Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles,Respectively

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA⁺ to CD45RO⁺ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.     

TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine implicated in the etiology of several autoimmune diseases, including rheumatoid arthritis (RA). TNF-α regulates diverse effector functions through the activation of TNF-α receptor (TNFR)1 and TNFR2. Although the detrimental role of this cytokine has been addressed in distinct disease settings, the effects of TNF-α on cytokine production by isolated CD4⁺ T helper type 1 (Th1) and Th17 cells, two T cell subpopulations that contribute to the pathogenesis of RA, have not been completely elucidated. Here, we show that TNF-α promotes a reduction and expansion in the frequency of both T cell subsets producing IFN-γ and IL-17, respectively. Selective blockade of TNFR1 or TNFR2 on Th1 and Th17 cells revealed that TNFR2 mediates the decrease in IFN-γ production, while signaling through both receptors augments IL-17 production. We also demonstrate that Th1, but not Th17 cells from RA patients present lower levels of TNFR1 compared to healthy controls, whereas TNFR2 expression on both T cell types is similar between patients and controls. Since TNF-α receptors levels in RA patients are not significantly changed by the therapeutic blockade of TNF-α, we propose that targeting TNFR2 may represent an alternative strategy to normalize the levels of key cytokines that contribute to RA pathogenesis.     

Plasma Extracellular Vesicles Play a Role in Immune System Modulation in Minimal Hepatic Encephalopathy

Minimal hepatic encephalopathy (MHE) is associated with changes in the immune system including an increased pro-inflammatory environment and altered differentiation of CD4+ T lymphocytes. The mechanisms remain unknown. Changes in extracellular vesicle (EV) cargo including proteins and miRNAs could play a main role as mediators of immune system changes associated with MHE. The aim was to assess whether plasma EVs from MHE patients played a role in inducing the pro-inflammatory environment and altered differentiation of CD4+ T lymphocyte subtypes in MHE patients. We characterized the miRNA and protein cargo of plasma EVs from 50 cirrhotic patients (27 without and 23 with MHE) and 24 controls. CD4+ T cells from the controls were cultured with plasma EVs from the three groups of study, and the cytokine release and differentiation to CD4+ T-cell subtypes were assessed. Plasma EVs from MHE patients had altered miRNA and protein contents, and were enriched in inflammatory factors compared to the controls and patients without MHE. EVs from MHE patients modulated the expression of pro-inflammatory IL-17, IL-21, and TNF-α and anti-inflammatory TGF-β in cultured CD4+ T lymphocytes, and increased the proportion of Th follicular and Treg cells and the activation of Th17 cells. In conclusion, plasma EVs could play an important role in the induction of immune changes observed in MHE.     

Opposing Roles of DCs and iNKT Cells in the Induction of Foxp3 Expression by MLN CD25+CD4+ T Cells during IFNγ-Driven Colitis

We have previously shown that a deficiency of CD1d-restricted invariant natural killer T (iNKT) cells exacerbates dextran sulfate sodium (DSS)-induced colitis in Yeti mice that exhibit IFNγ-mediated hyper-inflammation. Although iNKT cell-deficiency resulted in reduced Foxp3 expression by mesenteric lymph node (MLN) CD4⁺ T cells in DSS-treated Yeti mice, the cellular mechanisms that regulate Foxp3 expression by CD25⁺CD4⁺ T cells during intestinal inflammation remain unclear. We found that Foxp3⁻CD25⁺CD4⁺ T cells expressing Th1 and Th17 phenotypic hallmarks preferentially expanded in the MLNs of DSS-treated Yeti/CD1d knockout (KO) mice. Moreover, adoptive transfer of Yeti iNKT cells into iNKT cell-deficient Jα18 KO mice effectively suppressed the expansion of MLN Foxp3⁻CD25⁺CD4⁺ T cells during DSS-induced colitis. Interestingly, MLN dendritic cells (DCs) purified from DSS-treated Yeti/CD1d KO mice promoted the differentiation of naive CD4⁺ T cells into Foxp3⁻CD25⁺CD4⁺ T cells rather than regulatory T (Treg) cells, indicating that MLN DCs might mediate Foxp3⁺CD25⁺CD4⁺ T cell expansion in iNKT cell-sufficient Yeti mice. Furthermore, we showed that Foxp3⁻CD25⁺CD4⁺ T cells were pathogenic in DSS-treated Yeti/CD1d KO mice. Our result suggests that pro-inflammatory DCs and CD1d-restricted iNKT cells play opposing roles in Foxp3 expression by MLN CD25⁺CD4⁺ T cells during IFNγ-mediated intestinal inflammation, with potential therapeutic implications.     

