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BACKGROUND Numerous studies have been conducted on whether CD28 rs3116496 polymorphism affected cancer susceptibility, and these findings have been controversial. Thus, the purpose of this study was to assess the relationship between rs3116496 and susceptibility to cancer. MATERIAL AND METHODS The research published as of October 25, 2018 were comprehensively searched in PubMed, Embase, Cochrane Library and Chinese Wanfang database, CNKI, CBM. Statistical calculations performed using Stata12.0. RESULTS Overall analyses found that rs3116496 was a risk factor for cancer (C versus T, OR=1.14, 95% CI: 1.01-1.29, PH=0.003), and the heterogeneity was moderate (I²=53.3%). In subgroup analysis results by cancer types, the analysis showed that rs3116496 was a risk factor for breast cancer and leukemia. In the subgroup analysis by ethnicity, rs3116496 was a risk factor for cancer in the Asian population. After PHWE

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.Immunology and Cell Biology advance online publication, 23 December 2014; doi:10.1038/icb.2014.108. ispartof: Immunology and Cell Biology vol:93 issue:4 pages:417-23 ispartof: location:United States status: published

Phosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their distinct localization. In this study, we analyze the contribution of PIP5Kβ to T cell activation and show that CD28 induces the recruitment of PIP5Kβ to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kβ are pivotal for the PIP5Kβ regulatory functions in response to CD28 stimulation.

This study investigated whether changes in the cellular composition of blood during exercise could partly account for observations of exercise-induced changes in lymphocyte oxidative stress markers. Markers of oxidative stress were assessed before and after 60 min of intense treadmill running. Samples were collected from 16 men (means ± SD: age 33 ± 13 yr; body mass index 23.8 ± 2.5 kg/m2; maximal oxygen uptake 59.7 ± 5.2 ml·kg−1·min−1). Peripheral blood lymphocytes were assayed for protein carbonyl concentration, and plasma was assessed for lipid peroxides and antioxidant capacity. In a separate study, intracellular thiol concentration was determined in lymphocyte subsets from eight characteristically similar men by flow cytometry, of which T-cell memory populations were further identified on the basis of CD27, CD28, and CD45RA expression. Total lymphocyte protein carbonyls were transiently increased with exercise and returned to baseline within 15 min ( P < 0.001). This change was accompanied by an increase in plasma lipid peroxides ( P < 0.05) and total antioxidant capacity ( P < 0.001). Correlation analyses showed that lymphocyte protein carbonyl content was not related to changes in the cellular composition of peripheral blood during exercise. Natural killer cells (CD3−CD56+) and late-differentiated/effector memory cells (CD4+/CD8+CD27−CD28−/CD45RA+), which mobilized most with exercise, showed high intracellular thiol content ( P < 0.001). High thiol content suggests a lower oxidative load carried by these lymphocytes. Thus vigorous exercise resulted in a transient increase in lymphocyte oxidative stress. Results suggest this was unrelated to the alterations in the cellular composition of peripheral blood.

The cDNAs of three turkey CD markers, CD4, CD8alpha and CD28, were identified by screening a turkey cDNA library. The coding regions of the chicken and turkey genes are highly conserved, with 91.3-96.1% nucleotide (nt) and 84.2-95.5% amino acid (aa) identity. Identity was less conserved between avian CD markers and their mammalian homologues, ranging from 44.7 to 59.8% and 22.4 to 50.4% at the nt and aa levels, respectively. Anti-chicken CD8alpha and CD28 monoclonal antibodies were demonstrated to specifically cross-react with turkey CD8alpha and CD28, respectively.

The balance between antiapoptotic and proapoptotic proteins of the Bcl-2 family is critical in determining the fate of T cells in response to death stimuli. Proapoptotic genes, such as bax, are generally regulated by the p53 family of transcription factors, whereas NF-kappaB subunits can activate the transcription of antiapoptotic Bcl-2 members. Here, we show that CD28 activation protects memory T cells from irradiation-induced apoptosis by both upregulating bcl-xL and inhibiting bax gene expression. We found that p73, but not p53, binds to and trans-activates the bax gene promoter in irradiated T cells. The activation of RelA/NF-kappaB subunit in CD28 costimulated T cells and its binding onto the bax gene promoter results in suppression of bax transcription and decrease in both p73 and RNA polymerase II recruitment in vivo. RelA recruitment on the bax gene promoter is also accompanied by the lost of p300 binding and the parallel appearance of histone deacetylase-1-containing complexes. These findings identify RelA/NF-kappaB as a critical regulator of T-cell survival by affecting the balance of Bcl-2 family members.

