T Cell Transcriptome in Chromosome 22q11.2 Deletion Syndrome.

Phenotypic variations of chromosome 22q11.2 deletion syndrome (22qDS) have unclear explanations. T cell lymphopenia in 22qDS related to varying degrees of thymic hypoplasia contributes to the phenotypic heterogeneity. No phenotype correlation with genotype or deletion size is known for lymphopenia. We investigated gene expression in human T cells of participants with and without 22qDS and T cells of participants with 22qDS with low or normal T cells. Peripheral blood was collected from participants aged 5-8 y. Immune function was checked. RNA sequencing was completed on isolated T cells, and differential gene expression profiles of T cells between 22qDS and healthy control subjects were established. A total of 360 genes were differentially expressed (q < 0.05) between T cells of patients with 22qDS (n = 13) and healthy control subjects (n = 6) (log2 fold change range, -2.0747, 15.6724). We compared gene expression between participants with 22qDS with low (n = 7) and normal T cell counts (n = 6), finding 94 genes that were differentially expressed (q < 0.05) (log2 fold change range, -4.5445, 5.1297). Twenty-nine genes correlated with T cell counts and markers CD3, CD4, CD8, and CD45RA+CD4 (R ≥ 0.8). We found significantly differentially expressed genes in participants with 22qDS compared with healthy control subjects and in participants with 22qDS with low T cell counts compared with those with normal T cell counts. Several enriched pathways suggest a role of T cells in defective communication between T cells and the innate immune system in 22qDS. Among these, the liver X receptor/retinoid X receptor pathway was noted to show several differentially expressed genes affecting participants with 22qDS compared with healthy control subjects and more so those with low T cell counts than in those with normal T cell counts.

Transcription factor activation and protein phosphorylation patterns are distinct for CD28 and ICAM-1 co-stimulatory molecules.

We examined signaling differences between two co-stimulatory molecules, CD28 and ICAM-1 by analyzing transcription factors and proteins that are activated downstream of these co-stimulations. We observed that FAST-1, a crucial protein in the TGFß signaling pathway, was activated by only ICAM-1 co-stimulation, and not by CD28. We also observed that receptor tyrosine kinases Csk, Dtk, FGFR1 and ROR2 were phosphorylated upon CD28 co-stimulation and IGF-1R, HGFR, MuSK and EphA8 were phosphorylated upon ICAM-1 co-stimulation. Together, these findings suggest that these two co-stimulators induce the activation of different sets of proteins, suggesting that each co-stimulatory molecule has its unique signaling profile.

Engagement of CD45 alters early signaling events in human T cells co-stimulated through TCR + CD28.

Previously our lab has shown that co-stimulation of human T cells through different co-stimulatory molecules tune differentiation to different phenotypes. An open question is where in the signaling pathways induced by the co-stimulation do differences occur that contribute to outcome of differentiation. In this project, we investigate the early signaling process by comparing events that follow engagement of CD45 alone or in association with a co-stimulatory molecule: CD28. CD45 plays a crucial role to initiate T cell signaling by dephosphorylating a negatively regulatory tyrosine residue in Src family kinases such as Lck. First, we observed that engagement of CD45 alone induced signaling in T cells. We observed that TCR/CD3 stimulation with CD45 promoted prolonged Lck association with TCR/CD3 complex and Lck remained associated during TCR/CD3 + CD28 + CD45 stimulation as well. We concluded that Lck association is dependent on TCR/CD3 and CD45 engagement. Hence, CD45 altered early signaling events in T cells.

Cytokine Biomarkers of Disease Activity and Therapeutic Response after Initiating Methotrexate Therapy in Patients with Juvenile Idiopathic Arthritis.

