Search

Introduction:Particulate air pollution, containing nanoparticles, enhances the risk of pediatric allergic diseases that is potentially associated with disruption of neonatal immune system. Previous studies have revealed that maternal exposure to carbon black nanoparticles (CB-NP) disturbs the development of the lymphoid tissues in newborns. Interestingly, the CB-NP-induced immune profiles were observed to be different depending on the gestational period of exposure. It is important to identify the critical exposure period to prevent toxic effects of nanoparticles on the development of the immune system. Therefore, the present study was aimed to investigate the effect of CB-NP on the development of neonatal lymphoid tissues in mice, depending on the gestational period of exposure.Methods:Pregnant ICR mice were treated with a suspension of CB-NP (95 μg/kg body weight) by intranasal instillation; the suspension was administered twice during each gestational period as follows: the pre-implantation period (gestational days 4 and 5), organogenesis period (gestational days 8 and 9), and fetal developmental period (gestational days 15 and 16). The spleen and thymus were collected from offspring mice at 1, 3, and 5-days post-partum. Splenocyte and thymocyte phenotypes were examined by flow cytometry. Gene expression in the spleen was examined by quantitative reverse transcription-polymerase chain reaction.Results:The numbers of total splenocytes and splenic CD3−B220−phenotype (non-T/non-B lymphocytes) in offspring on postnatal day 5 were significantly increased after exposure to CB-NP during the organogenesis period compared with other gestational periods of exposure and control (no exposure). In contrast, expression levels of mRNA associated with chemotaxis and differentiation of immune cells in the spleen were not affected by CB-NP exposure during any gestational period.Conclusion:The organogenesis period was the most susceptible period to CB-NP exposure with respect to lymphoid tissue development. Moreover, the findings of the present and previous studies suggested that long-term exposure to CB-NP across multiple gestational periods including the organogenesis period, rather than acute exposure only organogenesis period, may more severely affect the development of the immune system.

Resistance training adaptively increases the muscle strength associated with protein anabolism. Previously, we showed that the combined intake of astaxanthin, &beta, carotene, and resveratrol can accelerate protein anabolism in the skeletal muscle of mice. The purpose of this study was to investigate the effect of anabolic nutrient-rich foods on muscle adaptation induced by resistance training. Twenty-six healthy men were divided into control and intervention groups. All participants underwent a resistance training program twice a week for 10 weeks. Astaxanthin-, &beta, carotene-, and resveratrol-rich foods were provided to the intervention group. Body composition, nutrient intake, maximal voluntary contraction of leg extension, oxygen consumption, and serum carbonylated protein level were measured before and after training. The skeletal muscle mass was higher after training than before training in both groups (p &lt, 0.05). Maximal voluntary contraction was increased after training in the intervention group (p &lt, 0.05), but not significantly increased in the control group. Resting oxygen consumption was higher after training in the intervention group only (p &lt, 0.05). As an oxidative stress marker, serum carbonylated protein level tended to be lower immediately after exercise than before exercise in the intervention group only (p = 0.056). Intake of astaxanthin-, &beta, carotene-, and resveratrol-rich foods supported resistance training-induced strength and metabolic adaptations.

Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software "Cancer Cell Finder" that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

Astaxanthin, a natural antioxidant, exists in non-esterified and esterified forms. Although it is known that astaxanthin can improve exercise endurance and cause metabolic improvement in skeletal muscle, the effects of the two different forms are unclear. We investigated the effects of the different forms of astaxanthin on endurance in mice. Eight-week-old ICR mice were divided into four groups: control; astaxanthin extracted from Haematococcus pluvialis in an esterified form; astaxanthin extracted from Phaffia rhodozyma in a non-esterified form; and astaxanthin synthesized chemically in a non-esterified form. After 5 weeks of treatment, each group was divided into sedentary and exercise groups. In the group fed astaxanthin from Haematococcus, the running time to exhaustion was longest, and the plasma and tissue concentrations of astaxanthin were significantly higher than those in the other groups. Astaxanthin from Haematococcus increased 5'-adenosine monophosphate-activated protein kinase levels in the skeletal muscle. Although the mice in the Haematococcus group ran for longer, hexanoyl lysine adduct levels in the skeletal muscle mitochondria were similar in the control and Haematococcus groups. Our results suggested that esterified astaxanthin promoted energy production and protected tissues from oxidative damage during exercise owing to its favorable absorption properties, leading to a longer running time.

In our previous paper, we reported on the preparation of some cationic amphiphilic Ir complexes (2c, 2d) containing KKGG peptides that induce and detect cell death of Jurkat cells. Mechanistic studies suggest that 2c interacts with anionic molecules and/or membrane receptors on the cell surface to trigger an intracellular Ca2+ response, resulting in the induction of cell death, accompanied by membrane disruption. We have continued the studies of cell death of Jurkat cells induced by 2c and found that xestospongin C, a selective inhibitor of an inositol 1,4,5-trisphosphate receptor located on the endoplasmic reticulum (ER), reduces the cytotoxicity of 2c, suggesting that 2c triggers the release of Ca2+ from the ER, leading to an increase in the concentration of cytosolic Ca2+, thus inducing cell death. Moreover, we synthesized a series of new amphiphilic cationic Ir complexes 5a–c containing photoreactive 3-trifluoromethyl-3-phenyldiazirine (TFPD) groups, in an attempt to identify the target molecules of 2...

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ–producing cells. CD8 T cell–mediated rejection of IL-1R−/− cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow–derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R–mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts.

Objective The antioxidant factors, astaxanthin, β-carotene, and resveratrol, have a potential effect on protein synthesis in skeletal muscle and a combined intake may have a greater cumulative effect than individual intake. The aim of this study was to investigate the combined effects on skeletal muscle mass and protein metabolic signaling during the hypertrophic process from atrophy in mice. Methods Male ICR mice were divided into five dietary groups consisting of seven animals each: normal, astaxanthin, β-carotene, resveratrol, and all three antioxidants. Equal concentrations (0.06% [w/w]) of the respective antioxidants were included in the diet of each group. In the mixed group, three antioxidants were added in equal proportion. One leg of each mouse was casted for 3 wk to induce muscle atrophy. After removal of the cast, the mice were fed each diet for 2 wk. The muscle tissues were collected, weighed, and examined for protein metabolism signaling and oxidative damage. Results The weight of the soleus muscle was increased in the astaxanthin, β-carotene, and resveratrol groups to a greater extent than in the normal group; this was accelerated by intake of the mixed antioxidants (P = 0.007). Phosphorylation levels of mammalian target of rapamycin and p70 S6 K in the muscle were higher in the mixed antioxidant group than in the normal group (P = 0.025; P = 0.020). The carbonylated protein concentration was lower in the mixed antioxidant group than in the normal group (P = 0.021). Conclusions These results suggested that a combination of astaxanthin, β-carotene, and resveratrol, even in small amounts, promoted protein synthesis during the muscle hypertrophic process following atrophy.