Polysaccharides from European Black Elderberry Extract Enhance Dendritic Cell Mediated T Cell Immune Responses

European black elderberry (Sambucus nigra L.) is a popular way to treat common colds or influenza infections. Mechanistically, this might be due to a direct antiviral effect or a stimulatory effect on the immune system of the host. Here, we evaluated the modulatory effects of black elderberry derived water extract (EC15) and its polysaccharide enriched fractions (CPS, BOUND, and UNBOUND) in comparison to a conventional alcoholic extract (EE25), regarding the phenotypical and functional properties of dendritic cells (DCs), which are essential cells to induce potent T cell responses. Interestingly, the water extract and its polysaccharide fractions potently induced DC maturation, while the ethanol extract did not. Moreover, the capacity to stimulate T cells by these matured DCs, as assessed using MLR assays, was statistically higher when induced by the water extracted fractions, compared to immature DCs. On the other hand, the ethanol extract EE25 did not induce T cell stimulation. Finally, the cytokine expression profiles of these DC—T cell cocultures were assessed and correlated well with increased T cell stimulation. Also, the expression of inflammatory cytokines, such as IL-6, TNF-α, and IFN-γ was highly increased in the presence of the elderberry water extract EC15, and the polysaccharide enriched CPS, BOUND, and UNBOUND fractions, but not by EE25. Thus, from these data, we conclude that the polysaccharides present in water-derived elderberry fractions induce potent immune-modulatory effects, which represents the basis for a strong immune-mediated response to viruses including influenza.     

Peripheral Blood T Cells of Patients with IPAH Have a Reduced Cytokine-Producing Capacity

Pulmonary arterial hypertension (PAH) is rare disease that is categorized as idiopathic (IPAH) when no underlying cause can be identified. Lungs of most patients with IPAH contain increased numbers of T cells and dendritic cells (DCs), suggesting involvement of the immune system in its pathophysiology. However, our knowledge on circulating immune cells in IPAH is rather limited. We used flow cytometry to characterize peripheral blood DCs and T cells in treatment-naive IPAH patients, compared with connective-tissue disease-PAH (CTD-PAH) patients and healthy controls (HCs). At diagnosis, T-helper (Th) cells of IPAH patients were less capable of producing TNFα, IFNγ, IL-4 and IL-17 compared to HCs. IPAH patients showed a decreased frequency of Th2 cells and significantly enhanced expression of the CTLA4 checkpoint molecule in naive CD4⁺ T cells and both naive and memory CD8⁺ T cells. Frequencies and surface marker expression of circulating DCs and monocytes were essentially comparable between IPAH patients and HCs. Principal component analysis (PCA) separated IPAH patients—but not CTD-PAH patients—from HCs, based on T-cell cytokine profiles. At 1-year follow-up, the frequencies of IL-17⁺ production by memory CD4⁺ T cells were increased in IPAH patients and accompanied by increased proportions of Th17 and Tc17 cells, as well as decreased CTLA4 expression. Treatment-naive IPAH patients displayed a unique T-cell phenotype that was different from CTD-PAH patients and was characterized by reduced cytokine-producing capacity. These findings point to involvement of adaptive immune responses in IPAH, which may have an implication for the development of therapeutic interventions.     

CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4⁺ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4⁺FoxP3⁺CD25⁺ regulatory T cells (Tregs) were known to suppress function of effector CD4⁺ T cells and contribute to resolution of disease. It has been recently reported that some CD4⁺ T-cell subsets demonstrate shared phenotypes with another CD4⁺ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4⁺ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.     

