Physiologically Based Simulations of Deuterated Glucose for Quantifying Cell Turnover in Humans.

In vivo [6,6-(2)H2]-glucose labeling is a state-of-the-art technique for quantifying cell proliferation and cell disappearance in humans. However, there are discrepancies between estimates of T cell proliferation reported in short (1-day) versus long (7-day) (2)H2-glucose studies and very-long (9-week) (2)H2O studies. It has been suggested that these discrepancies arise from underestimation of true glucose exposure from intermittent blood sampling in the 1-day study. Label availability in glucose studies is normally approximated by a "square pulse" (Sq pulse). Since the body glucose pool is small and turns over rapidly, the availability of labeled glucose can be subject to large fluctuations and the Sq pulse approximation may be very inaccurate. Here, we model the pharmacokinetics of exogenous labeled glucose using a physiologically based pharmacokinetic (PBPK) model to assess the impact of a more complete description of label availability as a function of time on estimates of CD4+ and CD8+ T cell proliferation and disappearance. The model enabled us to predict the exposure to labeled glucose during the fasting and de-labeling phases, to capture the fluctuations of labeled glucose availability caused by the intake of food or high-glucose beverages, and to recalculate the proliferation and death rates of immune cells. The PBPK model was used to reanalyze experimental data from three previously published studies using different labeling protocols. Although using the PBPK enrichment profile decreased the 1-day proliferation estimates by about 4 and 7% for CD4 and CD8+ T cells, respectively, differences with the 7-day and 9-week studies remained significant. We conclude that the approximations underlying the "square pulse" approach-recently suggested as the most plausible hypothesis-only explain a component of the discrepancy in published T cell proliferation rate estimates

Rapid turnover of T cells in acute infectious mononucleosis.

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

Accelerated in vivo proliferation of memory phenotype CD4+ T-cells in human HIV-1 infection irrespective of viral chemokine co-receptor tropism.

CD4(+) T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+) cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+) compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+) T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul) participated. CCR5-expression defined a CD4(+) subpopulation of predominantly CD45R0(+) memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+) vs CCR5(-); healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4(+) T-cells (predominantly CD45RA(+) naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+)CD45R0(+)CD4(+) memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4(+) T-cell loss is primarily driven by non-specific immune activation

Profound and selective lymphopaenia in primary lymphatic anomaly patients demonstrates the significance of lymphatic-lymphocyte interactions

IntroductionThe lymphatic system has a pivotal role in immune homeostasis. To better understand this, we investigated the impact of Primary Lymphatic Anomalies (PLA) on lymphocyte numbers and phenotype.MethodsThe study comprised (i) a retrospective cohort: 177 PLA subjects from the National Primary Lymphatic Anomaly Register with clinical and laboratory data, and (ii) a prospective cohort: 28 patients with PLA and 20 healthy controls. Patients were subdivided using established phenotypic diagnostic categories and grouped into simplex (localised tissue involvement only) and systemic (involvement of central lymphatics). Further grouping variables included genital involvement and the likelihood of co-existent intestinal lymphangiectasia. Haematology laboratory parameters were analysed in both cohorts. In the prospective cohort, prospective blood samples were analysed by flow cytometry for markers of proliferation, differentiation, activation, skin-homing, and for regulatory (CD4+Foxp3+) T cells (Treg).ResultsIn patients with PLA, lymphopaenia was frequent (22% of subjects), affected primarily the CD4+ T cell subset, and was more severe in subjects with systemic versus simplex patterns of disease (36% vs 9% for lymphopaenia; 70% vs 33% for CD4+ cells). B cells, NK cells and monocytes were better conserved (except in GATA2 deficiency characterised by monocytopaenia). Genital oedema and likelihood of concomitant intestinal lymphangiectasia independently predicted CD4+ T cell depletion. Analysing CD4+ and CD8+ T cells by differentiation markers revealed disproportionate depletion of naïve cells, with a skewing towards a more differentiated effector profile. Systemic PLA conditions were associated with: increased expression of Ki67, indicative of recent cell division, in naïve CD4+, but not CD8+ T cells; increased levels of activation in CD4+, but not CD8+ T cells; and an increased proportion of Treg. Skin-homing marker (CCR10, CLA and CCR4) expression was reduced in some patients with simplex phenotypes.DiscussionPatients with PLA who have dysfunctional lymphatics have a selective reduction in circulating lymphocytes which preferentially depletes naïve CD4+ T cells. The presence of systemic disease, genital oedema, and intestinal lymphangiectasia independently predict CD4 lymphopaenia. The association of this depletion with immune activation and increased circulating Tregs suggests lymphatic-lymphocyte interactions and local inflammatory changes are pivotal in driving immunopathology

Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique

Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4-18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water which yielded estimates in excess of 3 days. To investigate this disparity we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n=4) and deuterium-labeled glucose (n=9), a compound with more rapid labeling kinetics. To interpret results we developed a novel mechanistic model. We applied this model to both previously-published (n=5) and newly-generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (R=0.26, from published values). Analysis of heavy water datasets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit-time, were poorly-defined. Analysis of glucose-labeling data yielded more precise estimates of half-life, 0.79 ± 0.25 days (19 hours), and marrow transit-time, 5.80 ± 0.42 days. Substitution of this marrow transit-time in the heavy water analysis gave a better-defined blood half-life, 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing R to vary yielded a best-fit value, R=0.19. Reanalysis of the previously-published model and data also revealed the origin of their long estimates for neutrophil half-life, an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than one day

Immunosenescence and Cytomegalovirus: where do we stand after a decade?

AbstractSince Looney at al. published their seminal paper a decade ago it has become clear that many of the differences in T cell immunological parameters observed between young and old people are related to the age-associated increasing prevalence of infection with the persistent beta-herpesvirus HHV-5 (Cytomegalovirus). Ten years later, studies suggest that hallmark age-associated changes in peripheral blood T cell subset distribution may not occur at all in people who are not infected with this virus. Whether the observed changes are actually caused by CMV is an open question, but very similar, rapid changes observed in uninfected patients receiving CMV-infected kidney grafts are consistent with a causative role. This meeting intensively discussed these and other questions related to the impact of CMV on human immune status and its relevance for immune function in later life.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs

The fate and lifespan of human monocyte subsets in steady state and systemic inflammation.

In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells

Report from the second cytomegalovirus and immunosenescence workshop.

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are