It is thought that CD28 plays a crucial role in the maintenance of regulatory T cell (Treg) pool size through promoting the development and proliferation of these cells. However, recently we found that the dependency on CD28 co-stimulation for their development is different between Treg subsets, thymus-derived Tregs (tTregs, CD28-dependent) and peripherally-derived Tregs (pTregs, CD28-independent), suggesting that CD28 may also have differential influences on the homeostasis of each Treg subset. Here, we demonstrated that both Treg subsets were reduced in secondary lymphoid organs of CD28 deficient mice, and that this reduction was due to impaired proliferation in both Treg subsets by the intrinsic CD28 defect. However, we found that the massive proliferation of both Treg subsets under lymphopenic condition was regulated by CD28, whereas the proliferative activity of tTregs but not pTregs in the steady state was dependent on CD28. Also, experiments using mutant CD28 knock-in mice revealed that proliferation of pTregs under lymphopenic condition required only the Lck-NFκB pathway of CD28, whereas tTregs required an additional unknown pathway. These findings indicate that the dependency on CD28 for proliferation in each Treg subset differs depending on the environment.

p53 mutations are frequently detected in malignant gastric cancers. However, the molecular mechanisms by which loss of p53 function promotes gastric cancer are not clear. We utilized Gan mice (K19-Wnt1/C2mE), which have functional p53 and develop intestinal-type gastric tumors, to investigate the role of p53 in gastric cancer progression by knocking out p53. We found that gastric epithelial cells acquire tumorigenicity in the subcutis of C57BL/6 mice as a result of Wnt activation, COX-2 activation and p53 deficiency. With repeated allograft transfers, these gastric epithelial cells gradually acquired the properties of malignant gastric cancer. Loss of p53 conferred cell stemness and induced epithelial to mesenchymal transition (EMT) in gastric epithelial cells, and these properties were further enhanced by the in vivo microenvironment, ultimately leading to gastric cancer formation and metastasis. We also found that the in vivo microenvironment enhanced activation of the COX-2 pathway, which further contributed to cancer progression. With this system, we have succeeded in recapitulating the development of malignant gastric cancer from gastric epithelial cells in a normal immune environment.

The IL-7R plays critical roles in lymphocyte development and homeostasis. Although IL-7R expression is strictly regulated during lymphocyte differentiation and the immune response, little is known regarding its in vivo regulation. To address this issue, we established a mouse line with targeted deletion of the conserved non-coding sequence 1 (CNS1) element found 3.6 kb upstream of the IL-7Rα promoter. We report that IL-7Rα is expressed normally on T and B cells in thymus and bone marrow of CNS1−/− mice except for in regulatory T cells. In contrast, these mice show reduced IL-7Rα expression in conventional CD4 and CD8 T cells as well as regulatory T, NKT, and γδ T cells in the periphery. CD4 T cells of CNS1−/− mice showed IL-7Rα upregulation in the absence of growth factors and IL-7Rα downregulation by IL-7 or TCR stimulation, although the expression levels were lower than those in control mice. Naive CD4 and CD8 T cells of CNS1−/− mice show attenuated survival by culture with IL-7 and reduced homeostatic proliferation after transfer into lymphopenic hosts. CNS1−/− mice exhibit impaired maintenance of Ag-stimulated T cells. Furthermore, IL-7Rα upregulation by glucocorticoids and TNF-α was abrogated in CNS1−/− mice. This work demonstrates that the CNS1 element controls IL-7Rα expression and maintenance of peripheral T cells, suggesting differential regulation of IL-7Rα expression between central and peripheral lymphoid organs.

Title: The therapeutic efficacy of CD90-targeted liposomes in pulmonary carcinoma. Background: In refractory lung cancer tissue, it has been reported that cell populations exist that exhibit resistance to existing therapeutics. These cells have characteristics such as high stemness and tumorigenic potential, and they are thought to contribute to relapse and metastasis. Differentiation inducer 4[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl) carbamoyl] benzoic acid (Am80) has a growth inhibitory effect on pulmonary carcinoma cells, but it has been effective only in a high dose that causes damage to normal cells. We aimed to achieve a dose reduction by specific targeting of anaplastic pulmonary carcinoma cells and modification of antibodies on the liposome surface specific for membrane protein of undifferentiated cells because undifferentiated pulmonary cells have unique membrane proteins. CD90 is one of the undifferentiated markers expressed in pulmonary cells. Methods and Findings: In this study, we examined the usefulness evaluation of Am80 liposomes conjugated with anti-CD90 antibody using Calu-6, a CD90-positive undifferentiated human pulmonary carcinoma cell line. After the preparation of Am80 liposomes, the characterization and cell specificity, therapeutic efficacy of them were assessed. Am80 liposomes conjugated with antibody selectively combined with the surface of Calu-6 and had a significant influence on the inhibition of cell and tumor growth. The twice-weekly intratumoral administration of Am80 liposomes conjugated antibody (0.1 mg/kg as Am80) inhibited tumor growth to 3.64 ± 1.10 compared to empty liposomes (5.89 ± 1.61) in relative tumor volume at day 28. Conclusions: We revealed that the cellular uptake of Am80 liposomes conjugated with anti- CD90 antibody by anaplastic pulmonary carcinoma cells was highly effective and efficient.

