Naive Cell Population Research Articles

Droplet Microfluidic Technology for the Early and Label-Free Isolation of Highly-Glycolytic, Activated T-Cells.

A label-free, fixation-free and passive sorting method is presented to isolate activated T-cells shortly after activation and prior to the display of activation surface markers. It uses a recently developed sorting platform dubbed “Sorting by Interfacial Tension” (SIFT) that sorts droplets based on pH. After polyclonal (anti-CD3/CD28 bead) activation and a brief incubation on chip, droplets containing activated T-cells display a lower pH than those containing naive cells due to increased glycolysis. Under specific surfactant conditions, a change in pH can lead to a concurrent increase in droplet interfacial tension. The isolation of activated T-cells on chip is hence achieved as flattened droplets are displaced as they encounter a micro-fabricated trench oriented diagonally with respect to the direction of flow. This technique leads to an enrichment of activated primary CD4+ T-cells to over 95% from an initial mixed population of naive cells and cells activated for as little as 15 min. Moreover, since the pH change is correlated to successful activation, the technique allows the isolation of T-cells with the earliest activation and highest glycolysis, an important feature for the testing of T-cell activation modulators and to determine regulators and predictors of differentiation outcomes.

Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children.

Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.IMPORTANCE HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.

Overcoming immune dysfunction in the elderly: trained immunity as a novel approach.

People with advanced age have a higher susceptibility to infections and exhibit increased mortality and morbidity as the ability of the immune system to combat infections decreases with age. While innate immune cells display functional defects such as decreased phagocytosis, chemotaxis and cytokine production, adaptive immune cells exhibit reduced receptor diversity, defective antibody production and a sharp decline in naive cell populations. Successful responses to vaccination in the elderly are critical to prevent common infections such as influenza and pneumonia, but vaccine efficacy decreases in older individuals compared with young adults. Trained immunity is a newly emerging concept that showed that innate immune cells possess non-specific immunological memory established through epigenetic and metabolic reprogramming upon encountering certain pathogenic stimuli. Clinical studies suggest that trained immunity can be utilized to enhance immune responses against infections and improve the efficiency of vaccinations in adults; however, how trained immunity responses are shaped with advanced age is still an open question. In this review, we provide an overview of the age-related changes in the immune system with a focus on innate immunity, discuss current vaccination strategies for the elderly, present the concept of trained immunity and propose it as a novel approach to enhance responses against infections and vaccinations in the elderly population.

T Cell Receptor Cross-Reactivity between Similar Foreign and Self Peptides Influences Naive Cell Population Size and Autoimmunity

T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naive CD4(+) T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant because systemic expression of a self peptide reduced the size of a naive cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naive T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations and might allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection.

Single Naive CD4+ T Cells from a Diverse Repertoire Produce Different Effector Cell Types during Infection

A naive CD4(+) T cell population specific for a microbial peptide:major histocompatibility complex II ligand (p:MHCII) typically consists of about 100 cells, each with a different T cell receptor (TCR). Following infection, this population produces a consistent ratio of effector cells that activate microbicidal functions of macrophages or help B cells make antibodies. We studied the mechanism that underlies this division of labor by tracking the progeny of single naive T cells. Different naive cells produced distinct ratios of macrophage and B cell helpers but yielded the characteristic ratio when averaged together. The effector cell pattern produced by a given naive cell correlated with the TCR-p:MHCII dwell time or the amount of p:MHCII. Thus, the consistent production of effector cell subsets by a polyclonal population of naive cells results from averaging the diverse behaviors of individual clones, which are instructed in part by the strength of TCR signaling.

Negative Selection and Peptide Chemistry Determine the Size of Naive Foreign Peptide–MHC Class II-Specific CD4+ T Cell Populations

Naive CD4(+) T cell populations that express TCRs specific for different foreign peptide-MHC class II complex (pMHCII) ligands can vary in size over several orders of magnitude. This variation may explain why immune responses to some peptides are stronger than others. In this study, we used a sensitive pMHCII-tetramer-based cell enrichment method to study the derivation of two naive foreign pMHCII-specific naive CD4(+) T cell populations that differed in size by 8-fold in normal mice. Analysis of mice in which thymic negative selection was impaired revealed that the smaller population underwent more clonal deletion than the larger population. In addition, large naive cell populations tended to recognize peptides with tryptophan residues as TCR contacts. Thus, the foreign pMHCII that tend to be recognized by large naive populations induce minimal clonal deletion and contain certain amino acids with the capacity to interact favorably with TCRs.