STUDY OBJECTIVE: To evaluate the relationship between plasma cytokine levels with disease activity and therapeutic response in patients with juvenile idiopathic arthritis (JIA) after initiating methotrexate (MTX) therapy. DESIGN: Single-center observational prospective cohort study. SETTING: Outpatient pediatric rheumatology clinic at a tertiary care academic pediatric hospital. PATIENTS: The study included 61 patients diagnosed with JIA who started therapy with standard-dose MTX 15 mg/m2 /week. At 3 months, treating physicians were given the option of maintaining the MTX dose, increasing the MTX dose, or adding etanercept (ETN), based on their clinical judgment. MEASUREMENTS AND MAIN RESULTS: Patients were evaluated at baseline, 3 months (51 patients), and 6 months (35 patients). Plasma samples from each visit were analyzed for interleukin (IL)-1α, IL-1ß, IL-1Ra, IL-6, and tumor necrosis factor-α (TNF-α). Cytokine concentrations were evaluated for relationships with disease activity using the 71-joint count Juvenile Arthritis Disease Activity Score (JADAS). Therapeutic response was assessed by changes in JADAS. Failure to respond to standard-dose MTX was defined as the need for the addition of ETN or a MTX dose increase at or before the 3-month visit. Increased disease severity at baseline was associated with increased IL-6 (p=0.01) and TNF-α (p=0.008) levels. Initiation of MTX was associated with reductions in IL-1α (p=0.009), IL-1ß (p=0.01), IL-1Ra (p=0.007), and IL-6 (p=0.03) levels; however, reductions in JADAS were only associated with reductions in IL-6 (p=0.009) and TNF-α levels (p=0.02). Compared with responders, patients failing to respond to standard-dose MTX had increased TNF-α levels at baseline (p=0.02) and at 3 months (p=0.005). Reductions in JADAS by 6 months were observed following either the addition of ETN (p=0.009) or an increase in MTX dose (p=0.007), but the addition of ETN was associated with a median 7-fold increase in TNF-α levels (p=0.003) that corresponded with clinical response. CONCLUSION: Plasma cytokine levels were responsive to MTX therapy in patients with JIA, but only TNF-α and IL-6 levels were consistently associated with disease activity and therapeutic response. Increased TNF-α levels at baseline were associated with failure to respond to standard-dose MTX and the need for more aggressive drug therapy. Initiation of ETN resulted in increased TNF-α levels that corresponded with therapeutic response, suggesting a potential clinical benefit of monitoring TNF-α levels as a pharmacodynamic marker of etanercept activity.

DNA methylation levels associated with race and childhood asthma severity.

OBJECTIVE: Asthma is a common chronic childhood disease worldwide. Socioeconomic status, genetic predisposition and environmental factors contribute to its incidence and severity. A disproportionate number of children with asthma are economically disadvantaged and live in substandard housing with potential indoor environmental exposures such as cockroaches, dust mites, rodents and molds. These exposures may manifest through epigenetic mechanisms that can lead to changes in relevant gene expression. We examined the association of global DNA methylation levels with socioeconomic status, asthma severity and race/ethnicity. METHODS: We measured global DNA methylation in peripheral blood of children with asthma enrolled in the Kansas City Safe and Healthy Homes Program. Inclusion criteria included residing in the same home for a minimum of 4 days per week and total family income of less than 80% of the Kansas City median family income. DNA methylation levels were quantified by an immunoassay that assessed the percentage of 5-methylcytosine. RESULTS: Our results indicate that overall, African American children had higher levels of global DNA methylation than children of other races/ethnicities (p = 0.029). This difference was more pronounced when socioeconomic status and asthma severity were coupled with race/ethnicity (p = 0.042) where low-income, African American children with persistent asthma had significantly elevated methylation levels relative to other races/ethnicities in the same context (p = 0.006, Hedges g = 1.14). CONCLUSION: Our study demonstrates a significant interaction effect among global DNA methylation levels, asthma severity, race/ethnicity, and socioeconomic status.

Immunoendocrine alterations following Marine Corps Martial Arts training are associated with changes in moral cognitive processes.

