Ginsenoside Rg3 Alleviates Complete Freund's Adjuvant-Induced Rheumatoid Arthritis in Mice by Regulating CD4+CD25+Foxp3+Treg Cells.

Ginsenoside Rg3 (GRg3) is one of the major bioactive ingredients of ginseng, which is not only used as a herbal medicine but also used as a functional food to support body functions. In this study, the beneficial effects of GRg3 on rheumatoid arthritis (RA) mice was evaluated from anti-inflammatory and immunosuppressive aspects. The footpad swelling rate, pathological changes of the ankle joint, and levels of tumor necrosis factor α, interleukin 6, interleukin 10, and tumor necrosis factor ß were used to assess the anti-inflammatory effect of GRg3 on RA mice. Flow cytometric analysis of CD4+CD25+Foxp3+Treg cell percentage and metabolomic analysis based on gas chromatography-tandem mass spectrometry were used to assess the immunosuppressive effect and underlying mechanisms. GRg3 exhibited anti-inflammatory and immunosuppressive effects on RA mice. The potential mechanisms were related to regulate the pathways of oxidative phosphorylation and enhance the function of CD4+CD25+Foxp3+Treg cells to maintain peripheral immune tolerance of RA mice. These findings can provide a preliminary experimental basis to exploit GRg3 as a functional food or an effective complementary for the adjuvant therapy of RA.

Combined CD44- and CD25-Targeted Near-Infrared Photoimmunotherapy Selectively Kills Cancer and Regulatory T Cells in Syngeneic Mouse Cancer Models.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a mAb conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is a surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, CD25-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Treg), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44- and CD25-targeted NIR-PIT also resulted in some complete remissions. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.

The Effect of Antibody Fragments on CD25 Targeted Regulatory T Cell Near-Infrared Photoimmunotherapy.

Regulatory T (Treg) cells play a major role in immune suppression permitting tumors to evade immune surveillance. Depletion of intratumoral Treg cells can result in tumor regression. However, systemic depletion of Tregs may also induce autoimmune adverse events. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cell-specific cancer therapy that locally kills specific cells in the tumor. Antibody-photoabsorber (IRDye700DX) conjugates (APC) are injected and bind to the tumor, and subsequent administration of NIR light to the tumor results in rapid cell death only in targeted cells. CD25-targeted NIR-PIT has been shown to induce spatially selective depletion of tumor-associated Treg cells. In this study, we compared the efficacy of an antibody fragment, anti-CD25-F(ab')2, and a full antibody, anti-CD25-IgG, as agents for NIR-PIT. Tumor-bearing mice were divided into four groups: (1) no treatment; (2) anti-CD25-IgG-IR700 i.v. only; (3) anti-CD25-F(ab')2-IR700 i.v. with NIR light exposure; and (4) anti-CD25-IgG-IR700 i.v. with NIR light exposure. Although both CD25-targeted NIR-PITs resulted in significant tumor growth inhibition, the anti-CD25-F(ab')2-IR700 based NIR-PIT was superior to the anti-CD25-IgG-IR700 NIR-PIT. The anti-CD25-F(ab')2-IR700 demonstrated faster clearance from the body than the anti-CD25-IgG-IR700. Sustained circulation of anti-CD25-IgG-IR700 may block IL-2 binding on the activated effector T-cells decreasing immune response. In conclusion, anti-CD25-F(ab')2 based NIR-PIT was more effective in reducing tumor growth than anti-CD25-IgG based NIR-PIT. Absence of the Fc portion of the APC leads to faster clearance and therefore promotes a superior activated T cell response in tumors.

Polysaccharides from Hedyotis diffusa enhance the antitumor activities of cytokine-induced killer cells.