Ficus carica Polysaccharides Promote the Maturation and Function of Dendritic Cells

Various polysaccharides purified from plants are considered to be biological response modifiers and have been shown to enhance immune responses. Ficus carica L. is a Chinese traditional plant and has been widely used in Asian countries for its anti-tumor properties. Ficus carica polysaccharides (FCPS), one of the most essential and effective components in Ficus carica L., have been considered to be a beneficial immunomodulator and may be used in immunotherapy. However, the immunologic mechanism of FCPS is still unclear. Dectin-1 is a non-toll-like pattern recognition receptor, predominately expressed on dendritic cells (DCs). Activation of DCs through dectin-1 signaling can lead to the maturation of DC, thus inducing both innate and adaptive immune responses against tumor development and microbial infection. In our study, we found that FCPS could effectively stimulate DCs, partially through the dectin-1/Syk pathway, and promote their maturation, as shown by the up-regulation of CD40, CD80, CD86, and major histocompatibility complex II (MHCII). FCPS also enhanced the production of cytokines by DCs, including IL-12, IFN-γ, IL-6, and IL-23. Moreover, FCPS-treated DCs showed an enhanced capability to stimulate T cells and promote T cell proliferation. Altogether, these results demonstrate that FCPS are able to activate and maturate DCs, thereby up-regulating the immunostimulatory capacity of DCs, which leads to enhanced T cell responses.     

Reduced Expression of PD-1 in Circulating CD4+ and CD8+ Tregs Is an Early Feature of RRMS

Altered regulatory T cell (Treg) function could contribute to MS. The expression of activating and inhibitory receptors influences the activity of Tregs. Our aim was to investigate T cell phenotypes in relapsing–remitting MS (RRMS) patients at an early phase of the disease. We examined the influence of demographic parameters on the distribution of CD4+ and CD8+ T cell subclasses by generalized linear modeling. We also studied the expression of the following markers—CTLA-4, GITR, PD-1, FoxP3, Helios, CD28, CD62L, CD103—on T cell subsets from peripheral blood with a 14-color flow cytometry panel. We used an antibody array to define the profiles of 34 Th1/Th2/Th17 cytokines in the serum. Expression of PD-1 and GITR on CD4+ and CD8+ Tregs was decreased in RRMS patients. The proinflammatory factors IFN-γ, IL-17, IL-17F, TGFβ-1, TGFβ-3, IL-1SRII, IL-12 p40, sgp130, IL-6sR were significantly increased in RRMS patients. Therefore, a deficiency of PD-1 and GITR immune checkpoints on CD4+ and CD8+ Tregs is a feature of RRMS and might underlie impaired T cell control.     

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide

Naїve CD4⁺ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4⁺CD45RA⁺ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFβ mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC₂ receptor increased significantly with respect to the VPAC₁ receptor from day 4 of CD4⁺CD45RA⁺ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4⁺CD45RA⁺ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.     

Characterization of the Transient Deficiency of PKC Isozyme Levels in Immature Cord Blood T Cells and Its Connection to Anti-Allergic Cytokine Profiles of the Matured Cells

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4⁺ and CD8⁺ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4⁺ and CD8⁺ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1–2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially ‘corrected’ after birth.     

IL-33 Enhances IFNγ and TNFα Production by Human MAIT Cells: A New Pro-Th1 Effect of IL-33

Mucosal-associated invariant T (MAIT) cells represent a distinct T cell population restricted by the MHC-class-I-related molecule, MR1, which recognizes microbial-derived vitamin B2 (riboflavin) metabolites. Their abundance in humans, together with their ability to promptly produce distinct cytokines including interferon γ (IFNγ) and tumor necrosis factor α (TNFα), are consistent with regulatory functions in innate as well as adaptive immunity. Here, we tested whether the alarmin interleukin 33 (IL-33), which is secreted following inflammation or cell damage, could activate human MAIT cells. We found that MAIT cells stimulated with IL-33 produced high levels of IFNγ, TNFα and Granzyme B (GrzB). The action of IL-33 required IL-12 but was independent of T cell receptor (TCR) cross-linking. MAIT cells expressed the IL-33 receptor ST2 (suppression of tumorigenicity 2) and upregulated Tbet (T-box expressed in T cells) in response to IL-12 or IL-33. Electronically sorted MAIT cells also upregulated the expression of CCL3 (Chemokine C-C motif ligand 3), CD40L (CD40 Ligand), CSF-1 (Colony Stimulating Factor 1), LTA (Lymphotoxin-alpha) and IL-2RA (IL-2 receptor alpha chain) mRNAs in response to IL-33 plus IL-12. In conclusion, IL-33 combined with IL-12 can directly target MAIT cells to induce their activation and cytokine production. This novel mechanism of IL-33 activation provides insight into the mode of action by which human MAIT cells can promote inflammatory responses in a TCR-independent manner.     

