Galectin-3 Ablation Enhances Liver Steatosis,but Attenuates Inflammation and IL-33-Dependent Fibrosis in Obesogenic Mouse Model of Nonalcoholic Steatohepatitis

The importance of Galectin-3 (Gal-3) in obesity-associated liver pathology is incompletely defined. To dissect the role of Gal-3 in fibrotic nonalcoholic steatohepatitis (NASH), Gal-3-deficient (LGALS3^−/−) and wild-type (LGALS3^+/+) C57Bl/6 mice were placed on an obesogenic high fat diet (HFD, 60% kcal fat) or standard chow diet for 12 and 24 wks. Compared to WT mice, HFD-fed LGALS3^−/− mice developed, in addition to increased visceral adiposity and diabetes, marked liver steatosis, which was accompanied with higher expression of hepatic PPAR-γ, Cd36, Abca-1 and FAS. However, as opposed to LGALS3^−/− mice, hepatocellular damage, inflammation and fibrosis were more extensive in WT mice which had an elevated number of mature myeloid dendritic cells, proinflammatory CD11b⁺Ly6C^hi monocytes/macrophages in liver, peripheral blood and bone marrow, and increased hepatic CCL2, F4/80, CD11c, TLR4, CD14, NLRP3 inflammasome, IL-1β and NADPH-oxidase enzymes mRNA expression. Thus, obesity-driven greater steatosis was uncoupled with attenuated fibrotic NASH in Gal-3-deficient mice. HFD-fed WT mice had a higher number of hepatocytes that strongly expressed IL-33 and hepatic CD11b⁺IL-13⁺ cells, increased levels of IL-33 and IL-13 and up-regulated IL-33, ST2 and IL-13 mRNA in liver compared with LGALS3^−/− mice. IL-33 failed to induce ST2 upregulation and IL-13 production by LGALS3^−/− peritoneal macrophages in vitro. Administration of IL-33 in vivo enhanced liver fibrosis in HFD-fed mice in both genotypes, albeit to a significantly lower extent in LGALS3^−/− mice, which was associated with less numerous hepatic IL-13-expressing CD11b⁺ cells. The present study provides evidence of a novel role for Gal-3 in regulating IL-33-dependent liver fibrosis.     

Astaxanthin protects against renal fibrosis through inhibiting myofibroblast activation and promoting CD8+ T cell recruitment

BackgroundRenal fibrosis is a common pathological hallmark of chronic kidney disease, and no effective treatment is clinically available to manage its progression. Astaxanthin was recently found to be anti-fibrotic, but its effect on renal fibrosis remains unclear.MethodsC57BL/6J mice were subjected to unilateral ureteral obstruction and intragastrically administered astaxanthin. Histopathology and immunohistochemistry were performed to evaluate renal fibrosis. Flow cytometry was used to examine lymphocyte accumulation in the fibrotic kidneys. Western blotting, real-time qPCR, and immunofluorescence were performed to cover the underlying mechanism concerning astaxanthin treatment during renal fibrosis.ResultsOral administration of astaxanthin effectively alleviates renal fibrosis in mice. In vitro, astaxanthin inhibited fibroblast activation by modulating Smad2, Akt and STAT3 pathways and suppressed epithelial-to-mesenchymal transition in renal tubular epithelial cells through Smad2, snail, and β-catenin. Moreover, astaxanthin significantly induced the rapid accumulation of CD8⁺ T cells in fibrotic kidneys, which was accompanied by elevated expression of IFN-γ. Accordingly, the depletion of CD8⁺ T cells strongly diminished the protective effect of astaxanthin. Further investigation showed that astaxanthin increased the population of CD8⁺ T cells by upregulating the expression of CCL5 in macrophages.ConclusionsThese findings highlight the beneficial effect of astaxanthin on fibroblast activation, epithelial-to-mesenchymal transition, and CD8⁺ T cell recruitment during renal fibrosis.General significanceThese data indicate that astaxanthin could serve as a therapeutic strategy to treat renal fibrotic conditions.     

CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vβ4⁺ CD8⁺ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8⁺ T cells and MHV68 epitope specific CD8⁺ T cells to the lungs—despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vβ4⁺ CD8⁺ T cell expansion, eliminated MHV68-induced fibrosis—further implicating the activated Vβ4⁺ CD8⁺ T cell population in the induction of fibrosis. We further addressed the role that CD8⁺ T cells play in the induction of fibrosis by depleting CD8⁺ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vβ4⁺ CD8⁺ T cells as mediators of fibrotic disease in IFNγR-/- mice.     

Interleukin 35 (IL-35) and IL-37: Intestinal and peripheral expression by T and B regulatory cells in patients with Inflammatory Bowel Disease

The aim of the study was to characterize and to quantify peripheral and tissue. IL-35— and IL-37—producing cells in Inflammatory Bowel Disease (IBD) patients. We studied a total of 38 active UC, 31 inactive UC, 17 active CD, and 13 inactive CD and 50 non-inflamed tissues as control group. Gene expression was measured by real time polymerase chain reaction (RT-PCR) and protein expression was evaluated in tissue by immunohistochemistry and in peripheral blood mononuclear cells by flow cytometry. Higher levels of IL-35 was produced by intestinal regulatory B cells and circulating regulatory CD4⁺ and CD8⁺ T cells in active vs. inactive disease or healthy donors (P < 0.05). The IL-37 was conspicuously synthesized by circulating B cells, active natural killer cells and monocytes. These results suggest that down-regulation of inflammation in active IBD patients might be based on the increased expression of IL-35 and IL-37.     

Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Fingolimod Increases CD39-Expressing Regulatory T Cells in Multiple Sclerosis Patients

Background

Multiple sclerosis (MS) likely results from an imbalance between regulatory and inflammatory immune processes. CD39 is an ectoenzyme that cleaves ATP to AMP and has been suggested as a novel regulatory T cells (Treg) marker. As ATP has numerous proinflammatory effects, its degradation by CD39 has anti-inflammatory influence. The purpose of this study was to explore regulatory and inflammatory mechanisms activated in fingolimod treated MS patients.

Methods and Findings

Peripheral blood mononuclear cells (PBMCs) were isolated from relapsing-remitting MS patients before starting fingolimod and three months after therapy start. mRNA expression was assessed in ex vivo PBMCs. The proportions of CD8, B cells, CD4 and CD39-expressing cells were analysed by flow cytometry. Treg proportion was quantified by flow cytometry and methylation-specific qPCR. Fingolimod treatment increased mRNA levels of CD39, AHR and CYP1B1 but decreased mRNA expression of IL-17, IL-22 and FOXP3 mRNA in PBMCs. B cells, CD4⁺ cells and Treg proportions were significantly reduced by this treatment, but remaining CD4⁺ T cells were enriched in FOXP3⁺ cells and in CD39-expressing Tregs.

Conclusions

In addition to the decrease in circulating CD4⁺ T cells and CD19⁺ B cells, our findings highlight additional immunoregulatory mechanisms induced by fingolimod.     

Excretory/Secretory Products from Trichinella spiralis Adult Worms Ameliorate DSS-Induced Colitis in Mice

Background

Many evidences show the inverse correlation between helminth infection and allergic or autoimmune diseases. Identification and characterization of the active helminth-derived products responsible for the beneficial effects on allergic or inflammatory diseases will provide another feasible approach to treat these diseases.