Recombinant Chinese hamster ovary (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Although recent advances in 3D culture of rCHO cells is preferred to 2D monolayer culture for highly productive and robust expression of therapeutic proteins, there exists still limitation for efficient protein production. Therefore, a new cell culture system is essentially required for an efficient protein production. Here, we report on a new 3D cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein by CHO cells. Particularly, cocultivation of CHO spheroids with bovine aortic endothelial cells (BAEC) as a feeder layer cells was essential to stably increase a protein production. We investigated the co-culture mechanism of functional enhancement with respect to the cell–cell interactions. Functional comparison between 2D and 3D co-cultures suggested the preferred configuration as spheroid for higher protein production. Specifically, to estimate the effect of respective cell constitution in co-cultured spheroids on the protein production per CHO cell, the number of viable cells in cell proliferation was determined with culture periods. These studies demonstrated the significant role of micropatterned BAEC as a feeder layer for the retained formation of CHO spheroids, resulting in predominantly enhanced production of proteins, although the functional enhancement of CHO cells was obtained by co-culture with BAECs in both 2D and 3D configurations. Thus, heterotypic cell communications that play indispensable roles in increasing CHO functions should be properly obtained in 3D cell configurations. Significantly, these spheroids in the serum-free medium drastically enhanced protein expression level up to sevenfold compared with CHO monospheroids, suggesting that a suitable culture conditions for heterotypic cell–cell interactions would allow improved protein secretion to occur unimpeded.

Purpose The interaction between the inducible costimulatory molecule (ICOS) and ICOS ligand (ICOSL) has been implicated in the differentiation and functions of T cells. The purpose of the present study was to determine the role of ICOS-ICOSL in the immune privilege of corneal allografts. Methods Expression of ICOS and ICOSL mRNA from mouse eyes was assessed by RT-PCR. Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of ICOS-/- BALB/c recipients and BALB/c wild-type (WT) recipients treated with anti-ICOSL mAb, and graft survival was assessed. A separate set of WT and ICOS-/- BALB/c mice received an anterior chamber injection of C57BL/6 splenocytes, and induction of allospecific anterior chamber-associated immune deviation (ACAID) was assessed. In vitro, cornea was incubated with T cells from WT and ICOS-/- BALB/c mice, and destruction of corneal endothelial cells (CECs) and the population of Foxp3+ CD25+ CD4+ T cells was assessed. Results Inducible costimulatory molecule ligand mRNA was constitutively expressed in the cornea, iris-ciliary body, and retina. Allograft survival in ICOS-/- recipients and WT recipients treated with anti-ICOSL mAb was significantly shorter than in control recipients. Anterior chamber-associated immune deviation was induced less efficiently in ICOS-/- mice. Destruction of CECs by alloreactive ICOS-/- T cells was enhanced compared with WT T cells. After coincubation with allogeneic corneal tissue, the proportion of regulatory T cells was significantly greater among WT T cells than in ICOS-/- T cells. Conclusions The expression of ICOSL in the cornea and the ICOS-mediated induction of Foxp3+ CD4+ regulatory T cells may contribute to successful corneal allograft survival.

It has been reported that splenic immune responses play pivotal roles in the development of allergic diseases; however, the precise role of the spleen remains unclear. Herein, we demonstrated a novel role of the spleen in the pathogenesis of food allergy (FA). We found that mast cells (MCs) developed from progenitor cells present in spleen during an antigen-specific T-cell response in vitro. In a Th2 response-mediated FA model, significant expansion of MCs was also observed in spleen. The incidence of allergic diarrhea was profoundly reduced in splenectomized mice, whereas adoptive transfer of in vitro-induced splenic MCs into these mice restored allergic symptoms, suggesting that the splenic MCs functioned as the pathogenic cells in the development of FA. The in vitro-generated MCs required not only IL-3 but also IFN-γ, and treatment of FA-induced mice with anti-IFN-γ antibody suppressed expansion of MCs in spleen as well as diarrhea development, highlighting that IFN-γ in the spleen orchestrated the development of FA, which was followed by a Th2 response in the local lesion. Overall, we propose that the role of the spleen in the development of FA is to provide a unique site where antigen-specific T cells induce development of pathogenic MCs.

It has been proposed that sustained ICOS expression in chronic inflammatory immune conditions, such as autoimmunity and allergy, contributes to symptom exacerbation. Therefore modulation of ICOS gene expression could be a potential therapeutic strategy for such immune diseases. However, the precise molecular mechanisms controlling ICOS gene expression remain poorly understood. In this study, we explored transcription factors involving in ICOS gene expression and examined their roles in a physiological situation. Microarray analysis revealed that one AP-1 molecule, Fos-related antigen-2 (Fra2), was highly correlated with ICOS expression. Ectopic expression of Fra2 and other AP-1 molecules upregulated ICOS expression on T cells. We identified an AP-1-responsive site (AP1-RE) within the ICOS promoter region and demonstrated AP-1 actually binds to AP1-RE upon TCR/CD28 stimulation. Meanwhile, we found several cytokines could upregulate ICOS expression on both naive and effector T cells in a manner independent of TCR/CD28 stimulation. These cytokine stimuli induced AP-1 binding to AP1-RE. Together, our results indicate AP-1 transcription factors are involved in ICOS gene expression downstream of both TCR/CD28 signaling and cytokine receptor signaling, and suggest AP-1 activation via cytokine receptor signaling may be one of the mechanisms maintaining high level ICOS expression in chronic inflammatory immune responses.

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4(+) antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4(+) T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRbeta crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high "avidity" effector and memory T cells in response to pathogen are discussed.

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4−CD8− cells initiate expression of TCRβ. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRβ expression manifest in wild-type thymocytes, occurring even in Rag− or CD3ε− knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the “β” checkpoint for pre-TCR/TCR-dependent thymic differentiation.

Previously, we reported that allogeneic skin grafts were rapidly rejected by CD28 and CD40 ligand double deficient mice mediated by CD8+ T cells. These results indicated that some elements in addition to CD28- and CD40-mediated costimulation provide stimulatory signals for the activation of donor-specific CD8+ T cells. In this report, we investigated the role of inflammation associated with transplantation on costimulation-independent priming of CD8+ T cell during graft rejection. B6 RAG1 KO mice were transplanted with BALB/c-skin and adoptively transferred with syngeneic CD8+ T cells the same day or 50 days after transplantation. When blockade of CD28- and CD40-mediated costimulation failed to prevent acute rejection of freshly transplanted skin grafts, it efficiently delayed rejection of well-healed skin grafts. These results showed that factors associated with transplantation have essential roles in inducing costimulation blockade-resistant allograft rejection. Costimulation blockade failed to prevent acute graft-infiltration of NK cells and increasing expression of intragraft IL-12 and IL-15. These factors may trigger the graft-infiltration and priming of CD8+ T cells to induce costimulation blockade-resistant allograft rejection.

