Pembrolizumab Interferes with the Differentiation of Human FOXP3+-Induced T Regulatory Cells, but Not with FOXP3 Stability, through Activation of mTOR.

Programmed cell death 1 (PD-1) is critical for T regulatory cells (Tregs) to maintain peripheral tolerance to self-antigens. In the tumor microenvironment, interaction between PD-1 and its ligands supports tumor immune evasion. Pembrolizumab blocks interactions of PD-1 with its ligands, enhancing antitumor and clinical responses. We and others have reported that pembrolizumab does not affect function or phenotype of thymic-derived Tregs; however, little is known about its effect on extrathymic differentiation of peripheral Tregs. In this study, we investigated the effect of pembrolizumab on in vitro-induced Tregs (iTregs). Our work showed that PD-1 blockade interferes with iTreg differentiation and has no potential effect on the stability of FOXP3 after differentiation. Additionally, we found that both nontreated and pembrolizumab-treated iTregs were suppressive. However, pembrolizumab-treated iTregs were relatively less suppressive in higher Treg ratios and failed to produce IL-10 compared with their nontreated counterparts. Different methods including transcriptomic analyses confirmed that the downregulation of FOXP3 was mediated by activating mTOR and STAT1 and inhibiting MAPK pathways, shifting the iTreg polarization in favor of Th1 and Th17 subsets. To confirm the role of mTOR activation, we found that rapamycin diminished the effect of pembrolizumab-mediated downregulation of FOXP3. Ingenuity pathway analysis revealed that pembrolizumab-treated iTregs showed upregulation of genes promoting DNA repair and immune cell trafficking, in addition to downregulation of genes supporting cellular assembly and organization. To our knowledge, this is the first study to show that pembrolizumab interferes with differentiation of human FOXP3+ iTregs and to disclose some of the molecular pathways involved.

An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - Are your sorted cells getting SICS?

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term "sorter induced cell stress" (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted "CD4+" T-cells and "live" cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 µm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed.

Can the immune system still be efficient in the elderly? An immunological and immunoendocrine therapeutic perspective.

The continuous global increase in life expectancy represents a central challenge for our society and impacts public social security systems, families and individuals. One of the most striking changes that occur during normal human aging is immunosenescence, a progressive and overall diminution of immune functions that affect all cells and organs of the innate and adaptive immune system. As a hallmark of human aging, the progressive involution of the thymus leads to a disturbed balance and function of naïve, memory and effector T cells, thus promoting a latent pro-inflammatory status in the elderly. Together with chronic infections such as cytomegalovirus, that accumulate during life, this situation manifests in clinically relevant implications such as poor overall immune responses, decreased ability to control infectious disease and diminished response to vaccinations. Interestingly, this process parallels changes in the hormonal balance of aging subjects. In this review, we summarize recently published intriguing results from a very active and growing field of biomedical research and discuss some clinical consequences as well as possible ways of immune- and/or hormone-based interventions to delay or reverse immunosenescence.

Techniques in gerontology: cell lines as standards for telomere length and telomerase activity assessment.

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.

Naive T cells in the elderly: are they still there?

One of the most striking changes in the primary lymphoid organs during human aging is the progressive involution of the thymus. As a consequence, the rate of naïve T cell output dramatically declines with age and the peripheral T cell pool shrinks. These changes lead to increased incidence of severe infections and decreased protective effect of vaccinations in the elderly. Little is, however, known of the composition and function of the residual naïve T cell repertoire in elderly persons. To evaluate the impact of aging on the naïve T cell pool, we investigated the quantity, phenotype, function, composition, and senescence status of CD45RA(+)CD28(+) human T cells--a phenotype generally considered as naïve cells--from both young and old healthy donors. We found a significant decrease in the number of CD45RA(+)CD28(+) T cells in the elderly, whereas the proliferative response of these cells is still unimpaired. In addition to their reduced number, CD45RA(+)CD28(+) T cells from old donors display significantly shorter telomeres and have a restricted TCR repertoire in nearly all 24 Vbeta families. These findings let us conclude that naïve T cells cannot be classified with conventional markers in old age.

Disruption of vascular endothelial homeostasis by tobacco smoke: impact on atherosclerosis.

The World Health Organization (WHO) predicts that by 2020 tobacco will become the largest single health problem worldwide and will cause an estimated 8.4 million deaths annually (http://www5.who.int/tobacco/). Although the impact of smoking on human health is well defined from the medical point of view, surprisingly little is known about the mechanisms by which tobacco smoke mediates its disastrous effects. Here, we demonstrate that tobacco smoke dramatically changes vascular endothelial cell and tissue morphology, leading to a loss of endothelial barrier function within minutes. Long-term exposure of endothelial cells to tobacco smoke extracts induces necrosis that may trigger a pro-inflammatory status of the vessel wall. Pre-incubation of the extracts without cells for 6 h at 37 degrees C led to a complete loss of activity. Further, the endothelium could be rescued by changing to fresh medium even at times when the extracts had lost their activity. Finally, we show that N-acetyl cysteine and statins inhibit the adverse tobacco smoke effects.