Methods and Findings

Colitis was induced in C57BL/6 mice by giving 3% DSS orally for 7 days. During this period, the mice were treated daily with the excretory/secretory products from T. spiralis adult worms (AES) intraperitoneally. The severity of colitis was monitored by measuring body weight, stool consistency or bleeding, colon length and inflammation. To determine the T. spiralis AES product-induced immunological response, Th1, Th2, Th17 and regulatory cytokine profiles were measured in lymphocytes isolated from colon, mesenteric lymph nodes (MLN), and the spleen of treated mice. The CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Tregs) were also measured in the spleens and MLN of treated mice. Mice treated with AES significantly ameliorated the severity of the DSS-induced colitis indicated by the reduced disease manifestations, improved macroscopic and microscopic inflammation correlated with the up-regulation of Treg response (increased regulatory cytokines IL-10, TGF-beta and regulatory T cells) and down-regulation of pro-inflammatory cytokines (IFN-gamma, IL-6 and IL-17) in the spleens, MLN and colon of treated mice.

Conclusions

Our results provide direct evidences that T. spiralis AES have a therapeutic potential for alleviating inflammatory colitis in mice. This effect is possibly mediated by the immunomodulation of regulatory T cells to produce regulatory and anti-inflammatory cytokines and inhibit pro-inflammatory cytokines.     

CD8+ Regulatory T Cells,and Not CD4+ T Cells,Dominate Suppressive Phenotype and Function after In Vitro Live Mycobacterium bovis-BCG Activation of Human Cells

Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG), the only currently available vaccine against tuberculosis, has been reported to induce regulatory T cells in humans. The activity of regulatory T cells may not only dampen immunogenicity and protective efficacy of tuberculosis-vaccines, but also hamper diagnosis of infection of tuberculosis, when using immune (e.g. IFNγ-release) assays. Still, in settings of infectious diseases and vaccination, most studies have focused on CD4⁺ regulatory T cells, and not CD8⁺ regulatory T-cells. Here, we present a comparative analysis of the suppressive phenotype and function of CD4⁺ versus CD8⁺ T cells after in vitro live BCG activation of human cells. Moreover, as BCG is administered as a (partly) live vaccine, we also compared the ability of live versus heatkilled BCG in activating CD4⁺ and CD8⁺ regulatory T cell responses. BCG-activated CD8⁺ T cells consistently expressed higher levels of regulatory T cell markers, and after live BCG activation, density and (co-)expression of markers were significantly higher, compared to CD4⁺ T cells. Furthermore, selection on CD25-expression after live BCG activation enriched for CD8⁺ T cells, and selection on co-expression of markers further increased CD8⁺ enrichment. Ultimately, only T cells activated by live BCG were functionally suppressive and this suppressive activity resided predominantly in the CD8⁺ T cell compartment. These data highlight the important contribution of live BCG-activated CD8⁺ Treg cells to immune regulation and emphasize their possible negative impact on immunity and protection against tuberculosis, following BCG vaccination.     

Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10⁹ CFU/ml) or high (1 × 10¹¹ CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4⁺ ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3⁺ CD4⁺ CD8⁻ T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4⁺ ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3⁺ CD4⁺ CD8⁻ cells and Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells. The percentage of ileal intraepithelial CD3⁺ CD4⁻ CD8⁺ cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4⁺ ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4⁺ ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3⁺ CD4⁺ CD8⁻ T cells, the expansion of Peyer''s patch CD3⁺ CD4⁻ CD8⁻ and CD3⁻ CD4⁻ CD8⁺ cells, and the attenuation of F4⁺ ETEC-induced increase in CD3⁺ CD4⁺ CD8⁺ T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4⁺ ETEC challenge, thus eliciting a strong proinflammatory response.     

IL-15 Augments TCR-Induced CD4+ T Cell Expansion In Vitro by Inhibiting the Suppressive Function of CD25High CD4+ T Cells

Due to its critical role in NK cell differentiation and CD8⁺ T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4⁺ T cells. The increased levels of IL-15 found in several CD4⁺ T cell-driven (auto-) immune diseases prompted us to examine how IL-15 influences murine CD4⁺ T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4⁺ and CD8⁺ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4⁺ T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15Rα was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15Rβ, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4⁺ T cell suppression by a gradually expanding CD25^HighCD4⁺ T cell subset that expresses Foxp3 and originates from CD4⁺CD25⁺ Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology.     