ICOS is the third member of the CD28 family molecules and plays a critical role in many T cell-dependent immune responses. Although accumulated data suggest that ICOS costimulatory signals play an important role in Th2-mediated immune responses, the molecular basis for this selective differentiation mechanism is largely unknown. To clarify this mechanism, we used DO11.10 TCR transgenic ICOS−/− mice and evaluated the nature of ICOS costimulatory signals during the process of Ag-specific activation and differentiation of naive CD4+ T cells. Results obtained from these experiments demonstrated that Ag stimulation of naive CD4+ T cells in the absence of an ICOS signal resulted in impaired Th2 development. Unlike previous reports, we found that primary IL-4 production by these T cells was intact and that IL-4R sensitivity of these T cells was reduced as evidenced by a profound defect in IL-4-induced Stat6 phosphorylation and the early induction of GATA-3. The fact that ICOS ligation of wild-type T cells significantly enhanced IL-4-induced Stat6 phosphorylation and primary GATA-3 induction, but not IL-4 transcription, of naive CD4+ T cells was consistent with the results obtained from ICOS−/− T cell experiments. These observations led us to propose that the predominant effect of ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4R-mediated signaling.

The activation of naive T cells requires two signals from the antigen presenting cells (APC). Firstly, an antigen specific signal which is triggered by the binding of the T cell receptor (TCR) to the peptide-MHC complex, and secondly, antigen nonspecific signals initiated through a set of co-signalling receptors. Co-signalling molecules are cell-surface glycoproteins that play essential roles for the communication of a T cell with virtually all other host cells by modulating and fine-tuning TCR signals. On the basis of their functional outcome, co-signalling molecules can be divided into co-stimulators and co-inhibitors, which promote or suppress T-cell activation, respectively. By expression at the appropriate time and location, co-signalling molecules positively and negatively control the priming, growth, differentiation and functional maturation of a T-cell response. In this article, I overview property of co-signaling molecules in the CD28- and TNFR family and discuss their potential functional relationships with each other. In addition, role of these co-signalling molecules in various diseases, such as autoimmune diseases, graft rejection, allergy, inflammatory bowel disease, and cancer, and the therapeutic potential of targeting these molecules to enhance or curtail an ongoing immune response in these diseases are discussed.

The ICOS-B7RP-1-mediated T cell costimulatory pathway has been implicated crucial for T cell activation and differentiation. In this study, we investigated the role of this costimulation in the regulation of immune responses to parasitic infections by using blocking antibody against B7RP-1 as well as ICOS-deficient mice. The administration of anti-B7RP-1 monoclonal antibody (mAb) significantly suppressed the footpad swelling in susceptible BALB/c mice upon Leishmania major infection. The observation was consistent not only with the significant suppression of IL-4, IL-5 and IL-10 secretion from lymph node cells, which were derived from L. major -infected mice, but also with the significant reduction of total serum IgE and IgG 1 in anti-B7RP-1 mAb-treated BALB/c mice. Infection of ICOS-deficient mice with L. major also suggested the impaired Th2 immune responses in the absence of this costimulation. The immunological function of ICOS-B7RP-1 costimulatory pathway in infection was further confirmed by infecting anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice with Nippostrongylus brasiliensis . The characteristic elevation of total serum IgE and eosinophilia upon N. brasiliensis infection was suppressed by blocking this costimulation. Moreover, the protection to N. brasiliensis adult worms was suppressed in anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice. These results suggest the crucial role of this costimulatory pathway in the regulation of Th2-biased T cell differentiation and in host immune responses against L. major and N. brasiliensis infections.

The engagement of CD28 on T cells provides an essential costimulatory signal for T-cell activation and differentiation. Recent studies suggest that CTLA4Ig inhibits T-cell activation in vitro and in vivo, prevents acute and chronic allograft rejection, and induces tolerance in some experimental transplantation models.The present study examined the role of the CD28 costimulatory pathway in the induction of experimental anti-glomerular basement membrane (GBM) glomerulonephritis (GN). An accelerated type of anti-GBM GN was induced in wild-type mice and CD28-deficient (KO) mice. After 7 and 14 days, functional parameters, such as serum creatinine, amount of proteinuria, and serum mouse IgG levels were assessed. Histological studies were performed simultaneously to examine glomerular changes by light microscopy and immunoglobulin deposition by immunofluorescence staining. Flow cytometric analysis was undertaken to assess I-A(b) antigen expression on spleen cells.Anti-GBM GN induction was almost completely prevented in CD28-KO mice. CD28-KO mice had impaired ability to evoke B-cell activation, and developed lower mouse anti-rabbit IgG antibody titers in their serum than wild-type C57BL/6 mice. Furthermore, glomerular deposition of mouse anti-rabbit IgG was not detectable in CD28-KO mice after immunization with anti-GBM antibody.This is the first demonstration that the CD28 costimulatory pathway plays a critical role in autologous antibody production in anti-GBM GN, and suggests that effective inhibition of CD28-dependent autologous antibody production could be useful in the treatment of antibody-dependent GN in humans.

Suppressor of cytokine signaling (SOCS)3 has been characterized as a negative feedback regulator in cytokine-mediated Janus kinase signal transducer and activator of transcription signaling. However, this study shows that T cells from transgenic mice expressing SOCS3 exhibit a significant reduction in interleukin (IL)-2 production induced by T cell receptor cross-linking when T cells are costimulated with CD28. Decreased protein expression in SOCS3+/− mice enhanced CD28-mediated IL-2 production, clearly indicating the correlation between expression level of SOCS3 and IL-2 production ability. The SOCS3 protein interacted with phosphorylated CD28 through its SH2 domain but not the kinase inhibitory region. In addition, a point mutation in the SOCS3 SH2 domain attenuated the inhibition of CD28 function in IL-2 promoter activation. Committed T helper (Th)2 cells exclusively expressed SOCS3 and production of Th2 cytokines, such as IL-4 and IL-5, was much less dependent on CD28 costimulation compared with interferon γ and IL-2 production in Th1 cells. Consistent with this notion, the expression level of SOCS3 in early T cell activation influenced the ability of IL-2 production induced by CD28 costimulation. Therefore, the SOCS3 may play an alternative role in prohibiting excessive progression of CD28-mediated IL-2 production.

