Interleukin (IL)-7 and IL-15 are cytokines implicated in homeostatic control of the peripheral CD8 T-cell pool. We compared the effects of IL-7 and IL-15 on survival and proliferation of purified human CD8+ T-cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl-2 up-regulation. Surprisingly, although minimal proliferation of naïve CD8+ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL-15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8+ T cells through several cell divisions resulted in up-regulation of telomerase and the maintenance of telomere length. These data show that IL-7 and IL-15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset-dependent manner, and have implications for the homeostatic expansion of the naïve CD8+ T-cell pool.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-15/inmunología , Interleucina-7/inmunología , Telómero/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación , Antígenos Comunes de Leucocito/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores CCR7 , Receptores de Quimiocina/metabolismo , Subgrupos de Linfocitos T/inmunología , Telomerasa/metabolismoRESUMEN
Studies involving in-vivo labelling of lymphocyte DNA with 6,6-2H2-glucose to track T-cell turnover have contributed to understanding lymphocyte homeostasis in health and disease. Applying such studies in tropical settings (where diseases that affect T-cells are prevalent) requires protocol modifications including non-intravenous label administration, applicability in outpatient facilities, and T-cell sorting methods independent of a fluorescence activated cell sorter (FACS). Such protocols were validated in UK pilot studies and applied in The Gambia. Healthy adult subjects (n=12) were recruited from three Gambian villages. 6,6-2H2-glucose was administered orally in an outpatient clinic and T-cell subpopulations isolated from peripheral blood using plastic adherence, and Multisorttrade mark magnetic cell sorting (MACStrade mark) to obtain CD8+CD45R0+, CD8-CD45R0+, CD8+CD45R0- and CD8-CD45R0- subsets. To achieve high cell purity and yield, CD45R0- cells were reincubated with CD45R0 beads. T-cell proliferation and disappearance were quantified using gas chromatography mass spectrometry. Results were consistent with those of other studies showing higher turnover in memory (CD45R0+) than in naïve (CD45R0-) T-cell subsets, and an association between recent cell proliferation and susceptibility to cell death. Cell kinetics research is applicable in tropical settings, and can contribute to further understanding the regulation of adaptive immunity in response to infections and other insults.
Asunto(s)
Proliferación Celular , Linfocitos T/citología , Adolescente , Adulto , Femenino , Gambia , Homeostasis , Humanos , Masculino , Proyectos Piloto , Linfocitos T/fisiologíaRESUMEN
Information about the kinetic behavior and lifespan of lymphocytes is crucial to understanding the mechanisms that regulate processes such as immunologic memory. We have used in vivo labeling of dividing cells with 6,6-(2)H(2)-glucose, combined with cell sorting and gas-chromatography-mass spectrometry for deuterium enrichment, in order to analyze the kinetics of human total, naive, or memory B lymphocytes, separated from peripheral blood using monoclonal antibodies. We show that total blood B cells of young adults divide at an average rate of 1.9% (+/-1.0%) per day and at a similar though slightly slower rate, 1.5% (+/-1.3%) per day, in the elderly. Separation of naive and memory B cells according to expression of CD27 indicates that naive peripheral blood B cells divide slowly (0.46% per day), while memory cells proliferate more rapidly (2.66% per day). These data are compatible with the view that B-cell memory may be maintained by clones of proliferating B cells.
Asunto(s)
Linfocitos B/citología , Senescencia Celular/inmunología , Memoria Inmunológica , Adulto , Factores de Edad , Anciano , Linfocitos B/inmunología , Células Sanguíneas/inmunología , Proliferación Celular , Separación Celular , Deuterio , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Cinética , Masculino , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/análisisRESUMEN
The age-associated decline in immunocompetence is paralleled by changes in the proportions of PBL subpopulations. In turn, the size and composition of the peripheral lymphocyte pool is determined by input from the thymus and bone marrow and by the balance of proliferation and death in each lymphocyte subpopulation. We compared the kinetics of lymphocyte subtypes in young (seven of eight CMV seronegative) and healthy elderly human subjects (six of seven CMV seropositive), using deuterated glucose DNA labeling in vivo to measure rates of T cell proliferation and disappearance. For CD45RO(+) cells of both CD4(+) and CD8(+) subtypes and for CD4(+)CD45RA(+) cells the kinetics of proliferation and disappearance were remarkably similar between elderly and young subjects. In the young, the kinetics of CD8(+)CD45RA(+) cells with a naive phenotype resembled those of CD4(+)CD45RA(+) cells. However, CD8(+)CD45RA(+) T cells from the elderly exhibited a predominantly primed phenotype, and for this subset, although the proliferation rate was similar to that of other CD45RA(+) cells, the disappearance rate of labeled cells was greatly decreased compared with that of all other T cell subsets. Our data provide a direct demonstration that there are no substantial changes in in vivo kinetics for most T cell populations in healthy elderly compared with young subjects. However, primed CD8(+)CD45RA(+) cells show unusual kinetic properties, indicating the persistence of these cells in the blood and dissociation of proliferation from disappearance.