TcVac3 Induced Control of Trypanosoma cruzi Infection and Chronic Myocarditis in Mice

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8⁺T cell response with type-1 cytokine (IFN-γ⁺TNF-α>IL-4⁺IL-10) and cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ⁺CD8⁺T cells, a predominance of immunoregulatory IL-10⁺/CD4⁺T and IL10⁺/CD8⁺T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8⁺T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.     

Persistent activation of omentum influences the pattern of muscular lesion in the mdx diaphragm

The mdx (X chromosome-linked muscular dystrophy) mouse develops a multi-staged disorder characterized by muscle degeneration and reactive fibrosis. Skeletal muscles of mdx mice are not equally susceptible to degeneration. The aim of this study was to verify whether the intense remodeling of the mdx diaphragm could be attributed to influences from the peritoneal microenvironment and omentum, a lymphohematopoietic tissue rich in progenitor cells and trophic factors. At ages corresponding to increased muscular regeneration (12?weeks) and activation of fibrosis (24?weeks), the mdx omentum exhibited (1) morphological and functional characteristics of activation with enlarged milk-spots, an accumulation of CD4⁺, CD8⁺ and CD19⁺B220⁺ B lymphocytes; (2) the formation of clusters positive for proliferating cell nuclear antigen, mainly in B220⁺-rich areas organized in a follicular structure with a germinative center without any challenge by external antigen inducers; (3) clusters with cells positive for fibroblast growth factor-2, numerous Sca-1⁺CD3^-CD19^-Mac-1^- progenitor cells and increased CD4⁺, CD8⁺ and CD3⁺NK1.1⁺ cells in the peritoneal cavity. Omentectomy reduced areas with F4/80⁺ inflammatory infiltrate the activity of matrix metalloproteases 9 and 2, collagen deposition and areas with regenerating myofibers in the diaphragm. Thus, persistent activation of the omentum influences the pattern of inflammation and regeneration of the mdx diaphragm partly via the activation of progenitor cells and the production of growth factors that influence the physiopathology of the muscular tissue remodeling.     

Interleukin-4 and interferon-γ discordantly regulate collagen biosynthesis by functionally distinct lung fibroblast subsets

Pulmonary fibrosis is a potentially fatal consequence of treatments for malignancy and is an increasing problem in bone marrow transplant patients and in cases of allogeneic lung transplant. The fibrotic response is characterized by increases in lung fibroblast number and collagen synthesis. This laboratory previously isolated stable, functionally distinct, murine lung fibroblast subsets (Thy-1⁺ and Thy-1⁻) to study the contribution of fibroblast subpopulations in lung fibrosis. The fibroblast fibrotic response may be induced by cytokines secreted by infiltrating cells such as T lymphocytes and mast cells. In the current study two key regulatory cytokines, interferon-γ (IFN-γ) and interleukin-4 (IL-4), were investigated for their effects on the collagen synthesis of murine lung fibroblast subsets. IL-4 and IFN-γ are putatively characterized as fibrogenic and anti-fibrogenic cytokines, respectively, and are found in repairing lung tissue. Stimulation with recombinant IL-4 induced a 100% increase in total collagen production only by Thy-1⁺ fibroblasts. Types I and III collagen mRNA were increased in the Thy-1⁺ fibroblasts, unlike the Thy-1⁻ subset. In contrast, IFN-γ decreased constitutive collagen production by more than 50% in Thy-1⁺ and Thy-1⁻ fibroblasts. Interestingly, the two subsets utilized their collagen production machinery (collagenase, tissue inhibitors of metalloproteinases) differently to further regulate collagen turnover in response to IL-4 and IFN-γ. Overall, our data support the hypothesis that IL-4 is fibrogenic and IFN-γ is anti-fibrogenic. Moreover, selective expansion of IL-4 responsive fibroblasts (e.g., Thy-1⁺) may be important in the transition from repair to chronic fibrosis. In addition, these data suggest that an inflammatory response dominated by IL-4-producing Th₂ lymphocytes and/or mast cells will promote fibrosis development. © 1996 Wiley-Liss, Inc.     