Hedyotis diffusa is a well-known traditional Chinese herbal medicine. The polysaccharides extracted from H. diffusa (HDP) exhibit a range of pharmacological activities. Transfusion of cytokine-induced killer (CIK) cell is one type of adoptive cellular immunotherapy, which is becoming an important method of cancer immunotherapy. In this present study, we investigate the immunostimulatory effect of HDP on CIK cells. CIK cells were generated by culturing and stimulating peripheral blood monocytes of healthy volunteers. They were treated with HDP at three different concentrations (10, 50, and 100 µg/mL). The effect of HDP on CIK cell populations, intracellular cytokine production, and apoptosis was examined by flow cytometry. The antitumor effect of HDP on CIK cells was determined by cytotoxicity assay. Furthermore, the effect of HDP on the antitumor activity of CIK cells in a mouse model was investigated. HDP increased the percentage of CD3+CD56+ CIK cells but did not significantly change the percentage of CD4+, CD8+, or CD4+CD25+ CIK cells. The HDP-treated CIK cells showed a greater ability to kill tumor cells, as well as higher production of interferon-γ and tumor necrosis factor-α, compared with the no-HDP-treated CIK cells. The HDP-treated CIK cells also found a lower apoptosis level in vitro. Moreover, HDP combined with CIK cells had a stronger inhibitory effect on tumor growth in the mouse model compared with the CIK or HDP treatment alone. In conclusion, the results indicated that HDP enhanced the antitumor activity of CIK cells and could be used for cancer immunotherapy combined with CIK cell therapy.

Vitamin D3 supplementation and the IL-2/IL-2R pathway in multiple sclerosis: Attenuation of progressive disturbances?

Vitamin D3 upregulates IL-2 receptor alpha (IL2RA, CD25)-expression on CD4+ T cells in vitro. We investigated effects of 48-weeks vitamin D3 supplements on CD25-expression by CD4+ T cells of patients with multiple sclerosis (MS). There was no significant difference between the vitamin D3 (n=30) and placebo group (n=23) in IL2RA mRNA-expression by PBMC. Likewise, CD25 cell surface-expression by conventional or regulatory T cells (Treg) did not differ between groups, although Treg CD25-expression and circulating soluble-CD25 levels decreased significantly in the placebo but not vitamin D3-group. We speculate that vitamin D3 may promote the maintenance of CD25-related immune homeostasis in MS.

Decreased Levels of Spleen Tissue CD4+CD25+Foxp3+ Regulatory T Lymphocytes in Mice Exposed to Berberine.

The effects of isoquinoline alkaloid berberine (BER) on spleen tissue CD4+CD25+Foxp3+ regulatory T (Treg) cells were evaluated in BALB/c mice. Here, BER was administered daily by intraperitoneal injection at doses of 5 mg/kg and 10 mg/kg for 14 days. Following the exposure, mice spleen cellularities, IL-10 production by splenocytes, and spleen Treg/CD4+ cell profiles were studied in all the test groups of animals. The results showed that a high dose of BER (10 mg/kg) could decrease both the absolute and relative percentages of spleen Treg cells as well as decrease the production of IL-10 by splenocytes in the treated mice (p<0.05). BER at 5 mg/kg did not appear to affect any of these parameters. Based on the finding here, it would seem that BER has effective immunostimulatory properties, which contradicts the results from other studies indicating immunosuppressive effects of BER. Depending on the doses of BER used, it might have a broad spectrum from immunosuppressive to stimulatory effects. Further studies, including more doses, are required to better evaluate the effects of this natural product. Mechanistic studies are required, particularly in case of redox state of the immune cells, to elucidate and determine how BER functions to impart the toxicity effects demonstrated here and in other studies.

An Oriental Medicine, Hyungbangpaedok-San Attenuates Motor Paralysis in an Experimental Model of Multiple Sclerosis by Regulating the T Cell Response.

