Addition Of Exogenous IL-2 Research Articles

Interleukin-10 enhances IL-33-mediated MC activation and modulates the development of food allergy

Abstract We recently demonstrated an unexpected, proinflammatory role for the pleiotropic cytokine, IL-10, in promoting mast cell (MC) responses during food allergy. IL-10 enhanced MC proliferation and survival and promoted IgE-dependent responses to enteric ovalbumin (OVA) challenge. However, whether the effects of IL-10 on MCs extend beyond IgE-mediated signaling is not clear. To determine whether IL-10 can prime MC activation mediated by IgE-independent stimuli, we assessed the effects of rIL-10 on IL-33-stimulated bone marrow-derived MCs (BMMCs) and examined the development of food allergy in IL-10-depleted ST2−/− mice. IL-10 co-culture significantly enhanced the activity and cytokine production of both IgE and IL-33-activated MCs. Furthermore, the production of MC-derived cytokines such as IL-13 was suppressed in IL-33-stimulated IL-10−/− BMMCs, which could be restored by addition of exogenous IL-10. IL-10 enhanced MC FcɛRI and ST2 expression. Further, ST2−/− BMMCs demonstrated that IL-10 enhanced IgE-mediated activation in the absence of autocrine IL-33 signaling. To assess the role of IL-10 in vivo, food allergy was measured in WT and ST2−/− mice subjected to antibody-mediated IL-10 depletion. IL-10-depleted WT mice exhibited a significant attenuation in allergic diarrhea and MC-mediated responses to OVA challenge. Similarly, ST2−/− mice also exhibited a profound suppression of MC-mediated responses, demonstrating the importance of IL-33 in MC regulation. IL-10 depletion had no additional effects in ST2−/− mice. However, IL-10/ST2−/− mice exhibited a further decrease in OVA-IgE and MC activation compared to ST2−/− mice. Our data reveal a novel role for IL-10 in enhancing both IgE and IL-33-mediated MC responses. This project was supported by funds from the National Institutes of Health grants: NIAID R15AI107668 (CBM)

P294 AMT-101, a gut selective oral IL-10 fusion: results from a Phase 1b study in patients with active Ulcerative Colitis

Abstract Background IL-10 is a pleiotropic anti-inflammatory cytokine that restores mucosal homeostasis. Genetic deficiencies in IL-10 cause intestinal inflammation in mice and are associated with early onset IBD in humans. In mice, this can be reversed by addition of exogenous IL-10. Subcutaneous use of recombinant human IL-10 treatment in patients with Crohn’s Disease led to symptomatic and endoscopic improvement, but dose limiting systemic toxicity including anemia and thrombocytopenia was observed and appeared to be mediated by induction of gamma-interferon. AMT-101 is an oral, gut selective, fusion protein of IL-10 and a carrier protein that mediates transcytosis through intestinal enterocytes into the lamina propria (LP). Pre-clinical studies demonstrated that AMT-101 efficiently transits the epithelial cell barrier, targets mucosal immune cells in the LP, and results in improved colitis disease severity without systemic exposure. Methods A multiple ascending dose Ph1b POC trial was conducted in 16 patients with active UC where patients were randomized 3:1 to receive AMT-101 at doses of 1, 3, 10, or 30 mg or PBO, orally, once daily for 14 days. Primary and secondary outcome measures included safety and PK. Exploratory endpoints included changes in fecal calprotectin, histology, and stool microbiome. Results AMT-101 was safe and well tolerated; all AEs were mild to moderate and self limiting. No systemic toxicities associated with prior administration of IL-10 (e.g.anemia or thrombocytopenia) were observed. Systemic levels of AMT-101 were below limit of quantitation, given the GI restricted design. 1 mg and 3 mg AMT-101 led to placebo-adjusted mean reductions of FCP of 44% and 27%, respectively, in patients with baseline FCP>150 μg/g. Paired biopsy samples were centrally read in a blinded manner using the Geboes scoring system. Improvements were observed in 60% (6/10) of patients on AMT-101, compared with 0% (0/2) patients treated with placebo. Microbiome analyses revealed changes in phyla abundance in the 1mg and 3mg treatment cohorts (Bacteroidetes: 28.3% ± 31.4 to 51.3% ± 21.6; Firmicutes: 21.8% ± 12.7 to 30% ± 7.3), including Roseburia hominis and Faecalibacterium prausnitzii species. Conclusion The results of this Phase 1b study confirm that once daily, oral AMT-101 was safe and well tolerated without SAEs previously observed with systemic IL-10 and without systemic exposure, by design. Doses at 10 mg or less suggest potential clinical efficacy paired with an enhancement of favorable enteric commensal bacteria after only 14 days of treatment. These findings support AMT-101 as actively exerting an immunomodulatory effect in the intestinal lamina propria and support ongoing Ph2 trials of AMT-101.