SWAP-70-like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for Rho GTPases that is upregulated in Th2 cells, but whose physiological function is unclear. We show that SLAT(-/-) mice displayed a developmental defect at one of the earliest stages of thymocyte differentiation, the double-negative 1 (DN1) stage, leading to decreased peripheral T cell numbers. SLAT(-/-) peripheral CD4(+) T cells demonstrated impaired TCR/CD28-induced proliferation and IL-2 production, which was rescued by the addition of exogenous IL-2. Importantly, SLAT(-/-) mice were grossly impaired in their ability to mount not only Th2, but also Th1-mediated lung inflammatory responses, as evidenced by reduced airway neutrophilia and eosinophilia, respectively. Levels of Th1 and Th2 cytokine in the lungs were also markedly reduced, paralleling the reduction in pulmonary inflammation. This defect in mounting Th1/Th2 responses, which was also evident in vitro, was traced to a severe reduction in Ca(2+) mobilization from ER stores, which consequently led to defective TCR/CD28-induced translocation of nuclear factor of activated T cells 1/2 (NFATc1/2). Thus, SLAT is required for thymic DN1 cell expansion, T cell activation, and Th1 and Th2 inflammatory responses.

We report the fabrication and characterization of neutravidin-conjugated silica nanobeads doped with a ruthenium-complex luminophore and functionalized with anti-human CD3 and anti-human CD28, and an acid-sensitive polymer. We observed that the nanobeads were readily delivered into Jurkat T leukemia cells by endocytosis, transported into lysosomes, and subsequently into the cytoplasm as revealed by pH-sensitive luminescence. Since signs of cytotoxicity were not observed, the reported nanobeads could be an excellent and non-toxic building block for efficient intracellular transporters.

Contains fulltext : 51645.pdf (Publisher’s version ) (Closed access) Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.

The co-stimulatory molecule CD28 is essential for activation of helper T cells. Despite this critical role, it is not known whether CD28 has functions in maintaining T cell responses following activation. To determine the role for CD28 after T cell priming, we generated a strain of mice where CD28 is removed from CD4+ T cells after priming. We show that continued CD28 expression is important for effector CD4+ T cells following infection; maintained CD28 is required for the expansion of T helper type 1 cells, and for the differentiation and maintenance of T follicular helper cells during viral infection. Persistent CD28 is also required for clearance of the bacterium Citrobacter rodentium from the gastrointestinal tract. Together, this study demonstrates that CD28 persistence is required for helper T cell polarization in response to infection, describing a novel function for CD28 that is distinct from its role in T cell priming. DOI: http://dx.doi.org/10.7554/eLife.03180.001, eLife digest Invasion by a bacterium or virus typically activates a mammalian host's immune system to eliminate the pathogen. The cells of the so-called ‘innate immune system’ are the body's first line of defense against infection, and these cells patrol the organs and tissues in an effort to locate and eliminate pathogens quickly. The innate immune response is rapid and non-specific, but often cannot completely clear an infection. When necessary, innate immune cells will escalate the immune response by activating the second branch of the immune system, called the ‘adaptive immune system’. This specifically targets and eradicates an invading pathogen. T cells are essential components of the adaptive immune system, and these cells can be readily distinguished from other types of cell by proteins called T cell receptors (or TCRs) found on their surface. There are also different types of T cell, each with a specific function. T helper cells, for example, help other adaptive immune cells to mature and activate, which involves these immune cells proliferating and developing into more specialized cells. For a T cell to activate, two events must occur at the same time. First, the TCR must recognize and bind to a fragment of the pathogen that is presented to it by an innate immune cell. And second, ‘co-stimulatory molecules’ present on the surfaces of both the T cell and the same innate immune cell must interact. Using these two signals to activate a T cell helps to ensure the adaptive immune response is not ‘unleashed‘ unnecessarily. Co-stimulatory molecules have become popular targets for therapies aimed at treating autoimmune disorders—where the immune system attacks and destroys the body's own tissues. One of the most well studied co-stimulatory molecules expressed by T cells is called CD28; however, it remained unknown whether CD28 is involved in any processes after T cell activation. Now, Linterman et al. reveal that the CD28 co-stimulatory molecule plays a number of roles in addition to T cell activation. For example, a newly developed mouse model showed that CD28 must remain on the surface of T helper cells after they have been activated for these cells to effectively specialize. Linterman et al. also discovered that CD28 helps different T helper cell subtypes to develop. Linterman et al. demonstrate that CD28 is critical throughout a host's response to infection, and suggest that if CD28 is lost on activated T cells (which happens during aging, HIV infection and autoimmune diseases) the responses of T helper cells become limited. Furthermore, these findings reveal that treatments that target the CD28 co-stimulatory molecule will also affect on-going immune responses. DOI: http://dx.doi.org/10.7554/eLife.03180.002

