Treg Formation Research Articles

Molecular mechanisms of control of differentiation of regulatory t-lymphocytes by exogenous melatonin

We investigated the role of epiphyseal hormone melatonin in the differentiation of naive CD4+T cells into regulatory T cells (Treg). The hormone at physiological and pharmacological concentrations inhibited Treg differentiation, decreasing both the proportion of CD4+FOXP3+ cells in the culture and the level of TGF‑β, the key cytokine for this T cell subpopulation. The inhibitory effect of exogenous melatonin was due to its interaction with the membrane receptors MT1 and MT2. At the same time, the signals realized through RORa — the nuclear receptor for melatonin — stimulated Treg formation; however, they were considerably weaker than the signals from the membrane receptors and were overlapped by the latter. Since the Treg subpopulation plays an important role in physiological and pathological processes in the body, the revealed effects of melatonin should be taken into account in its therapeutic use.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

Molecular Mechanisms of Control of Differentiation of Regulatory T-Lymphocytes by Exogenous Melatonin.

We investigated the role of epiphyseal hormone melatonin in the differentiation of naive CD4+ T cells into regulatory T cells (Treg). The hormone at physiological and pharmacological concentrations inhibited Treg differentiation, decreasing both the proportion of CD4+FOXP3+ cells in the culture and the level of TGF-β, the key cytokine for this T cell subpopulation. The inhibitory effect of exogenous melatonin was due to its interaction with the membrane receptors MT1 and MT2. At the same time, the signals realized through RORα-the nuclear receptor for melatonin-stimulated Treg formation; however, they were considerably weaker than the signals from the membrane receptors and were overlapped by the latter. Since the Treg subpopulation plays an important role in physiological and pathological processes in the body, the revealed effects of melatonin should be taken into account in its therapeutic use.

Unregulated antigen-presenting cell activation by T cells breaks self tolerance

T cells proliferate vigorously following acute depletion of CD4+ Foxp3+ T regulatory cells [natural Tregs (nTregs)] and also when naive T cells are transferred to syngeneic, nTreg-deficient Rag1-/- hosts. Here, using mice raised in an antigen-free (AF) environment, we show that proliferation in these two situations is directed to self ligands rather than food or commensal antigens. In both situations, the absence of nTregs elevates B7 expression on host dendritic cells (DCs) and enables a small subset of naive CD4 T cells with high self affinity to respond overtly to host DCs: bidirectional T/DC interaction ensues, leading to progressive DC activation and reciprocal strong proliferation of T cells accompanied by peripheral Treg (pTreg) formation. Likewise, high-affinity CD4 T cells proliferate vigorously and form pTregs when cultured with autologous DCs in vitro in the absence of nTregs: this anti-self response is MHCII/peptide dependent and elicited by the raised level of B7 on cultured DCs. The data support a model in which self tolerance is imposed via modulation of CD28 signaling and explains the pathological effects of superagonistic CD28 antibodies.

Upregulation of CD81 in trophoblasts induces an imbalance of Treg/Th17 cells by promoting IL-6 expression in preeclampsia.

The disturbance of maternal immune tolerance to a semiallogeneic fetus is recognized as one of the key pathologies of preeclampsia (PE), in which an imbalance between the inflammation-limiting regulatory T cells (Tregs) and the inflammation-mediating Th17 cells plays an essential role. Previously, we reported that the abnormal upregulation of tetraspannin CD81 in trophoblast cells (fetal component) participated in the pathogenesis of PE. However, as one of the potential immune regulatory molecules, whether CD81 induces PE by interfering with the balance of the maternal immune system has not yet been clarified. Thus, we investigated the relationship between the upregulation of CD81 in trophoblast cells and the imbalance of Treg and Th17 cells in mothers. Here, we demonstrated that upregulation of CD81 in trophoblast cells was accompanied by a decrease in Treg cells and an increase in Th17 cells in both the basal plate (placental maternal side) and peripheral blood of patients with PE. In vitro culture of naïve T cells with medium from the CD81-overexpressing trophoblast cell line HTR-8 resulted in enhanced differentiation of T cells into Th17 cells and decreased the formation of Tregs, which was dependent on the paracrine signaling of IL-6 in trophocytes, induced by CD81. In a CD81-induced PE rat model, we found a significant shift of T cell differentiation towards Th17 cells, and administration of IL-6 antibody mitigated the PE phenotype and the imbalance of the Treg/Th17 cells. These results define a vital regulatory cascade involving trophocyte-derived CD81, IL-6, and maternal Treg/Th17 cells in the pathogenesis of PE and suggests new therapeutic approaches based on CD81 and IL-6 downregulation to prevent human PE.