Long-term consequences of the delay between virologic failure of highly active antiretroviral therapy and regimen modification

Current treatment guidelines recommend immediate modification of antiretroviral therapy in HIV-infected individuals with incomplete viral suppression. These recommendations have not been tested in observational studies or large randomized trials. We evaluated the consequences of delayed modification following virologic failure. We used prospective data from two clinical cohorts to estimate the effect of time until regimen modification following first regimen failure on all-cause mortality. The impact of regimen type was also assessed. As the effect of delayed switching can be confounded if patients with a poor prognosis modify therapy earlier than those with a good prognosis, we used a statistical methodology - marginal structural models - to control for time-dependent confounding. A total of 982 patients contributed 3414 person-years of follow-up following first regimen failure. Delay until treatment modification was associated with an elevated hazard of all-cause mortality among patients failing a reverse transcriptase inhibitor-based regimen (hazard ratio per additional 3 months delay = 1.23, 95% confidence interval: 1.08, 1.40), but appeared to have a small protective effect among patients failing a protease inhibitor-based regimen (hazard ratio per additional 3 months delay = 0.93, 95% confidence interval: 0.87, 0.99). Delay in modification after failure of regimens that do not contain a protease inhibitor is associated with increased mortality. Protease inhibitor-based regimens are less dependent on early versus delayed switching strategies. Efforts should be made to minimize delay until treatment modification in resource-poor regions, where the majority of patients are starting reverse transcriptase inhibitor-based regimens and HIV RNA monitoring may not be available.

UC blood infection with clinical strains of Mycobacterium tuberculosis: a novel model

Use of unrelated cord blood transplantation (UCBT) is increasing, yet high rates of mortality secondary to infection remain a problem. We investigated the utility of using umbilical cord blood (UCB) as a model to study a naive cell population challenged by Mycobacterium tuberculosis. Mononuclear cells were isolated from nine UCB samples and infected with each of four distinct strains of M. tuberculosis. The isolates used were two highly transmissible clinical strains, the virulent laboratory strain H37Rv and a unique strain isolated from only one case (i.e. non-virulent). CFU were assessed at 3 h post-infection (day 0) and at day 7 to generate growth curves. Viability of the mononuclear cells was assessed prior to infection, 3 h post-infection and at days 3, 5 and 7 post-infection. IFN-gamma and TNF-alpha levels were determined at 24 h post-infection. All three of the virulent strains demonstrated rapid growth in UCB cells that was significantly faster than the growth rate observed for the non-virulent unique isolate. There was no significant decrease in UCB cell viability after the 7-day incubation period regardless of infecting isolate. UCB cells secreted IFN-gamma in response to infection, with no significant difference related to infection with different isolates. However, there was a significant increase in the amount of TNF-alpha elicited following infection with the non-virulent isolate compared with the virulent isolates. These results show that UCB can be used as a model to study infection, hopefully leading to new therapies for neonates and UCBT recipients.

Functional and phenotypic variations in human T cells subjected to retroviral-mediated gene transfer.

The insertion of suicide genes in donor T lymphocytes constitutes the basis of new approaches aiming at the treatment of the graft-versus-host disease (GVHD), a frequent complication in recipients of allogeneic haematopoietic grafts. In this study we investigated the impact that the ex vivo manipulation required for the retroviral transduction of T cells had on the functionality and differentiation of these cells. Compared to fresh T cells, samples that had been subjected to standard activation (1 microg/ml of both anti-CD3i and anti-CD28i MoAbs) followed by transduction with vectors encoding for the HSV-tk and tNGFR genes maintained the proliferative response to an allogeneic stimulus. These cells, however, had a significantly lower cytotoxic response to allogeneic cells compared to fresh samples. When the concentration of anti-CD3i was reduced to up to 1000-fold (1 ng/ml), similar T-cell transductions were obtained, while the cytotoxicity of the ex vivo manipulated samples was significantly recovered, when assessed either at 7 or 14 days of culture. In all instances, a similar functionality was observed in transduced samples not subjected to immunomagnetic cell sorting, compared to purified fractions enriched in NGFR(+) and NFGR(-) cells. The analysis of CD45RA and CCR7 markers in samples transduced under standard stimulatory conditions showed a differentiation of fresh CD8(+) CD45RA(+)/CCR7(+) naive cells to cells having a predominant central CD45RA(-)/CCR7(+) and effector CD45RA(-)/CCR7(-) memory phenotype. However, when samples were activated with low doses of anti-CD3i, a significant population of naive cells became apparent. Although activation with high doses of anti-CD3i/anti-CD28i resulted in a similar phenotype in both NGFR(+) and NFGR(-) populations, the naive population observed in samples activated with low concentrations of anti-CD3i was almost restricted to the NGFR(-) population. These results show that reducing the stimulation mediated by anti-CD3i in protocols of T-cell retroviral gene transfer significantly helps to preserve the cytotoxic capacity of these cells to allogeneic cells, without affecting the susceptibility of these cells to the retroviral vector. In addition, we observed that modulating the activation of transduced T cells implies the generation of changes in the differentiation of CD8(+) cells, although we could not establish a direct relationship between the CD45RA/CCR7 phenotype of these cells and their cytotoxic reactivity to an allogeneic stimulus.

