Suppression of Th1 and Th17, but not Th2, responses in a CD8(+) T cell-mediated model of oral tolerance.
ABSTRACT: The role of CD8(+) T cells in oral tolerance remains unclear. To address this, we developed a model to induce CD8(+) Tregs by feeding the major histocompatibility complex class I immunodominant epitope of OVA, OVA((257-264)). OVA((257-264)) feeding induced tolerance similar to that observed in OVA protein-fed mice, capable of suppressing the production of Th1 and Th17 cytokines and inhibiting a Th1-driven delayed-type hypersensitivity response following immunization with whole OVA (wOVA) protein. OVA((257-264)) peptide-induced suppression could be transferred to naive mice with CD8(+) cells, but not CD8-depleted cells, isolated from mesenteric lymph nodes of peptide-fed mice. Interestingly, while capable of inhibiting Th1 and Th17 responses, OVA((257-264)) feeding could not suppress any feature of a Th2 inflammatory response, though OVA protein feeding could, suggesting that these cells function through a different mechanism than their CD4(+) counterparts generated in response to feeding with wOVA. Thus, CD8(+) T cells are functionally capable of mediating tolerance to Th1 and Th17 responses.
Project description:In the present study, we evaluated adjuvant potential of Poria cocos polysaccharide (PCP) on the Th1-type immune responses of C57/BL6 mice against ovalbumin (OVA). We first determined the effect of PCP on maturation of murine bone marrow derived dendritic cells (BMDCs), PCP significantly upregulated surface expression of MHCII, CD40, CD80, CD86 and enhanced production of IL-6 and IL-12p40. In addition, PCP affected receptor-mediated endocytosis, but not pinocytosis in BMDCs. Furthermore, OVA + PCP immunization induced specific cytotoxic CD8+ T cell killing of OVA (257-264) peptide pulsed cell. When mice were immunized subcutaneously in a week interval with OVA + PCP. Serum were collected for measuring OVA-specific antibody and splenocytes were harvested for analyzing CD69, IFN-? ELISpot and cytokines production. The result indicated that OVA-specific IgG, IgG2a and IgG1 antibody levels in serum were significantly elevated by PCP compared with control. PCP increased OVA-specific IFN-?-secreting CD8+, CD4+ T cells, promoted CD8+ T cell proliferation and up-regulated Th-1 type (IFN-?, IL-2) cytokine production. In conclusion, data suggest that PCP enhanced cellular immune response and possess potential as a vaccine adjuvant for Th1 immune response.
Project description:The CD8 co-receptor can modulate CD8(+) T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-gamma and IL-4 exert opposing effects on the expression of CD8alpha mRNA and surface CD8 protein during CD8(+) T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin (OVA)(257-264)-specific TCR-transgenic OT-I CD8(+) T cells activated with OVA(257-264)-coated antigen presenting cells or polyclonal stimuli, and on wild type CD8(+) T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-gamma or IFN-gamma R gene. When WT or IFN-gamma-deficient OT-I CD8(+) T cells were analyzed 9 days after co-injection with control or IL-4-expressing OVA(+) tumor cells into RAG-2(-/-)gamma c(-/-) mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-gamma-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD8(low) cells displayed markedly impaired binding of OVA(257-264)/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-gamma in tuning the CD8 co-receptor during primary CD8(+) T cell activation both in vitro and in vivo.
Project description:The ultimate goal of antitumor vaccines is to develop memory CD8(+) cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. We previously demonstrated that the ovalbumin (OVA)-specific CD4(+) T cell-based (OVA-T(EXO)) vaccine generated using OVA-pulsed dendritic cell (DC(OVA))-released exosomes (EXO(OVA)) stimulate CTL responses via IL-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4(+) OVA-T(EXO) cells or OVA-T(EXO) cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2K(b)/OVA(257-264) tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10(OVA). We demonstrated that CD4(+) OVA-T(EXO) cells stimulated more efficient CTL responses compared to DC(OVA). By assessing primary and recall CTL responses in mice immunized with OVA-T(EXO) or with OVA-T(EXO) lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-T(EXO). Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10(OVA) tumors. Overall, this work suggests that a novel CD4(+) T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination.