Asunto(s)
Envejecimiento/inmunología , Subgrupos de Linfocitos T/citología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , División Celular , Células Clonales/citología , Deuterio/farmacocinética , Femenino , Glucosa/farmacocinética , Humanos , Inmunocompetencia , Antígenos Comunes de Leucocito/análisis , Recuento de Linfocitos , Masculino , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/análisisRESUMEN
During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lymphocytes are produced. To investigate the dynamics of clonal expansion, we measured cell proliferation during AIM using deuterated glucose to label DNA of dividing cells in vivo, analyzing cells according to CD4, CD8 and CD45 phenotype. The proportion of labeled CD8(+)CD45R0(+) T lymphocytes was dramatically increased in AIM subjects compared to controls (mean 17.5 versus 2.8%/day; p<0.005), indicating very rapid proliferation. Labeling was also increased in CD4(+)CD45R0(+) cells (7.1 versus 2.1%/day; p<0.01), but less so in CD45RA(+) cells. Mathematical modeling, accounting for death of labeled cells and changing pool sizes, gave estimated proliferation rates in CD8(+)CD45R0(+) cells of 11-130% of cells proliferating per day (mean 47%/day), equivalent to a doubling time of 1.5 days and an appearance rate in blood of about 5 x 10(9) cells/day (versus 7 x 10(7) cells/day in controls). Very rapid death rates were also observed amongst labeled cells (range 28-124, mean 57%/day),indicating very short survival times in the circulation. Thus, we have shown direct evidence for massive proliferation of CD8(+)CD45R0(+) T lymphocytes in AIM and demonstrated that rapid cell division continues concurrently with greatly accelerated rates of cell disappearance.
Asunto(s)
Mononucleosis Infecciosa/inmunología , Linfocitos T/fisiología , Enfermedad Aguda , Adolescente , Adulto , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Humanos , Inmunofenotipificación , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/fisiología , Activación de Linfocitos , MasculinoRESUMEN
The ability to measure, describe and interpret T cell kinetics is pivotal in understanding normal lymphocyte homeostasis and diseases that affect T cell numbers. Following in vivo labeling of dividing cells with 6,6-D(2)-glucose in eight healthy volunteers, peripheral blood T cells were sorted by CD4, CD8 and CD45 phenotype. Enrichment of deuterium in DNA was measured by gas chromatography-mass spectrometry. A novel model of T cell kinetics, allowing for heterogeneity within T cell pools, was used to analyze data on acquisition and loss of label and calculate proliferation and disappearance rates for each subpopulation. Proliferation rates for CD45RO(+)CD8(+) cells and CD45RO(+)CD4(+) cells were 5.1% and 2.7% /day, respectively (equivalent doubling times: 14 and 26 days). CD45RA(+)CD8(+) lymphocytes and CD45RA(+)CD4(+) lymphocytes had slower proliferation rates, 0.5% and 0.6% / day, respectively (doubling time about 4 months). Disappearance rates of labeled cells were similar for all cell types (7%-12% / day) and exceeded corresponding proliferation rates. This disparity may be understood conceptually in terms of either phenotypic heterogeneity (rapid versus slow turnover pools), or history (recently divided cells are more likely to die). The new kinetic model fits the data closely and avoids the need to postulate a large external source of lymphocytes to maintain equilibrium.
Asunto(s)
Modelos Inmunológicos , Linfocitos T/citología , Linfocitos T/inmunología , Adulto , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , División Celular , Deuterio , Femenino , Humanos , Cinética , Antígenos Comunes de Leucocito/metabolismo , MasculinoRESUMEN
Hemophagocytic lymphohistiocytosis (HLH) composes a group of rare heterogenous disorders characterized by uncontrolled accumulation and infiltration of activated T lymphocytes and macrophages. Cytotoxic T and natural killer cell activity is significantly reduced or absent in these patients. Mutations in the important mediator of lymphocyte cytotoxicity perforin were identified in a number of HLH individuals. Here we report a novel missense mutation thr435met in the conserved Ca(2+) binding domain of perforin in a patient with HLH. Prediction of the 3-dimensional structure of the thr435met perforin mutant using comparative molecular modeling indicates that the protein's ability to bind Ca(2+), and therefore its cytolytic function, would be strongly compromised. In addition, this patient exhibited abnormal CD45 splicing caused by a C77G mutation in the gene encoding CD45 (PTPRC). Our findings suggest a combined role for perforin mutation and abnormal CD45 splicing as significant contributory factors in the pathogenesis of HLH.