Mechanisms underlying immune tolerance caused by recombinant Echinococcus granulosus antigens Eg mMDH and Eg10 in dendritic cells.

Mice immunized with recombinant Echinococcus granulosus antigens Eg10 and Eg mMDH do not show elevated resistance to E. granulosus infection but show aggravated infection instead. To gain a deeper insight in the immune tolerance mechanisms in mice immunized with Eg10 and Eg mMDH, this study simulated the immune tolerance process in vitro by culturing bone marrow-derived dendritic cells (BMDCs) in the presence of Eg10 or Eg mMDH. Scanning electron microscopy revealed that Eg10- and Eg mMDH-treated DCs exhibited immature cell morphology, while addition of LPS to the cells induced changes in cell morphology and an increase in the number of cell-surface protrusions. This observation was consistent with the increased expression of the cell-surface molecules MHCII and CD80 in Eg10- and Eg mMDH-treated DCs pretreated with LPS. DCs exposed to the two antigens had a very weak ability to induce T-cell proliferation, but could promote the formation of Treg cells. Introduction of the indoleamine 2,3-dioxygenase (IDO) inhibitor, 1-methyl tryptopha (1-MT) enhanced the ability of the antigens to induce T cells and inhibited the induction of Treg cells. Eg mMDH-treated DCs showed a strong response to 1-MT: the DCs had high mRNA levels of IDO, IL-6, and IL-10, while 1-MT decreased the expression. In contrast, DCs treated with Eg10 did not show significant changes after 1-MT treatment. Eg mMDH inhibited DC maturation and promoted IDO expression, which, on the one hand, decreased the ability of DCs to induce T-cell proliferation, resulting in T-cell anergy, and on the other hand, induced the formation of Tregs, resulting in an immunosuppressive effect. In contrast, the escape mechanisms induced by Eg10 did not primarily depend on the IDO pathway and might involve other mechanisms that need to be further explored.