The preventive and therapeutic mechanisms in multiple sclerosis are not clearly understood. We investigated whether Hyungbangpaedok-san (HBPDS), a traditional herbal medicine, has a beneficial effect in experimental autoimmune encephalomyelitis (EAE) mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG 35-55). Onset-treatment with 4 types of HBPDS (extracted using distilled water and 30%/70%/100% ethanol as the solvent) alleviated neurological signs, and HBPDS extracted within 30% ethanol (henceforth called HBPDS) was more effective. Onset-treatment with HBPDS reduced demyelination and the recruitment/infiltration and activation of microglia/macrophages in the spinal cord of EAE mice, which corresponded to the reduced mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), iNOS, and chemokines (MCP-1, MIP-1α, and RANTES) in the spinal cord. Onset-treatment with HBPDS inhibited changes in the components of the blood-brain barrier such as astrocytes, adhesion molecules (ICAM-1 and VCAM-1), and junctional molecules (claudin-3, claudin-5, and zona occludens-1) in the spinal cord of EAE mice. Onset-treatment with HBPDS reduced the elevated population of CD4+, CD4+/IFN-γ+, and CD4+/IL-17+ T cells in the spinal cord of EAE mice but it further increased the elevated population of CD4+/CD25+/Foxp3+ and CD4+/Foxp3+/Helios+ T cells. Pre-, onset-, post-, but not peak-treatment, with HBPDS had a beneficial effect on behavioral impairment in EAE mice. Taken together, HBPDS could alleviate the development/progression of EAE by regulating the recruitment/infiltration and activation of microglia and peripheral immune cells (macrophages, Th1, Th17, and Treg cells) in the spinal cord. These findings could help to develop protective strategies using HBPDS in the treatment of autoimmune disorders including multiple sclerosis.

Astragaloside IV attenuates allergic inflammation by regulation Th1/Th2 cytokine and enhancement CD4(+)CD25(+)Foxp3 T cells in ovalbumin-induced asthma.

Astragaloside IV is the chief ingredient of Radix Astragali, which has been used in the Traditional Chinese Medicine as a major component of many polyherbal formulations for the repair and regeneration of injured organ and tissues. We tested the anti-asthmatic effects of AST IV and the possible mechanisms. BALB/c mice that were sensitized and challenged to ovalbumin (OVA) were treated with AST IV (40mg/kg and 20mg/kg) 1h before they were challenged with OVA. Our study demonstrated that AST IV inhibited OVA-induced increases in eosinophil count; interleukin (IL)-4 level were recovered in bronchoalveolar lavage fluid increased IFN-γ and IL-10 levels in bronchoalveolar lavage fluid. Histological studies demonstrated that AST IV substantially inhibited OVA-induced eosinophilia in lung tissue. Flow cytometry studies demonstrated that AST IV substantially increased CD4(+)CD25(+)Foxp3 T cells (Treg). Furthermore quantitative real-time (qPCR) studies demonstrated that AST IV substantially enhanced Foxp3 mRNA expression in lung tissue. These findings suggest that AST IV may effectively ameliorate the progression of airway inflammation and could be used as a therapy for patients with allergic inflammation.

Effect of narrow band-ultraviolet B on CD4(+) CD25(high) FoxP3(+) T-lymphocytes in the peripheral blood of vitiligo patients.

BACKGROUND: It is widely believed that an imbalance between activated CD8(+) T cells and regulatory T cells (Tregs) exists in patients with vitiligo. Although there is evidence that narrow band ultraviolet (NB-UVB) irradiation can induce Tregs' number and activity, but up to our knowledge, none of the published studies involved the possible effect of NB-UVB on Tregs in vitiligo. OBJECTIVE: To evaluate the effect of NB-UVB on circulating CD4(+) CD25(high) FoxP3(+) regulatory T cells (FoxP3(+) Tregs) in vitiligo. METHODS: This prospective analytic study included 20 patients with active non-segmental vitiligo and 20 healthy controls. The patients were exposed to NB-UVB therapy three times per week for 30 sessions. Blood sampling before and after NB-UVB phototherapy was done to evaluate circulating CD4(+) CD25(high) Tregs and Foxp3(+) Tregs. RESULTS: The CD4(+) CD25(high) Tregs% and FoxP3(+) Tregs% were significantly higher in vitiligo patients compared with controls. NB-UVB therapy decreased both of them in patients, but they did not reach those of controls. Each of circulating CD4(+) CD25(high) Tregs% and FoxP3(+) Tregs% didn't correlate with either extent or activity of vitiligo before or after NB-UVB. CONCLUSION: Tregs functional defect is probably having an impact on NSV. NB-UVB may improve the function of Tregs. Understanding the mechanisms through which NB-UVB exert its effect on reducing the number of circulating Tregs would help open up the paths for future therapeutic options.