Eomesodermin promotes a central memory phenotype in CD8+ T cells through the induction of IL-10.

Abstract Following an immune response, a fraction of antigen-specific T cells persist as memory cells. Of these, central memory CD8+ T cells (TCM), which express secondary lymphoid organ-homing molecules and self-renew efficiently, are critical for maintaining long term memory. High levels of the T-box transcription factor Eomesodermin (Eomes) are associated with the TCM phenotype, but the mechanisms by which Eomes controls different aspects of TCM function are poorly understood. Using Eomes-knockout (EKO) CD8+ T cells, we demonstrate that Eomes regulates the expression of a number of TCM-associated markers including Bcl6, Ly6C and CD62L. In addition, we found that Eomes promotes the expression of IL-10 in activated CD8+ T cells. Interestingly, the addition of exogenous IL-10 was sufficient to upregulate CD62L expression in WT cells. Taken together, our data suggest that Eomes uses IL-10 to promote at least part of the central memory phenotype in CD8+ T cells. Consistent with this model, we find that TCM are secreting IL-10 in vivo, without additional re-stimulation. These findings warrant a modified model of TCM differentiation which includes distinct roles for Eomes and IL-10 in a T cell-intrinsic program driving TCM formation.

Critical role for OX40 signaling in Thymic and Peripheral Treg Expansion

Abstract Regulatory T-cells (Treg) play a pivotal role in suppressing self-reactive effector T-cells (Teff) and help maintain the critical balance between self-tolerance and autoimmunity. Restoration of homeostatic balance between Teff/Treg cells has been shown to ameliorate many autoimmune diseases including type-1 diabetes. Recently, we have reported a novel strategy for the selective expansion of Tregs using OX40L (a TNF-superfamily ligand) and Jagged (JAG)-1 (a Notch family ligand) treatment independent of canonical TCR-signaling, but mediated through specific interaction between OX40L-OX40 and JAG1-Notch3 preferentially expressed on Tregs. OX40L-JAG1 treatment of NOD mice with soluble OX40L and JAG1 increased proliferation of peripheral Tregs and delayed the onset of diabetes. In the present study, we show that OX40L treatment significantly increased Tregs in the thymus by inducing proliferation of thymic Tregs independent of TCR-stimulation upon addition of exogenous IL-2. OX40−/− mice had reduced numbers of thymic Tregs compared to wild type (WT) controls. The reduction in thymic Treg numbers likely due to impaired ability of proliferation in response to OX40L. However, we did not observe any change in IL-2 sensitivity of thymocytes in OX40−/− mice. Mechanistic studies suggested that OX40L-IL-2 induced thymic Treg proliferation was mediated through NF-kB and AKT-mTOR-S6 signaling. Further, phenotypic characterization studies revealed a significant decrease in TIGIT+Treg population, often associated with Treg activation, in both thymus and spleen of OX40−/− mice compared to WT controls. Taken together, these results suggest a critical role for OX40L/OX40 signaling in the development as well as function of Tregs.

A novel, intergenic enhancer contributes to Il2 gene expression via chromosomal looping.