What We're Reading

APLNR model (from PDB structure 5VBL)A two-cell genome-wide CRISPR deletion screen of melanoma was established that detected genes whose loss made tumors resistant to T cells. Loss of the G protein–coupled receptor APLNR, which interacts with JAK1 and modulates responsiveness to IFNγ, was found to make tumor cells more resistant to T-cell antitumor responses.Patel SJ, …, Restifo NP. Nature 2017 Aug 31; 548:537–42.Mismatch repair is a neoantigen generator (from Xiao & Freeman, Cancer Discovery 2015)Patients with mismatch-repair deficiencies in 24 different tumor types were assessed for responses to anti–PD-1 treatment. Tumors across multiple cancer types were sensitive to blockade therapy: 53% of patients experienced regression, with 21% complete responses. Tumor-specific T cells and clonotypes increased during these responses, affirming this deficiency as a biomarker predicting responsiveness independent of tumor type.Le DT, …, Diaz LA Jr. Science 2017 Jul 28; 357:409–13.Ovarian cancer (from Konecny et al. Clin Cancer Res 2011)Multiple tumor sites in a patient with high-grade serous ovarian cancer were sampled during debulking surgery after a treatment-free period. Progressing tumors excluded immune cells, whereas regressing and stable tumors were infiltrated with local, oligoclonally expanded T cells. Thus, multiple tumor immune microenvironments can coexist within the same patient, perhaps explaining the often heterogenous responses of metastatic lesions.Jiménez-Sánchez, A, …, Miller ML. Cell 2017 Aug 24; 170:927–38.DCs in micrometastatic liver (from Fig. 1)A particular type of dendritic cell (DC) (CD11b+CD11c+MHC-II+CD24+CD64lowF4/80low) was found early in sites of metastatic pancreatic cancer that supported the development of Tregs and the suppression of CD8+ T cells, which encouraged metastatic growth. These DCs expressed MGL2 and PD-L2, through which this subset could be targeted or blocked to reduce Treg formation and promote activation of CD8+ T cells, respectively.Kenkel JA, …, Engleman EG. Cancer Res 2017 Aug; 77:4158–70.Evading detection in Italy (by Mentnafunangann via Wikimedia Commons)Exposure to TGFβ in vitro or in vivo can convert natural killer (NK) cells into ILC1s and intILC1s, which, unlike NK cells, were unable to control local tumor growth and metastasis. The ILC1s produce more TNF and less IFNγ than NK cells, which interferes with antitumor activity and is responsible, in part, for the reduced innate immunosurveillance mediated by TGFβ.Gao Y, …, Smyth MJ. Nat Immunol 2017 Sep; 18:1004–15.Lymph node flow (from Sunshineconnelly via Wikimedias Commons)Lymph-node antigen concentration is greatest near sinuses and lesser in the paracortex. DC2s, located in the antigen-rich regions, present antigen to CD4+ T cells, whereas DC1s, located in antigen-scarcer paracortical regions, present antigen to CD8+ T cells. Thus, the localization of the distinct DC subsets and nature of antigen dispersal makes CD8 responses more sensitive than CD4 responses to even modest reductions in antigen.Gerner MY, …, Germain RN. J Exp Med 2017 Aug 28. doi: 10.1084/jem.20170335.

A distinct subset of plasmacytoid dendritic cells induces activation and differentiation of B and T lymphocytes

Plasmacytoid dendritic cells (pDCs) are known mainly for their secretion of type I IFN upon viral encounter. We describe a CD2hiCD5+CD81+ pDC subset, distinguished by prominent dendrites and a mature phenotype, in human blood, bone marrow, and tonsil, which can be generated from CD34+ progenitors. These CD2hiCD5+CD81+ cells express classical pDC markers, as well as the toll-like receptors that enable conventional pDCs to respond to viral infection. However, their gene expression profile is distinct, and they produce little or no type I IFN upon stimulation with CpG oligonucleotides, likely due to their diminished expression of IFN regulatory factor 7. A similar population of CD5+CD81+ pDCs is present in mice and also does not produce type I IFN after CpG stimulation. In contrast to conventional CD5-CD81- pDCs, human CD5+CD81+ pDCs are potent stimulators of B-cell activation and antibody production and strong inducers of T-cell proliferation and Treg formation. These findings reveal the presence of a discrete pDC population that does not produce type I IFN and yet mediates important immune functions previously attributed to all pDCs.

Synthetic RORγ agonists regulate multiple pathways to enhance antitumor immunity

ABSTRACTRORγt is the key transcription factor controlling the development and function of CD4+ Th17 and CD8+ Tc17 cells. Across a range of human tumors, about 15% of the CD4+ T cell fraction in tumor-infiltrating lymphocytes are RORγ+ cells. To evaluate the role of RORγ in antitumor immunity, we have identified synthetic, small molecule agonists that selectively activate RORγ to a greater extent than the endogenous agonist desmosterol. These RORγ agonists enhance effector function of Type 17 cells by increasing the production of cytokines/chemokines such as IL-17A and GM-CSF, augmenting expression of co-stimulatory receptors like CD137, CD226, and improving survival and cytotoxic activity. RORγ agonists also attenuate immunosuppressive mechanisms by curtailing Treg formation, diminishing CD39 and CD73 expression, and decreasing levels of co-inhibitory receptors including PD-1 and TIGIT on tumor-reactive lymphocytes. The effects of RORγ agonists were not observed in RORγ−/− T cells, underscoring the selective on-target activity of the compounds. In vitro treatment of tumor-specific T cells with RORγ agonists, followed by adoptive transfer to tumor-bearing mice is highly effective at controlling tumor growth while improving T cell survival and maintaining enhanced IL-17A and reduced PD-1 in vivo. The in vitro effects of RORγ agonists translate into single agent, immune system-dependent, antitumor efficacy when compounds are administered orally in syngeneic tumor models. RORγ agonists integrate multiple antitumor mechanisms into a single therapeutic that both increases immune activation and decreases immune suppression resulting in robust inhibition of tumor growth. Thus, RORγ agonists represent a novel immunotherapy approach for cancer.