Codonopsis pilosula polysaccharide attenuates cecal ligation and puncture sepsis via circuiting regulatory T cells in mice.

BACKGROUND: Codonopsis pilosula polysaccharide (CPPS) isolated from one of the Chinese herbs is known to have a variety of immunomodulatory activities. However, it is not clear whether CPPS can exert an effect on the immune functions of regulatory T cells (Tregs). This study was carried out to investigate the effect of CPPS on the immune function of peripheral blood Tregs in sepsis induced by cecal ligation and puncture (CLP). METHODOLOGY AND PRINCIPAL FINDINGS: BALB/c mice were randomly divided into five groups: sham group, CLP group, CLP with CPPS (40, 100, and 250 mg/kg) treatment group, and they were killed on days 1, 2, 3, and 4 after CLP, respectively, with eight animals at each time point. Magnetic microbeads were used to isolate peripheral blood Tregs and CD4 T cells. Phenotypes of Tregs, such as Toll-like receptor 4 (TLR4) and Foxp3, were analyzed by flow cytometry, and coculture medium cytokines levels were determined with enzyme-linked immunosorbent assay. The levels of TLR4 and the expression of Foxp3 in the Treg from CLP group were markedly increased in comparison to the sham group. Administration of CPPS could significantly decrease the TLR4 level and inhibited the expression of Foxp3 on Tregs in sepsis mice. At the same time, proliferative activity and expression of interleukin 2 and interleukin 2Rα on CD4 T cells were restored. In contrast, anti-TLR4 antibody could block the effect of CPPS on Treg immune function. CONCLUSIONS: Codonopsis pilosula polysaccharide might suppress excessive Tregs, at least in part, via TLR4 signaling on Tregs and trigger a shift of TH2 to TH1 with activation of CD4 T cells in sepsis induced by CLP.

The clinical and immunological significance of GAD-specific autoantibody and T-cell responses in type 1 diabetes.

Antigen-specific interventions are desirable approaches in Type 1 Diabetes (T1D) as they can alter islet-specific autoimmunity without systemic side effects. Glutamic acid decarboxylase of 65 kDa (GAD65) is a major autoantigen in type 1 diabetes (T1D) and GAD-specific autoimmunity is a common feature of T1D in humans but also in mouse models of the disease. In humans, administration of the GAD65 protein in an alum formulation has been shown to reduce C-peptide decline in recently diagnosed patients, however, these observations were not confirmed in subsequent phase II/III clinical trials. As GAD-based immune interventions in different formulations have successfully been employed to prevent the establishment of T1D in mouse models of T1D, we sought to analyze the efficacy of GAD-alum treatment and the effects on the GAD-specific immune response in two different mouse models of T1D. Consistent with the latest clinical trials, mice treated with GAD-alum were not protected from diabetes, although GAD-alum induced a GAD-specific Th2-deviated immune response in transgenic rat insulin promoter-glycoprotein (RIP-GP) mice. These observations underline the importance of a thorough, preclinical evaluation of potential drugs before the initiation of clinical trials.