Abstract IL-2 is a key immunoregulatory cytokine with pleotropic effects on many immune cell types. Its expression is highly regulated in T cells through multiple signaling pathways, chromatin remodeling, DNA methylation, and cooperative transcription factor binding. Recently, we identified an evolutionarily conserved intergenic enhancer 83 kb upstream of the Il2 gene that augments transcription from the Il2 promoter and upstream regulatory region (URR) by >50-fold in recombinant reporter assays. Upon TCR/CD28 co-stimulation of primary CD4+ T cells, a long-range chromosomal loop is established between the −83 kb CNS and the endogenous Il2 promoter, and both regions become epigenetically marked by histone acetylation and methylation. To determine the relative contribution of the URR vs. this new distal element to inducible transcription of the Il2 gene, we deleted the URR or the −83 kb CNS in mice using CRISPR/CAS genome editing. Deletion of the URR (~500 bp beyond the TATA element) completely abolished the Il2 transcription by CD4+ T cells, while a ~500 bp deletion of the −83 kb CNS resulted in a 2- to 4-fold decrease in IL-2 production. Young mice deficient for the −83 kb enhancer exhibited no major defects in lymphoid development, however, mutant CD4+ T cells showed reduced proliferative capacity in vitro. Furthermore, TGFB-induced iTreg differentiation from mutant conventional CD4+ precursors was strongly impaired, but could be fully restored by addition of exogenous IL-2. These results show that induction of Il2 depends upon cooperation between the promoter-URR and at least one intergenic enhancer, and suggest mechanisms by which graded expression of Il2 might be achieved through ‘modular’ recruitment of distinct enhancer elements.

Altered Natural Killer Cell Function in HIV-Exposed Uninfected Infants.

HIV-exposed uninfected (HEU) infants have higher rates of severe and fatal infections compared with HIV-unexposed (HUU) infants, likely due to immune perturbations. We hypothesized that alterations in natural killer (NK) cell activity might occur in HEU infants and predispose them to severe infections. Case-control study using cryopreserved peripheral blood mononuclear cells (PBMCs) at birth and 6 months from HEU infants enrolled from 2002 to 2009 and HUU infants enrolled from 2011 to 2013. NK cell phenotype and function were assessed by flow cytometry after 20-h incubation with and without K562 cells. The proportion of NK cells among PBMCs was lower at birth in 12 HEU vs. 22 HUU (1.68 vs. 10.30%, p < 0.0001) and at 6 months in 52 HEU vs. 72 HUU (3.09 vs. 4.65%, p = 0.0005). At birth, HEU NK cells demonstrated increased killing of K562 target cells (p < 0.0001) and increased expression of CD107a (21.65 vs. 12.70%, p = 0.047), but these differences resolved by 6 months. Stimulated HEU NK cells produced less interferon (IFN)γ at birth (0.77 vs. 2.64%, p = 0.008) and at 6 months (4.12 vs. 8.39%, p = 0.001), and showed reduced perforin staining at 6 months (66.95 vs. 77.30%, p = 0.0008). Analysis of cell culture supernatants indicated that lower NK cell activity in HEU was associated with reduced interleukin (IL)-12, IL-15, and IL-18. Addition of recombinant human IL-12 to stimulated HEU PBMCs restored IFNγ production to that seen in stimulated HUU cultures. NK cell proportion, phenotype, and function are altered in HEU infants. NK cell cytotoxicity and degranulation are increased in HEU at birth, but HEU NK cells have reduced IFNγ and perforin production, suggesting an adequate initial response, but decreased functional reserve. NK cell function improved with addition of exogenous IL-12, implicating impaired production of IL-12 by accessory cells. Alterations in NK cell and accessory cell function may contribute to the increased susceptibility to infection in HEU infants.

Mesenchymal Stem Cells Induced In Vitro Generated Regulatory-T Cells: Potential Soldiers of Transplantation Biology

Introduction: CD4+ regulatory T-cells that constitutively express CD25 have a potent mechanism of action of immunomodulation and hence hold a great therapeutic potential for the treatment of autoimmune diseases and tolerance induction in the context of solid organ transplantation. Methods: For in vitro generation of Tregs volunteer donor’s adipose tissue was resected and mesenchymal stem cells (MSC) were generated. MSC were co-cultured with post-renal transplantation recipient’s peripheral blood derived peripheral blood mononuclear cells (PBMC) and with irradiated PBMC from same recipient with the addition of exogenous IL-2. Controls were carried out without use of MSC. In vitro generated Tregs were subjected for quantitative and qualitative analysis of Tregs specific markers. Results: Quantitative analysis revealed increased cell counts of Tregs in comparison to PBMC. Tregs specific markers evaluation using flocytometer showed rise in all Treg specific cell population: CD4+25+127Neg with mean 7.6 ± 6.1%, CD4+FoxP3+ mean of 21.7 ± 15.3% and CD4+CTLA-4+ mean of 16.9 ± 13.7%. Conclusion: We conclude that MSC induce in vitro generation of Tregs. In vitro generated Tregs hold a great promise as “magic bullet” for various disease prevention and/or treatment. Keywords: Regulatory-T cells, In vitro generation, mesenchymal stem cells, co-culture, IL-2.