Maternal exposure to environmental factors is implicated as a major factor in the development of the immune system in newborns. Newborns are more susceptible to microbial infection because their immune system is immature. Development of lymphocytes reflects an innate program of lymphocyte proliferation. The aim of this study was to investigate the effects of maternal exposure to carbon black nanoparticle (CB-NP) during early gestation on the development of lymphoid tissues in infantile mice. Pregnant ICR mice were treated with a suspension of CB-NP (95 μg kg(-1) time(-1)) by intranasal instillation on gestational day 5 and 9. Spleen tissues were collected from offspring mice at 1, 3, 5, and 14 days postpartum. Splenocyte phenotypes were examined by investigating the pattern of surface molecules using flow cytometry. Gene expression in the spleen was examined by quantitative RT-PCR. CD3(+) (T), CD4(+) and CD8(+) cells were decreased in the spleen of 1-5-day-old offspring in the treated group. Expression level of Il15 was significantly increased in the spleen of newborn male offspring, and Ccr7 and Ccl19 were increased in the spleen of female offspring in the CB-NP group. Splenic mRNA change profiles by CBNP were similar between male and female offspring. This article concluded that exposure of pregnant mothers to CB-NP partially suppressed the development of the immune system of offspring mice. The decrease in splenic T cells in the treated group recovered at 14 days after birth. This is the first report of developmental effect of nanoparticle on the lymphatic phenotype.

The functional role of inducible costimulator (ICOS)-mediated costimulation was examined in an in vivo model of alloantigen-driven Th1 or Th2 cytokine responses, the parent-into-F1 model of acute or chronic graft-vs-host disease (GVHD), respectively. When the Ab specific for mouse ICOS was injected into chronic GVHD-induced mice, activation of B cells, production of autoantibody, and development of glomerulonephritis were strongly suppressed. In contrast, the same treatment enhanced donor T cell chimerism and host B cell depletion in acute GVHD induced host mice. Blocking of B7-CD28 interaction by injection of anti-B7-1 and anti-B7-2 Abs inhibited both acute and chronic GVHD. These observations clearly indicate that the costimulatory signal mediated by CD28 caused the initial allorecognition resulting in the clonal expansion of alloreactive T cells, whereas the costimulatory signal mediated by ICOS played a critical role in the functional differentiation and manifestation of alloreactive T cells. Furthermore, treatment with anti-ICOS Ab selectively suppresses Th2-dominant autoimmune disease.

We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4’s actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2.

T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [3H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [3H]thymidine incorporation.

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peri-transplant costimulatory blockade (CB) to promote long-term graft acceptance. After vascularized MHC-mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 post-transplant by 6.5 Gy irradiation or by administration of anti-CD4 plus anti-CD8 mAb. Long-term surviving allografts were gradually rejected after lymphodepletion (MST=74 ± 5 days post-irradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44high effector/memory T cells in lymphatic organs and strong recovery of donor-reactive T cell responses, indicating lymphopenia-induced-proliferation and donor alloimmune responses occurring in the host. T regulatory (CD4+Foxp3+) cell and B cell numbers as well as donor-specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by lymphopenia-induced proliferation of donor-reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.

Engagement of CD28 on T cells provides a co-stimulatory signal necessary for T cell activation and differentiation. Recent findings suggest that priming of T helper (Th)2 cells is more dependent on CD28 activation that Th1 cells. The present study examines whether mice that lack expression of CD28 as a result of gene targeting are capable of generating a Th2 response characteristic during infection with the intravascular trematode parasite Schistosoma mansoni. Mutant and control mice were either inoculated in the footpad with S. mansoni eggs (a potent inducer of a Th2 response) or infected percutaneously with the parasite. Draining lymph nodes (after footpad injection) or spleen cells (after natural infection) were harvested at 12 days and 8 weeks, respectively, and examined for cytokine responses to egg antigens. CD28-deficient mice (-/-) generated diminished egg antigen-driven interleukin (IL)-4 and IL-5 production (by 5- to 17-fold, respectively) compared to CD28-expressing (+/+) littermates. In contrast, lymphocyte proliferation and interferon (IFN)-gamma production to egg antigens were equivalent for mutant and control mice. Infected CD28-/- mice also had reduced immunoglobulin secretion. Serum levels of parasite antigen-specific IgG1 and polyclonal IgE were significantly diminished in CD28-/- compared to CD28+/+ mice. Lack of CD28 expression had no effect on granuloma formation around eggs trapped in the liver, but increased susceptibility of mice to primary schistosomiasis infection. These studies indicate that CD28 activation contributes to T cell priming required for generation of a Th2 response to an intravascular dwelling helminth parasite.

Recognition of self major histocompatability complex (MHC)-presented antigen (MHC:Ag) by the T cell antigen receptor (TCR) is by itself not sufficient to induce T cell proliferation. Rather, to be fully activated T cells require both a TCR-generated signal and a "costimulatory" signal. This important costimulatory signal is not completely understood. Recent evidence suggests that this costimulatory signal is generated by the interaction of the T cell CD28 receptor with the B7 counterreceptor found on antigen-presenting cells. Regulation of costimulation may well prove to be more complex than was previously imagined based on the discovery that CD28 and B7 are each members of larger gene families. The present review highlights recent advances in the understanding of the CD28 and B7 receptor families with an emphasis on controversies in the field. Certain forms of immunopathology that might result from the aberrant regulation of CD28 and/or B7 expression are also discussed.

Cyclometalated iridium(III) complexes have received considerable attention and are important candidates for use as luminescent probes for cellular imaging because of their potential photophysical properties. We previously reported that fac-Ir(atpy)(3)4 (atpy = 2-(5'-amino-4'-tolyl)pyridine) containing three amino groups at the 5'-position of the atpy ligand shows a maximum red emission (at around 600 nm) under neutral and basic conditions and a green emission (at 531 nm) at acidic pH (pH 3-4). In this Article, we report on the design and synthesis of a new pH-sensitive cyclometalated Ir(III) complex containing a 2-(5'-N,N-diethylamino-4'-tolyl)pyridine (deatpy) ligand, fac-Ir(deatpy)(3)5. The complex exhibits a considerable change in emission intensity between neutral and slightly acidic pH (pH 6.5-7.4). Luminescence microscopic studies using HeLa-S3 cells indicate that 5 can be used to selectively stain lysosome, an acidic organelle in cells. Moreover, complex 5 is capable of generating singlet oxygen in a pH-dependent manner and inducing the death of HeLa-S3 cells upon photoirradiation at 377 or 470 nm.