4-1BB Signaling Enhances Primary and Secondary Population Expansion of CD8+ T Cells by Maximizing Autocrine IL-2/IL-2 Receptor Signaling

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is primarily expressed on activated T cells and is known to enhance proliferation of T cells, prevent activation-induced cell death, and promote memory formation of CD8⁺ T cells. In particular, it is well acknowledged that 4-1BB triggering preferentially enhances the expansion of CD8⁺ T cells rather than CD4⁺ T cells, but the underlying mechanism remains unclear. Here we found that 4-1BB triggering markedly increased IL-2Rα (CD25) and IL-2 expressions of CD8+ T cells but minimally for CD4⁺ T cells. Proliferation of CD8⁺ T cells was moderately enhanced by direct 4-1BB triggering in the absence of signaling through IL-2Rα/IL-2 interactions, but further promoted in the presence of IL-2Rα/IL-2 interactions. Among the TNFRSF members including OX40, GITR, CD30, and CD27, 4-1BB was superior in the ability to induce IL-2Rα expression on CD8+ T cells. When the primary and secondary expansions of CD8⁺ T cells in vivo were examined by adoptively transferring OVA-specific CD8⁺ T cells along with the treatment with agonistic anti-4-1BB and/or antagonistic anti-CD25 F(ab’)₂ mAb, 4-1BB triggering enhanced both primary and secondary expansion of CD8⁺ T cells in vivo, and the 4-1BB effects were moderately suppressed in primary expansion while completely abolished in secondary expansion of OVA-specific CD8⁺ T cells by blocking IL-2Rα. These results suggest that 4-1BB-mediated increases of IL-2Rα and IL-2 prolong the effects of transient TCR- and 4-1BB-mediated signaling in CD8⁺ T cells, and that 4-1BB triggering preferentially enhances the expansion of CD8⁺ T cells through the amplification of autocrine IL-2/IL-2R signaling loop.     

Helminth Infection and Commensal Microbiota Drive Early IL-10 Production in the Skin by CD4+ T Cells That Are Functionally Suppressive

The skin provides an important first line of defence and immunological barrier to invasive pathogens, but immune responses must also be regulated to maintain barrier function and ensure tolerance of skin surface commensal organisms. In schistosomiasis-endemic regions, populations can experience repeated percutaneous exposure to schistosome larvae, however little is known about how repeated exposure to pathogens affects immune regulation in the skin. Here, using a murine model of repeated infection with Schistosoma mansoni larvae, we show that the skin infection site becomes rich in regulatory IL-10, whilst in its absence, inflammation, neutrophil recruitment, and local lymphocyte proliferation is increased. Whilst CD4⁺ T cells are the primary cellular source of regulatory IL-10, they expressed none of the markers conventionally associated with T regulatory (T_reg) cells (i.e. FoxP3, Helios, Nrp1, CD223, or CD49b). Nevertheless, these IL-10⁺ CD4⁺ T cells in the skin from repeatedly infected mice are functionally suppressive as they reduced proliferation of responsive CD4⁺ T cells from the skin draining lymph node. Moreover, the skin of infected Rag^-/- mice had impaired IL-10 production and increased neutrophil recruitment. Finally, we show that the mechanism behind IL-10 production by CD4⁺ T cells in the skin is due to a combination of an initial (day 1) response specific to skin commensal bacteria, and then over the following days schistosome-specific CD4⁺ T cell responses, which together contribute towards limiting inflammation and tissue damage following schistosome infection. We propose CD4⁺ T cells in the skin that do not express markers of conventional T regulatory cell populations have a significant role in immune regulation after repeated pathogen exposure and speculate that these cells may also help to maintain skin barrier function in the context of repeated percutaneous insult by other skin pathogens.     