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3+ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3+CD103+CD38− stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3+CD103−CD38+ labile-phenotype Tregs in the thymus and increased autoreactive CD4+ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4+ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.

The tryptophan metabolite picolinic acid suppresses proliferation and metabolic activity of CD4+ T cells and inhibits c-Myc activation.

Tryptophan metabolites, including kynurenine, 3-hydroxyanthranilic acid, and picolinic acid, are key mediators of immunosuppression by cells expressing the tryptophan-catabolizing enzyme indoleamine2,3-dioxygenase. In this study, we assessed the influence of picolinic acid on cell viability and effector functions of CD4(+)T cells following in vitro activation with agonistic anti-CD3/anti-CD28 antibodies. In contrast to kynurenine and 3-hydroxyanthranilic acid, exposure of T cells with picolinic acid did not affect cell viability, whereas proliferation and metabolic activity were suppressed in a dose-dependent manner. On the other hand, cytokine secretion and up-regulation of cell surface activation markers were not or only weakly inhibited by picolinic acid. Picolinic acid exposure induced a state of deep anergy that could not be overcome by the addition of exogenous IL-2 and inhibited Th cell polarization. On the molecular level, important upstream signaling molecules, such as the MAPKs ERK and p38 and the mammalian target of rapamycin target protein S6 ribosomal protein, were not affected by picolinic acid. Likewise, NFAT, NF-κB, and AP-1 promoter activity in Jurkat T cells was not influenced by exposure to picolinic acid. Whereas transcriptional levels of v-myc avian myelocytomatosis viral oncogene homolog were not affected by picolinic acid, phosphorylation at Ser62 was strongly reduced in picolinic acid-exposed T cells following activation. In conclusion, picolinic acid mediates a unique immunosuppressive program in T cells, mainly inhibiting cell cycle and metabolic activity, while leaving other effector functions intact. These functional features are accompanied by reduced phosphorylation of v-myc avian myelocytomatosis viral oncogene homolog. It remains to be determined whether this effect is mediated by direct inhibition of ERK activity or whether indirect mechanisms apply.

Immunization with Recombinant Adenoviral Vectors Expressing HCV Core or F Proteins Leads to T Cells with Reduced Effector Molecules Granzyme B and IFN-γ: A Potential New Strategy for Immune Evasion in HCV Infection.

Multispecific, broad, and potent T cell responses have been correlated with viral clearance in hepatitis C virus (HCV) infection. However, the majority of infected patients develop chronic infection, suggesting that natural infection mostly leads to development of inefficient T cell immunity. Multiple mechanisms of immune modulation and evasion have been shown in HCV infection through various investigations. This study examined the generation and modulation of T cell responses against core and frameshift (F) proteins of HCV. A single immunization of mice with replication incompetent recombinant adenovirus vectors encoding for F or core antigens induces poor T cell responses and leads to generation of CD4+ and CD8+ T cells with low granzyme B (GrB) expression. These T cells have impaired GrB enzyme activity and are unable to kill peptide loaded target cells. The low intracellular expression of GrB is not due to degranulation of cytotoxic granules containing cytotoxic T cells. Addition of exogenous IL-2 in in vitro cultures leads to partial recovery of GrB production, whereas immunization with the Toll-like receptor (TLR) agonist poly I:C leads to complete restoration of GrB expression in both CD4+ and CD8+ T cells. Thus, a possible new strategy of T cell modulation is recognized wherein effector T cells are caused to be dysfunctional by HCV-derived antigens F or core, and strategies are also delineated to overcome this dysfunction. These studies are important in the investigation of prophylactic vaccine and immunotherapy strategies for HCV infection.

Regulation of CD4+ T cell activation and effector function by the histone acetyltransferase Myst1 (IRM6P.662)

Abstract Chromatin remodeling events are central molecular mechanisms that serve to govern CD4+ T cell function through regulation of gene accessibility. Myst1 is a histone acetyltransferase responsible for the acetylation of the protein tail of histone 4 at lysine residue 16, and is thought to participate in the activation of gene transcription. Studies of murine and human CD4+ T cells indicate that Myst1 is upregulated upon T cell receptor stimulus. Deletion of the gene encoding Myst1 resulted in a significant proliferation defect in these cells that is unaffected by the addition of exogenous IL-2. Myst1-deficient T cells also exhibit modulations in cytokine production and lineage commitment following skewing to various effector lineages in vitro. Additionally, signal transduction via STAT5 may play a role in Myst1 expression, as downregulation of STAT5a/b expression via siRNA transfection significantly decreased Myst1 expression levels in T cells. These results suggest an important role for the STAT5-Myst1 axis in governing the activation and effector function of CD4+ T cells.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies.