We have proposed a system that recognizes text characters from the region pointed at with a fingertip for the visually handicapped. For example, in a supermarket, the blind user can pick up and touch the goods. However, the user cannot read the characters printed on the surface of the object. He/she cannot exactly know what the object is. It is very inconvenient for him/her. The proposed system will help the user to recognize the characters on the object. The system detects the ring which the user wears on the index finger and calculates the position of the user's fingertip of the index finger. Some acrylic infrared-reflective beads are arranged on the ring and it is easy to detect them from an infrared image. After fingertip detection, a region in which characters to be recognized is generated according to the position of the fingertip. The characters are extracted within the region using image processing. Thus, the redundant character recognition is avoided. The extracted characters are combined as a text and the text is read aloud by OCR software and free reading software for the user. Some experiments show the basic validity of the proposed method and system.

T lymphocytes have been implicated in the onset of many autoimmune diseases; however, the mechanisms underlying T-cell activation toward self antigens are poorly understood. To study whether T-lymphocyte costimulation can overcome the immunologic unresponsiveness observed in an in vivo model, we have created transgenic mice expressing the costimulatory mouse molecule B7-1, a ligand for the CD28 receptor, on pancreatic beta cells. We now report that triple-transgenic mice expressing both B7-1 and a viral glycoprotein on their beta cells, along with T cells expressing the viral-glycoprotein-specific transgenic T-cell receptor, all develop insulitis (lymphocytic infiltration of the pancreatic islets) and diabetes. In striking contrast, the T cells in double-transgenic mice expressing the same viral glycoprotein (but no B7) on their pancreatic beta cells and the transgenic T-cell receptor on their T cells, reported earlier, remain indifferent to the glycoprotein-expressing beta cells. In fact, all three transgenes are required to initiate immune-mediated destruction of the beta cells. Mice expressing any of the transgenes alone, or any two in combination, maintain normal islet architecture and never spontaneously develop insulitis or diabetes. Our results show that aberrant B7 expression on peripheral tissues may play an important role in the activation of self-reactive T cells and further suggest that abnormal expression of costimulatory receptors may be involved in various T-cell-mediated autoimmune diseases.

In the present study, we examined the sequential changes of diethylnitrosamine (DEN)-induced hepatocarcinogenesis in Wistar rats. After 14 weeks of DEN treatment, hyperplastic nodules developed as a consequence of the appearance of renewed hepatocytes, degenerated hepatocytes, oval cells and fibrotic changes. Total bilirubin and alanine aminotransferase levels were significantly higher in the DEN group compared to the control group throughout the experimental period. Our data may prove beneficial to future analyses of chemopreventive compounds during various stages of hepatocarcinogenesis in rats.

Mixed hematopoietic chimerism provides a powerful means of achieving transplantation tolerance. We investigated the efficacy of combined blockade of the CD40/CD154 and CD28/B7 costimulation pathways to induce sustained mixed chimerism in cynomolgus monkeys following major histocompatibility complex-mismatched bone marrow (BM) transplants. A nonmyeloablative conditioning regimen of busulfan, intravenous and intraosseous ifosfamide, and anti-thymocyte globulin was used. BM transplantation was followed by a one-week course of CTLA4-Ig/anti-CD154 monoclonal antibodies. Three recipients achieved a wide range of transient chimerism (10.8-79.8%). A rapid proliferation of host effector memory (CD28(low)CD95(high)) CD8(+) T cells was observed in conditioned animals whether or not they received allogeneic BM, and this expansion occurred concurrently with the loss of chimerism in BM recipients. CD8(+) T cells from the recipients had increased reactivity to donor stimulators vs. third-party stimulators. Additional immunosuppression with tacrolimus or deoxyspergualin after transplantation delayed post-transplant proliferation of effector memory CD8(+) T cells but did not promote chimerism. A one-month course of costimulatory blockade also did not prevent marrow rejection. These studies demonstrate that combined CD40/CD154 and CD28/B7 costimulatory blockade supports transient mixed chimerism induction following nonmyeloablative conditioning in primates, but is insufficient to overcome host immune resistance likely mediated by effector memory CD8(+) T cells.

Although there is growing evidence that NKT cells play an important role in various immune responses through the invariant T cell receptor, other cell surface molecules responsible for their function are not fully understood. Here we study the role of ICOS, the third member of the CD28 family of costimulatory receptors, in in vivo and in vitro NKT cell responses. To establish its in vivo role in systems dependent on NKT cells, we examined the development of Con A-induced hepatitis in ICOS knockout (ICOS(-/-)) mice. We demonstrated that hepatic injury in ICOS(-/-) mice was greatly suppressed as evidenced by the reduced elevation of serum transaminases, reduced apoptosis of hepatocytes and mild histopathological changes. In investigating the cause of this defect, we first found that the NKT cell population is significantly reduced in the liver and spleen of ICOS(-/-) mice. We made and analyzed mixed bone marrow chimera mice with bone marrow cells from ICOS(+/+) and ICOS(-/-) mice, and demonstrated that the defect in ICOS-mediated costimulation results in a significant defect in the development of NKT cells, especially of Valpha14i NKT cells, in the thymus. When we examined the function of residual NKT cells in ICOS(-/-) mice, we found that their cytokine production following stimulation with alpha-galactosylceramide (alpha-GalCer) was strongly impaired. Based on these findings, we propose that ICOS-mediated costimulation may play a critical role in both the development and the optimal function of NKT cells, and that defective ICOS-mediated costimulation may result in impaired Con A-induced hepatitis in ICOS(-/-) mice.