Different properties allow HIV-1 and SIV to establish a chronic disease, e.g. genome insertion into long living quiescent T cells, high genome mutations and evasion of the immune system. The lentiviral Nef protein undertakes important functions of evading the immune system. One of these functions is the downregulation of CD4 receptors from infected cell surfaces. Downregulation of the main entry receptor of HIV-1 prevents superinfection and related cell death, enhances release and infectivity of viral particles and might weaken the immune response, as it is an important costimulatory factor of TCR-mediated T cell activation. Although it is well known that CD4 downmodulation is a key function which is highly conserved among HIV-1 and SIV Nef proteins, the mechanism of downmodulation for a broad panel of different HIV-1 group and SIV Nefs remained elusive. Thus the first goal of this study was to investigate the CD4 downregulation pathway. As available studies so far point towards an endocytosis enhancement probability I evaluated the capacity of this pathway for a broad panel of nef alleles. Furthermore, I chose a test system which allows me to measure intracellular retained CD4 receptors, as another possible downregulation mechanism, which is predominantly thought to be used for MHC-I downregulation. I also measured overall downregulation capabilities of each HIV-1 and SIV nef allele to be able to put the obtained values into an overall context. Besides CD4, CD28 is another Nef-mediated downregulated surface receptor of infected host cells. This receptor as well plays important roles in immune responses by providing co-stimulatory signals for T cell activation. It was suggested that the mechanisms of Nef-mediated CD4 and CD28 downmodulation are related (Swigut et al., 2001), so the second goal of this study was to investigate CD28 downregulation mechanisms.

The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining.

To evaluate the long-term effects of drospirenone (DRSP)/ethinylestradiol (EE) alone, metformin alone, and DRSP/EE-metformin on CD4(+)CD28(null) T lymphocytes frequency, a cardiovascular risk marker, in patients with hyperinsulinemic polycystic ovary syndrome (PCOS).Randomized clinical trial.Ninety three patients with hyperinsulinemic PCOS were age matched and body mass index matched and randomized to receive a 6 months daily treatment with DRSP (3 mg)/EE (0.03 mg), or metformin (1500 mg), or DRSP/EE combined with metformin.CD4(+)CD28(null) T-cell frequencies.The DRSP/EE and metformin groups did not show any significant change in the CD4(+)CD28(null) frequency compared to the baseline. Interestingly, a statistically significant decrease in CD4(+)CD28(null) frequency occurred after 6 months of DRSP/EE-metformin (median 3-1.5; P.01). Of note, this statistically significant association was confirmed after adjusting for baseline values in DRSP/EE-metformin group by analysis of covariance (P.05).In women with hyperinsulinemic PCOS, combined therapy with DRSP/EE and metformin may reduce cardiovascular risk.

