Andrew Worth

Rapid Turnover of Effector-Memory CD4 T Cells in Healthy Humans

Memory T cells can be divided into central–memory (TCM) and effector–memory (TEM) cells, which differ in their functional properties. Although both subpopulations can persist long term, it is not known whether they are maintained by similar mechanisms. We used in vivo labeling with deuterated glucose to measure the turnover of CD4+ T cells in healthy humans. The CD45R0+CCR7− TEM subpopulation was shown to have a rapid proliferation rate of 4.7% per day compared with 1.5% per day for CD45R0+CCR7+ TCM cells; these values are equivalent to average intermitotic (doubling) times of 15 and 48 d, respectively. In contrast, the CD45RA+CCR7+ naive CD4+ T cell population was found to be much longer lived, being labeled at a rate of only 0.2% per day (corresponding to an intermitotic time of approximately 1 yr). These data indicate that human CD4+ TEM cells constitute a short-lived cell population that requires continuous replenishment in vivo.

In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection

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‐D2‐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 ∼ 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (± standard deviation) proliferation rate was 4·3 ± 2·4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 ± 7·6 × 106 cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6·9 ± 4·0%/day; half‐life (T½) < 10 days]. Healthy elderly subjects (n = 8) had lower proliferation and production rates (P = 2·5 ± 1·0%/day and 7·3 ± 3·7 × 106 cells/l/day, respectively; P = 0·04). Similar rates were seen in patients chronically infected with human T‐cell lymphotropic virus type I (HTLV‐I) (P = 3·2 ± 1·9%/day). In acute infectious mononucleosis (n = 5), NK cell numbers were increased but kinetics were unaffected (P = 2·8 ± 1·0%/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.

Measurement and modeling of human T cell kinetics

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‐D2‐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.

Direct Measurement of T Cell Subset Kinetics In Vivo in Elderly Men and Women

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.

In vivo T lymphocyte dynamics in humans and the impact of human T-lymphotropic virus 1 infection

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 1012 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.

Neutralization of Plasmodium falciparum Merozoites by Antibodies against PfRH5

There is intense interest in induction and characterization of strain-transcending neutralizing Ab against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) as a target of broadly neutralizing Abs, but there is little information regarding the functional mechanism(s) of Ab-mediated neutralization. In this study, we report that vaccine-induced polyclonal anti-PfRH5 Abs inhibit the tight attachment of merozoites to erythrocytes and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 mAbs, we provide evidence of the following: 1) the ability to block the PfRH5–basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; 2) neutralizing mAbs bind spatially related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; 3) a brief exposure window of PfRH5 is likely to necessitate rapid binding of Ab to neutralize parasites; and 4) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly neutralizing anti-malaria Abs and further encourage anti-PfRH5–based malaria prevention efforts.

Human cytomegalovirus-specific CD8+ T-cell expansions contain long-lived cells that retain functional capacity in both young and elderly subjects

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 Vβ 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 Vβ 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.

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 5×109 cells/day (versus 7×107 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.

The antigen-specific memory CD8+ T-cell response induced by BCG in cattle resides in the CD8+γ/δTCR−CD45RO+ T-cell population

Tuberculosis (TB) remains a worldwide leading cause of death among infectious diseases. Development of safer and more efficacious vaccines requires a basic understanding of the protective mechanisms induced by BCG. Here we show that vaccination of cattle with BCG induces CD8+gamma/deltaTCR-CD45RO+ T-cells that can produce IFN-gamma, up-regulate transcription and expression of perforin, lyse BCG-infected monocyte-derived macrophages (MoMvarphi) and contribute to a reduction in the number of intracellular mycobacteria. We also observed BCG-induced CD8+ responses in vivo. After infection of cattle with Mycobacterium bovis, CD8+gamma/deltaTCR-CD45RO+ cells responded more strongly to M. bovis-infected MoMvarphi than to BCG-infected MoMvarphi. These results indicate that the antigen-specific CD8+ memory response resides in the CD8+gamma/deltaTCR-CD45RO+ cell population.

Thioether-bonded constructs of Fab'gamma and Fc gamma modules utilizing differential reduction of interchain disulfide bonds

We describe a two-stage preparation of chemically engineered Ab constructs, employing as modules Fab′γ from mAb or rAb, and Fc from human normal IgG1. A multivalent, optionally multispecific F(ab′)n core is formed in stage one, and one or more Fc modules added in stage two. Examples include bispecific Fab2Fc2 (for simplicity, primes and Greek letters are omitted from names of final constructs) and trivalent Fab3Fc2, which are designed to kill neoplastic cells. An essential element in the construction is the availability of the Fab′ in two reduced forms, Fab′(-sulfhydryl (SH))5 and Fab′-SH. The first is obtained by full reduction of the interchain disulfide bonds (SS) in the F(ab′)2 fragment of IgG. Fab′-SH is obtained by disulfide-interchange reactions on Fab′(-SH)5, whereby the γ-light SS is reconstituted, an unusual intrachain SS forms in the γ-chain hinge, and one hinge SH remains. F(ab′)2 and F(ab′)3 cores are built using partially reduced modules, being given intermodular thioether links that resist reduction. These cores are then fully reduced, making available SH groups for addition of the Fcγ modules. In the final constructs, all intermodular links embody tandem thioether bonds arising at hinge-region cysteines. Cytotoxic activities of representative constructs, and some enhancements deriving from multiple modules, are assessed. In guinea pigs, catabolism of Fab2Fc2 yielded a t1/2 similar to that of human IgG1, although the serum Fab2Fc2 revealed some proteolytic breakdown not shown by the IgG1. Immunotherapy of a guinea-pig leukemia confirmed the ability of these constructs to kill target cells in vivo.