Combined physical and psychological stress events have been associated with exacerbated endocrine responses and increased alterations in immune cell trafficking when compared to exercise stress alone. Military training programs are rigorous in nature and often purposefully delivered in environments combining high levels of both physical and mental stress. The objective of this study was to assess physiological and cognitive changes following U.S. Marine Corps Martial Arts training. Seven active-duty, male Marines were observed during a typical Marine Corps Martial Arts training session. Immune parameters, including immunomodulatory cytokines, and hormone concentrations were determined from blood samples obtained at baseline, immediately post training (IP) and at 15min intervals post-training to 1h (R15, R30, R45, R60). Assessments of cognitive moral functioning (moral judgment and intent) were recorded at intervals during recovery. There were significant fluctuations in immunoendocrine parameters. Peak endocrine measures were observed within the IP-R15 time interval. Distributions of circulating immune cells were significantly altered with neutrophils and all lymphocyte subsets elevated at IP. IFN-γ and IL-17a exhibited small, non-significant, parallel increases over the recovery period. Moral functioning was informed by different social identities during the recovery resulting in changes in moral decision-making. These data demonstrate that the Marine Corps Martial Arts Program induces significant alterations in lymphocyte and leukocyte distributions, but does not shift the balance of Th1/Th2 cytokines or induce a systemic inflammatory response. The program does, however, induce alterations in moral decision-making ability associated with the observed endocrine responses, even suggesting a potential interaction between one's social identities and endocrine responses upon moral decision-making.

Timely blockade of ICAM-1.LFA-1 interaction prevents disease onset in a mouse model of emphysema.

AIM: It is becoming apparent that emphysema is partly driven by self-reactive T cells inducing inflammatory damage. Thus, T cells become targets for therapy similar to other autoimmune diseases. Costimulatory blockade therapy targets disease-specific T cells, rendering them ineffective by blocking a necessary costimulatory event on the T-cell surface. This therapy is tested here in mouse emphysema. MATERIALS & METHODS: Peptides representing contact domains of counter receptors LFA-1 and ICAM-1 were used as blockade therapy in elastase-induced emphysema. RESULTS: When administered during the first week after disease induction, blockade prevented lung destruction, reduced leukocyte infiltration and inhibited the decrease in T-cell CD4:CD8 ratio, also common in human emphysema. CONCLUSION: Costimulatory blockade therapy can affect the progress of emphysema.

FCER2 (CD23) asthma-related single nucleotide polymorphisms yields increased IgE binding and Egr-1 expression in human B cells.

CD23 is the low-affinity Fc receptor for IgE. When expressed on B cells, CD23 appears to play a role in regulation of IgE synthesis. Polymorphisms within FCER2, the gene encoding CD23, have been associated with atopy, increased risk of exacerbations in patients with asthma, and high serum IgE levels. A single-nucleotide polymorphism (rs2228137) present in exon 4 of FCER2 encodes a nonsynonymous amino acid change (R62W) and is the subject of the present analysis. Human B cell stable transfectants were established to characterize the functional relevance of the R62W SNP. We demonstrate that CD23b-R62W-expressing human B cells bind IgE with greater affinity than wild-type cells and display differences in kinetics of CD23-mediated ERK1/2 activation that may be responsible for the increased levels of Egr-1 mRNA observed after stimulation through CD23. Finally, the R62W SNP seems to alter the tertiary or quaternary structure of CD23 because in the absence of N-glycosylation the CD23b-R62W-expressing cells appear to be less sensitive to endogenous proteases. These observations may have implications in mechanisms responsible for the atopic phenotypes observed in patients with asthma who possess this genotype.

Omalizumab may decrease IgE synthesis by targeting membrane IgE+ human B cells.