Immunoregulatory effects of glycyrrhizic acid exerts anti-asthmatic effects via modulation of Th1/Th2 cytokines and enhancement of CD4(+)CD25(+)Foxp3+ regulatory T cells in ovalbumin-sensitized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhizic acid (GA) is the main bioactive ingredient of licorice (Glycyrrhiza glabra), and has been found to be associated with multiple therapeutic properties. AIM OF THE STUDY: In this study, we investigated immunoregulatory effects of glycyrrhizic acid on anti-asthmatic effects and underlying mechanisms. MATERIALS AND METHODS: Asthma model was established by ovalbumin-induced. A total of 60 mice were randomly assigned to six experimental groups: control, model, dexamethasone (2 mg/kg) and GA (10 mg/kg, 20 mg/kg, 40 mg/kg). Airway resistance (Raw) were measured by the forced oscillation technique, histological studies were evaluated by The hematoxylin and eosin (HE) staining, Th1/Th2 and Th17 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA), and CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was evaluated by Flow Cytometry (FCM), the forkhead/winged helix transcription factor (Foxp3) was evaluated by western blotting. RESULTS: Our study demonstrated that, compared with model group, GA inhibited OVA-induced increases in Raw and eosinophil count; interleukin (IL)-4, IL-5, IL-13 levels were recovered in bronchoalveolar lavage fluid compared; increased IFN-γ level in bronchoalveolar lavage fluid; histological studies demonstrated that GA substantially inhibited OVA-induced eosinophilia in lung tissue and airway tissue compared with model group. Flow cytometry studies demonstrated that GA substantially enhanced Tregs compared with model group. CONCLUSION: These findings suggest that GA may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.

Beneficial therapeutic effect of Chinese herbal Bushen formula on CHB patients with mildly elevated alanine aminotransferase by down-regulating CD4+CD25+T cells.

ETHNOPHARMACOLOGICAL RELEVANCE: To investigate the clinical effects of the Bushen formula on chronic hepatitis B (CHB) patients with mildly elevated alanine aminotransferase (ALT) (1-2 times ULN) and its relevant immune mechanism. MATERIALS AND METHODS: Fifty CHB patients were enrolled, and they were categorized into two groups according to infection state, including CHB carrier group(24 patients) and mildly elevated ALT (1-2 times ULN) group (26 patients), and 12 healthy volunteers were collected as normal control group. In addition, patients with mildly elevated ALT were taking the Bushen formula for 6 months. Biochemical assessment, viral load quantitation and HBV markers were examined at the Department of Clinical Laboratory, Shanghai Shuguang Hospital. Peripheral blood mononuclear cells were separated from patients and healthy volunteers. T cells were separated from healthy blood, and Hepatitis B core antigen (HBcAg) was added to the culture media. The percentage of CD4+CD25+T cells and the expression levels of IFN-γ and IL-4 were examined by flow cytometry and the expression level of FoxP3 was examined by real-time PCR and flow cytometry. The Chinese medicine symptom complex score was calculated. RESULTS: The percentage of CD4+CD25+T cells and the expression level of Foxp3 in two CHB patient groups were higher than that in the normal control group, and the percentage of CD4+CD25+T cells and the expression level of Foxp3 in the CHB carrier group were highest. The experiment in vitro showed that both the percentage of CD4+CD25+T cells and the expression of Foxp3 were elevated by HBcAg in CD4+T cells separated from normal peripheral blood. In patients with mildly elevated ALT, the serum ALT level was significantly decreased from 81.77±18.24U/L to 64.92±20.60U/L and the serum HBV DNA level was significantly decreased from 6.54±0.95 (log) to 5.15±2.22 (log), and symptom scores were significantly decreased from 6.19±3.99 to 2.77±1.66 after administration of the Bushen formula. In addition, we found that the frequency of CD4+CD25+T cells correlated negatively with the expression level of IFN-γ in CD4+T cells from CHB patients with mildly elevated ALT, and the percentage of CD4+CD25+T cells was decreased from 11.07±4.30% to 8.70±3.49% and the expression level of IFN-γ in CD4+T cells was increased from 13.98±3.25% to 15.85±3.43% after administration of the Bushen formula. CONCLUSION: In CHB patients, the percentage of CD4+CD25+T cells and the expression of Foxp3 in peripheral blood were increased, which can be repeated in HBcAg-stimulated healthy CD4+T cells. The Bushen formula has positive effects on CHB patients with mildly elevated ALT (1-2 times ULN) by reducing serum ALT and HBV DNA levels, which is relevant with the decreasing of the percentage of CD4+CD25+T cells and the increasing of the expression level of IFN-γ in CD4+T cells.