High level HPV-16 E7 oncoprotein expression correlates with reduced pRb-levels in cervical biopsies.

High-risk human papillomaviruses (HPVs) are major etiological agents of cervical cancer. Despite excellent epidemiological evidence for a direct role of HPV-16 in cervical carcinogenesis, molecular pathways underlying carcinogenesis in vivo remain obscure. The E7 gene is required for immortalization and maintenance of the transformed phenotype in vitro; however, little is known about its role for tumorigenesis in vivo. The E7 gene codes for an unstable protein the abundance of which in cervical biopsies is unknown. We show here that E7 protein levels strongly increase during cervical carcinogenesis, underlining its fundamental role in cervical cancer. The E7 protein was found predominantly in the nucleus and to a minor extent in the cytoplasm in the cervical cancer cell line Ca Ski in vitro and in invasive cervical carcinoma in situ, suggesting that nuclear resident E7 plays a major role in cervical carcinogenesis in humans. The retinoblastoma protein (pRb) is a major E7-target in vitro. We show here that pRb expression is initially upregulated in LSIL and disappears in later stages concomitant with increased E7 levels, suggesting that E7-driven degradation of pRb is involved in cervical tumorigenesis in humans.

Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts.

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by high-resolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

Progression of arteriovenous bypass restenosis in mice exposed to a 50 Hz magnetic field.

The controversy over whether magnetic fields (MF) produced by electrical wiring and appliances contribute to diseases such as cancer has been debated in the literature for more than 2 decades. These extremely low frequency fields at 50 or 60 Hz are omnipresent in the industrialized world and have been linked to various forms of cancer by epidemiological studies. Little has been published investigating any possible role of MF and cardiovascular disease, and this is the first study looking specifically at the effect of exposure to high-intensity MF on the development and progression of restenosis. A mouse arteriovenous bypass model was used, and mice were exposed to MF for periods of 1, 2, or 3 weeks. Neointima formation, infiltration of mononuclear cells, and heat shock protein 60 expression were all studied at the conclusion of the exposure regimen. Animals exposed to the MF for 1 week showed significantly smaller neointima formation compared with control mice exposed to a null field, although this difference was not observed in mice exposed for 2 or 3 weeks. No difference was found between mice exposed to MF and controls in any of the other parameters investigated.

Development and evaluation of an in vitro model for the analysis of cigarette smoke effects on cultured cells and tissues.

INTRODUCTION: Smokers have an increased risk for a variety of diseases. Among the most prominent is atherosclerosis, the leading cause of death in the Western world. Although this conjunction is accepted knowledge, the basic biological mechanisms and the identities of the active tobacco smoke constituents surprisingly are still unknown. One reason for this is the lack of accurate in vitro models. METHODS: Cell culture experiments, including cell morphology and cell death analyses, high-performance liquid chromatography, and liquid chromatography coupled to mass spectrometry via an electrospray ionization interface allowing collision-induced dissociation analyses, were applied. RESULTS AND DISCUSSION: In this study, we present and validate an in vitro model that has proven to be useful for standardized studies of cellular and histological effects of cigarette smoke. The system consists of a cigarette smoke sampling device in which water-soluble cigarette smoke constituents pass over from the gas phase into the aqueous phase resulting in nicotine concentrations identical to the in vivo concentrations, suggesting in vivo similar conditions for gas-to-liquid compound exchange.

Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age.

The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age.

IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells.

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcgammaRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.

Interferon-gamma induces neuroendocrine-like differentiation of human prostate basal-epithelial cells.

BACKGROUND: Prostatic neuroendocrine (NE) cells are intraglandular hybrid epithelial-neural-endocrine cells that express and secrete numerous hormones and neuropeptides, which presumably regulate growth, differentiation, and secretory activity of the prostatic epithelium. This specialized cell type appears to differentiate from a common basal/precursor/stem cell that also gives rise to the secretory epithelium. In order to elucidate mechanisms of NE-differentiation the effects of type 1 (alpha, beta) and type 2 (gamma) interferons (IFNs) on human prostate basal cells (PrECs) were evaluated. METHODS AND RESULTS: Application of alpha/beta IFN increased the expression of the cell-cycle inhibitor p21(CIP1) and inhibited DNA synthesis, while only IFN-gamma led to increased apoptosis, cell-cycle inhibitor p27(KIP1) upregulation, and differentiation of PrECs into NE-like cells. In vitro differentiated NE-like cells expressed the glycolytic enzyme neuron-specific enolase (NSE) and chromogranin A (CgA), known markers of NE-cells in vivo in the prostate. These NE-like cells also changed cytokeratin (CK) expression patterns by upregulating CK 8/18, predominantly found in terminally-differentiated secretory luminal/NE epithelial cells. CONCLUSIONS: IFN-gamma produced locally in the prostate by basal cells and, under proinflammatory conditions, by infiltrating lymphocytes could support NE cell differentiation and play a role in NE differentiation processes of tumor cells in hormone-refractory prostate cancer.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Age-related differences in phenotype and function of CD4+ T cells are due to a phenotypic shift from naive to memory effector CD4+ T cells.

Based on the combined expression of CD27 and CD28, a putative model of T cell differentiation has been previously proposed. We used CD27 and CD28 expression in order to comparatively study the size, cytokine production capacity and proliferative response of CD4+ T cell sub-populations from healthy young and elderly volunteers. Elderly persons had a lower percentage of CD27+CD28+ but a higher percentage of CD27-CD28+ and CD27-CD28-CD4+ T cells than the young persons. CD27-CD28-CD4+ T cells were present, although at relatively low numbers, in the vast majority of the healthy elderly donors but were only sporadically detected in young persons. Each CD4+ T cell sub-population exhibited a distinct phenotype and cytokine production profile, which were not affected by age. When purified CD27+CD28+ were stimulated by staphylococcal enterotoxin B, they proliferated to a greater extent than CD27-CD28+ and CD27-CD28-CD4+ T cells. However, we did not observe age-related differences in proliferative response of each sub-population. We concluded that although the size of the different sub-populations differed between the young and the old group, the functional characteristics of each sub-population were the same in both age groups. This suggests that on a per cell basis there is no functional impairment of CD4 memory T cells in elderly persons. Consequently, potential differences in the function of the total CD4+ T cell population are most likely due to different composition of repertoire.

Age-related loss of naïve T cells and dysregulation of T-cell/B-cell interactions in human lymph nodes.

In this study we analysed the effects of age on T and B lymphocytes in human lymph nodes by comparing lymphocyte subsets in paraffin sections from lymph node tissue taken from healthy young and elderly people. We demonstrate that the relative number of CD8(+) T cells decreases with age but that the relative number of CD4(+) T cells does not. There is also a very pronounced age-dependent loss of CD45RA(+) naive T cells. The number and size of follicles and the relative number of CD20(+) B cells are similar in young and elderly donors. For polymerase chain reaction analysis of the T-cell receptor (TCR) repertoire the TCR-gamma gene rearrangements were used as a marker of clonality. This is a reliable tool to detect not only clonal TCR-gammadelta populations but also TCR-alphabeta populations. Young donors with clonal T-cell expansions in their lymph node tissue do, however, have a higher number of CD20(+) B cells, a higher relative size of germinal centres compared to the follicle mantles and a higher number of immunoglobulin M-expressing cells than young donors without evidence of clonal T-cell expansions. Corresponding changes are not observed in elderly donors with clonal T-cell expansions in their lymph node tissue. In summary our findings demonstrate characteristic effects of aging on human lymph node tissue, the most striking feature being the depletion of naive T cells and the apparent dysregulation of T-cell/B-cell interactions in old age.

CD25-expressing CD8+ T cells are potent memory cells in old age.

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Neuroendocrine differentiation of human prostatic primary epithelial cells in vitro.

BACKGROUND: Dispersed prostatic neuroendocrine cells are involved in growth regulation of the prostate and are considered to play a role in the pathogenesis of prostate carcinoma and benign prostatic hyperplasia (BPH). They are meant either to be derived from the neural crest during embryogenesis or by direct differentiation of the cells from locally present precursor cells. METHODS: An in vitro model was developed for human prostatic epithelial and neuroendocrine cell differentiation. Minced explants from radical prostatectomies were seeded on collagen I-coated plates. RESULTS: The majority of outgrowing cells were basal cells, positive for cytokeratin markers K 5/14 and CD 44, as determined by confocal laser scanning microscopy. A small fraction of interdispersed single cells expressing c-kit, which is found on pluripotent precursors, was identified by immunofluorescence. From these basal cells, in vitro differentiation of cells with neuroendocrine morphology could be achieved within 3 days. These were at rest, i.e., non-bromodeoxyuridine incorporating cells and characteristically coexpressed K 5/14, K 18, and the neuroendocrine marker chromogranin A. Luminal cells staining for K 8 or 18 were not observed. CONCLUSION: Neuroendocrine differentiation of adult prostatic cells was achieved in vitro, favoring the hypothesis that neuroendocrine cells are derived from peripheral precursor cells. The acceleration of this differentiation pathway may be the reason for the increased presence of neuroendocrine cells in areas of epithelial hyperplasia in BPH.