BACKGROUND: Omalizumab, is a humanized anti-IgE monoclonal antibody used to treat allergic asthma. Decreased serum IgE levels, lower eosinophil and B cell counts have been noted as a result of treatment. In vitro studies and animal models support the hypothesis that omalizumab inhibits IgE synthesis by B cells and causes elimination of IgE-expressing cells either by induction of apoptosis or induction of anergy or tolerance. METHODS: We examined the influence of omalizumab on human tonsillar B cell survival and on the genes involved in IgE synthesis. Tonsillar B cells were stimulated with IL-4 plus anti-CD40 antibody to induce class switch recombination to IgE production in the presence or absence of omalizumab. Cell viability was assessed and RNA extracted to examine specific genes involved in IgE synthesis. CONCLUSIONS: We found that omalizumab reduced viable cell numbers but this was not through induction of apoptosis. IL-4R and germline Cϵ mRNA levels were decreased as well as the number of membrane IgE+ cells in B cells treated with omalizumab. These data suggest that omalizumab may decrease IgE synthesis by human B cells by specifically targeting membrane IgE-bearing B cells and inducing a state of anergy.

The role of CD23 in IgE dependent signaling: implications from pharmacogenetics.

The association of immunoglobulin E (IgE) with allergic diseases and asthma is well established. IgE binds to two receptors on various immune and inflammatory cells. The lower-affinity IgE Fc receptor, CD23, has multiple functions in enhancing the regulation of IgE production itself, and that of various pro-inflammatory activities and mediators. The data in this report are derived from an analysis of variation in the CD23 gene that leads to a coding exchange and to a single nucleotide polymorphism (SNP) associated with the substitution of an arginine residue for a tryptophan residue in the protein structure of CD23. This genetic variation is associated with three findings identified in this report. First, the tryptophan exchange is associated with greater expression of RNA for the interleukin (IL)-4 receptor alpha chain and greater expression of RNA for egr-1 transcription factor, both of which are proinflammatory gene products that influence allergy-related immune functions. Second, the exchange is associated with cell surface expression of IL-4R. Third, an analysis of potential arginine-to-tryptophan exchanges in the entire human genome has identified a number of interesting exchanges in immunologic genes of interest for their role in allergic responses. A discussion of these three findings is presented.

CD23-mediated cell signaling in human B cells differs from signaling in cells of the monocytic lineage.

CD23 is the low affinity receptor for IgE and in B cells CD23 has been proposed to play a role in the regulation of IgE synthesis. CD23 is expressed also on other cell types including monocytes/macrophages, eosinophils, follicular dendritic cells and intestinal epithelial cells none of which is capable of expressing IgE. The diverse nature of the expressing cells suggests that either the CD23-mediated signal transduction pathway may be different among the cell types or biological outcomes differ in different cells in response to the same signaling pathway. To address this issue, the CD23 signaling pathway was analyzed and compared in primary tonsillar B cells and in the monocytic cell lines U937 and THP-1. Activation of the tyrosine kinase Fyn and the serine/threonine kinase Akt were only observed in B cells. These results suggest that the CD23-mediated signal transduction pathways in human B cells and human monocytes are different.

Two different modes of costimulation predispose human T lymphocytes to differential responses in the presence of HDL or oxidized LDL.