Interleukin-12: basic principles and clinical applications.

The most important advance in the last 10 years in our understanding of how to direct the immune response by vaccination or immunotherapy has been the description and subsequent refinement of the T helper type 1/2 (Th 1/2) paradigm. (Mosmann and Coffman 1989). This paradigm has provided the framework necessary to formulate basic questions related to defining the cues pathogens provide that shape the immune response. A major advance in this area came with the discovery of interleukin-12 (IL-12) (Kobayashi et al. 1989; Stern et al. 1990) and the subsequent demonstration that IL-12 promotes the development of Th 1 cells in vitro (Hsieh et al. 1993; Manetti et al. 1993). Thus, in naive T cell populations exposure to antigen in the presence of IL-12 for several days, followed by restimulation with antigen alone, led to the development of interferon (IFN)-γ producing T cells. Moreover, it was found that one could link together IL-12, the innate immune response, pathogens, and Th 1 cell development. This was done by showing that bacteria, such as Listeria monocytogenes, induced Th l cell development and that this occurred by stimulation of macrophages to produce IL-12 (D’Andrea et al. 1992; Hsieh et al. 1993). This observation has led to the description of a common pathway leading from the innate immune response to adaptive immunity, in which intracellular pathogens stimulate macrophages to produce IL-12, which promotes the development of Th 1 cells from a naive cell population. This pathway can now be exploited to develop approaches for the design of new immunotherapies and vaccines.

Host leukocytes and their products in chronic kidney allograft rejection in rats.

Bilaterally nephrectomized Lewis recipients of Fisher 344 (F344) kidney allografts, treated with CyA (1.5 mg/kg/day x 10), develop progressive changes of chronic rejection. Treated F344-to-F344 acted as isograft controls. Proteinuria was determined sequentially. Grafts were harvested 8, 12 and 16 weeks after transplantation (n = 9/group/time period). Infiltrating host cells and their products were assessed in chronically rejecting grafts by histology and immunostaining using mAbs for monocyte/macrophages, T-cells, ICAM-1, LFA-1 and cytokines. For in vitro binding studies, snap-frozen sections of transplanted kidneys were incubated with monocytes/macrophages and lymphocytes isolated from peripheral blood (PBL) of naive animals. For in vivo migration studies, naive cell populations were labeled with Bis-Benzamide and transferred i.v. to grafted animals at weeks 8, 12 and 16 (n = 3/group); grafts were harvested 24 h later and cell localization assessed under immunofluorescence. Increasing numbers of ED1 + monocytes/macrophages in allografted kidneys peaked at 16 weeks, localizing preferentially in glomeruli, where IL-1, IL-6 and TNF-alpha expression had also become intense and correlated with progressive glomerulosclerosis. Binding studies corroborated these results. In vitro, a few monocytes/macrophages bound to glomeruli and vessels at 8 weeks; by 12 weeks, binding to glomeruli was high (72% of cells). In vivo, large numbers of transferred labelled monocytes/macrophages were found in kidney allografts at 12 weeks (23%, isografts; < 7%, P < 0.01). In contrast T cells (primarily CD4+) were a consistent feature in allografts elevated as compared to isografts and correlating with in vitro and in vivo binding patterns; associated cytokines included IL-2, IFN- and TNF-alpha. Functional data followed these results: urine protein excretion by allograft recipients increased from baseline at 8 weeks (12 mg/day) to > 50 mg/day at 16 weeks at which point animals were beginning to die of renal failure; proteinuria in isografted rats did not increase during this time period. These results suggest that monocyte/macrophage and CD4+ T cells and their products are important in chronic kidney allograft rejection, contributing to the progressive sclerosis and fibrosis.