Learning from cancer: Using D-lactate for therapeutic immunosuppression

Abstract Accumulation of lactic acid in the tumor microenvironment contributes to local immunosuppressive conditions that weaken anti-tumor immunity. In mammals including humans, lactate is present almost entirely as the L-lactate optical isomer. We hypothesized that D-lactate could have similar immune modulatory effects that maybe exploited for therapeutic immunosuppression. 5–40 mM of added sodium D- and L-lactate caused marked impairment of murine, and human, CD4 and CD8 T cell proliferation in vitro, with stronger effects of D-lactate than L-lactate. Neither D- nor L-lactate affected cell viability or apoptosis. Adding 20 mM D-lactate to CD4+ Foxp3− T cells under polarizing conditions increased Foxp3+ induced regulatory T cell (iTreg) formation by 46.2 ±31.5% (P<0.03, n=4). L-lactate also augmented Treg induction but to a lesser degree. Next, we adoptively transferred 1×106 conventional T cells, with or without 1.25×105 Tregs, into Rag1−/− mice, treated each group with 90 mmol/kg/d D-lactate or NaCl for 5 days. After 7 days, T cell proliferation was reduced by 62.1% using D-lactate treatment (P<0.03, n=8). While Treg suppressive function was unaffected, de-novo Foxp3+ Treg formation was increased in D-lactate (14.9 ±3.4%) vs. NaCl (10.6 ±2.3%) treated animals (P<0.01, n=8). Adding D-lactate to proliferating T cells increased oxygen consumption rates compared to NaCl controls, by 56.1 ±35.1% (p<0.05, n=4). In contrast, methyl-esters of L- and D-lactate resistant to either lactate dehydrogenase isoform did not reduce T cell homeostatic proliferation. Our data show that D-lactate has in vivo immunosuppressive effects and can induce de-novo iTregs. The process of lactate oxidation is likely required for its immune modulatory effects.

The contribution of the HSCs niche to bone marrow failure in the IL-2 deficient model

Abstract Bone marrow failure (BMF) is a devastating disease where regeneration of peripheral blood lineages is compromised, resulting in anemia and mortality. In IL-2 KO BALB/c mice, the balance between early myeloid and lymphoid progenitor cell populations is skewed resulting in insufficient myelopoiesis with accumulation of CD8 T cells in the BM. It is widely accepted that crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other’s behavior. However, it remains unknown how the BM niche in this mouse model contribute to the BMF. Multipotent stromal cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs) are the three types of BM stromal cells (BMSCs) that may serve as the distinct functional niche to maintain HSCs. Repots have shown the interplay between CD8 T cells and MSCs cause a change in myelopoiesis and that MSCs promote the formation of Tregs. Our data also indicates that IL-2 KO CD8 T cells are important for the expansion of CD4 T cells in the bone. Since IL-2 KO bones are extremely brittle and CD4 T cells produce RANKL, the ligand needed for osteoclast (OC) differentiation, we evaluated the frequency of BMSCs and OCs. Our preliminary data shows clear differences in BMSCs profiles of IL-2 KO mice, namely elevated stromal cell numbers and a shift in Sca-1 expression in ECs and CD51 expression in OBs. Additionally, OC precursor frequency is elevated in the BM, indicating an increase in bone resorption. The ability of the different subsets of the BMSCs to support the differentiation of HSCs in vitro and the role of CD8 T cells in promoting these changes in vivo is being currently evaluated. Overall, our preliminary data suggests that the change in the bone microenvironment may contribute to the BMF observed in these mice.

Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity.

Regulatory T cells (Tregs) expand in peripheral lymphoid organs and can produce immunosuppressive cytokines to support tumor growth. IL-10 abrogation efficiently induces Treg formation but dampens tumoral neuropilin-1 (Nrp-1) Treg signaling, which simultaneously augments Th1 and Th17 immunity. These effects are associated with the plasticity and stability of Tregs and effector T cell functions that can limit tumorigenesis. Within the tumor microenvironment, there appears to be a “mutual antagonism” between immunoenhancement and immunosuppression mechanisms, eventually leading to decreased metastasis. In contrast, tumor progression is paralleled by a reduction in Nrp-1-producing Tregs controlled by the IL-10 and TGF-β1 levels. However, Th1, Th17 and Treg immunity is primarily regulated by IL-10 or Nrp-1 and not TGF-β1 except when combined with IL-10. These results emphasize the important implications for the therapeutic use of Tregs. The number of Treg cells must be maintained in a healthy and dynamic homeostatic range to prevent malignant diseases. Moreover, Treg-mediated immunosuppression can be limited by reducing tumor-derived Treg Nrp-1 levels.

HDAC5 controls the functions of Foxp3(+) T-regulatory and CD8(+) T cells.

Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.

Butyrate and Mucosal Inflammation: New Scientific Evidence Supports Clinical Observation

Butyrate and Mucosal Inflammation: New Scientific Evidence Supports Clinical Observation

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide.

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4+CD25- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25+ cells prior to immunization, allowing the in vivo formation of Tregs from CD4+CD25- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4+CD25+ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4+CD25- T cells.

Distinct modes of antigen presentation promote the formation, differentiation, and activity of foxp3+ regulatory T cells in vivo.

How the formation and activity of CD4(+)Foxp3(+) regulatory T cells (Tregs) are shaped by TCR recognition of the diverse array of peptide:MHC complexes that can be generated from self-antigens and/or foreign Ags in vivo remains poorly understood. We show that a self-peptide with low (but not high) stimulatory potency promotes thymic Treg formation and can induce conventional CD4(+) T cells in the periphery to become Tregs that express different levels of the transcription factor Helios according to anatomical location. When Tregs generated in response to this self-peptide subsequently encountered the same peptide derived instead from influenza virus in the lung-draining lymph nodes of infected mice, they proliferated, acquired a T-bet(+)CXCR3(+) phenotype, and suppressed the antiviral effector T cell response in the lungs. However, these self-antigen-selected Tregs were unable to suppress the antiviral immune response based on recognition of the peptide as a self-antigen rather than a viral Ag. Notably, when expressed in a more immunostimulatory form, the self-peptide inhibited the formation of T-bet(+)CXCR3(+) Tregs in response to viral Ag, and Ag-expressing B cells from these mice induced Treg division without upregulation of CXCR3. These studies show that a weakly immunostimulatory self-peptide can induce thymic and peripheral Foxp3(+) Treg formation but is unable to activate self-antigen-selected Tregs to modulate an antiviral immune response. Moreover, a strongly immunostimulatory self-peptide expressed by B cells induced Tregs to proliferate without acquiring an effector phenotype that allows trafficking from the draining lymph node to the lungs and, thereby, prevented the Tregs from suppressing the antiviral immune response.

Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4(+) CD25(hi) FoxP3(+) T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-β1 mediates induction of Tregs. TGF-β1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-β1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-β1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC.