In addition to levels of high-density lipoprotein (HDL), oxidized (ox) low-density lipoprotein (LDL), the inflammatory process and certain genetic factors, T cells are crucial for expansion of atherosclerotic plaque. The interrelationships among these influences are still being defined. Here, we examined how HDL and oxLDL affect T cell function. T cells require two activation signals to achieve functional activity. The first signal is specific and is delivered by appropriately presented antigen. The second (costimulatory) signal can be received through any of several T cell surface proteins, the most widely studied of which are CD28 and LFA-1. We have identified ICAM-1, resident on T cells, as a costimulatory protein. Here, we describe differential effects when T cells were costimulated through either LFA-1 or ICAM-1 in the presence of HDL or oxLDL. In general, T cells costimulated through either LFA-1 or ICAM-1 in the presence of oxLDL were predisposed to a decrease in proliferation and increased apoptosis, although ICAM-1-costimulated cells were protected from apoptosis induced by lower levels of oxLDL. T cell subsets also were examined. In the presence of HDL, CD8(+) T cells increased proliferation when costimulated through LFA-1. HDL exerted no effect on proliferation of CD4(+) T cells whereas proliferation decreased in the presence of oxLDL. Naïve T cells proliferated better in response to costimulation through LFA-1 in the presence of HDL but proliferation of effector/memory cells was not altered in the presence of HDL. When T cells were costimulated through LFA-1, in the presence of either HDL or oxLDL synthesis of Th-1 but not Th-2 cytokines was increased. T cells costimulated through ICAM-1 increased Th-1 but not Th-2 cytokines but this was not altered in the presence of HDL or oxLDL. Thus, the nature of costimulation seems to influence T cell responses in the presence of the lipoproteins.

Costimulation of naive human CD4 T cells through intercellular adhesion molecule-1 promotes differentiation to a memory phenotype that is not strictly the result of multiple rounds of cell division.

The process by which naive T cells become activated, differentiate into effector cells and ultimately generate long-lived memory cells is dependent upon a number of factors, including the costimulatory signals received by the T cell. To best understand the multiple events involved, it is important to understand the potential contributions by individual signalling proteins using both in vitro and in vivo studies. Here, the potential for costimulation through intercellular adhesion molecule-1 (ICAM-1; CD54), resident on the surface of naive human T cells, to influence differentiation was investigated. Costimulation of naive T cells through ICAM-1 resulted in expansive cell division, high interleukin-2 production, and protection from apoptosis. Prolonged culture led to outgrowth of a subpopulation of cells with a highly differentiated CD45RA- CD11a(hi) CD27- phenotype. In this respect, costimulation through ICAM-1 was similar to costimulation through CD28 and different from costimulation through leucocyte function-associated antigen-1. The CD45RA- CD11a(hi) CD27- cells responded to suboptimal stimulation through the T-cell receptor alone with a more robust proliferative response compared with naive cells from the same subject. These cells also secreted higher levels of T helper type 1 cytokines in response to lower levels of stimulation than their naive counterparts. The surface phenotype and more sensitive response characteristics suggest the creation of a memory T-cell subpopulation as a result of costimulation through ICAM-1. Finally, generation of this memory population was the result of specific costimulatory signals, and not merely because of a high number of cell divisions. These data reveal a new role for resident ICAM-1 to influence the differentiation of naive T cells.

MIP-1alpha induces differential MAP kinase activation and IkappaB gene expression in human B lymphocytes.

The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) stimulates migration of B cells and affects B cell immunoglobulin production. However, the molecular mechanisms by which MIP-1alpha modulates these biologic effects have not been completely defined. Previously, we demonstrated that treatment of B cells with MIP-1alpha induced the transcription factor, nuclear factor (NF)-kappaB, to bind to DNA, concomitant with the degradation of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB activation. Here, we report that MIP-1alpha treatment of tonsil B cells induced IkappaB gene expression that was dependent on MIP-1alpha-mediated activation of a pathway(s) involving NF-kappaB and phosphatidylinositol-3 kinase (PI3K). The NF-kappaB pathway is understood to be controlled in an autoregulatory fashion, so expression of IkappaB is thought to provide a means by which B cells modulate this pathway after stimulation with MIP-1alpha. Although the idea of NF-kappaB autoregulation is not novel, this is the first report to suggest the regulation of B cell gene expression by MIP-1alpha. In addition, we observed the activation of Jun N-terminal kinase (JNK) and p38 mitogenic-activated protein kinase (MAPK), but not extracellular signal-related kinase (ERK) in response to MIP-1alpha. Although p38 and NF-kappaB activity were both necessary for B cell migration, IkappaB gene expression was not affected by p38 inhibition, suggesting that p38 is involved in a separate MIP-1alpha-mediated signal transduction pathway.