Ectonucleotidase CD38 Demarcates Regulatory,Memory-Like CD8+ T Cells with IFN-γ-Mediated Suppressor Activities

Regulatory CD8⁺ T cells are critical for self-tolerance and restricting excessive immune responses. The variety of immune functions they fulfill, the heterogeneity of their phenotype, and the mechanism of action are still poorly understood. Here we describe that regulatory CD8⁺ T cells exhibiting immunosuppressive actions in vitro and in vivo are recognized as CD38^high T cells and present in naive mice. CD38 is a glycosylated membrane protein with ectonucleotidase properties. CD8⁺CD38^high (CD44⁺CD122⁺CD62L^high) lymphocytes suppress CD4⁺ effector T-cell proliferation in an antigen-non specific manner via IFN-γ. While direct cell-to-cell contact is needed for this suppressor activity, it is independent of membrane-bound TGF-β and granzyme B release. IL-15 potentiates the suppressive activity of CD8⁺CD38^high T cells and controls their survival and expansion. In humans CD8⁺CD38^high T cells inhibit CD4⁺ effector T cell proliferation. In vivo, CD8⁺CD38^high, but not CD8⁺CD38⁻ T cells mitigate murine experimental autoimmune encephalomyelitis (EAE) by reducing the clinical score and delaying disease occurrence. EAE suppression is enhanced by pre-treatment of CD8⁺CD38^high T cells with IL-15. These findings add evidence that the expression of ectoenzyme receptor family members positively correlates with suppressor functions and identifies CD8⁺CD38^high T cells as potential inhibitors of excessive immune responses.     

T regulatory cell responses to immunization with a soluble egg antigen in Schistosoma mansoni-infected mice

The aim of the study is to characterize the phenotypes of CD4⁺ CD25⁺ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naïve C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI (P<0.01). The percentage of T regulatory cells (CD4⁺ CD25⁺) increased significantly (P<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-γ, IL-4, and TNF-α, showed significant decreases (P<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.     

Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity,which is suppressed by regulatory T cells in breast cancer

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8⁺ T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8⁺ T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8⁺ T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8⁺ T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8⁺ T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8⁺ T cells but could significantly enhance IL-2-mediated promotion of CD8⁺ T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8⁺ T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4⁺CD25⁺ regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8⁺ T cells, an effect suppressible by CD4⁺CD25⁺ Treg cells.     

Mechanism of γδ T-Cell-Mediated Inhibition of Stem Cell Differentiationin Vitro:Possible Relevance for Myelosuppression in HIV-Infected Individuals

We investigated whether γδ T cells contribute to the suppression of myelopoiesis in HIV infection. Freshly isolated γδ T cells from HIV seropositive patients suppressed CFU–GM growthin vitro.Preactivation of γδ T cells with IL-2 and/or IL-15 further reduced the number of CFU–GM. Natural killer cells and to a lower extent CD4⁺and CD8⁺cells also inhibited CFU–GM growth. In contrast to γδ T cells, this effect was not dependent on IL-15 or IL-2 preactivation. Moreover, no enhanced inhibitory effect of CD56⁺and CD4⁺cells was observed in HIV⁺subjects compared to HIV⁻donors. The myelosuppressive effect of supernatants of γδ T cells could be inhibited by antibodies against IFN-γ or TNF-α. Accordingly, we found increased numbers of TNF-α- or IFN-γ-secreting CD8⁺γδ T cells in HIV⁺patients. We conclude that the increased fraction of activated γδ T cells producing myelosuppressive cytokines might contribute to the dyshematopoiesis frequently observed in HIV-infected individuals.