ICOS and CD28 are expressed by T cells and are involved in costimulation of cytokine production in T helper (TH) cells. ICOS binds B7h expressed by several cell types, whereas CD28 binds B7.1 and B7.2 expressed by activated antigen presenting cells. This work investigated the role of B7h and B7.1 in TH17 and TH9 cell differentiation by assessing activity of recombinant B7h-Fc and B7.1-Fc on human naive TH cells activated in the presence of different combinations of exogenous cytokines. In the presence of TGF-β1 and IL-1β (TH17 promoting condition), B7h-Fc was more effective than B7.1-Fc in inducing IL-17A and IL-10 secretion, whereas B7.1-Fc was more effective in inducing IL-17F. Dual costimulation with B7h-Fc and B7.1-Fc displayed an intermediate pattern with predominance of IL-17F over IL-17A, secretion of high levels of IL-10, and secretion of IL-9 levels lower than those induced by B7.1-Fc alone. In the presence of TGF-β1 and IL-4 (TH9 promoting condition), B7h-Fc induced IL-17A only, whereas B7.1-Fc induced also IL-17F, IL-10, and high levels of IL-9. Experiments on memory TH cells showed that B7h-Fc mainly supported secretion of IL-17A and IL-10, whereas B7.1-Fc supported secretion of IL-17A, IL-17F, IL-10, and IL-9. These data indicate that B7h and B7.1 play different roles in modulation of TH17 and TH9 differentiation. This plasticity might be important in the immune response to pathogens and tumors, and in the development of autoimmune diseases, and should be taken in consideration in designing of immunotherapeutic protocols triggering ICOS or CD28.

The suppressive effect of the glucocorticoid dexamethasone (DEX) on purified CD4+ T cells was found to depend on the activation pathway. In contrast to anti-CD3- or PHA-induced T cell proliferation, the alternative pathway of T cell activation, i.e., through anti-CD2 and anti-CD28, appeared largely resistant to DEX. By titrating anti-CD28 or the protein kinase C (PKC) activator PMA in the DEX-sensitive systems, it was demonstrated that inhibition by DEX could be abrogated by enhancing the CD28 signal or by stimulation of the PKC-dependent pathway. Supraoptimal concentrations of PMA were inhibitory for proliferation and this effect was partly prevented by DEX. These data suggest that the outcome of the effect of DEX on CD4+ T cells is dependent on the activation pathway, in particular the role and composition of the transcription factor AP-J. Chemicals/CAS: dexamethasone, 50-02-2; phytohemagglutinin, 9008-97-3; protein kinase C, 141436-78-4; Antigens, CD28; Dexamethasone, 50-02-2; Interleukin-2; Interleukin-4, 207137-56-2; Protein Kinase C, EC 2.7.1.37; Tetradecanoylphorbol Acetate, 16561-29-8

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.

Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3′ region of the cAMP response modulator ( Crem ) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4 + T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4 + T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMP-driven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 ( Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/CREM and inhibition of NFATc1 and IL-2 induction.

The immunological synapse is a dynamic structure, formed between a T cell and one or more antigen-presenting cells, characterized by lipid and protein segregation, signaling compartmentalization, and bidirectional information exchange through soluble and membrane-bound transmitters. In addition, the immunological synapse is the site where signals delivered by the T-cell receptors, adhesion molecules, as well as costimulatory and coinhibitory receptors are decoded and integrated. Signaling modulation and tunable activation thresholds allow T cells to interpret the context in which the antigen is presented, recognize infectious stimuli, and finally decide between activation and tolerance. In this review, we discuss some strategies used by membrane receptors to tune activation signals in T cells.

International audience; CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of zeta-chain-associated protein of 70 kD, phospholipase-gamma, and protein kinase C- into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca(2+) mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-kappaB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion.

Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance. ispartof: Journal of Immunology vol:181 issue:2 pages:1034-1042 ispartof: location:United States status: published

Cells derived from the amniotic membrane of human placenta have been receiving particular attention because of their stem cell potentiality and immunomodulatory properties, which make them an attractive candidate source for cell therapy approaches. In this study, we isolated cells from the mesenchymal region of amnion and identified two subpopulations discordant for expression of the HLA-DR, CD45, CD14, and CD86 cellular markers. We therefore refer to the unfractionated cell population derived from this region as amniotic mesenchymal tissue cells (AMTC). We studied the suppressive and stimulatory characteristics of the unfractionated, HLA-DR-positive, and HLA-DR-negative AMTC populations and demonstrated that all three fail to induce an allogeneic T-cell response. However, unfractionated AMTC, which could inhibit T-cell allogeneic proliferation responses, induced proliferation of T cells stimulated via the T-cell receptor (TcR), in a cell-cell contact setting. We have shown that this stimulatory capacity can be attributed to the HLA-DR-positive AMTC subpopulation. Indeed, even though the HLA-DR-positive AMTC fraction surprisingly failed to induce proliferation of resting allogeneic T cells, they could cause strong proliferation of anti-CD3-primed allogeneic T cells. This stimulatory effect was not observed using the HLA-DR-negative AMTC fraction. The revelation that human amniotic mesenchyme possesses cell populations with both suppressive and stimulatory properties sheds additional light on the immunomodulatory functions of this tissue and may contribute to the clarification of some ongoing controversies associated with mesenchymal stromal cells of other sources, such as the presence of HLA-DR-positive cells and the suppressive versus stimulatory properties of these cells.Disclosure of potential conflicts of interest is found at the end of this article.

Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (

Bacterial superantigens (SAgs) constitute a large family of bacterial toxins that share the capacity to induce massive activation of the human immune system. Such a feature is based on the ability of these toxins to activate T cells that express Beta-chains of the T cell antigen receptor (TCR) containing variable regions (V) coded by specific families of VBeta genes. In addition, bacterial SAgs bypass the need for processing by antigen-presenting cells by directly binding to major histocompatibility complex class II molecules on the surface of these cells. Emerging work indicates that bacterial SAgs utilize not only the canonical pathways of TCR-mediated T cell activation but also other pathways. Here, we review the structural information on recognition of bacterial SAgs by T cells, the TCR signalling induced by this recognition event, and the effector functions that this recognition triggers. We analyze experimental evidence suggesting the existence of alternative receptors and coreceptors for bacterial SAgs, and outline future challenges in the research with these toxins.

Loss of Treg function appears to be a critical factor in the pathogenesis of human autoimmune diseases. Attention has focused on defects of CD4(+)CD25(high) Tregs, and techniques have been developed to determine their function. In contrast, the role of Tr1 regulatory T cells, which secrete the antiinflammatory cytokine IL-10, in autoimmune disease has not been well assessed. CD46 is a newly defined costimulatory molecule for T cell activation, and CD46-costimulated human T cells induce a Tr1 Treg phenotype with considerable amounts of IL-10 secretion. Here, we examined the role of Tr1 cells in patients with multiple sclerosis (MS) by stimulating CD4(+) T cells with anti-CD3 and -CD46 mAbs and measuring IL-10 secretion. There were striking defects in the induction of Tr1 cells with CD46 costimulation as measured by IL-10 but not IFN-gamma secretion in patients with MS compared with healthy subjects. This loss of Tr1 cell-associated IL-10 secretion was specific to CD46 and not CD28 costimulation and was associated with an altered regulation of the CD46-Cy2 isoform that differentially regulates T cell function in a CD46-transgenic murine model. These data demonstrate a second major Treg defect in human autoimmune disease associated with the CD46 pathway.

Background Human parvovirus B19 (B19) is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. Methods and Findings The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA–peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%–0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. Conclusion This is the first example to our knowledge of an “acute” human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation—analogous to that for cytomegalovirus infection—is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development., Although parvovirus B19 apparently causes an acute viral infection, it appears to induce a persistent activated CD8 + T cell response.