Cell-free Stem Cell-Derived Extract Formulation for Regenerative Medicine Applications

Stem cells for regenerative medicine purposes offer therapeutic benefits, but disadvantages are still ill defined. The benefit of stem cells may be attributed to their secretion of growth factors (GFs), cytokines (CKs), and extracellular vesicles (EVs), including exosomes. We present a novel cell-free stem cell-derived extract (CCM), formulated from human progenitor endothelial stem cells (hPESCs), characterized for biologically active factors using ELISA, nanoparticle tracking analysis and single particle interferometric reflectance imaging sensing. The effect on fibroblast proliferation and ability to induce stem cell migration was analyzed using Alamar Blue proliferation and Transwell migration assays, respectively. GFs including IGFBP 1, 2, 3, and 6, insulin, growth hormone, PDGF-AA, TGF-α, TGF-β1, VEGF, and the anti-inflammatory cytokine, IL-1RA were detected. Membrane enclosed particles within exosome size range and expressing exosome tetraspanins CD81 and CD9 were identified. CCM significantly increased cell proliferation and induced stem cell migration. Analysis of CCM revealed presence of GFs, CKs, and EVs, including exosomes. The presence of multiple factors including exosomes within one formulation, the ability to promote cell proliferation and induce stem cell migration may reduce inflammation and pain, and augment tissue repair.     

Sex-Based Selectivity of PPARγ Regulation in Th1, Th2, and Th17 Differentiation

Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been recognized to regulate adaptive immunity through Th17 differentiation, Treg functions, and T_FH responses. However, its role in adaptive immunity and autoimmune disease is still not clear, possibly due to sexual differences. Here, we investigated in vitro treatment study with the PPARγ agonist pioglitazone to compare Th1, Th2, and Th17 differentiation in male and female mouse splenic T cells. Pioglitazone treatment significantly inhibited various effector T cell differentiations including Th1, Th2, and Th17 cells from female naïve T cells, but it selectively reduced IL-17 production in male Th17 differentiation. Interestingly, pioglitazone and estradiol (E2) co-treatment of T cells in males inhibited differentiation of Th1, Th2, and Th17 cells, suggesting a mechanism for the greater sensitivity of PPARγ to ligand treatment in the regulation of effector T cell differentiation in females. Collectively, these results demonstrate that PPARγ selectively inhibits Th17 differentiation only in male T cells and modulates Th1, Th2, and Th17 differentiation in female T cells based on different level of estrogen exposure. Accordingly, PPARγ could be an important immune regulator of sexual differences in adaptive immunity.     

T Lymphocyte Antigen 4-Modified Dendritic Cell Therapy for Asthmatic Mice Guided by the CCR7 Chemokine Receptor

The CD80/CD86-CD28 axis is a critical pathway for immuno-corrective therapy, and the cytotoxic T lymphocyte antigen 4 (CTLA4) is a promising immunosuppressor targeting the CD80/CD86-CD28 axis; however, its use for asthma therapy needs further optimization. A human CTLA4 fused with the IgCγ Fc (CTLA4Ig) and mouse CC chemokine receptor type7 (CCR7) coding sequences were inserted into a recombinant adenovirus (rAdV) vector to generate rAdV-CTLA4Ig and rAdV-CCR7. The naive dendritic cells (DCs) were infected with these rAdVs to ensure CCR7 and CTLA4Ig expression. The therapeutic effects of modified DCs were evaluated. rAdV-CTLA4Ig and rAdV-CCR7 infected DCs improved all asthma symptoms. Inflammatory cell infiltration and cytokine analysis showed that rAdV-CTLA4Ig and rAdV-CCR7-modified DC therapy reduced the number of eosinophils and lymphocyte and neutrophil infiltration in the lung. Interestingly, assessment of the humoral immunity showed that the IL-4 and IFNγ levels of the rAdV-CTLA4Ig and rAdV-CCR7-modified DC-treated mice decreased significantly and did not reverse the Th1/Th2 balance. DCs expressing CCR7 displayed guidance ability for DC migration, primarily for DCs in the inflammatory lung. Additionally, the rAdVs caused an inflammatory response by inducing DC differentiation, inflammatory cell infiltration and changes in cytokines; however, mice transplanted with rAdV-green fluorescent protein (GFP)-infected DCs displayed no asthma manifestations. In conclusion, CTLA4Ig-modified DCs exhibited a therapeutic effect on asthma, and CCR7 may guide DC homing. The combination of these two molecules may be a model for precision-guided immunotherapy.     