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25- effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.

[Regulation effect of scorpio and scolopendra on CD4 + CD25 + FoxP3 + Treg cell in peripheral blood from rats with collagen-induced arthritis (CIA)].

OBJECTIVE: To study the regulation effect of Scorpio and Scolopendra on CD4 + CD25 + FoxP3 + Treg Cell in peripheral blood from rats with collagen-induced arthritis. METHODS: Sixty female Wistar rats were randomly divided into 6 groups, normal control group, model control group, low-dose, middle-dose and high-dose Scorpio and Scolopendra group,and the type II collagen group. Rats' arthritis was induced by collagen. The number of CD4 + CD25 + FoxP3 + Treg cell in peripheral blood was tested by flow cytometry, and the level of IL-2 in serum was detected by enzyme linked immunosorbent assay. RESULTS: Compared with the model groups,the levels of CD4 + CD25 + Treg cell and CD4 + CD25 + FoxP3 + Treg cell were increased obviously in the high and low dose of Scorpio and Scolopendra groups (P < 0.05 or P < 0.01), while the level of IL-2 in serum was decreased remarkably in the middle and low dose of Scorpio and Scolopendra groups (P < 0.05 or P < 0.01). CONCLUSION: It is realized that Scorpio and Scolopendra effectively treat RA by regulating the level of CD4 + CD25 + FoxP3 + Treg cell to restore immunological tolerance.

Condensed tannins from Botswanan forage plants are effective priming agents of γδ T cells in ruminants.

The potential impact of extracts from forage plants on γδ T cell activity in ruminants was evaluated using an in vitro immunoassay. This study investigated whether plant extracts could prime γδ T cells via up-regulation of CD25 (interleukin-2 receptor alpha). Purified Sephadex LH-20 fractions, isolated from Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius and Grewia flava, were screened against γδ T cells on kid, lamb and calf peripheral blood lymphocytes. Condensed tannins (CT) from G. flava significantly primed γδ T cells in kids up to 64.75% at 10 µg/mL, which was statistically significant relative to the negative control at 22.66% (p=0.004). CT from T. oleifolius also induced priming of γδ T cells in kids, while fractions from V. rotundifolium and V. verrucosum induced minimal priming of γδ T cells. In contrast, there was no significant priming of γδ T cells from lambs and calves for any of the tested fractions (p>0.05). These findings suggest that CT from a selected range of Botswanan forage plants can stimulate the immune system in vivo in selected ruminant species and may participate in enhancing host innate immune responses.

Deferiprone modulates in vitro responses by peripheral blood T cells from control and relapsing-remitting multiple sclerosis subjects.