The outcome of T-cell costimulation through intercellular adhesion molecule-1 differs from costimulation through leucocyte function-associated antigen-1.

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. The outcome of T-cell activation can be influenced by the nature of the costimulatory signal the T cell receives. We recently demonstrated the ability of stimulation through intercellular adhesion molecule-1 (ICAM-1), resident on the T-cell surface, to provide a second signal for T-cell activation, and have extended that work here to begin an examination of the functional outcome of this set of signals. Costimulation through ICAM-1 resulted in a greater percentage of cells having undergone more than three divisions when compared to costimulation through leucocyte function-associated antigen-1 (LFA-1). Costimulation through ICAM-1 also had an effect similar to costimulation through CD28 in its ability to down-regulate the cyclin dependent kinase inhibitor p27kip1. Costimulation through ICAM-1 provided greater protection from apoptosis than costimulation through LFA-1, especially in cells having divided more than three times. This was supported by the ability of costimulation through ICAM-1 to up-regulate the anti-apoptotic protein Bcl-2. Finally, costimulation through ICAM-1 or CD28 produced a greater number of T cells with a memory phenotype than costimulation through LFA-1.

Influence of carbohydrate ingestion on cytokine responses following acute resistance exercise.

The effect of carbohydrate supplementation (CHO) on interleukin 2 (IL-2) and interleukin 5 (IL-5) secretion following acute resistance exercise was examined in 9 resistance-trained males. Subjects completed a randomized, double-blind protocol with exercise separated by 14 days. The exercise consisted of a high-intensity, short rest interval squat workout. Subjects consumed 1.0 g x kg body mass(-1) CHO or an equal volume of placebo (PLC) 10 min prior to and 10 min following exercise. Blood was collected at rest (REST), immediately post exercise (POST), and at 1.5 h of recovery (1.5 h POST). Isolated peripheral blood mononuclear cells were stimulated with PHA and assayed for IL-2 and IL-5 secretion. IL-2 secretion was significantly decreased at POST for both the PLC and CHO groups. However, the degree of decrease was less in the CHO group (16%) than in the PLC group (48%), and this difference was statistically significant. These responses were transient, and the values returned to normal by 1.5 h POST. A mild and transient but significant decrease in IL-5 secretion by the PLC group was observed at POST (26%) compared to REST. No significant decrease was observed in IL-5 secretion for CHO from REST to POST (12%). These data support a possible effect of carbohydrate supplementation on IL-2 and IL-5 secretion following high-intensity resistance exercise.

Stimulation through intercellular adhesion molecule-1 provides a second signal for T cell activation.

Regulation of T cell activation requires two signals. First, appropriately presented Ag in the context of MHC interacts with the T cell Ag receptor-CD3 complex. The best-studied second signal is CD28, which resides on the T cell and responds to its counter receptor, B7. A second signal also can be delivered through LFA-1 residing on the T cell, responding to its counter receptor ICAM-1 residing on a different cell. Characterization of a second signal is tied to its ability to costimulate (along with stimulation through the TCR) proliferation, IL-2 secretion, and coactivation of phosphatidylinositol 3-kinase. We examined whether ICAM-1, residing on the T cell surface, could deliver a second signal into that T cell. Costimulation through CD3 plus ICAM-1 caused increased T cell proliferation, increased expression of the activation marker CD69, increased transcription through the IL-2 regulatory region, and increased secretion of selected Th1 but not Th2 cytokines. Costimulation through CD3 plus ICAM-1 caused synergistic activation of phosphatidylinositol 3-kinase. Finally, the combination of anti-CD3 plus anti-ICAM-1 (but not anti-CD3 alone) caused prolonged proliferation of naive T cells in a manner similar to costimulation through LFA-1 or CD28. Thus, we demonstrate for the first time that ICAM-1 resident on a T cell can deliver a costimulatory signal into that T cell.