To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.

TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses.

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction.

IntroductionActivation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology.MethodsAntigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays.ResultsIn AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis.ConclusionsThese data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0419-y) contains supplementary material, which is available to authorized users.

T‐cell suppression by red blood cells is dependent on intact cells and is a consequence of blood bank processing

Red blood cells (RBCs) suppress T-cell responsiveness through a mechanism requiring cell-cell contact. Questions remain as to whether this effect is an allogeneic response, related to cell death, or dependent on particular components of the RBCs. Peripheral T cells were isolated from healthy donors and exposed to stored allogeneic RBCs or autologous RBCs after processing. RBCs were lysed by hypotonic solvent to produce cellular ghosts. Tritiated thymidine proliferation assays were utilized. Cultures were saturated with interleukin (IL)-2 to determine whether impaired IL-2 synthesis played a role. T-cell proliferation was suppressed by both autologous and allogeneic RBCs. RBC membrane integrity does enhance T-cell suppression. T-cell death is not responsible for the suppressive changes. IL-2 synthesis is suppressed in RBC-exposed T cells but addition of exogenous IL-2 does not rescue proliferative capabilities. Proliferation of T cells was inhibited with RBC exposure but mitigated with the addition of fresh RBCs. T-cell suppression is enhanced by intact RBCs but this effect is unrelated solely to alloantigens. Neither apoptosis nor necrosis of T cells contributes to this phenomenon. IL-2 synthesis is suppressed after RBC exposure as a consequence of T-cell inhibition, but is not the primary cause of suppression. Fresh RBCs do not mediate T-cell suppression, indicating that changes in the RBC and development of the storage lesion may occur during initial blood bank processing.

Inhibitory Effects of Trypanosoma cruzi Sialoglycoproteins on CD4+ T Cells Are Associated with Increased Susceptibility to Infection

BackgroundThe Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens characterized by decreased IL-2 synthesis. Trypanosoma cruzi mucin (Tc Muc) has been implicated in this phenomenom. These molecules contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues.Methodology/Principal FindingsIn this study, we evaluated the ability of Tc Muc to modulate the activation of CD4+ T cells. Our data show that cross-linking of CD3 on naïve CD4+ T cells in the presence of Tc Muc resulted in the inhibition of both cytokine secretion and proliferation. We further show that the sialylated O-Linked Glycan residues from tc mucin potentiate the suppression of T cell response by inducing G1-phase cell cycle arrest associated with upregulation of mitogen inhibitor p27kip1. These inhibitory effects cannot be reversed by the addition of exogenous IL-2, rendering CD4+ T cells anergic when activated by TCR triggering. Additionally, in vivo administration of Tc Muc during T. cruzi infection enhanced parasitemia and aggravated heart damage. Analysis of recall responses during infection showed lower frequencies of IFN-γ producing CD4+ T cells in the spleen of Tc Muc treated mice, compared to untreated controls.Conclusions/SignificanceOur results indicate that Tc Muc mediates inhibitory efects on CD4+ T expansion and cytokine production, by blocking cell cycle progression in the G1 phase. We propose that the sialyl motif of Tc Muc is able to interact with sialic acid-binding Ig-like lectins (Siglecs) on CD4+ T cells, which may allow the parasite to modulate the immune system.

102 : Interleukin 10 control of the inflammasome restricts arthritic bone erosion

Members of the nucleotide-binding oligomerization domain receptor (NOD)-like receptor (NLR) family are implicated in the pathology of various auto-inflammatory and autoimmune diseases. While the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Signaling by pattern recognition receptors is often inhibited by the inflammatory cytokine interleukin (IL)-10. In antigen-induced arthritis, IL-10-deficient (IL-10KO) mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with an increase in tartrate resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1 β . When compared to wild type controls, mRNA from the inflamed synovium of IL-10KO mice showed increased expression of Il1b , Il33 and components of the NLRP3 inflammasome. Furthermore, synovial Nlrp3 and Caspase 1 ( Casp1) expression correlated with Acid phosphatase 5, tartrate resistance ( Acp5) (encoding TRAP) expression, while neutralization of IL-10 receptor signaling in wild type mice caused increased expression of Nlrp3 and Casp1 . In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. Thus, IL-10 regulation of the inflammasome may impact homeostatic bone turnover and limit the degree of bone erosions observed in inflammatory arthritis.