Vitamin A is not associated with exacerbations in multiple sclerosis

BackgroundVitamin A is a multifunctional vitamin that can inhibit the formation of Th17 cells, which are probably involved in the development of relapses in MS. Furthermore, it promotes Treg formation. Therefore, vitamin A can be hypothesized to be lower in patients than in healthy controls, and to decrease relapse risk in relapsing–remitting MS (RRMS) patients. ObjectiveTo compare vitamin A levels in MS patients and controls, and to investigate whether vitamin A levels are associated with relapse risk. MethodsIn a case-control study all-trans-retinol levels were compared between 31 RRMS patients and 29 matched controls.In a prospective longitudinal study in 73 RRMS patients, serum samples for all-trans-retinol measurements were taken every eight weeks. Associations between all-trans-retinol concentrations and relapse rates were calculated using Poisson regression with the individual serum levels as time-dependent variable. Associations between vitamin A and vitamin D were calculated. ResultsMean vitamin A levels were lower in patients (2.16μmol/l) than in controls (2.44μmol/l) but with borderline significance (p=0.05). In the longitudinal study, during follow-up (mean 1.7 years), 58 patients experienced a total of 139 relapses. Monthly moving averages of all-trans retinol levels were categorized into tertiles: a low (<2.9μmol/l), medium (2.9–3.7μmol/l) and high level (>3.7μmol/l). Relapse rates were not associated with serum all-trans retinol levels (p>0.2), in univariate nor in multivariate analysis.Serum concentrations of all-trans-retinol and 25-OH-vitamin D were positively correlated, although this correlation was weak (r=0.15). ConclusionWe did not find evidence for a role for vitamin A in the disease course of RRMS. We did find an association between vitamin A and D levels in the RRMS patients, possibly explained by dietary products that contain both fat-soluble vitamins.

Dendritic Cell–Dependent In Vivo Generation of Autoregulatory T Cells by Antidiabetogenic MHC Class II

Several mechanisms have been proposed to explain how certain MHC class II molecules afford dominant resistance to autoimmune diseases like type 1 diabetes (T1D). However, it remains unclear how protective MHC types can blunt autoreactive T cell responses directed against a diverse repertoire of autoantigenic epitopes presented by disease-promoting MHCs. In this study, we show that expression of I-E on dendritic cells (DCs) of NOD mice promotes the differentiation of MHC promiscuous autoreactive CD4(+) clonotypes into antidiabetogenic autoregulatory T cells. We expressed an I-Eα(kloxP) transgene in NOD mice and used cell type-specific I-E ablation to show that I-E-expressing DCs, but not B cells, promote the generation of autoreactive CD4(+)Foxp3(+) regulatory T cells (Tregs) and their accumulation in the pancreas-draining lymph nodes. There, these Tregs suppress the presentation of β cell Ags to naive autoreactive CD4(+) and CD8(+) T cells restricted by diabetogenic MHC molecules in an I-E-independent manner. Whereas selective removal of I-E on DCs abrogated autoregulatory Treg formation and T1D protection, selective removal of I-E on B cells was inconsequential. These results explain how certain MHC class II molecules can completely suppress antigenically complex autoimmune responses in an Ag-nonspecific manner.

Reishi Protein LZ-8 Induces FOXP3+Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice

LZ-8, an immunomodulatory protein isolated from Ganoderma lucidum (also known as Ling-Zhi or Reishi), has been shown to promote cell proliferation and IL-2 production in T cells. In this study, we show that LZ-8 induces the expansion of both murine and human CD4+ T cells into FOXP3+ regulatory T (Treg) cells. LZ-8 treatment was found to stimulate a 4-fold and a 10-fold expansion in the Treg populations of murine and human primary CD4+ T cells, respectively. In addition, the expression of CTLA-4 and IL-10 was induced in LZ-8-treated CD4+ T cells. Using neutralizing antibodies and gene-deficient T-cell lines, we also found that LZ-8 promotes Treg expansion through a CD45-mediated signaling pathway and that the CD18-dependent induction of IL-2 was involved in Treg formation and IL-10 production. The suppressive activity of LZ-8 was confirmed using a murine model of DSS-induced colitis; the disease was alleviated by the adoptive transfer of LZ-8-treated CD4+ T cells. In conclusion, a new regulatory function for LZ-8 was identified, and the molecular mechanisms underlying this function were elucidated.