Although it is well-known that the ICOS-ICOS ligand (ICOSL) costimulatory pathway is important for many immune responses, recent accumulated evidence suggests that dysregulation of this pathway may lead to and/or exaggerate autoimmune responses. ICOS is induced on the cell surface after T cell activation. Similarly, ICOSL is up-regulated on APCs by several mitogenic stimuli. However, the mechanism regulating expression of the ICOS-ICOSL pair, and the significance of controlling their expression for an appropriate immune response, is largely unknown. To gain a better understanding of the importance of fine control of the ICOS-ICOSL costimulatory pathway, we generated ICOS-transgenic (Tg) mice that have high constitutive expression of ICOS in all T cells. Using ICOS-Tg mice, we studied whether in vivo immune responses were affected. Unexpectedly, we first found that ICOS-Tg mice exhibited a phenotype resembling ICOS-deficient mice in their Ag-specific Ab response, such as a defect in class switch recombination. Further examination revealed that ICOSL expression of APCs was significantly suppressed in ICOS-Tg mice. Interestingly, suppression of ICOSL was induced by interaction of ICOSL with ICOS, and it seemed to be regulated at the posttranscriptional level. The suppressive effect of the ICOS-ICOSL interaction overcame the positive effect of CD40 or B cell activation factor of the TNF family (BAFF) stimulation on ICOSL expression. Together, our studies demonstrate a novel mechanism for the regulation of ICOSL expression in vivo and suggest that the ICOS costimulatory pathway is subject to negative feedback regulation by ICOSL down-regulation in response to ICOS expression.

Background. Immune regulatory CD4+CD25+ T (regulatory T; Treg) cells play a vital role in the induction and maintenance of self-tolerance. They are essential for the homeostasis of T cells, the prevention of autoimmunity, and the induction of tolerance to allogeneic donor grafts. However, the underlying mechanism of their functions remains mostly elusive. Therefore, we investigated here a crucial role of Treg cells in their response to alloantigen via the programmed death (PD)-1/PD-1 ligand (PD-L1) pathway. Methods. In vitro mixed lymphocyte reaction (MLR) assay, graft-versus-host disease (GvHD) and a skin transplantation model were used to evaluate the mechanisms of PD- 1/PD-L1 pathway. Results. Blockade of the PD-1/PD-L1 pathway using anti-PD-L1 monoclonal antibodies (mAb) is found to inhibit Treg cell's ability to suppress and restore CD4+CD25-T-cell proliferation in vitro. GvHD was lethal after adoptive transfer of allogeneic C57BL/6 (H-2K b ) spleen cells to NOD/SCID (H-2K d ) mice unless CD25+ T cells were also included. Strikingly, the suppression of GvHD by CD25+ cells was abrogated by anti-PD-L1 mAb administration. The abrogation of Treg-cell-mediated suppression could also be demonstrated in a Balb/c (H-2K d ) to B6/Rag-2KO (H-2K b ) skin-allograft model. Conclusions. The blockade of the PD-1/PD-L1 pathway abrogates Treg-mediated immunoregulation, thus suggesting that the PD-1/PD-L1 pathway is required for Treg suppression of the alloreactive responses of CD4 + CD25-T cells. This finding has important implications for clarifying the mechanisms of allograft rejection and GvHD.

Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-κB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-κB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-κB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-κB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.

To clarify the relationship between cellular immune status and nutritive condition, feeding program, blood profiles, and leukocyte populations were analyzed in two dairy herds experiencing frequent mastitis. Fourteen of the 35 lactating cows in herd A, and 18 of the 50 lactating cows in herd B scored positive on the California Mastitis Test (CMT), and 3 of the 73 lactating cows were CMT positive in herd C, which was the control. All herds were evaluated during five different milking stages, and blood was collected from five cows at each stage. With regard to feed content, the percentages of total digestible nutrients (TDN) and crude protein (CP) were found to be lower in herds A and B than in herd C. Levels of serum total cholesterol and blood urea nitrogen were lower in herds A and B than those in herd C. Neutrophil counts in herds A and B were increased compared to the neutrophil counts in herd C. On the other hand, the numbers of CD3(+) T cells and CD14-MHC class(+) cells were lower in herd A and B than in herd C. A decrease in peripheral lymphocytes and undernourishment were observed in the herds with frequent occurring mastitis.

We found that inducible costimulator (ICOS)-deficient spleen cells have a defect in the ability to induce Th2-mediated chronic graft-versus-host disease (GVHD), whereas they were fully capable of inducing Th1-mediated acute GVHD. In contrast, CD28-deficient spleen cells induced neither acute nor chronic GVHD. To define the mechanisms of how these two CD28 family molecules manifest distinct functions in GVHD, the kinetics of their surface expression by donor T cells in two types of GVHD were investigated. It was found that expression of ICOS by donor T cells dramatically up-regulated after adoptive transfer in both acute and chronic GVHD, but in acute GVHD this up-regulation was transient and quickly down-regulated. In contrast, donor T cells in chronic GVHD maintained high levels of ICOS expression throughout the experiment. These results suggested that ICOS-mediated costimulatory signals are predominantly active in Th2-dominant responses. Changing patterns of CD28 expression by donor T cell were identical in both GVHD as they exhibited slight but sustained up-regulation after transfer, suggesting that CD28 provides a costimulatory signal necessary for the initial T cell activation, regardless of whether in Th1 or Th2 responses. These results lead us to propose that the expression levels of costimulatory receptors are critical for determining the polarization of helper T cell function.

While the ligand of inducible co-stimulator (ICOS), B7 homologous protein, is widely expressed in somatic cells, B7-1 and B7-2 expression is mainly limited to lymphoid organs. Thus, the activation of T cells through ICOS without a CD28-mediated signal may occur in physiological situations. In order to gain a better understanding of the role of the ICOS co-stimulatory signal in immune responses, we studied the cellular response of T cells to beads coated with anti-ICOS or anti-CD28, plus sub-optimal anti-CD3 mAb. We demonstrate that while CD28 ligation induced expansion of both CD4+ and CD8+ populations, ICOS ligation only resulted in the expansion of CD8+ T cells, and induced apoptosis in the CD4+ T cell population. It was found that IL-2 is critically required for CD8+ T cell expansion triggered by ICOS ligation, whereas it had only a limited effect on the expansion of CD4+ T cells. This distinct reactivity of CD4+ and CD8+ T cell populations to exogenous IL-2 strongly correlates with the expression level of IL-2 receptor beta-chain, CD122, on T cells. Furthermore, we defined a small but distinct population of memory phenotype CD4+ T cells that constitutively express ICOS. Interestingly, while naive CD4+ T cells were unable to produce IL-2, ICOS-expressing T cells produced a substantial amount of IL-2 by stimulation with anti-ICOS/CD3 beads, suggesting that IL-2, which is indispensable for CD8+ T cell expansion, is produced by this ICOS-expressing T cell population. These results provide evidence indicating that the ICOS co-stimulatory signal plays a distinct role in the development of CD4+ and CD8+ T cell-mediated immune responses.