Project description:Age-associated decline in immunity to infection has been documented across multiple pathogens, yet the relative contributions of the aged priming environment and of lymphocyte-intrinsic defects remain unclear. To address the impact of the aging environment on T-cell priming, adult naïve OT-I TCR transgenic CD8 T cells, specific for the H-2Kb-restricted immunodominant OVA(257-264) epitope, were transferred into adult or old recipient mice infected with the recombinant intracellular bacterium Listeria monocytogenes carrying the chicken ovalbumin protein (Lm-OVA). We consistently found that adult OT-I CD8 expansion was reduced in aged recipient mice, and this correlated with numeric, phenotypic, and functional defects selectively affecting CD8?+ dendritic cells (DC). Following Lm-OVA infection, aged mice failed to accumulate CD8?+ DC in the spleen, and these cells expressed much lower levels of critical costimulatory molecules in the first three days following infection. Further, aged CD8?+ DC showed impaired uptake of the bacteria at very early time points following infection. Treatment of aged mice with Flt3 ligand (Flt3L) improved the number of DC present in the spleen prior to Lm-OVA infection, and improved, but did not reconstitute, OT-I expansion to Lm-OVA infection. These results suggest that age-associated changes in antigen uptake, pathogen sensing, and/or antigen presentation contribute to impaired adaptive immune responses to microbial pathogens with aging.
Project description:The targeting of natural tolerogenic liver sinusoidal endothelial cells (LSEC) by nanoparticles (NPs), decorated with a stabilin receptor ligand, is capable of generating regulatory T-cells (Tregs), which can suppress antigen-specific immune responses, including to ovalbumin (OVA), a possible food allergen. In this regard, we have previously demonstrated that OVA-encapsulating poly(lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles eliminate allergic airway inflammation in OVA-sensitized mice, prophylactically and therapeutically. A competing approach is a nanocarrier platform that incorporates pharmaceutical agents interfering in mTOR (rapamycin) or NF-κB (curcumin) pathways, with the ability to induce a tolerogenic state in nontargeted antigen-presenting cells system-wide. First, we compared OVA-encapsulating, LSEC-targeting tolerogenic nanoparticles (TNPs) with nontargeted NPs incorporating curcumin and rapamycin (Rapa) in a murine eosinophilic airway inflammation model, which is Treg-sensitive. This demonstrated roughly similar tolerogenic effects on allergic airway inflammation by stabilin-targeting NP<sup>OVA</sup> <i>versus</i> nontargeted NPs delivering OVA plus Rapa. Reduction in eosinophilic inflammation and TH2-mediated immune responses in the lung was accompanied by increased Foxp3<sup>+</sup> Treg recruitment and TGF-β production in both platforms. As OVA incorporates IgE-binding as well as non-IgE-binding epitopes, the next experiment explored the possibility of obtaining immune tolerance by non-anaphylactic T-cell epitopes. This was accomplished by incorporating OVA<sup>323-339</sup> and OVA<sup>257-264</sup> epitopes in liver-targeting NPs to assess the prophylactic and therapeutic impact on allergic inflammation in transgenic OT-II mice. Importantly, we demonstrated that the major histocompatibility complex (MHC)-II binding (former) but not the MHC-I binding (latter) epitope interfered in allergic airway inflammation, improving TNP<sup>OVA</sup> efficacy. The epitope-specific effect was transduced by TGF-β-producing Tregs. In the final phase of experimentation, we used an OVA-induced anaphylaxis model to demonstrate that targeted delivery of OVA and its MHC-II epitope could significantly suppress the anaphylaxis symptom score, mast cell release, and the late-phase inflammatory response. In summary, these results demonstrate comparable efficacy of LSEC-targeting <i>versus</i> pharmaceutical PLGA nanoparticles, as well as the ability of T-cell epitopes to achieve response outcomes similar to those of the intact allergens.