T cells are important mediators of autoimmune inflammation in relapsing-remitting multiple sclerosis (RRMS). Previous studies found that deferiprone, an iron chelator, suppressed disease activity in a mouse model of multiple sclerosis, and inhibition of T cell proliferation was implicated as a putative mechanism. The objective of the present study was to examine the effects of deferiprone on suppressing in vitro responses of T cells from control and RRMS subjects. Peripheral blood T cells were co-stimulated with anti-CD3+anti-CD28 and cultured with or without interleukin 2 (IL-2). Proliferating CD4+ T cells from control and RRMS subjects, cultured with or without IL-2, decreased in response to 75 µM deferiprone, although the extent of decreased proliferation of CD4+ T cells from RRMS subjects was less than for control subjects. Proliferating CD8+ T cells from control subjects, cultured with or without IL-2, also decreased in response to 75 µM deferiprone, and this decrease was seen in proliferating CD8+ T cells from RRMS cultured with IL-2. CD4+CD25+ and CD8+CD25+ cells from control subjects, cultured with or without IL-2, declined in 75 µM deferiprone, but the decrease was smaller than for the CD4+ and CD8+ proliferative responses. CD4+CD25+ and CD8+CD25+ cells from RRMS subjects showed more variability than for control subjects, but CD4+CD25+ cultured with IL-2 and CD8+CD25+ cells cultured without IL-2 significantly declined in 75 µM deferiprone. CD4+FoxP3+ and CD4+CD25+FoxP3+ cells tended to remain constant or increase. In summary, deferiprone induced declines in proliferative responses at a dosage that is within peak serum pharmacological concentrations.

Monoclonal antibody blockade of IL-2 receptor α during lymphopenia selectively depletes regulatory T cells in mice and humans.

Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (T(Regs); P = .0006). Monoclonal antibody (mAb)-mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted T(Regs) (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased T(Regs) (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti-IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced T(Reg) frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated T(Reg) depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti-IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.

Two different regulatory T cell populations that promote corneal allograft survival.

PURPOSE: To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts. METHODS: Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated. RESULTS: Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID. CONCLUSIONS: Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

Total alkaloids of Sophora alopecuroides increases the expression of CD4+ CD25+ Tregs and IL-10 in rats with experimental colitis.

Previous studies have demonstrated that the total alkaloids of Sophora alopecuroides (TASA), which contains many different ingredients like sophocarpine, matrine, oxymatrine, sophoridine, sophoramine, aloperine and cytosine, were able to protect colon against ulcers caused by 2,4,6-trinitrobenze sulphonic acid (TNBS)/ethanol treated models. In order to elucidate the mechanisms by which TASA exerts its effect of anti-inflammation and immunoregulation on rats with colitis, DAI (disease activity index) and histological grading of colitis were evaluated in the animal model. Moreover, the expression of CD4(+)CD25(+) regulatory T cells (Tregs) and IL-10 in rats with experimental colitis were observed by FCM, ELISA and RT-PCR in this study. Results showed that TASA (15, 30 or 60 mg/kg/day) significantly up-regulated CD4(+)CD25(+)Tregs (P = 0.02, P = 0.02, P = 0.03) and IL-10 levels (ELISA: P = 0.03, P = 0.02, P = 0.00; RT-PCR: P = 0.04, P = 0.02, P = 0.01) respectively and decreased the DAI and histological grading of colitis in the peripheral blood (PB) and colon of rat colitis models (3.44 +/- 1.53, 4.25 +/- 1.27, 4.42 +/- 1.24 and 3.50 +/- 1.42, 4.05 +/- 1.32, 4.51 +/- 1.55 vs. 7.18 +/- 1.32 and 7.38 +/- 1.52, P < 0.05, P < 0.01, respectively). Most interestingly, a negative correlation was demonstrated between the expression of CD4(+)CD25(+) Tregs and DAI (Pearson r(PB) = -0.677, P < 0.01; Pearson r(COLON) = -0.663, P < 0.01, n = 60), or histological grading of colitis (Pearson r(PB) = -0.725, P < 0.01; Pearson r(COLON) = -0.623, P < 0.01, n = 60). Simultaneously, a positive correlation existed between CD4(+)CD25(+) Tregs and IL-10 cytokine (IL-10 mRNA) in the colon and PB of rats (Pearson r(PB) = 0.789, P < 0.01, n = 60; Pearson r(COLON) = 0.678, P < 0.01, n = 60). These results may explain to some extent the mechanisms of TASA on treating rats with experimental colitis.