Regulatory CD8+ T Cells Associated with Erosion of Immune Surveillance in Persistent Virus Infection Suppress In Vitro and Have a Reversible Proliferative Defect

CD4(+) T cell help is critical for CD8(+) T cell memory and immune surveillance against persistent virus infections. Our recent data have showed the lack of CD4(+) T cells leads to the generation of an IL-10-producing CD8(+) T cell population during persistent murine γ-herpesvirus-68 (MHV-68) infection. IL-10 from these cells is partly responsible for erosion in immune surveillance, leading to spontaneous virus reactivation in lungs. In this study, we further characterized the generation, phenotype, and function of these IL-10-producing CD8(+) T cells by comparing with a newly identified IL-10-producing CD8(+) T cell population present during the acute stage of the infection. The IL-10-producing CD8(+) populations in acute and chronic stages differed in their requirement for CD4(+) T cell help, the dependence on IL-2/CD25 and CD40-CD40L pathways, and the ability to proliferate in vitro in response to anti-CD3 stimulation. IL-10-producing CD8(+) T cells in the chronic stage showed a distinct immunophenotypic profile, sharing partial overlap with the markers of previously reported regulatory CD8(+) T cells, and suppressed the proliferation of naive CD8(+) T cells. Notably, they retained the ability to produce effector cytokines and cytotoxic activity. In addition, the proliferative defect of the cells could be restored by addition of exogenous IL-2 or blockade of IL-10. These data suggest that the IL-10-producing CD8(+) T cells arising in chronic MHV-68 infection in the absence of CD4(+) T cell help belong to a subset of CD8(+) regulatory T cells.

Inhibition of accelerated rejection mediated by alloreactive CD4+ memory T cells and prolonged allograft survival by arsenic trioxide

The aim of this study was to evaluate and determine the potential mechanisms of As2O3 in accelerated rejection mediated by alloreactive CD4+ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4+ memory T cells) was performed on Day 0, and As2O3 was administered to recipient mice from Day 0 to 10. As a result, As2O3 could reduce the proliferation of allo-primed CD4+ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As2O3 group was prolonged from 5.8 ± 0.7 to 14.2 ± 2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As2O3 treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4+ T cells, CD4+ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As2O3 as a novel immunosuppressant targeting CD4+ memory T cells.

Inhibiting accelerated rejection mediated by alloreactive CD4+ memory T cells and prolonging allograft survival by 1α,25-dihydroxyvitamin D3 in nude mice

Donor-reactive memory T cells are major barriers to long-term survival of transplanted organs due to their capacity to accelerate rejection. In this study we investigated the ability of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] to inhibit accelerated rejection mediated by alloreactive CD4+ memory T cells and to prolong cardiac allograft survival in an adoptive T cell memory/heart transplant model of nude mice. In vitro, the proliferation of CD4+ memory T cells was significantly inhibited by 1,25(OH)2D3 and was restored following addition of exogenous IL-2. Compared with the control group, the mean survival time of cardiac allografts in the 1,25(OH)2D3 group was prolonged from 6.5±0.3 to 20.2±0.8days in vivo. Five days after transplantation, the levels of IL-2 and IFN-γ were reduced in the grafts and the recipient sera by 1,25(OH)2D3 treatment, while that of IL-10 increased. The proportions of CD4+ memory T cells and CD4+Foxp3+ T cells, both in recipient spleen and lymph nodes, were lowered by 1,25(OH)2D3 treatment when compared with the control group. Our data suggests that 1,25(OH)2D3 inhibits expansion of CD4+ memory T cells, possibly by inducing clonal anergy and/or clonal deletion, resulting in prolongation of cardiac allograft survival in nude mice. These results may provide a rational basis for exploiting 1,25(OH)2D3 as a novel immunosuppressant targeting CD4+ memory T cells.