Inducible costimulator (ICOS) is the third member of the CD28 superfamily, expressed on antigen-primed T-cells, enhancing Th2 differentiation. Anti-glomerular basement membrane (anti-GBM) glomerulonephritis results from multiple effects generated by both Th1 and Th2 cells. To evaluate the contribution of these T-cells to the progression of anti-GBM glomerulonephritis, we investigated the effect of double blockade of CD28 and ICOS signaling. Anti-GBM glomerulonephritis was induced in C57BL/6 mice, a Th1-prone strain. CD28 signaling was inhibited with the use of fusion proteins of cytolytic T-lymphocyte-associated antigen-4 (CTLA4 immunoglobulin) and ICOS signaling by the monoclonal antibody (mAb) for ICOS. Blood and urine samples were collected 5 and 14 days after induction of anti-GBM glomerulonephritis. Mice were killed to facilitate histopathologic analyses at the same time. Anti-GBM glomerulonephritis was prevented from functionally deteriorating (eg, proteinuria or increasing serum creatinine) by CTLA4 immunoglobulin or anti-activation-inducible lymphocyte immunomodulatory molecule (AILIM) mAb as an anti-ICOS mAb. Nevertheless, double or single blockade of ICOS signaling showed a weaker inhibitory effect than single blockade of CD28 signaling in terms of the serum immunoglobulin level and histopathologic change. There is no synergistic effect between CTLA4 immunoglobulin and anti-AILIM mAb when simultaneously administered in experimental anti-GBM glomerulonephritis. Double blockade of both CD28 signaling and ICOS signaling is effective for preventing functional deterioration in this model. However, CD28 single blockade is more effective than double blockade both serologically and histopathologically.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses antibody production through activation of a transcription factor, the aryl hydrocarbon receptor (AhR). To explore the genes that are involved in the suppression of antibody production by TCDD, we investigated TCDD-induced changes in gene expression in the CD4 T cells and B cells of C57BL/6 mice immunized with ovalbumin (OVA) plus alum as an adjuvant. Changes in gene expression were analyzed with Affymetrix oligonucleotide microarrays. The results showed that OVA-immunization alone up-regulated expression levels of many genes in the CD4 T cells as early as 3 h after immunization, with 55 up-regulated and 5 down-regulated. At 24 h, 42 genes were found to be up-regulated and 30 down-regulated. Fewer genes were affected in the B cells than in the CD4 T cells. In contrast to the up-regulation of genes induced by immunization in the CD4 T cells, administration of TCDD to mice 3 h prior to the immunization mainly caused down-regulation of genes in the CD4 T cells when compared with immunization alone, with 1 being up-regulated and 4 down-regulated at 3 h after immunization and 3 up-regulated and 34 down-regulated at 24 h. In particular, at 3 and 24 h, TCDD suppressed expression of three and seven genes, respectively, that were up-regulated by immunization. Another characteristic of the TCDD-induced changes in gene expression was the suppression of many genes encoding proteins that are involved in GTP-binding protein-linked signaling in CD4 T cells. These results suggest that the inhibition of immunization-induced gene expression and modulation of G-protein-linked signaling in CD4 T cells are responsible for the TCDD-induced suppression of antibody production.

The signal transduction of the cAMP/cAMP-dependent protein kinase [protein kinase A (PKA)] pathway through multiple receptors is critical for many processes in all cell types. In T cells, the engagement of both the TCR-CD3 complex and the CD28 co-stimulatory molecule also induces cAMP, and subsequently activates PKA. It is believed that elevation of cAMP levels in T cells is inhibitory of IL-2 production and T cell proliferation. However, the function and detailed signal transduction mechanisms of the cAMP/PKA pathway in naive T(h) cells are less well understood. In this study, we show that calcitonin gene-related peptide (CGRP) down-regulates IL-2 and IFN-gamma production and up-regulates IL-4 production to promote T(h)2 differentiation by moderate activation of the cAMP/PKA pathway via the CGRP receptor in the presence of a CD3/CD28 co-stimulation signal. The IL-4 production and transcriptional activation of T(h)2 cytokine mRNAs were also reproduced by the addition of a cAMP analogue, dibutyryl-cAMP, in CD3/CD28-stimulated naive T(h) cells. More interestingly, cAMP/PKA activation in naive T(h) cells stimulated with anti-CD3 plus anti-CD28 mAb is essential for inducing IL-4 production and promoting T(h)2 differentiation; in addition, NF-AT is a downstream effector of the cAMP/PKA signaling pathway. These findings indicate that the cAMP/PKA pathway transduces the critical activation signal to T(h)2 polarization by a CD3/CD28 co-stimulation signal and a PKA activating reagent.

We generated T cell receptor transgenic mice specific for the liver stages of the rodent malaria parasite Plasmodium yoelii and studied the early events in the development of in vivo effector functions in antigen-specific CD8(+) T cells. Differently to activated/memory cells, naive CD8(+) T cells are not capable of exerting antiparasitic activity unless previously primed by parasite immunization. While naive cells need to differentiate before achieving effector status, the time required for this process is very short. Indeed, interferon (IFN)-gamma and perforin mRNA are detectable 24 h after immunization and IFN-gamma secretion and cytotoxic activity are detected ex vivo 24 and 48 h after immunization, respectively. In contrast, the proliferation of CD8(+) T cells begins after 24 h and an increase in the total number of antigen-specific cells is detected only after 48 h. Remarkably, a strong CD8(+) T cell-mediated inhibition of parasite development is observed in mice challenged with viable parasites only 24 h after immunization with attenuated parasites. These results indicate that differentiation of naive CD8(+) T cells does not begin only after extensive cell division, rather this process precedes or occurs simultaneously with proliferation.

Commonly used inbred mouse strains express different combinations of integrated mouse mammary tumor proviruses (MMTV). This appendix summarizes the proviruses that have been detected. The reported functional properties of those MMTV proviral products which have been identified as superantigens are also summarized, including the ability to elicit primary or secondary T cell responses and to induce Vb-specific clonal deletion during T cell differentiation. In addition, the amino acid sequences of putative ORF gene products of different MMTV are compared.

The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.