CD28 is constitutively expressed on CD4 + cells, but its homologue CD152 is only weakly expressed after cell activation. To determine whether these 2 costimulatory molecules can be inserted into human immunodeficiency virus type 1 (HIV-1), virus was produced in CD28- and CD152-expressing Jurkat-derived cells. Both molecules were efficiently acquired by virions. Virus attachment and infectivity were more affected by CD152 than by CD28. Given that CD28/CD152-CD80/ CD86 interactions play a dominant role in antigen presentation, it can thus be proposed that the association between virus-anchored host CD28/CD152 and cell-surface CD80/ CD86 on target cells might have consequences for the transmission and pathogenesis of HIV-1.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:21:08Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:34:11Z : No. of bitstreams: 1 2-s2.0-4444271905.pdf: 448442 bytes, checksum: b7bce4a171bd524265b6639fd8817104 (MD5) Made available in DSpace on 2014-05-27T11:21:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-09-01 A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-γ and to restrict the growth of bacilli. TB Network Dept. of Biochemistry and Immunology University of São Paulo Department of Pathology Ribeirão Preto Sch. of Med. University of São Paulo Dept. of Microbiology and Immunology Biosciences Institute São Paulo State University Dept. of Biochemistry and Immunology Ribeirão Preto Sch. of Med. University of São Paulo, Avenida Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP Dept. of Microbiology and Immunology Biosciences Institute São Paulo State University

Human immunodeficiency virus type 1 (HIV-1) carries virus-encoded and host-derived proteins. Recent advances in the functional characterization of host molecules inserted into mature virus particles have revealed that HIV-1 biology is influenced by the acquisition of host cell membrane components. The CD28/B7 receptor/ligand system is considered one of the fundamental elements of the normal immune response. Two major cell types that harbor HIV-1 in vivo, i.e., monocytes/macrophages and CD4 + T cells, express the costimulatory molecules CD80 (B7.1) and CD86 (B7.2). We investigated whether CD80 and CD86 are efficiently acquired by HIV-1, and if so, whether these host-encoded molecules can contribute to the virus life cycle. Here we provide the first evidence that the insertion of CD80 and CD86 into HIV-1 increases virus infectivity by facilitating the attachment and entry process due to interactions with their two natural ligands, CD28 and CTLA-4. Moreover, we demonstrate that NF-κB is induced by CD80- and CD86-bearing virions when they are combined with the engagement of the T-cell receptor/CD3 complex, an event that is inhibited upon surface expression of CTLA-4. Finally, both CD80 and CD86 were found to be efficiently incorporated into R5- and X4-tropic field strains of HIV-1 expanded in cytokine-treated macrophages. Thus, besides direct interactions between the virus envelope glycoproteins and cell surface constituents, such as CD4 and some specific chemokine coreceptors, HIV-1 may attach to target cells via interactions between cell-derived molecules incorporated into virions and their natural ligands. These findings support the theory that HIV-1-associated host proteins alter virus-host dynamics.

CD28 is one of the most important costimulatory receptors necessary for full T lymphocyte activation. The CD28 receptor can enhance T cell antigen receptor (TCR) signals, as well as deliver independent signals. Indeed, CD28 engagement by B7 can generate TCR-independent signals leading to IκB kinase and NF-κB activation. Here we demonstrate that the TCR-independent CD28 signal leads to the selective transcription of survival (Bcl-xL) and inflammatory (IL-8andB cell activation factor, but not proliferative (IL-2), genes, in a NF-κB-dependent manner. CD28-stimulated T cells actively secrete IL-8, and Bcl-xL up-regulation protects T cells from radiation-induced apoptosis. The transcription of CD28-induced genes is mediated by the specific recruitment of RelA and p52 NF-κB subunits to target promoters. In contrast, p50 and c-Rel, which preferentially bind NF-κB sites on theIL-2gene promoter after anti-CD3 stimulation, are not involved. Thus, we identify CD28 as a key regulator of genes important for both survival and inflammation.

Although ezrin is tyrosine phosphorylated following TCR ligation, its biological role in T cell activation is not known. Here, we shhow that ezrin clusters at the immunological synapse upon T cell stimulation. Clustering of ezrin can be triggered by TCR ligation, or, more efficiently, by CD28 ligation. The clusters of ezrin at the immunological synapse include serine/threonine phosphorylated ezrin predominantly located within cell membrane lipid rafts. Based on these data, we propose that ezrin may play a role in the formation/stabilization of lipid raft signalosomes at the immunological synapse and therefore contribute to sustain TCR-dependent signalling.