Parathyroid hormone (PTH)-Induced Intracellular Ca2+Signalling in Naive and PTH-Desensitized Osteoblast-Like Cells (ROS 17/2.8): Pharmacological Characterization and Evidence for Synchronous Oscillation of Intracellular Ca2+*

We showed recently that the initial peak cytosolic ionized calcium ([Ca2+]i) response to PTH (2-min exposure) is preserved relative to the cAMP response in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) desensitized by 72-h exposure to PTH. We attempted in the present studies to determine the mechanisms for preservation of the [Ca2+]i response and to explore the effects of longer PTH rechallenges. The [Ca2+]i response to a 20-min perifusion with rat PTH [rPTH-(1-34)] was monitored by aequorin luminescence in both naive and PTH-desensitized ROS 17/2.8 cells. The responses of both naive and desensitized cells consisted of two phases: an initial peak, followed by an intermediate plateau that was sustained in the presence of PTH. We observed in the naive cell populations synchronous oscillations in [Ca2+]i concentration during this second phase (amplitude, 10-60 nM; frequency, 1-3/100 sec). These oscillations were maintained through extracellular calcium (EC Ca2+) entry; the initial peak was the result of Ca2+ release from intracellular stores. In desensitized cells, these two phases could not be clearly separated with respect to Ca2+ source, but, as we showed before, exhibited an enhanced dependence on EC Ca2+ entry for the response to PTH. Nevertheless, in the desensitized cells, the sustained [Ca2+]i response was diminished in magnitude and showed little oscillatory behavior. Brief exposure to neomycin sulfate, an inhibitor of phosphoinositide turnover, attenuated the PTH-induced [Ca2+]i rise in both naive and desensitized cells. Protein kinase-C activity did not appear to be required for either phase of the PTH-induced [Ca2+]i response. Exposure to cholera toxin attenuated the [Ca2+]i response to hormone in both naive and desensitized cells, more markedly in the latter. Cholera toxin treatment dramatically increased basal cAMP levels in both cell preparations; PTH-stimulated cAMP production was unchanged in naive cells, but increased nearly 4-fold in desensitized cells. We propose that the preserved PTH-induced peak [Ca2+]i rise in desensitized cells results primarily from the diminished regulation of EC Ca2+ entry by the cAMP response limb. The attenuated sustained oscillatory behavior observed in desensitized cells upon rechallenge with hormone may be the result of reduced phosphoinositide turnover and reduced Ca2+-stimulated Ca2+ release. Thus, the [Ca2+]i response to PTH in osteoblast-like cells is complex and modulable and seems to provide a number of ways to regulate intracellular metabolism under various conditions. We speculate that this plasticity of the [Ca2+]i response to PTH is related to the pleiotropic actions of the hormone on cells of the osteoblast lineage.

Expression of CD31 epitopes on human lymphocytes: CD31 monoclonal antibodies differentiate between naive (CD45RA+) and memory (CD45RA‐) CD4‐positive T cells

The CD31 antigen, a member of the immunoglobulin superfamily with a possible cell adhesion function, is expressed on approximately 50% of peripheral blood lymphoid cells at relatively low intensity (10-20% of the level on monocytes). In the accompanying paper we showed that a mAb, 5A2.G5, which identifies a glycosylation-dependent epitope of the CD31 antigen, bound to fewer lymphocytes than two other CD31 mAb, B2B1 and 2BD4, although the 3 antibodies bound equally well to monocytes. We have now analyzed the pattern of expression of epitopes of the CD31 antigen on lymphoid cell subpopulations using two-color immunofluorescence and flow cytometry. Large granular lymphocytes (CD16+), CD8-positive T cells and B cells (SMIg+) were mostly CD31-positive as indicated by the binding of mAb B2B1 and 2BD4. Single populations displaying some overlap with the negative control were obtained in each case. In contrast, CD4-positive T cells fell into two discrete populations with respect to CD31 antigen expression. mAb 5A2.G5 displayed weaker binding to all lymphoid cell types, indicating that the pattern of glycosylation of the CD31 antigen differs between lymphocytes (of all types) and cells of the myeloid lineages. The heterogeneity of CD31 antigen expression by CD4-positive cells was further examined by dual-labelling of purified CD4 cells with mAb B2B1 and CD45RA or CD29 mAb which identify naive and memory T cells respectively. The CD31 antigen was found to be preferentially expressed by the CD45RA-positive, naive cell population.