IDO and CD40 May Be Key Molecules for Immunomodulatory Capacity of the Primed Tonsil-Derived Mesenchymal Stem Cells

Background: Tonsil-derived mesenchymal stem cells (T-MSCs) were reported to have suppressive effect on T cells, yet much remains unknown about the underlying mechanisms supporting this effect. We investigated the underlying mechanism of the immunomodulatory effect of T-MSCs on immune cell proliferation and cytokine production. Methods: We isolated T-MSCs from human palatine tonsil and evaluated the immunomodulatory capacity using RT-PCR, ELISA, and flow cytometry. Additionally, we assessed the expression of various soluble factors and several costimulatory molecules to detect the priming effect on T-MSCs. Results: T-MSCs significantly inhibited the immune cell proliferation and cytokine expression (TNF-α and IFN-γ) in the direct co-culture, but there was no suppressive effect in indirect co-culture. Additionally, we detected a remarkably higher expression of indoleamine 2,3-dioxygenase (IDO) in the primed T-MSCs having co-expression CD40. Moreover, immune cells or CD4⁺ T cells showed lower TNF-α, IFN-γ, and IL-4 expression when the primed T-MSC were added; whereas those findings were reversed when the inhibitor for IDO (not IL-4) or CD40 were added. Furthermore, T-bet and GATA3 levels were significantly decreased in the co-cultures of the primed T-MSCs and CD4⁺ T cells; whereas those findings were reversed when we added the neutralizing anti-CD40 antibody. Conclusions: Primed T-MSCs expressing IDO and CD40 may have immunomodulatory capacity via Th1-mediated and Th2-mediated immune response.     

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14⁺ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.     

Deficiency of IL-27 Signaling Exacerbates Experimental Autoimmune Uveitis with Elevated Uveitogenic Th1 and Th17 Responses

Human uveitis is an autoimmune disease of the central nervous system that is characterized by ocular inflammation with the involvement of uveitogenic Th1 and Th17 responses. In experimental autoimmune uveitis (EAU), the animal model for human uveitis, both responses are proven to be critical in disease development. Therefore, targeting both Th1 and Th17 cells has therapeutic implication for disease resolution. IL-27 is a multifunctional cytokine that can either promote or inhibit T cell responses and is implicated in both autoimmune and infectious diseases. The aim of this study is to characterize the role of IL-27/IL-27R signaling in regulating uveitogenic Th1/Th17 responses in EAU. By immunizing IL-27Rα^−/− mice and their wild-type (WT) littermates for EAU, we demonstrated that IL-27 signaling deficiency exacerbated EAU with severe ocular inflammation and impairment of visual function. Furthermore, there was a significant increase in the eye-infiltrating Th1 and Th17 cells in IL-27Rα^−/− EAU mice compared to WT. Their retinal antigen-specific Th1 and Th17 responses were also significantly increased, as represented by the elevation of their signature cytokines, IFN-γ and IL-17A, respectively. We also observed the upregulation of another pathogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), from effector T cells in IL-27Rα^−/− EAU mice. Mechanistic studies confirmed that IL-27 inhibited GM-CSF production from Th17 cells. In addition, the induction of IL-10 producing type 1 regulatory T (Tr1) cells was impaired in IL-27Rα^−/− EAU mice. These results identified that IL-27 signaling plays a suppressive role in EAU by regulating multiple CD4⁺ cell subsets, including the effector Th1 and Th17 cells and the regulatory Tr1 cells. Our findings provide new insights for therapeutic potential in controlling uveitis by enhancing IL-27 signaling.