Project description:The objective of this study was to determine the effect of feeding a maternal diet supplemented with docosahexaenoic acid (DHA) during the suckling period on the development of the immune system and oral tolerance (OT) in offspring. Dams were randomized to consume one of two nutritionally adequate diets throughout the suckling period: control (N = 12, 0% DHA) or DHA (N = 8, 0.9% DHA) diet. At 11 days, pups from each dam were randomly assigned to a mucosal OT challenge: the placebo or the ovalbumin (OVA) treatment. At three weeks, plasma immunoglobulins and splenocyte cytokine production ex vivo were measured. OVA-tolerized pups had a lower Th2 (IL-13) response to OVA despite the presence of more activated T cells and memory cells (CD27+, all p < 0.05). Feeding a high DHA diet improved the ability of splenocytes to respond to mitogens toward a skewed Th1 response and led to a higher IL-10 and a lower TGF-? production after stimulation with OVA (all p < 0.05). Untolerized DHA-fed pups had lower plasma concentrations of OVA-specific immunoglobulin E (p for interaction < 0.05). Overall, feeding a high DHA maternal diet improves the tolerance response in untolerized suckled pups in a direction that is thought to be beneficial for the establishment of OT.
Project description:Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-? production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8?(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8?(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8?(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8?(+) DCs in vivo for efficient induction of CTL responses.
Project description:CD4+ T cell subsets including regulatory T cells (Tregs), Th1 and Th17 are critical for control and development of inflammation and autoimmunity. We investigated the in vitro and in vivo effects of silymarin, a well-known herbal medicine on differentiation and function of Tregs and Th1 and Th17 responses. For in vitro study, mice splenocytes treated with 20-30 ?g/ml silymarin were evaluated for gene expressions of specific transcription factors and cytokines of CD4+ T cell subsets using real-time PCR. Induction of Treg cell development in the presence of silymarin was performed on isolated naïve CD4+ T cells. Effect of silymarin-induced Tregs on T cell suppression was determined by CFSE labeling method. Results of this part showed that silymarin significantly decreased IFN?, ROR?t and IL-17 gene expressions and upregulated Foxp3, TGF-? and IL-10 mRNA. More silymarin-enhanced naïve CD4+ T cells differentiated to Tregs (67%) than the control (47%). Silymarin-induced Tregs reduced proliferation of naïve activated T cells (<50%). For in vivo study, mice were immunized with ovalbumin (Ova) on days 1 and 14. Silymarin (100 mg/Kg) was intraperitoneally administered two days before the first Ova challenge followed by on every day for two weeks. Splenocytes were then isolated for assessment of CD4+ T cell subsets and ex vivo analysis using flow cytometry. Treatment of Ova-immunized mice with silymarin increased Tregs (11.24?±?1.2%, p?<?0.01(but decreased Th1 (1.72?±?0.4%, p?<?0.001) and Th17 (1.07?±?0.04%, p?<?0.001) cells. Ex vivo Ova challenge of splenocytes from Ova-immunized mice treated with silymarin decreased proliferation of splenocytes, IFN? (2.76% of control) and IL-17 (<8%) along with increased TGF-? (59.7%) expressions in CD4+T-bet+, CD4+ROR?t+ and CD4+Foxp3+ T cells, respectively. In conclusion, silymarin promoted Treg differentiation and function and decreased Th1 and Th17 cells. Silymarin may differentially regulate CD4+ T cell responses which can provide potential benefits for its use as treatment of immune-related diseases. Graphical abstract ?.
Project description:The B7 family member programmed death-1 ligand (PD-L1) has been shown to play an inhibitory role in the regulation of T cell responses in several organs. However, the role of PD-L1 in regulating tolerance to self-Ags of the small intestine has not been previously addressed. In this study, we investigated the role of PD-L1 in CD8(+) T cell tolerance to an intestinal epithelium-specific Ag using the iFABP-tOVA transgenic mouse model, in which OVA is expressed as a self-Ag throughout the small intestine. Using adoptive transfer of naive OVA-specific CD8(+) T cells, we show that loss of PD-1:PD-L1 signaling, by either Ab-mediated PD-L1 blockade or transfer of PD-1(-/-) T cells, leads to considerable expansion of OVA-specific CD8(+) T cells and their differentiation into effector cells capable of producing proinflammatory cytokines. A fatal CD8(+) T cell-mediated inflammatory response develops rapidly against the small bowel causing destruction of the epithelial barrier, severe blunting of intestinal villi, and recruitment and activation of myeloid cells. This response is highly specific because immune destruction selectively targets the small intestine but not other organs. Collectively, these results indicate that loss of the PD-1:PD-L1 inhibitory pathway breaks CD8(+) T cell tolerance to intestinal self-Ag, thus leading to severe enteric autoimmunity.
Project description:There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.