Udgivelsesdato: 2000-Aug The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

CTLA-4 (CD152) engagement results in down-regulation of T cell activation. Two mechanisms have been postulated to explain CTLA-4 inhibition of T cell activation: negative signaling and competitive antagonism of CD28:B7-mediated costimulation. We assessed the contributions of these two mechanisms using a panel of T cell lines expressing human CTLA-4 with mutations in the cytoplasmic region. Under conditions of B7-independent costimulation, inhibition of IL-2 production following CTLA-4 engagement required the CTLA-4 cytoplasmic region. In contrast, under B7-dependent costimulation, inhibition of IL-2 production by CTLA-4 engagement was directly proportional to CTLA-4 cell surface levels and did not require its cytoplasmic region. Thus, CTLA-4 down-regulates T cell activation by two different mechanisms—delivery of a negative signal or B7 sequestration—that are operational depending on the levels of CTLA-4 surface expression. These two mechanisms may have distinct functional outcomes: rapid inhibition of T cell activation or induction of T cell anergy.

The BCL1 lymphoma in Balb/c mice can be successfully treated with bispecific (anti-CD3 x anti-idiotype) antibodies (BSABs). In these experiments, animals were injected intraperitoneally (IP) with 5 x 10(3) tumor cells (day 0) and treated with one single intravenous (IV) injection of 5 micrograms BSAB (day 9). Because cross-linking of the CD3 complex is not in itself sufficient to activate resting T cells, the therapeutic success was mainly based on the progressive retargeting of the relatively small cytotoxic T-lymphocyte effector cell pool already in existence in vivo. For this reason, the therapy lost its effectiveness at higher tumor loads. Two possibilities were explored to treat mice with a higher tumor load (10(5) tumor cells). First multiple injections of BSABs were used, but a dose-related monovalent anti-CD3 immunosuppression was induced. This approach was only beneficial when the immune system was able to recover from the previous injection of BSAB. As a second approach, CD28 costimulation together with BSABs were used in an attempt to activate resting T cells, ultimately enlarging the effector T-cell pool. However, we were repeatedly unsuccessful in attempts to improve our in vivo results using young, naive animals in which the majority of T cells are of the naive phenotype. Only when animals were primed to induce the memory T-cell type was a significantly better outcome observed, and then only with multiple injections of BSABs and anti-CD28 monoclonal antibodies (MoAbs), rather than with BSAB or anti-CD28 MoAb alone. These results suggest that this combination was able to activate memory and effector T cells and to focus them on the tumor, but was unable to activate naive T cells fully. The in vivo potency of the BSAB and CD28 costimulation was shown by the fact that one-tenth of the quantity of BSAB was required to cure animals with 20 times the tumor load.

Human CD45RA+ ('naive') and CD45RO+ ('memory') CD4+ T cells were compared with respect to their sensitivity to dexamethasone (DEX). In three different activation pathways, i.e. (i) immobilized anti-CD3, (ii) immobilized anti-CD3 plus soluble anti-CD28 and (iii) soluble anti-CD2 plus soluble anti-CD28, naive CD4+ T cells appeared more sensitive to DEX than memory CD4+ T cells. In the anti-CD3 system this difference in sensitivity was apparent at a suboptimal DEX concentration. Addition of anti-CD28 rendered the cells largely insensitive to DEX, indicating that the CD28 pathway is less dependent of the DEX-sensitive transcription factor AP-1. However, the alternative pathway of T cell activation through CD2/CD28 triggering was highly sensitive to DEX when naive cells were studied; in the case of memory cells, at least a 10-fold higher DEX concentration was needed to achieve a comparable inhibition. The strong inhibitory effect of DEX on naive CD4+ T cells stimulated via the alternative pathway was completely abrogated by activation of protein kinase C (PKC) with phorbol myristate acetate. Our data suggest that at least two different mechanisms contribute to DEX resistance, i.e. CD28 triggering and PKC activation, which may occur more effectively in memory cells making them less sensitive to DEX.

CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti-CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-gamma upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti-CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN-gamma and minute amounts of IL-4 and IL-10, suggesting that these "naive" cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-gamma as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells.